WO2019067887A1 - Enac inhibitor peptides and uses thereof - Google Patents

Abstract

The disclosure relates to epithelial sodium channel (ENaC) inhibitor compounds and peptides and uses thereof, e.g., in the treatment of a disorder related to sodium channel, such as, for example, cystic fibrosis, idiopathic pulmonary fibrosis, or chronic obstructive pulmonary disease.

Description

ENAC INHIBITOR PEPTIDES AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001 ] This application claims the benefit of U.S. Patent Application No. 62/565,727, filed on September 29, 2017, the entire contents of which are incorporated herein by reference.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on September 27, 2018, is named 102382-0061_SL.txt and is 50,283 bytes in size.

BACKGROUND

[0001] Epithelial mucosal surfaces are lined with fluids whose volume and composition are precisely controlled. In the airways, a thin film of airway surface liquid helps protect mammalian airways from infection by acting as a lubricant for efficient mucus clearance (Hobbs et al, J. Physiol. 591: 4377 (2013), Knowles et al, J. Clin. Invest. 109:571 (2002)). This layer moves cephalad during mucus clearance and excess liquid that accumulates as two airways converge is eliminated by Na'-moduiated airway surface liquid absorption with Na^* passing through the epithelial Na⁺ channel (ENaC) (Hobbs et al, supra: Knowles et al, supra. Critically, the mechanism, by which ENaC activity is regulated in the airways is poorly understood.

[0002] Recently, evidence has been accumulating that molecular regulators in the airway surface liquid can serve as volume sensing signals whose dilution or concentration can alter specific cell surface receptors that control ion transport rates to either absorb or secrete airway surface liquid as needed (Chambers et al, Respir. Physiol NeurohioL 159:256 (2007)). As one of the regulated targets, ENaC must be cleaved by intracellular furin-type proteases and/or extracellular channel activating proteases (CAPs) such as prostasin to be active and to conduct

Na⁺ (Planes et al, Ciirr. Top. Dev. Biol. 78:23 (2007); Rossier, Proc. Am. Thorac. Soc. 1 :4

(2004); Vallet et al, Nature 389:607 (1997); Chraibi et al, J. Gen. Physiol 1 11 : 127 (1998)).

ENaC can also be cleaved and activated by exogenous serine proteases such as trypsin, an action that is attenuated by the protease inhibitor aprotinin (Vallet et al, Nature 389:607

(1997)). When human bronchial epithelial cultures are mounted in Ussing chambers where native airway surface liquid is washed away, ENaC is predominantly active, suggesting that cell attached proteases are predominant (Bridges et al., Am. J. Physiol. Lung Cell. Mai. Physiol. 281 :L16 (2001); Donaldson et al, J Biol. Chem. 277:8338 (2002)). In contrast under thin film conditions, where native airway surface liquid is present, ENaC activity is reduced, suggesting that airway surface liquid contains soluble protease inhibitors (Myerburg et al, J. Biol. Chem. 281 :27942 (2006); Tarran et al, J. Gen. Physiol 127:591 (2006); Gaillard et al, Pfleugers Arch, 460: 1-17, 2010).

[0003] It has been shown that the Short Palate Lung and Nasal epithelial Clone (SPLUNCl) protein comprises up to 10% of the total protein in the airway surface liquid and can readily be detected in both nasal lavage and tracheal secretions (Bingle et al. Hum Mol Genet 11, 937, 2002; Campos et al, Am J Respir Cell Mol Biol 30, 184; Lindahl et al. Electrophoresis 22, 1795, 2001). SPLUNC l appears to be a volume sensing molecule since it can be secreted onto the mucosal surface of the superficial epithelia where ENaC is expressed (Barilett et al, J. Leukoc. Biol 83: 1201 (2008); Bingle et al, J. Pathol 205:491 (2005)). Furthermore, SPLUNCl has been demonstrated to contain a subdomain that functions as an inhibitor of ENaC through its N-terminal domain. SPULUCi fragments, which are described in WO10/138794 (corresponding US Pat. No. 9,549,967), WO13/043720 (corresponding US Pat. No. 9,127,040), and WO16/057795 (corresponding U.S. Pub. No. 2016-0102121), have been demonstrated to promote ENaC internalization.

[0004] There is an unmet need for peptide compounds which are thermally stable, enzymaticaily resistant to proteolysis and which are also biologically active at the target tissue site, for example, epithelial mucosal surfaces lining mammalian airways.

SUMMARY

[0005] This disclosure relate to peptides that mimic the properties of ENaC-reguiating activity of SPLUNC 1 , for example, by binding to ENaC and/or inhibiting ENaC-mediated ion transport to regulate sodium absorption. The peptides are stable, e.g., with regard to thermal stability and enzymatic stability, which makes them suitable for in vivo pharmacological applications even when administered to an environment rich in proteolytic enzymes. The peptides of the disclosure are soluble in aqueous solvents and non-toxic. The peptides are also less likely to interact with excipients compared to the longer SPLUNCl -derived peptides and therefore are easily bioavailabie compared to existing peptide therapies.

[0006] Particularly, described herein are short SPLUNCl peptides that have pharmacological profiles that are comparable, if not better than, SPLUNCl and longer SPLUNC-derived peptides (e.g., 8-mer, 18-mer or 23-mer oligopeptides derived from SPLUNCl). Partly due to their physiochemical characteristics such as size, charge, polarity, etc., the peptides of the disclosure can be specifically delivered in therapeutically effective amounts at targeted tissues, e.g., epithelial mucosal surfaces lining mammalian airways, using traditional delivery devices. For example, the peptides of the disclosure provide certain advantages for delivery to the airway in the fonn of an aerosol e.g., as a dry powder, and for formulation for administration using dry powder inhalers(DPI), metered-dose inhaler (MD1), nebulizers, and the like.

[0007] This disclosure further relates to the use of peptide compounds for treating diseases or disorders that are responsive to modulating sodium absorption, such as, e.g., lung diseases, including cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), and other respiratory malignancies (see, Fellner et al, Mol Cell Pediatr., 3(1): 16, 2016). As described in detail in the Examples section below, the peptides of the disclosure significantly improved survival (or reduced mortality) of animals suffering from cystic fibrosis (CF). Additionally, compared to untreated subjects, animals treated with the peptide compounds of the disclosure also had better outcomes, such as longer disease-free survival period (DFS) or longer time to death (TTD), It is thought that these in vivo pharmacological effects may be imparted, in part, by the airway surface liquid (ASL) homeostatic balance conferred by the peptides of the disclosure.

[0008] These and other aspects of the disclosure are set forth in more detail in the description of below.

[0009] In some embodiments, the disclosure relates to a compound of Formula lb and salts thereof, R'-PEP-R² (lb); wherein R¹ and R² are, independently, absent, or a blocking or a protecting group; and PEP is X1-X2-X3-X4-X5-X6-X7 (SEQ ID NO: 2), wherein Xi to X? are each, independently, an amino acid, wherein Xi is leucine (L), isoleucine (I), methionine (M), phenylalanine (F), or valine (V), or a conservatively substituted amino acid of any one of L, I, M, F, or V: X21S proline (P) or arginme (R) or a conservatively substituted amino acid of any one of P or R; X3 1S leucine (L), isoleucine (I), methionine (M), phenylalanine (F), or valine (V), or a conservatively substituted ammo acid of any one of L, I, M, F, or V: X4 is proline (P) or arginine (R) or a conservatively substituted amino acid of any one of P or R; Xs is (L), isoleucine (I), methionine (M), phenylalanine (F), or valine (V), or a conservatively substituted amino acid of any one of L, I, M, F, or V; Xe is aspartic acid (D), asparagine (N), glutamine (Q), glutamic acid (E), serine (S), lysine (K), arginme (R), or citnilline (Cit), or a conservative^ substituted amino acid of any one of D, N, Q, E, S, K, R, or Cit; and X? is absent, or glutamine (Q), arginine (R), asparagine (N), aspartic acid (D), glutamic acid (E), histidine (H), lysine (K), methionine (M), or serine (S) or a conservatively amino acid of any one of Q, R, N, D, E, H, K, M or S.

[0010] In some embodiments, the disclosure relates to a compound of Formula lb and salts thereof, R^!-PEP-R² (lb); wherein R¹ and R² are, independently, absent, or a blocking or a protecting group; and PEP is X1-X2-X3-X4-X5-X6-X7 (SEQ ID NO: 2), wherein Xi to X? are each, independently, an ammo acid, wherein X; is leucine (L), isoleucine (I), methionine (M), phenylalanine (F), or valine (V), or a conservatively substituted amino acid of any one of L, I, M, F, or V; X21S proline (P) or arginine (R) or a conservatively substituted ammo acid of anyone of P or R; Xi is leucine (L), isoleucine (I), methionine (M), phenylalanine (F), or valine (V), or a conservatively substituted amino acid of any one of L, I, M, F, or V; X4 is proline (P) or arginine (R) or a conservatively substituted amino acid of any one of P or R; Xs is (L), isoleucine (I), methionine (M), phenylalanine (F), or valine (V), or a conservatively substituted amino acid of any one of L, 1, M, F, or V: Xe is aspartic acid (D), asparagine (N), glutamine (Q), glutamic acid (E), serine (S), or a conservatively substituted amino acid of any one of D, N, Q, E, S; and X? is absent or glutamine (Q), arginine (R), asparagine (N), aspartic acid (D), glutamic acid (E), histidine (H), lysine (K), methionine (M), or serine (S) or a conservatively amino acid of any one of Q, R, N, D, E, H, K, M or S.

[0011] In some embodiments, the disclosure relates to a compound of Formula Ic and salts thereof, R'-PEP-R² (Ic); wherein R¹ and R² are, independently, absent, or a blocking or a protecting group; and PEP is X1-X2-X3-X4-X5-X6 (SEQ ID NO: 1), wherein Xi-Xe are as described above.

[0012] In some embodiments, the disclosure relates to a compound of Formula lb and salts thereof, R^!-PEP-R² (lb); wherein R¹ and R- are, independently, absent, or a blocking or a protecting group; and PEP is X1-X2-X3-X4-X5-X6-X7 (SEQ ID NO: 2), wherein X; to X? are each, independently, an amino acid, wherein Xi is leucine (L) or a conservatively-substituted amino acid thereof; X2 is proline (P) or a conservatively-substituted amino acid thereof; X3 is leucine (L) or a conservatively-substituted amino acid thereof; X4 is proline (P) or a conservatively-substituted amino acid thereof; X5 is leucine (L) or a conservatively-substituted amino acid thereof; Xe is aspartic acid (D) or a conservatively-substituted amino acid thereof; and X7 is present and is an amino acid which is Q, R, N, D, E, H, K, M or S or a conservatively substituted ammo acid of Q, R, N, D, E, H, K, M or S.

[0013] In some embodiments, the disclosure relates to a compound of Formula lb and salts thereof, R'-PEP-R² (lb); wherein R^! and R² are, independently, absent, or a blocking or a protecting group; and PEP is X1-X2-X3-X4-X --X&--X7 (SEQ ID NO: 2), wherein Xi to X7 are each, independently, an amino acid, wherein Xi is leucine (L) or a conse atively-substituted amino acid thereof; X2 is proline (P) or a conservatively-substituted amino acid thereof; ¾ is leucine (L) or a conservatively-substituted amino acid thereof; X4 is proline (P) or a conservatively-substituted amino acid thereof; X5 is leucine (L) or a conservatively-substituted amino acid thereof; Xe is aspartic acid (D) or a conservatively-substituted amino acid thereof; and X? is absent or glutamine (Q) or a conservatively-substituted amino acid thereof. Preferably, in compounds of Formula lb, X2 or XA or both X2 and X4 are proline.

Θ014] In some embodiments, the disclosure relates to a compound of Formula lb and salts thereof, R¹ PEP R² (lb); wherein R¹ and R² are both absent, in which case, the compound is a peptide (PEP) consisting essentially of or consisting the sequence X1-X2-X3-X4-X5-X6-X7 (SEQ ID NO: 2), wherein Xi to X? are each, are described above, in some embodiments, Xi to X7 are each, independently, an amino acid, wherein X i is L, I, M, F, or V; X2 is P or R; X3 is L, 1, M, F, or V; \^' i is P or R; Xj is L, 1, M, F, or V; Xe is D, N, Q, E or S; and X7 is Q, R. N, D, E, H, K . M or S,

Θ015] In another embodiment, the disclosure relates to a compound of Formula lb and salts thereof, R'-PEP-R² (lb); wherein at least one of R¹ and R² is a blocking or a protecting group and PEP is X1-X2-X3-X4-X5-X6-X7 (SEQ ID NO: 2), wherein Xi to X7 are each, as described previously. In some embodiments, R¹ and R², each, individually comprise a blocking or protecting group selected from acetylation at the N-terminus, amidation at the C-terminus, or both acetylation at the N-terminus and amidation at the C-terminus. In another embodiment, R^! and R², each, independently, comprise a blocking or protecting group comprising one to about four non-proteinogenic amino acids. In one particular embodiment, R^! and R², each, independently, comprise a blocking or protecting group selected from D -amino acids (e.g., D- aianine), Norieucine (Nle), 4-hydroxyproline (HYP), 3,4-dehydro-L-proime (DHP), aminoheptanoic acid (AHP), (2R,5S)-5-phenyl- pyrrolidine-2-carboxylic acid (2PP), L-a- methy] serine (MS), N-methylvaline (MV), 6-aminohexanoic acid (6-AHP), and 7- aminoheptanoic acid (7-AHP). Preferably, R¹ and R² each, independently, comprise one or two D-alanine residues.

Θ016] In some embodiments, the disclosure relates to a compound of Formula lb and salts thereof, R^!-PEP-R² (lb); wherein R¹ and R- are, independently, absent, or a blocking or a protecting group; and PEP is selected from the group consisting of LPLPLDQ (SEQ ID NO: 4) and LPIPLDQ (SEQ ID NO: 7).

[0017] In some embodiments, the disclosure relates to a compound of Formula Ic and salts thereof, R'-PEP-R² (Ic); wherein R¹ and R² are, independently, absent, or a blocking or a protecting group; and PEP is X1-X2-X3-X4-X5-X6 (SEQ ID NO: 1 ), wherein Xi to Xe are each, independently, an amino acid, wherein Xi is L, I, M, F, or V, or a conservatively substituted amino acid of any one of L, I, M, F, or V ; X2 is P or R or a conservatively substituted amino acid of any one of P or R; X3 is L, I, M, F, or V, or a conservatively substituted amino acid of any one of L, I, M, F, or V; X4 1S P or R or a conservatively substituted amino acid of any one of P or R; X5 is L, I, M, F, or V, or a conservatively substituted amino acid of any one of L, I, M, F, or V; and X& is D, N, Q, E, S, K, R, or Cit, or a conservatively substituted amino acid of any one of D, N, Q, E, S, K, R, or Cit.

[0018] In some embodiments, the disclosure relates to a compound of Formula Ic and salts thereof, R^!-PEP-R² (Ic); wherein R¹ and R² are, independently, absent, or a blocking or a protecting group; and PEP is X1-X2-X3-X4-X5-X6 (SEQ ID NO: I ), wherein, Xi is L or a conservatively-substituted amino acid thereof; X2 is P or a conservatively-substituted amino acid thereof; X3 is L or a conservatively-substituted amino acid thereof; X4 is P or a conservatively-substituted amino acid thereof; Xs is L or a conservatively-substituted amino acid thereof; and Xe is D or a conservatively-substituted amino acid thereof. Preferably, in compounds of Formula Ic, X2 or X₄ or both X2 and X4 are proline.

[0019] In some embodiments, in compounds of Formula Ic, R¹ and R² are both absent, in which case, the compound is a peptide (PEP) consisting essentially of or consisting the sequence Xi- X2-X3-X4-X5-X6 (SEQ ID NO: 1), wherein Xi to Xe are each, independently, as described above. In some embodiments, Xi to Xe are each, independently, an amino acid, wherein Xi is L, I, M, F, or V; X2 is P or R; X₃ is L, I, M, F, or V; XA is P or R; X₅ is L, L M, F, or V; and X₆ is D, N, Q, E or S.

[0020] In another embodiment, the disclosure relates to a compound of Formula Ic and salts thereof, R'-PEP-R² (Ic); wherein at least one of R¹ and R² is a blocking or a protecting group and PEP is X1-X2-X3-X4-X5-X& (SEQ ID NO: 1 ), wherein Xi to X& are each, as described previously. In some embodiments, R^! and R², each, individually comprise a blocking or protecting group selected from acetyiation at the -terminus, amidation at the C-terminus, or both acetyiation at the N -terminus and amidation at the C-terminus. In another embodiment, R¹ and R², each, independently, comprise a blocking or protecting group comprising one to about four non-proteinogenic amino acids. In one particular embodiment, R^! and R², each, independently, comprise a blocking or protecting group selected from D -amino acids (e.g., D- alanine), Norleucine (\ lo). 4-hydroxyproline (HYP), 3,4-dehydro-L-proline (DHP), aminoheptanoic acid (AHP), (2R,5S)-5-phenyl- pyrrolidine-2-carboxylic acid (2PP), L-a- methylserine (MS), N-methyl valine (MV), 6-aminohexanoic acid (6-AHP), and 7- aminoheptanoic acid (7-AHP), preferably D-alanine.

[0021] In some embodiments, the disclosure relates to a compound of Formula lb and salts thereof, R '-PEP-R² (Ic); wherein R¹ and R² are, independently, absent, or a blocking or a protecting group; and PEP is selected from the group consisting of LPLPLD (SEQ ID NO: 3) and LPIPLD (SEQ ID NO: 6).

[0022] In some embodiments, the disclosure relates to a compound selected from the group consisting of: (a) aLPLPLDa (SEQ ID NO: 12); (b) aLPLPLDQa (SEQ ID NO: 13); (c) aLPIPLDa (SEQ ID NO: 14); (d) aLPIPLDQa (SEQ ID NO: 15); (e) aaLPLPLDaa (SEQ ID NO: 16); (f) aaLPLPLDQaa (SEQ ID NO: 17); (g) aaLPIPLDaa (SEQ ID NO: 18); and (h) aaLPIPLDQaa (SEQ ID NO: 19); wherein "a" is a non-proteinogenic amino acid selected from the group consisting of D-amino acids (e.g., D-alanine) (D-Ala); Norleucine (Nle); 4- hydroxyproline (HYP); 3,4-dehydro-L-proline (DHP): aminoheptanoic acid (AHP); (2R,5S)-

5- phenyl- pyrrolidine-2-carboxylic acid (2PP); L-a-methyl serine (MS); N-methyivaline (MV)

6- aminohexanoic acid (6-AHP); and 7-aminoheptanoic acid (7-AHP). Preferably, the disclosure relates to a compound selected from the group consisting of: (a) aLPLPLDa (SEQ ID NO: 12); (b) aLPLPLDQa (SEQ ID NO: 13); (c) aLPIPLDa (SEQ ID NO: 14); (d) aLPIPLDQa (SEQ ID NO: 15); (e) aaLPLPLDaa (SEQ ID NO: 16); (f) aaLPLPLDQaa (SEQ ID NO: 17); (g) aaLPIPLDaa (SEQ ID NO: 18); and (h) aaLPIPLDQaa (SEQ ID NO: 19); wherein "a" is D-alanine.

[0023] In some embodiments, the disclosure relates to a peptide consisting essentially of Xi- X2-X3-X4-X5-X6-X7 (SEQ ID NO: 2), wherein Xi to X? are each, are described above. In some embodiments, X i to X? are each, independently, an amino acid, wherein Xi is L, I, M, F, or V; X < is P or R; X3 is L, I, M, F, or V; X i is P or R; Xs is L, I, M, F, or V; Xe is D, R, N, Q, E, K, S or Cit; and X? is Q, R, N, D, E, H, K, M or S. Preferably, X2 or X4 or both X2 and X4 of SEQ ID NO: 2 are proline (P). Especially, the disclosure relates to a peptide consisting essentially of X1-X2-X3-X4-X5-X6-X7 (SEQ ID NO: 2), wherein Xi is L, I, M, F, or V; X > Is P; X3 is L, I, M, F, or V; X i is P; X₅ is L, I, M, F, or V; Xe is D, R, N, Q, E, K, S or Cit; and X₇ is Q, R, N, D, E, H, K, M, or S.

[0024] In some embodiments, the disclosure relates to a peptide consisting essentially of X i- X2-X3-X4-X5-X6-X7 (SEQ ID NO: 2), wherein Xi to X? are each, are each, independently, an amino acid, wherein Xi is L, I, M, F, or V; X2 is P or R; X3 is L, I, M, F, or V; X4 is P or R; X5 is L, I, M, F, or V; Xe is D, N, Q, E, S; and Χτ is Q, R, N, D, E, H, K, M or S, Preferably, X2 or X4 or both Xi and X4 of SEQ ID NO: 2 are proline (P). Especially, the disclosure relates to a peptide consisting essentially of X1-X2-X3-X4-X5-X6-X7 (SEQ ID NO: 2), wherein Xi is L, I, M, F, or V; X2 is P; Xi is L, I, M, F, or V: X₄ is P; Xs is L, I, M, F, or V; Xe is D, N, Q, E, or S; and τ is Q, R . N, D, E, H, K, M, or S.

[0025] In some embodiments, the disclosure relates to a peptide consisting essentially of Xi- X2-X3-X4-X5-X6 (SEQ ID NO: 1 ), wherein Xi to Xe are each, are described above. In some embodiments, X: to Xe are each, independently, an amino acid, wherein Xi is L, I, M, F, or V; X2 is P or R; X₃ is L, I, M, F, or V; X₄ is P or R; X₅ is L, I, M, F, or V; and X& is D, R, N, Q, E, K, S or Cit, Preferably, X2 or X4 or both X2 and X4 of SEQ ID NO: 1 are proline (P). Especially, the disclosure relates to a peptide consisting essentially of X1-X2--X3--X4--X5--X6 (SEQ ID NO: 1), wherein Xi is L, I, M, F, or V; X2 is P; X₃ is L, I, M, F, or V: XA is P; X₅ is L, I, M, F, or V; and Xe is D, R, N, Q, E, K, S or Cit.

[0026] In some embodiments, the disclosure relates to a peptide consisting essentially of Xi- X2-X3-X4-X5-X6 (SEQ ID NO: 1), wherein Xi to Xe are each, independently, an amino acid, w herei n Xi is L, I, M, F, or V; X2 is P or R; X₃ is L, I, M, F, or V: X₄ is P or R; Xj is L, I, M, F, or V; and Xe is D, N, Q, E, or S. Preferably, X2 or X4 or both X2 and 4 of SEQ ID NO: 1 are proline (P). Especially, the disclosure relates to a peptide consisting essentially of X1-X2- Χ3-ΧΨ--Χ5-Χ6 (SEQ ID NO: 1), wherein Xi is L, I, M, F, or V; X2 is P; X3 is L, I, M, F, or V; X₄ is P; Xs is L, I, M, F, or V; and Xe is D, N, Q, E, or S.

[0027] In some embodiments, the disclosure relates to a kit comprising, in one or separate packages, at least one compound or peptide of the disclosure, optionally together with instructions for using the kit.

Θ028] In some embodiments, the disclosure relates to method of inhibiting the activation of a sodium channel or inhibiting sodium absorption through a sodium channel or reducing the level of a sodium channel present on the surface of a cell, comprising contacting a sodium channel with a compound or a peptide of the disclosure. In some embodiments, the sodium channel is contacted with a compound of Formula lb or Ic, e.g., a compound selected from the group consisting of: (a) aLPLPLDa (SEQ ID NO: 12); (b) aLPLPLDQa (SEQ ID NO: 13); (c) aLPIPLDa (SEQ ID NO: 14); (d) aLPIPLDQa (SEQ ID NO: 15); (e) aaLPLPLDaa (SEQ ID

NO: 16); (f) aaLPLPLDQaa (SEQ ID NO: 1 7); (g) aaLPIPLDaa (SEQ ID NO: 18); and (h) aaLPIPLDQaa (SEQ ID NO: 19); wherein "a" is a non-proteinogenic D-amino acid (e.g., D- aianine) (D-Ala). In another embodiment, the sodium channel is contacted with a composition comprising the peptide of the disclosure which consists essentially of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the sodium channel is an epithelial sodium channel (ENaC), e.g., a sodium channel is present in an isolated cell in an epithelial cell culture or in an animal. In some embodiments, the sodium channel is present in an animal that has a respiratory disease. Preferably, the animal is a human subject. In some embodiments, the contacting step involves administering the compound or peptide to the animal and the activation of the sodium channel is inhibited by at least 20%, at least 50%, at least 90% or more.

[0029] In some embodiments, the disclosure relates to a method for treating a disorder responsive to inhibition of sodium absorption across an epithelial mucosal surface in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound or a peptide of the disclosure. In some embodiments, the method includes administering to the subject, an effective amount of a compound of Formula lb or Ic, e.g., a compound selected from the group consisting of: (a) aLPLPLDa (SEQ ID NO: 12); (b) aLPLPLDQa (SEQ ID NO: 13); (c) aLPIPLDa (SEQ ID NO: 14); (d) aLPIPLDQa (SEQ ID NO: 15); (e) aaLPLPLDaa (SEQ ID NO: 16); ( i) aaLPLPLDQaa (SEQ ID NO: 17); (g) aaLPIPLDaa (SEQ ID NO: 18); and (h) aaLPIPLDQaa (SEQ ID NO: 19); wherein "a" is a non-proteinogenic D-amino acid (e.g., D-alanine) (D-Ala). In some embodiments, the method of treatment includes administering to the subject, an effective amount of a composition comprising a peptide consisting essentially of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the disorder is a lung disorder, e.g., cystic fibrosis (CF), idiopathic pulmonary fibrosis (IPF), asthma, acute or chronic bronchitis, or chronic obstructive pulmonary disease (COPD). In another embodiment, the disorder is non- cystic fibrosis bronchiectasis. In another embodiment, the disorder is a kidney disorder or a gastrointestinal disorder. Preferably, the subject is a human subject.

[0030] In another embodiment, the disclosure relates to a method for regulating salt balance, blood volume, and/or blood pressure in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound or a peptide of the disclosure. In some embodiments, the method includes administering to the subject, an effective amount of a compound of Formula lb or Ic, e.g., a compound selected from the group consisting of: (a) aLPLPLDa (SEQ ID NO: 12); (b) aLPLPLDQa (SEQ ID NO: 13); (c) aLPIPLDa (SEQ ID

NO: 14); (d) aLPIPLDQa (SEQ ID NO: 15); (e) aaLPLPLDaa (SEQ ID NO: 16); (f) aaLPLPLDQaa (SEQ ID NO: 17); (g) aaLPIPLDaa (SEQ ID NO: 18); and (h) aaLPIPLDQaa

(SEQ ID NO: 19); wherein "a" is a non-proteinogenic D-amino acid (e.g., D-alanine) (D-Ala).

In some embodiments, the method includes administering to the subject, an effective amount of a composition comprising a peptide consisting essentially of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the therapeutically effective amount of the compound or the peptide, when administered to the subject, reduces the level of a sodium channel present on the surface of a cell in the subject. Preferably, the subject is a human subject.

[0031 ] In another embodiment, the disclosure relates to a method for treating a lung disorder, a gastrointestinal disorder, a kidney disorder, or a cardiovascular disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound or a peptide of the disclosure. In some embodiments, the method includes administering to the subject an effective amount of a compound of Formula lb or Ic, e.g., a compound selected from the group consisting of: (a) aLPLPLDa (SEQ ID NO: 12); (b) aLPLPLDQa (SEQ ID NO: 13); (c) aLPIPLDa (SEQ ID NO: 14); (d) aLPIPLDQa (SEQ ID NO: 15); (e) aaLPLPLDaa (SEQ ID NO: 16); (f) aaLPLPLDQaa (SEQ ID NO: 17); (g) aaLPIPLDaa (SEQ ID NO: 18); and (h) aaLPIPLDQaa (SEQ ID NO: 19); wherein "a" is a non-proteinogenic D-amino acid {e.g., D-alanine) (D-Ala). In some embodiments, the method includes administering to the subject, an effective amount of a composition comprising a peptide consisting essentially of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, In some embodiments, the disorder is a lung disorder selected from cystic fibrosis, non-cy stic fibrosis bronchiectasis, chronic obstructive pulmonary disease, acute or chronic bronchitis, and asthma. In another embodiment, the disorder is a gastrointestinal disorder which is inflammatory bowel disease. Preferably, the subject is a human subject.

[0032] In some embodiments, the disclosure relates to a method of improving the outcome of a subject suffering from, a disease mediated by ENaC activity, comprising administering to the subject an therapeutically effective amount of a pharmaceutical composition comprising the a compound or peptide of the disclosure, e.g., a compound selected from the group consisting of: (a) aLPLPLDa (SEQ ID NO: 12); (b) aLPLPLDQa (SEQ ID NO: 13); (c) aLPIPLDa (SEQ ID NO: 14); (d) aLPIPLDQa (SEQ ID NO: 15); (e) aaLPLPLDaa (SEQ ID NO: 16); (f) aaLPLPLDQaa (SEQ ID NO: 17); (g) aaLPIPLDaa (SEQ ID NO: 18); and (h) aaLPIPLDQaa (SEQ ID NO: 19); wherein "a" is a non-proteinogenic D-amino acid {e.g., D-alanine) (D-Ala) or a composition comprising a peptide consisting essentially of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the disease mediated by ENaC activity is a lung disorder selected from the group consisting of cystic fibrosis, non-cystic fibrosis bronchiectasis, chronic obstructive pulmonary disease, acute or chronic bronchitis, and asthma. In some embodiments, the disclosure relates to improving an outcome which is disease-free survival period (DPS), or time to death (TTD), forced expiratory volume in one second (FEV1 ) or percent predicted FEV1 in the subject suffering from the lung disorder. Preferably, the subject is a human subject.

[0033] In another embodiment, the disclosure relates to a method for treating dry eye, dry mouth, vaginal dryness, or rhinosinusitis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound or peptide of the disclosure, e.g., a compound selected from the group consisting of: (a) aLPLPLDa (SEQ ID NO: 12): (b) aLPLPLDQa (SEQ ID NO: 13); (c) aLPIPLDa (SEQ ID NO: 14); (d) aLPIPLDQa (SEQ ID NO: 15); (e) aaLPLPLDaa (SEQ ID NO: 16); (t) aaLPLPLDQaa (SEQ ID NO: 17): (g) aaLPIPLDaa (SEQ ID NO: 18); and (h) aaLPIPLDQaa (SEQ ID NO: 19); wherein "a" is a non-proteinogenic D-amino acid (e.g., D-alanine) (D-Ala) or a composition comprising a peptide consisting essentially of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. Preferably, the subject is a human subject.

[0034] In some embodiments, the disclosure relates to a method of increasing innate immunity in a subject and/or reducing the susceptibility of the subject to a pathogen, comprising administering to the subject, a therapeutically effective amount of a compound or peptide of the disclosure, e.g., a compound selected from the group consisting of: (a) aLPLPLDa (SEQ ID NO: 12); (b) aLPLPLDQa (SEQ ID NO: 13); (c) aLPIPLDa (SEQ ID NO: 14); (d) aLPIPLDQa (SEQ ID NO: 15); (e) aaLPLPLDaa (SEQ ID NO: 16); (f) aaLPLPLDQaa (SEQ ID NO: 17); (g) aaLPIPLDaa (SEQ ID NO: 18); and (h) aaLPIPLDQaa (SEQ ID NO: 19); wherein '^"a" is a non-proteinogenic D-amino acid (e.g., D-alanine) (D-Ala) or a composition comprising a peptide consisting essentially of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, Preferably, the subject is a human subject and the pathogen is selected from bacteria, viruses, fungi, and protists.

[0035] In some embodiments, the disclosure relates to a compound or peptide of the disclosure, e.g., a compound selected from the group consisting of: (a) aLPLPLDa (SEQ ID NO: 12); (b) aLPLPLDQa (SEQ ID NO: 13); (c) aLPIPLDa (SEQ ID NO: 14); (d) aLPIPLDQa (SEQ ID

NO: 15); (e) aaLPLPLDaa (SEQ ID NO: 16); (f) aaLPLPLDQaa (SEQ ID NO: 17); (g) aaLPIPLDaa (SEQ ID NO: 18); and (h) aaLPIPLDQaa (SEQ ID NO: 19); wherein "a" is a non-proteinogenic D-amino acid (e.g., D-ala ine) (D-Ala) or a composition comprising a peptide consisting essentially of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ

ID NO: 2, for use in treatment of a lung disorder, a gastrointestinal disorder, a kidney disorder, a cardiovascular disorder, dry eye, dry mouth, vaginal dryness, or rhinosinusitis. Preferably, the disorder is a lung disorder selected from the group consisting of COPD, IPF, and CF or a disorder selected from dry eyes, dry mouth, vaginal dryness and/or chronic rhinosinusitis. [0036] In another embodiment, the disclosure relates to use of a compound or a peptide of the disclosure, e.g., a compound selected from the group consisting of: (a) aLPLPLDa (SEQ ID NO: 12): (b) aLPLPLDQa (SEQ ID NO: 13); (c) aLPIPLDa (SEQ ID NO: 14); (d) aLPIPLDQa (SEQ ID NO: 15); (e) aaLPLPLDaa (SEQ ID NO: 16); (f) aaLPLPLDQaa (SEQ ID NO: 17); (g) aaLPIPLDaa (SEQ ID NO: 18); and (h) aaLPIPLDQaa (SEQ ID NO: 19); wherein "a" is a non-proteinogenic D-amino acid (e.g., D-alanine) (D-Ala) or a composition comprising a peptide consisting essentially of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, for the manufacture of a medicament for the treatment of a lung disorder, a gastrointestinal disorder, a kidney disorder, a cardiovascular disorder, dry eye, diy mouth, vaginal dryness, or rhinosinusitis. Preferably, the disorder is a lung disorder selected from the group consisting of COPD, IPF, and CF or a disorder selected from dry eyes, dry mouth, vaginal dryness and/or chronic rhinosinusitis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The details of one or more embodiments of the disclosure are set forth in the accompanying drawings/tables and the description below. Other features, objects, and advantages of the disclosure will be apparent from the drawings/tables and detailed description, and from the claims.

[0038] FIG. 1 shows results of the assessment of ENaC internalization by peptide compounds of the present disclosure.

[0039] FIG. 2 shows results of the assessment of thermal stability of peptide compounds of the present disclosure.

[0040] FIG. 3 shows results of the assessment of protease resistance of the peptide compounds of the present disclosure

[0041] FIG. 4 shows results of the assessment of effect of the peptide compounds of the present disclosure on survival of a bENaC-Tg mouse model .

[0042] FIG. 5 shows results of the assessment of the effect of the peptide compounds of the present disclosure on the survival of cells in vitro.

DETAILED DESCRIPTION

[0043] The present disclosure will now be described in more detail with reference to the accompanying drawings, in which preferred embodiments of the disclosure are shown. This disclosure may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art to which this disclosure belongs. The terminology used in the description of the disclosure herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. All publications, patent applications, patents, patent publications and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and/or paragraph in which the reference is presented.

[0045] As used in the description of the disclosure and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Also as used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0046] Where a range of values is provided in this disclosure, it is intended that each intervening value between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure. For example, if a range of 1 μΜ to 8 μ.Μ is stated, it is intended that 2 μ.Μ, 3 μΜ, 4 μ,Μ, 5 μΜ, 6 μΜ, and 7 μΜ are also explicitly disclosed.

[0047] The word "about" means a range of plus or minus 10% of that value, e.g., "about 5" means 4.5 to 5.5, "about 100" means 90 to 100, etc., unless the context of the disclosure indicates otherwise, or is inconsistent with such an interpretation. For example in a list of numerical values such as "about 49, about 50, about 55, "about 50" means a range extending to less than half the interval(s) between the preceding and subsequent values, e.g., more than 49.5 to less than 52.5. Furthermore, the phrases "less than about" a value or "greater than about" a value should be understood in view of the definition of the term "about" provided herein.

[0048] This disclosure relates to inhibitors of epithelial Na⁺ channel (ENaC). In some embodiments, the ENaC inhibitors are compounds, such as peptides and peptide derivatives, which mimic the ENaC-regulating activity of SPLUNC1. The peptides are preferably stable, e.g., thermally stable and proteolytically stable as described herein . A thermally stable peptide can be, for example, stable for up to 14 days or more at 40 °C. Alteraateiy or additionally, the peptide compounds of the disclosure are also proteolytically stable. Typically the proteolytically stable peptide will not be substantially degraded (about 20% of the peptide, preferable 10% or less or 5% or less of the peptide is degraded) when incubated with a physiological concentration of protease for a period of about 30 minutes, 60 minutes or 90 minutes. In a particular example, the peptide is stable against human neutrophil elastase (HNE), an enzyme which degrades SPLUNC1 and impairs airway epithelial defense against bacteria. See, Jiang et al. PLoS One, 8(5):e64689, 2013. For instance, in an in vitro assay, a 90 minute exposure resulted in about 60% loss of stability of the longer naturally occurring S 18 peptide of SPLUNC, while there was no appreciable loss (e.g., a loss of less than 5%) of stability of the peptide compounds of the disclosure. Based on these results, it is believed that the peptide compounds of the disclosure will have better stability and improved bioavailability compared to the natural SPLUNC 1 protein or the naturally occurring S I 8 peptide of SPLUNC 1.

[0049] As used herein, the term "peptide" includes a natural peptide comprising a linear chain or branched chain of amino acids, peptidomimetics, as well as pharmaceutically acceptable salts thereof. A peptide comprises a plurality of ammo acid residues, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more amino acid residues which are bonded to each other via covalent bonds, e.g. , a peptide bond. "Amino acid residue" means the individual amino acid units incorporated into the peptides of the disclosure. As used herein, the term "amino acid" includes the 22 amino acids that are proteinogenic amino acids and non-proteinogenic ammo acids. The term "proteinogenic amino acid," is used in the field of biochemistry to refer to the 22 amino acids that are incorporated into eukaryotic and/or prokaryotic proteins during translation, such as: (a) histidine (His: H); (b) isoleucine (lie; 1); (c) leucine (Leu: L); (d) Lysine (Lys; K); (e) methionine (Met; M); (f) phenylalanine (Phe; F); (g) threonine (Tiir; T); (h) tryptophan (Tip; W); (i) valine (Val; V); (j) arginine (Arg; R); (k) cysteine (Cys; C); (1) glutamine (Gin; Q); (m) glycine (Gly; G); (n) proline (Pro; P); (o) serine (Ser; S); (p) tyrosine (Tyr; Y); (q) alanine (Ala; A); (r) asparagine (Asn; N); (s) aspartic acid (Asp; D): (t) glutamic acid (Glu; E); (u) selenocysteine (Sec; U); (v) pyrrolysine (Pyl; O). The term "non-proteinogenic amino acid" is used in the field of biochemistry to refer to naturally occurring and non-naturally occurring amino acids that are not proteinogenic amino acids, such as (1) citrulline (Cit); (2) cystine; (3) gama-amino butyric acid (GABA); (4) ornithine (Orn); (5) theanine; (6) homocysteine (Hey); (7) thyroxine (Thx); and amino acid derivatives such as betaine; carnitine; camosine creatine; hydroxytryptophan; hydroxyproline (Hyp); N-acetyl cysteine; S-Adenosyl methionine (SAM- e); taurine; tyramine, D- amino acids such as D-alanine (D-Ala); Norleucine (Nie); 4- hydroxyproline (HYP); 3,4-dehydro-L-proline (DHP); aminoheptanoic acid (AHP); (2R,5S)-

5- phenyl- pyrrolidine-2-carboxylic acid (2PP); L-a-methylserine (MS); N-methylvaline (MV);

6- aminohexanoic acid (6-AHP); and 7-aminoheptanoic acid (7- AHP). Θ050] A "conservative" amino acid substitution, as used herein, generally refer to substitution of one amino acid residue with another amino acid residue from within a recognized group which typically changes the structure of the peptide by biological activity of the peptide is substantially retained. Conservatively substituted amino acids can be identified using a variety of well know methods, such as a blocks substitution matrix (BLOSUM), e.g., BLOSUM62 matrix. BLOSUM is a substitution matrix used for sequence alignment of proteins, wherein an alignment score is used to map out relationship between evolutionarily divergent protein sequences. They are based on local alignments. For instance, a BLOSUM62 substitution matrix for naturally -occurring amino acids is presented in the web at ncbi(dot)nlm(dot)nih(dot)gov/class/fieldguide BLOSUM62.txt, which is incorporated by reference herein, A conservative amino acid substitution can be the replacement of a first amino acid amino with a structurally or chemically similar amino acid analog. Many suitable amino acid analogs are known in the art, and representative examples include, e.g., p- Acetylphenylalanine, m-Acetylphenylalanine, O-allyltyrosine, Phenylselenocysteine, p~ Propargyloxyphenylalanine, p-Azidophenylalanine, p-Boronophenylalanine, 0~ methyltyrosine, p-Aminophenylalanine, p-Cyanophenylalanine, m-Cyanophenylalanine, p- Fluorophenylalanine, p-lodophenylalanine, p-Bromophenylaianine, p-Nitrophenylalanine, L- DOPA, 3-Aminoty osine, 3-Iodotyrosine, p-Isopropylphenylalanine, 3-(2-Naphthyl)alanine, biphenylalanine, homoglutamine, D-tyrosine, p-Hydroxyphenyllactic acid, 2-Aminocaprylic acid, bipyridylalanine, HQ-alanine, p-Benzoylphenylaianine, o-Nitrobenzylcysteine, o- Nitrobenzylserine, 4,5-Dimethoxy-2-Nitrobenzyiserine, o-Nitrobenzyllysine, o- Nitrobenzyltyrosine, 2-Nitrophenylalanme, dansyl alanine, p-Carboxymethylphenylalanine, 3- Nitrotyrosine, sulfotyrosine, acetyllysine, methylhistidine, 2-Aminononanoic acid, 2- Aminodecanoic acid, pyrrolysine, Cbz-lysine, Boc-lysine, allyioxycarbonyllysine, arginosuccinic acid, citrulline, cysteine sulfinic acid, 3,4-dihydroxyphenylalanine, homocysteine, liomoserine, ornithine, 3-monoiodotyrosine, 3,5-diiodotryosine, 3,5,5,- tniodothyronine, and 3,3',5,5'-tetraiodothyronine. The term includes modified or unusual amino acids e.g., D-amino acids, hydroxy-lysine, 4-hydroxyproiine, N-Cbz-protected amino acids, 2,4-dian inobutyric acid, homoarginine, norleucine, N-methylaminobutyric acid, naphthylalanine, phenylglycine, -phenylproline, tert-leucine, 4-aminocyclohexylaianine, N- methyl -norleucine, 3 ,4-dehydroproline, N,N -dimethylaminoglycine, N-methylaminoglycine, 4-aminopiperidine-4-carboxylic acid, 6-aminocaproic acid, trans-4-(aminomethyl)- cyclohexanecarboxylic acid, 2-, 3-, and 4-(aminomethyl)-benzoic acid, 1 - aminocyclopentanecarboxylic acid, l-animocyclopropanecarboxyiic acid, and 2-benzyl-5- aminopentanoic acid; functionalized amino acids, e.g., alkyne-functionalized, azide- funciionalized, ketone-functionalized, aminooxy -functionalized amino acids and the like. See Liu et al, Ann. Rev. Biochem. 79:413, 2010; Kim. et al, Curr. Opin. Chem. Biol 17:412, 2013.

[0051 ] Conservative amino acid substitutions of proteinogenic amino acids with other proteinogenic amino acids are generally from within the groups in Table 1 :

[0052] Table 1, Exemplary amino acid substitutions

[0053] Preferred amino acid substitutions are the substitution of on amino acid residue with another ammo acid residues from within the following groups:

Group I: I, L, M, F, and V;

Group II: P and R;

Group III: N, Q, E, D, and S;

Group IV: R, N, D, Q, E, H, K, M, and S; and Group V: A, N, S, T, and V.

[0054] To identify or design peptide sequences of the disclosure other than those specifically disclosed herein, amino acid substitutions may be based on any characteristic known in the art, including the relative similarity or differences of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicitv, charge, size, and the like. For example, the hydropathic index of amino acids may be considered. The importance of the hydropathic ammo acid index in conferring interactive biologic function on a protein is generally understood in the art (see, Kyte et al, JMol Biol. 757: 105 (1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn, defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Ammo acid have recognized hydropathic index values based on their hydrophobicity and charge characteristics, e.g., isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (~ 0.9); tyrosine (-1.3); proline (-1 .6); histidme (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). Amino acid substitutions can be made to preserve the general hydropathic character of the original ammo acid, for example by replacing isoleucine with valine. Similarly, amino acid substitutions can be made on the basis of hydrophilicity See U.S. Pat. No. 4,554, 101 at Table 2, which assigns the following hydrophilicity values to the amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 + 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± I); alanine (-0,5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). Accordingly, the hydrophilicity of the amino acid (or amino acid sequence) may be considered when identifying additional peptides beyond those specifically disclosed herein.

[0055] Ammo acid substitutions in the compounds and peptide disclosed herein are typically selected to so as to substantially preserve the ENaC inhibition activity of the peptide. Guidance for substitutions, insertion, or deletion may be based on alignments of amino acid sequences of different variant proteins or proteins from different species. For example, at certain residue positions that are fully conserved, substitution, deletion or insertion is generally disfavored, while at other positions where one or more residues are not conserved, an amino acid change can be tolerated. Residues that are semi -conserved may tolerate changes that preserve charge, polarity, and/or size. For example, a compound or peptide comprising the ammo acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 20 may have 1 , 2, 3 or 4 ammo acid substitutions, at position 1, 2, 3, 4, 5, 6, 7, and/or 8 wherein the substituted amino acid may be any one of the known 22 proteinogenic amino acids, and the compound or peptide maintains an ENaC inhibiting activity. Particularly, compound or peptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 20 may have 1, 2, 3 or 4 ammo acid substitutions, at position 1, 2, 3, 4, 5, 6, 7, and/or 8 wherein the ammo acid at position 1 (L) is substituted to I, M, F, or V; the am ino acid at position 2 (P) is substituted to R; the amino acid as position 3 (L) is substituted to I, M, F, or V; the amino acid at position 4 (P) is substituted to R; the amino acid at position 5 (L) is substituted to I, M, F, or V; the amino acid at position 6 (D) is substituted to R, N, Q, E, K, and S; the amino acid at position 7 (Q) is substituted to R, N, D, E, H, K, M, and S and/or the amino acid at position 8 (T) is substituted to A, N or V.

[0056] The disclosure relates to compounds of Formula I and salts thereof,

R^PEP-R² (I) wherein R^! and R² are, independently, a blocking or a protecting group or absent, and PEP is X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO: 20) or a functional fragment of SEQ ID NO: 20; wherein

Xi is leucine (L), isoleucine (I), methionine (M), phenylalanine (F), or valine (V), or a conservatively substituted amino acid of any one of L, I, M, F, or V;

X2 is proline (P) or arginine (R) or a conservatively substituted ammo acid of any one of P or R;

X3 is leucine (L), isoleucine (I), methionine (M), phenylalanine (F), or valine (V), or a conservatively substituted amino acid of any one of L, I, M, F, or V;

X4 is proline (P) or arginine (R) or a conservatively substituted amino acid of any one of P or ;

X5 1S leucine (L), isoleucine (I), methionine (M), phenylalanine (F), or valine (V), or a conservatively substi tuted amino acid of any one of L, I, M, F, or V;

Xe is aspartic acid (D), asparagine (N), glutamine (Q), glutamic acid (E), serine (S), lysine (K), arginine (R), or citnilline (Cit), or a conservatively substituted amino acid of any one of D, N, Q, E, S, K, R, or Cit; X? is absent, or glutamine (Q), arginine (R), asparagine (N), aspartic add (D), glutamic acid (E), histidine (H), lysine (K), methionine (M), or serine (S) or a conservatively substituted amino acid of any one of Q, R, N, D, E, H, K, M or S; and

Xs is absent, or alanine (A), asparagine (N), serine (S), threonine (T) or valine (V) or a conservatively substituted amino acid of any one of A, N, S, T, or V.

[0057] Preferably, the disclosure relates to compounds of Formula 1 and salts thereof,

R^PEP-R² (I)

wherein R¹ and R² are, independently, a blocking or a protecting group or absent, and PEP is X1-X2-X3-X4--X --X&--X7--X8 (SEQ ID NO: 20) or a functional fragment of SEQ ID NO: 20, wherein,

Xi is leucine or a conservatively substituted amino acid thereof;

X?. is proline or a conservatively substituted amino acid thereof;

X3 is leucine or a conservatively substituted amino acid thereof:

X₄ is proline or a conservatively substituted amino acid thereof;

X5 is leucine or a conservatively substituted amino acid thereof;

Xe is aspartic acid or a conservatively substituted amino acid;

X? is absent, or glutamine or a conservatively substituted amino acid thereof; and

Xs is absent, or A, N, or V or a conservatively substituted amino acid of any one of A, N, or V.

[0058] Accordingly, the disclosure relates to compounds of Formula la and salts thereof,

R^Xi-Xi-Xs-Xa-Xs-Xe-XT-Xs-R la),

wherein R¹, R², and Xi to Xe are as defined in Formula I, and X7 is Q, R, N, D, E, H, , M or S or a conservatively substituted amino acid of any one of Q, R, N, D, E, H, K, M or S; and Xs is or A, N, or V or a conservatively substituted amino acid of any one of A, N, or V.

[0059] The disclosure preferably relates to compounds of Formula I or la, wherein Xi is L, I, M, F, or V: \ > is P or R; X3 is L, I, M, F, or V: X > is proline P or R; Xs is L, 1, M, F, or V; Xe is D, N, Q, E, or S; X7 Q, R, N, D, E, I I. K, M, or S; and Xs is A, N or V. Preferably, X2 or X i or

[0060] Accordingly, the disclosure relates to compounds of Formula la, wherein Xi is L, I, M, F, or V; X2 is P; X3 is L, I, M, F, or V; X₄ is P; Xs is L, I, M, F, or V; Xe is D, R, N, Q, E, K, S or Cit; X? is Q, R, N, D, E, H, K, M, or S; and Xs is A, N or V. For example, PEP in Formula I or la can be LPLPLDQA (SEQ ID NO: 9) or LPIPLDQN (SEQ ID NO: 10) or LPIPLDQV (SEQ ID NO: 1 1) or a functional fragment thereof. Particularly, PEP is an ENaC inhibitor peptide.

[0061] In another aspect, in the compounds of Formula la, above, the peptides disclosed under Table 1 of U.S. Pub. No, 2017-0226180 (spanning pages 4 to 8 of US 2017-0226180) are excluded from the peptides and compounds of this disclosure.

[0062] The disclosure also relates to compounds of Formula lb and salts thereof,

R^l-Xi-X2-X3-X4-X5-X6-X7-R² (lb), wherein K . K and Xi to Xe are as defined in Formula I. and X? is Q, R, N, D, E, H, K, M or S or a conservatively substituted amino acid of any one of Q, R, N, D, E, H, K, M or S.

[0063] The disclosure relates to compounds of Formula lb, wherein Xi is L, I, M, F, or V; X2 is P or R : X3 is L, 1, M, F, or V; X₄ is P or R; Xj is L, I, M, F, and V; Xe is D, N, Q, E, or S; and X? is Q, R, N, D, E, H, K, M, or S. Preferably, X2 or X4 or both X2 and X4 are P.

Θ064] Particularly, in compounds of Formula lb, Xi is L, I, M, F, or V; X2 is P; X3 is L, I, M, F, or V; i is P: Xs is L, I, M, F, or V; Xe is D, R, N, Q, E, K, S or Cit; and X₇ is Q, R, N, D,

E, H, K, M, or S. For example, PEP in Formula lb can be LPLPLDQ (SEQ ID NO: 4) or LPIPLDQ (SEQ ID NO: 7) or a functional fragment thereof. Particularly, PEP is an ENaC inhibitor peptide.

[0065] The disclosure further relates to compounds of Formula lc and salts thereof,

R^1~Xi^~X₂-X3-X4-X5^~X6~R² (lc),

wherein R¹, R², and Xi to Xe are as defined in Formula I.

[0066] The disclosure also relates to compounds of Formula Ic, wherein Xi is L, I, M, F, or V; X2 is P or R; X₃ is L, 1, M, F, or V; X i is P or R; Xj is L, 1, M, F, or V: and Xe is D or N, Q, E, or S. Preferably, X2 or X4 or both X2 and X4 are P.

[0067] Particularly, in compounds of Formula Ic, Xi is L, I, M, F, or V; X2 is P; X3 is L, I, M,

F, or V; X₄ is P; Xs is L, I, M, F, or V; and Xe is D, R, N, Q, E, K, S or Cit. For example, PEP in Formula Ic can be LPLPLD (SEQ ID NO: 3) or LPIPLD (SEQ ID NO: 6) or a functional fragment thereof.

[0068] In preferred aspects of compound of Formula I, la, lb or Ic, at least one of R¹ and R² is a blocking or protecting group, and more preferably both R¹ and R² are blocking or protecting groups. Suitable blocking groups are generally known in the art and include, without limitation, additional amino acids attached to the amino and/or carboxyl terminal residues of the peptide. When the peptide component of the compound (PEP in Formulas 1) has an ammo acid sequence that is found in naturally occurring protein, it is preferred that an additional amino acids that serve as a blocking or protecting group (R^! and/or R²) are not the same amino acid that is immediately adjacent to the PEP sequence as it is found in the naturally occurring protein. For example, R¹ and/or R² can include one to about 4 non-protemogenic ammo acids, such as a D-amino acid (e.g., D-alanine), Norleucine (Nle); 4-hydroxyproline (HYP); 3,4- dehydro-L-proline (DHP); aminoheptanoic acid (AHP); (2R,5S)-5-phenyl- pyrrolidine-2- carboxylic acid (2PP): L-a-methyl serine (MS); N-methylvaline (MV); 6-aminohexanoic acid (6-AHP); 7-aminoheptanoic acid (7-AHP), and the like, in some preferred aspects of compounds of Formula I, la, lb or Ic, R¹ and R² are each, independently, one to about 4 non- proteinogenic amino acids, preferably one or two non-proteinogenic amino acids, such as D- alanine. Alternatively, blocking groups such as pyroglutamic acid or other molecules known in the art can be attached to the amino terminal, carboxyl terminal or both the amino terminal and carboxyl terminal residues, or the amino group at the ammo terminus and/or carboxyl group at the carboxyl terminus can be replaced with a different moiety. Additionally, the peptide terminus can be modified, e.g., by acetyiation of the N-terminus and/or amidation of the C-terminus. Likewise, the peptides can be covalently or non-covalently coupled to pharmaceutically acceptable "carrier'¹ proteins prior to administration.

[0069] In particular examples, the compound is RZ-LPLPLD-R² (SEQ ID NO: 21) or R¹- LPIPLD-R- (SEQ ID NO: 22), wherein, R¹ and R² are each, independently, a blocking or a protecting group as described herein. In other examples, the compound is R'-LPLPLDQ-R² (SEQ ID NO: 23) or R^!-LPIPLDQ~R² (SEQ ID NO: 24), wherein, R^! and R² are each, independently, absent, or a blocking or a protecting group as described herein. In other examples, the compound is R^l-LPLPLDQT-R² (SEQ ID NO: 25) or R'-LPIPLDQT-R² (SEQ ID NO: 26), wherein, R¹ and R² are each, independently, absent, or a blocking or a protecting group as described herein. In preferred examples, R¹ and R² are each one or two non- proteinogenic amino acids, such as a D-amino acids (e.g., D-alanine), Norleucine (Nle); 4- hydroxyproline (HYP); 3,4-dehydro-L-proline (DHP); aminoheptanoic acid (AHP); (2R,5S)-

5- phenyl- pyrroiidine-2-carboxylic acid (2PP); L-a-methylserine (MS); N-methylvaline (MV);

6- aminohexanoic acid (6-AHP); 7-aminoheptanoic acid (7-AHP), and the like.

[0070] The disclosure further relates to peptides comprising, consisting essentially of, or consisting of the sequence set forth in a) Xi-X2-X3-X4-Xs-X6 (SEQ ID NO: 1), b) X1-X2-X3- X4-X5-X6-X7 (SEQ ID NO: 2), or c) X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO: 20) wherein, in each case, individually, Xi is leucine (L), isoleucine (I), methionine (M), phenylalanine (F), or valine (V), or a conservatively substituted amino acid of any one of L, I, M, F, or V;

X2 is proline (P) or arginine (R) or a conservatively substituted amino acid of any one of P or R;

X3 is leucine (L), isoleucine (I), methionine (M), phenylalanine (F), or valine (V), or a conservative^ substituted amino acid of any one of L, I, M, F, or V;

X* is proline (P) or arginine (R) or a conservatively substituted amino acid of any one of P or R;

X.-3 is leucine (L), isoleucine (I), methionine (M), phenylalanine (F), or valine (V), or a conservatively substituted amino acid of any one of L, I, M, F, or V;

Xe is aspartic acid (D), asparagine (N), glutamine (Q), glutamic acid (E), serine (S), lysine (K), arginine (R), or citrulline (Cit), or a conservatively substituted amino acid of any^¬ one of D, N, Q, E, S, K, R, or Cit;

X? is glutamine (Q), arginine (R), asparagine (N), aspartic acid (D), glutamic acid (E), histidine (H), lysine (K), methionine (M), or serine (S) or a conservatively substituted amino acid of any one of Q, R, N, D, E, H, K, M or S; and

Xs is alanine (A), asparagine (N), serine (S), threonine (T) or valine (V) or a conse vatively substituted amino acid of any one of A, N, S, T, or V; and active fragments and salts thereof. Preferably, in the peptide compounds of SEQ ID NO: 20, above, Xg is A, N, or V, or a conservatively substituted amino acid of any one of A, N, or V.

[0071] The disc!osure further relates to peptides comprising, consisting essentially of, or consisting of the sequence set forth in a) Xi-X2-X3-X -X5-X6 (SEQ ID NO: I), b) Xi-X₂-X3- X4-X5-X6-X7 (SEQ ID NO: 2), or c) 1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO: 20) wherein, in each case, individually,

Xi is leucine or a conservatively substituted amino acid thereof;

X2 is proline or a conservatively substituted ammo acid thereof;

X3 is leucine or a conservatively substituted amino acid thereof:

X₄ is proline or a conservatively substituted amino acid thereof;

X5 is leucine or a conservatively substituted amino acid thereof;

Xe is aspartic acid or a conservatively substituted amino acid;

X7 is glutamine or a conservatively substituted amino acid thereof; and

Xs is alanine (A), asparagine (N) or valine (V) or a conservatively substituted amino acid of any one of A, N or V; and active fragments and salts thereof. [0072] In some embodiments, X2 or X4 or both X2 and X4 in the peptide (PEP) sequence SEQ ID NO: 1, SEQ ID NO: 2 and/or SEQ ID NO: 20 are prolines (P). Preferably, in the peptide sequence, Xi is L, I, M, F, or V; X2 is P; X3 is L, I, M, F, or V; X i is P; Xs is L, L M, F, or V; Xe is D, N, Q, E, , K, S or Cit; X?, when present, is Q, R, N, D, E, H, K, M, or S; and Xs, when present, is A, N, or V.

[0073] In certain embodiments, the peptide has at least 80% amino acid sequence identity to LPLPLDQA (SEQ ID NO: 9) or LPIPLDQN (SEQ ID NO: 10) or LPIPLDQV (SEQ ID NO: 11) over the length of SEQ ID NO: 9, 10 or 1 1.

[0074] In preferred aspects, the peptides that contain amino acid sequences that are found in naturally occurring SPLUNC1 protein, including the peptides LPVPLD (SEQ ID NO: 27), LPVPLDQ (SEQ ID NO: 28) and LPVPLDQT (SEQ ID NO: 29) are excluded from the peptides and compounds of this disclosure.

[0075] In another aspect, the peptides disclosed under Table 1 of U.S. Pub. No. 2017-0226180 (spanning pages 4 to 8 of US 2017-0226180) are excluded from the peptides and compounds of this disclosure.

[0076] In another embodiment, the disclosure relates to peptides containing conservative amino acid substitutions. The conservative amino acid substitution can be made using proteinogenic amino acids or non-proteinogenic amino acids. In some embodiments, the conservative mutation is made with an amino acid analogs, such as, p-Acetylphenylalanine, m- Acetylphenylalanine, O-allyltyrosine, Phenylselenocysteine, p-Propargyloxyphenylalanine, p- Azidophenylalanine, p-Boronophenylalanine, O-methyltyrosine, p-Aminophenylalanine, p~ Cyanophenylalanine, m-Cyanophenylalanine, p-Fluorophenylalanine, p-Iodophenylalanine, p- Bromophenylalanine, p-Nitrophenylalanine, L-DOPA, 3-Aminotyrosine, 3-Iodotyrosine, p- Isopropylphenylalanine, 3-(2-Naphtliyl)alanine, biphenylalanine, homoglutamine, D-tyrosine, p-Hydroxyphenyllactic acid, 2-Aminocaprylic acid, bipyridylalanine, HQ~alanine, p- Benzoylphenylalanine, o-Nitrobenzylcysteine, o-Nitrobenzylserine, 4,5-Dimethoxy-2- Nitrobenzylserine, o-Nitrobenzyllysine, o-Nitrobenzyltyrosine, 2-Nitrophenylalanine, dansylalanine, p-Carboxyniethylphenylalanine, 3-Nitrotyrosine, sulfotyrosine, acetyllysine, methylhistidine, 2-Aminononanoic acid, 2-Aminodecanoic acid, pyrrolysine, Cbz-lysine, Boc- lysine, allyloxycarbonyllysine, arginosuccinic acid, citrulline, cysteine sulfinic acid, 3,4- dihydroxyphenylalanine, homocysteine, homoserine, ornithine, 3-monoiodotyrosine, 3,5- diiodotryosine, 3,5,5, -triiodothyronine, and 3,3',5,5'-tetraiodothyronine. The term includes modified or unusual amino acids e.g., D-amino acids, hydroxylysine, 4-hydroxyproline, N- Cbz-protected amino acids, 2,4-diaminobutyric acid, homoarginine, norleucine, N- methylaminobutyric acid, naphthylalanine, phenylglycine, -phenylproline, tert-leucine, 4- aminocyclohexylalanine, N-methyl-norleucine, 3,4-dehydroproline, N,N- dimethyiammogiycme, N-metiiylaminoglycine, 4-aminopiperidine-4-carboxylic acid, 6- aminocaproic acid, trans-4-(aminomethyl)-cyclohexanecarboxylic acid, 2-, 3-, and 4- (aminomethyi)-henzoic acid, 1 -aminocyclopentanecarboxylic acid, 1 - aminocyclopropanecarboxylic acid, and 2-benzyl-5-aminopentanoic acid; functionalized amino acids, e.g. , alkyne-functionalized, azide-functionalized, ketone-functionalized, aminooxy-functionalized amino acids and the like. See Liu et al , Ann. Rev. Biochem. 79:413, 2010; Kim et al., Curr. Opin. Chem. Biol. 17:412, 2013.

[0077] The disclosure relates to peptides that comprise, consist essentially of, or consist of Xi- X2-X3-X4-X5-X6 (SEQ ID NO: I), wherein Xi is L, I, M, F, or V or a conservatively substituted amino acid of any one of L, I, M, F, or V; Xi is P or R or a conservatively substituted amino acid of any one of P or R; X3 is L, I, M, F, or V or a conservatively substituted amino acid of any one of L, I, M, F, or V; X4 is P or R or a conservatively substituted amino acid of any one of P or R; X5 is L, I, M, F, or V or a conservatively substituted amino acid of any one of L, I, M, F, or V; and Xe is D, R, N, Q, E, S, K, or Cit, or a conservati ely substituted amino acid of any one of D, R, N, Q, E, S, K, or Cit; and active fragments and salts thereof.

[0078] In more specific embodiments, the peptide comprises, consists essentially of, or consists of X1-X2-X3-X4-X5-X6 (SEQ ID NO: 1), wherein Xi is leucine (L) or a conservatively- substituted amino acid thereof; X2 is proline (P) or a conservatively-substituted amino acid thereof; X3 is leucine ( I .) or a conservatively-substituted amino acid thereof; X i is proline (P) or a conservatively-substituted amino acid thereof; X5 is leucine (L) or a conservatively- substituted amino acid thereof; and Xe is aspartic acid (D) or a conservatively-substituted amino acid thereof, and active fragments and salts thereof.

[0079] In some embodiments, in the peptide (PEP) sequence X1-X2-X3-X4-X5-X6 (SEQ ID NO: 1), X2 or X₄ or both X2 and X4 are prolines (P). Preferably, in the peptide sequence SEQ ID NO: 1, X] is L, I, M, F, or V; X2 is P; X₃ is L, L M, F, or V; X₄ is P; X₅ is L, I, M, F, or V; and X is D, R, N, Q, E, K, S or Cit. For example, the peptide can comprise, consist essentially of, or consist of LPLPLD (SEQ ID NO: 3) or LPIPLD (SEQ ID NO: 6) or a functional fragment thereof.

[0080] In certain embodiments, the peptide has at least 80% amino acid sequence identity to

LPLPLD (SEQ ID NO: 3) or LPIPLD (SEQ ID NO: 6) over the length of SEQ ID NO: 3 or 6.

[0081 ] This disclosure also relates to peptides that comprise, consist essentially of, or consist of X1-X2-X3-X4-X5-X6-X7 (SEQ ID NO: 2), wherein Xi is L, I, M, F, or V or a conservatively substituted amino acid of any one of L, I, M, F, or V; X2 is P or R or a conservatively substituted amino acid of P or R; X3 is L, I, M, F, or V or a conservatively substituted amino acid of any one of L, I, M, F, or V; X4 is P or R or a conservatively substituted amino acid of any one of P or R; X5 is L, I, M, F, or V or a conservatively substituted ammo acid of any one of L, I, M, F, or V; Xe is D, R, N, Q, E, K, S or Cit or a conservatively substituted amino acid of D, R, N, Q, E, K, S or Cit; and X? is Q, R, N, D, E, H, K, M, or S or a conservatively substituted ammo acid of any one of Q, R, N, D, E, H, K, M, or S; and active fragments and salts thereof.

[0082] In more specific embodiments, the peptide comprises, consists essentially of, or consists of X1-X2-X3-X4-X5-X6-X7 (SEQ ID NO: 2), wherein Xi is leucine (L) or a conservatively-substituted amino acid thereof; X2 is proline (P) or a conservatively-substituted amino acid thereof; X3 is leucine (L) or a conservatively-substituted amino acid thereof; X4 is proline (P) or a conservatively-substituted amino acid thereof; Xs is leucine (L) or a consereatively-substituted amino acid thereof; Xe is aspartic acid (D) or a conservatively- substituted amino acid thereof; X? is glutamine (Q) or a conservatively-substituted amino acid thereof, and active fragments and salts thereof.

[0083] In some embodiments, in the peptide (PEP) sequence X1-X2-X3-X4-X5-X6-X7 (SEQ ID NO: 2), X2 or X4 or both X2 and X4 are prolines (P). Preferably, in the peptide sequence SEQ ID NO: 2, Xi is L, I, M, F, or V; X2 is P; X3 is L, I, M, F, or V; X₄ is P; Xj is L, I, M, F, or V; and Xe is D, R, N, Q, E, K, S or Cit; and Χτ is Q, R, N, D, E, H, K, M, or S. For example, the peptide can comprise, consist essentially of, or consist of LPLPLDQ (SEQ ID NO: 4) or LPIPLDQ (SEQ ID NO: 7) or a functional fragment thereof.

[0084] In certain embodiments, the peptide has at least 80% amino acid sequence identity to LPLPLDQ (SEQ ID NO: 4) or LPIPLDQ (SEQ ID NO: 7) over the length of SEQ ID NO:4 or

H

[0085] The disclosure also relates to peptides that comprise, consist essentially of, or consist of Xi-X2-X3-X4-Xj-Xe-X7-Xs (SEQ ID NO: 20), wherein Xns L, I, M, F, or V or a conservative^ substituted amino acid of any one of L, I, M, F, or V; X2 1S P or R or a conservative!}_' substituted amino acid of any one of P or R; X3 is L, I, M, F, or V or a conservatively substituted amino acid of any one of L, I, M, F, or V; X4 is P or R or a conservatively substituted amino acid of any one of P or R; Xs is L, I, M, F, or V or a conservatively substituted ammo acid of any one of L, I, M, F, or V; Xe is D, R, N, Q, E, K, S or Cit or a conservatively substituted amino acid of any one of D, R, N, Q, E, K, S or Cit; X? is

Q, R, N, D, E, H, K, M, or S or a conservatively substituted amino acid of any one of Q, R, N,

D, E, H, K, M, or S; and Xs is A, N, S, T, or V or a conservatively substituted amino acid of any one of A, N, S, T, or V; and functional fragments and salts thereof. Preferably, Xs is A, N, or V, or a conservatively substituted amino acid of any one of A, N, or V.

[0086] In more specific embodiments, the peptide comprises, consists essentially of, or consists X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO: 20) wherein Xi is leucine (L) or a conservatively-substituted amino acid thereof; X2 is proline (P) or a conservatively-substituted amino acid thereof: X3 is leucine (L) or a conservatively-substituted ammo acid thereof; X4 is proline (P) or a conservatively-substituted amino acid thereof; Xs is leucine (L) or a conservatively-substituted amino acid thereof; Xe is aspartic acid (D) or a conservatively- substituted amino acid thereof; X? is glutamine (Q) or a conservatively-substituted amino acid thereof; and Xs is A, N, or V, and functional fragments and salts thereof,

[0087] In some embodiments, in the peptide (PEP) sequence X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO: 20), X2 or X, or both X2 and X4 are prolines (P). Preferably, in the peptide sequence SEQ ID NO: 20, X; is L, I, M, F, or V; X₂ is P; X₃ is L, I, M, F, or V; X₄ is P; Xs is L, I, M, F, or V; and ¾ is D, R, N, Q, E, K, S or Cit; Xi is Q, R, N, D, E, H, K, M, or S; and Xs is A, N, S, T, or V. For example, the peptide can comprise, consist essentially of, or consist of LPLPLDQT (SEQ ID NO: 5) or LPIPLDQT (SEQ ID NO: 8) or a functional fragment thereof.

[0088] In certain embodiments, the peptide has at least 80% amino acid sequence identity to LPLPLDQT (SEQ ID NO: 5) or LPIPLDQT (SEQ ID NO: 8) over the length of SEQ ID NO:5 or 8.

[0089] Variant, mutant and modified forms of the peptides, as described herein, can be prepared and represent additional aspects of this disclosure.

ΘΘ90] Especially, the disclosure relates to peptides consisting essentially of or consisting of the ammo acid sequence set forth in LPLPLD (SEQ ID NO: 3); LPLPLDQ (SEQ ID NO: 4);

LPLPLDQT (SEQ ID NO: 5); LPIPLD (SEQ ID NO: 6); LPIPLDQ (SEQ ID NO: 7); or

LPIPLDQT (SEQ ID NO: 8); including, protected or modified forms thereof.

[0091 ] In some aspects, the peptides comprising X1-X2-X3-X4-X5-X6 (SEQ ID NO: 1 ) or Xi-

X7-X3-X4-XS-X6-X7 (SEQ ID NO: 2) or X1-X2-X3-X4-X5-X6-X7-XS (SEQ ID NO: 20) or a functional fragment thereof can include additional amino acids at the amino and/or carboxy terminal. In these aspects, it is generally preferred that the N -terminus or the C-terminus or both the N- and the C-termini of the peptide does not contain a corresponding amino acid of

SPLUNC 1 (e.g., human BPI fold-containing family A member 1 (SPLUNC l) having

UNIPROT Accession No. Q9NP55 (version 1 , deposited October 1 , 2000)). For example, a sequence alignment indicates that the LPLPLD (SEQ ID NO: 3), LPIPLD (SEQ ID NO: 6),

LPLPLDQ (SEQ ID NO: 4) and LPIPLDQ (SEQ ID NO: 7) can be aligned with the sequence FGGLPVPLDQ TLPL (SEQ ID NO: 30; matching amino acids in hold) which is found in human SPLUNC1. Accordingly, under one embodiment, peptides of the present disclosure having the amino acid sequence SEQ ID NOs: 1 or 2 do not contain any additional amino acids in the N-terminus or the C-terminus which is identical to and which further corresponds to the amino acid of human SPLUNCl protein, e.g., G at N-l, G at N-2, F at N-3, etc.; T at C+l, L at C+2, P at C+3, etc. of SEQ ID NO: 4 or SEQ ID NO: 7. The N-minus (N-) used herein is well-known in the art to mean an amino acid preceding the N-terminus of a given sequence, while the integer (e.g., 1 ) is the positioning of said amino acid relative to the N-terminus. Likewise, the C-plus (C+) nomenclature means an amino acid following the C-terminus of a given sequence, while the integer (e.g., 1) is understood the positioning of said amino acid relative to the C-terminus.

[0092] The disclosure further relates to functional fragments of the ENaC inhibitor peptides disclosed above. As used herein, a ^"'functional fragment" of a peptide substantially retains at least one biological activity normally associated with that peptide (e.g., binding to or inhibiting a sodium channel). In particular embodiments, the "functional fragment" substantially retains all of the activities possessed by the unmodified peptide. By "substantially retains" biological activity, it is meant that the peptide retains at least about 20%, 30%, 40%, 50%, 60%, 75%, 85%, 90%, 95%, 97%, 98%, 99%, or more, e.g., 99.9%, of the biological activity of the native polypeptide. In another embodiment, the functional fragment has a higher level of activity, about 110%, 120%, 130%, 150%, 175%, 200%, 300%, 350%, 500%, or more, than the native peptide. A "nonfunctional" peptide is one that exhibits little or essentially no detectable biological activity normally associated with the peptide (e.g., at most, only an insignificant amount, e.g., less than about 10% or even 5%). Biological activities such as protein binding and sodium channel inhibitory activity can be measured using assays that are well known in the art and as described herein, e.g., using a Surface Plasmon Resonance (SPR) assay.

[0093] The disclosure further relates to variants of the ENaC inhibitor peptides comprising the ammo acid sequence set forth in LPLPLD (SEQ ID NO: 3); LPLPLDQ (SEQ ID NO: 4); LPLPLDQT (SEQ ID NO: 5); LPIPLD (SEQ ID NO: 6); LPIPLDQ (SEQ ID NO: 7); or LPIPLDQT (SEQ ID NO: 8). The term "variant" as used herein refers to a biomolecule (e.g., polypeptide or nucleic acid) whose sequence that differs from that of a parent sequence by virtue of at least one modification. Accordingly, variant polypeptides comprise at least one modification of an amino acid residue; variant polynucleotides comprise at least one modification of a nucleic acid residue. [0094] In some embodiments, the disclosure includes variant polypeptides having at least one amino acid modification compared to the parent polypeptide, e.g., from about one to about five amino acid modifications compared to the parent, e.g., differing in amino acid sequence by one, two, three, four, or five amino acids, compared to ammo acid sequence set forth in LPLPLD (SEQ ID NO: 3); LPLPLDQ (SEQ ID NO: 4); LPLPLDQT (SEQ ID NO: 5); LPIPLD (SEQ ID NO: 6); LPIPLDQ (SEQ ID NO: 7): or LPIPLDQT (SEQ ID NO: 8).

[0095] Under an alternate embodiment, the variant ENaC inhibitor peptide may comprise an amino acid sequence which is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%, or greater % identity to, for example, the following polypeptide sequences: LPLPLD (SEQ ID NO: 3); LPLPLDQ (SEQ ID NO: 4); LPLPLDQT (SEQ ID NO: 5); LPIPLD (SEQ ID NO: 6); LPIPLDQ (SEQ ID NO: 7); or LPIPLDQT (SEQ ID NO: 8).

[0096] As is known in the art, a number of different programs can be used to identify whether a polypeptide has sequence identity or similarity to a known sequence. Sequence identity or similarity may be determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith et a!.. Adv. Appl. Math. 2:482 ( 1981 ), by the sequence identity alignment algorithm of Needleman et al. , J Mol. Biol. 48A43 ( 1970), by the search for similarity method of Pearson et a!,., Proc. Natl. Acad. Sci. USA- 55 :2444 (1988), by computerized implementations of these algorithms (G AP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI). See, Devereux et at, Nucl. Acid Res. 72:387 (1984),

[0097] An example of a useful algorithm is PILEUP. PILE-UP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng et ah, J Mol Evol. 55 :351 (1987); the method is similar to that described by Higgins et al , CABIOS 5 : 15 1 (1989). Another example of a useful algorithm is the BLAST algorithm, described in Altschul et al, J. Mol. Biol 215 :403 (1990) and Karlin et al, Proc. Natl. Acad. Sci. USA 90:5873 (1993). A particularly useful BLAST program is the WU- BLAST-2 program which was obtained from Altschul et al, Meth. Enzymol 266:460 ( 1996). WU-BLAST-2 uses several search parameters, which are preferably set to the default values. The parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity. An additional useful algorithm is gapped BLAST as reported by Altschul et al, Nucleic Acids Res. 25:3389 ( 1997).

[0098] A percentage amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "longer" sequence in the aligned region. The "longer" sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Biast-2 to maximize the alignment score are ignored).

[0099] The alignment may include the introduction of gaps in the sequences to be aligned. In addition, for sequences which contain either more or fewer amino acids than the peptides specifically disclosed herein, it is understood that in some embodiments, the percentage of sequence identity will be determined based on the number of identical amino acids in relation to the total number of amino acids. Thus, for example, sequence identity of sequences shorter than a sequence specifically disclosed herein, will be determined using the number of amino acids m the shorter sequence, in some embodiments. In percent identity calculations relative weight is not assigned to various manifestations of sequence variation, such as insertions, deletions, substitutions, etc.

[00100] In some embodiments, only identities are scored positively (+1 ) and all forms of sequence variation including gaps are assigned a value of "0," which obviates the need for a weighted scale or parameters as described below for sequence similarity calculations. Percent sequence identity can be calculated, for example, by dividing the number of matching identical residues by the total number of residues of the "shorter" sequence in the aligned region and multiplying by 100. The "longer" sequence is the one having the most actual residues in the aligned region.

[00101] In another embodiment, included herein are mutant ENaC inhibitor peptides comprising mutation in the core polypeptide sequence set forth in LPLPLD (SEQ ID NO: 3); LPLPLDQ (SEQ ID NO: 4); LPLPLDQT (SEQ ID NO: 5); LPIPLD (SEQ ID NO: 6); LPIPLDQ (SEQ ID NO: 7); or LPIPLDQT (SEQ ID NO: 8), In some embodiments, the mutation is a deletion or addition of 1-3 ammo acids. Preferably, the mutation does not change the biological activity of the mutein compared to a wild-type protein (e.g., SPLUNCl). A "deletion," as used herein, refers to changes in an amino acid or nucleotide sequence resulting in the removal of one or more amino acid residues or nucleotides, respectively, to the parent molecule. "Addition," as used herein, refers to changes in an ammo acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively, to the parent molecule. [00102] In another embodiment, included herein are homologs to the aforementioned

ENaC inhibitor peptide or polynucleotides encoding such polypeptides (described below). The term "homology," as used herein, refers to a polypeptide sequence derived from a common ancestral gene. There may be partial homology or complete homology, A partially complementary sequence that at least partially mimics a parent sequence (e.g., LPLPLD (SEQ ID NO: 3); LPLPLDQ (SEQ ID NO: 4); LPLPLDQT (SEQ ID NO: 5): LPIPLD (SEQ ID NO: 6); LPIPLDQ (SEQ ID NO: 7); or LPIPLDQT (SEQ ID NO: 8} with respect to its biological activity. In certain embodiments, the peptide mimics the sodium channel binding domain of a parent PLUNC protein. The sodium channel binding domain is the minimal fragment of the PLUNC protein required to have substantially the same binding activity to the sodium channel as the full length PLUNC protein. The term "substantially the same binding activity" refers to an activity that is at least about 50% of the binding activity of the full length protein, e.g., at least about 60%, 70%, 80%, or 90%, or more, of the binding activity. In some embodiments, the peptide has at least the same binding activity as the full length PLUNC protein. In some embodiments, the sodium channel is ENaC, e.g. , human ENaC, In another embodiment, the sodium channel is one that is similar in sequence and/or structure to ENaC, such as acid-sensing ion channels (ASIC).

[00103] The disclosure further relates to modified peptides. For example, the ENaC inhibitor peptide can be cyclized. As another example, the ENaC inhibitor peptide can have one or more amino acid modifications. An ENaC inhibitor peptide can include one or more D- amino acids. Modifications of interest that do not alter primary sequence include chemical derivatization of polypeptides, e.g., acetylation, or carboxylation. Also included are modifications of glycosylation, e.g. those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g. by exposing the polypeptide to enzymes which affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Also embraced are peptides that have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.

[00104] In some preferred aspects, the peptides of the disclosure include amino- and/or carboxyl-terminal blocking groups or protecting groups. Suitable blocking groups can include, without limitation, additional unrelated amino acids attached to the amino and/or carboxvl terminal residues of the peptide to be administered. This can be done either chemically during the synthesis of the peptide or using recombinant DNA technology or other suitable methods.

For example, one or two or more non-proteinogenic amino acids, such as a D-amino acid e.g.,

D-aianme, can be added to the amino terminus, the carboxy terminus or both the ammo and carboxy termini . Alternatively, blocking groups such as pvroglutamic acid or other molecules known in the art can be attached to the amino terminal, carboxyl terminal or both the amino terminal and carboxy terminal residues, or the amino group at the amino terminus and/or carboxyl group at the carboxyl terminus can be replaced with a different moiety. Additionally, the peptide terminus can be modified, e.g., by acetylation of the N-terminus and/or amidation of the C-terminus. Likewise, the peptides can be covalently or non-covalently coupled to pharmaceutically_' acceptable "carrier" proteins prior to administration.

[00105] Also provided in the disclosure are ENaC inhibitor peptides or compounds of

Formula I thai have been modified using ordinaiy molecular biological techniques and synthetic chemistry so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent. For instance, the ENaC inhibitor peptide or compound of Formula I may be joined to a wide variety of other oligopeptides or proteins for a variety of purposes. By providing for expression of the subject peptides, various post-translational modifications may be achieved, e.g., famesylation or prenylation. In another embodiment, the ENaC inhibitor peptide or compounds of Formula I can be bound to a lipid group at a terminus, so as to be able to be bound to a lipid membrane, such as a liposome.

[00106] The disclosure further relates to ENaC inhibitor peptides or compounds of

Formula I containing other types of modifications including, e.g., (1) end-cappings of the terminal of the peptides, such as amidation of the C-terminus and/or acetylation or deamination of the N-terminus; (2) introducing peptidomimetic elements in the structure; and (3) cyclization, in which the cyclization of the peptide can occur through natural amino acids or non-naturally-occurring building blocks.

[0100] The disclosure relates to peptidomimetics that have the same sidechains as one or more

ENaC inhibitor peptides or compounds of Formula I described herein.

[0101 ] The disclosure further relates to "peptoids" derived from one or more above-described

ENaC inhibitor peptides or compounds of Formula I. A peptoid is a polypeptide containing one or more N-substituted glycine residues. An N-substituted amino acid residue has a standard amino acid side-chain pendant from the N, rather than from the a-carbon. For example NVal has a 2-propyl group pendant from its N. N-alkyl glycine residues are common peptoid building blocks, which may mimic standard amino acids, such as Val (2-propyl), Leu (isobutyl) or He

(2-butyl) among the 19 other standard R-group-containing amino acids, or which may contain virtually any R-group, for example any lower alkyl (C-l -6) group, alcohol, amine, organic acid

(carboxy 1-containmg) group, etc. By including N-modified glycine residues, the peptoid is made resistant to proteolysis and may be functional) zed in a manner that increases the peptide's bioavailability or tissue localization. U.S. Pat. No. 6,075, 121 describes peptoid structures and methods of producing peptoids. U.S. Pat. No. 6,887,845 describes N-palmitoyl derivatized surfactant protein-B, The palmitoyl moieties are thought to interact with cellular lipid bilayers, thereby affecting the bioavailability of the polypeptide.

[0102] In some embodiments, the peptides of the disclosure may comprise one or more additional residues at the amino- and/or carboxyl-terminal ends. In some embodiments, the additional residue is D-alanine (D-A!a); Norleucine (Me); 4-hydroxyprohne (HYP); 3,4- dehydro-L-proline (DHP); aminoheptanoic acid (AHP); (2R,5S)-5-phenyl- pyrrolidine-2- carboxylic acid (2PP); L-a-methylserine (MS); N-methyivaline (MV); 6-aminohexanoic acid

(6-AHP); or 7-aminoheptanoic acid (7-AHP). Representative examples of modified ENaC inhibitor peptides or compounds of Formula I include, e.g., peptides having the following structures: aLPLPLDa (SEQ ID NO: 12); aLPLPLDQa (SEQ ID NO: 13); aLPLPLDQTa (SEQ

ID NO: 31); aLPIPLDa (SEQ ID NO: 14); aLPIPLDQa (SEQ ID NO: 15); aLPIPLDQTa (SEQ

ID NO: 32); aaLPLPLDaa (SEQ ID NO: 16); aaLPLPLDQaa (SEQ ID NO: 1 7); aaLPLPLDQTaa (SEQ ID NO: 33); aaLPIPLDaa (SEQ ID NO: 18); aaLPIPLDQaa (SEQ ID

NO: 19); aaLPIPLDQTaa (SEQ ID NO: 34), wherein, a is an additional residue selected from

D-alanine (D-Ala); Norleucine (Nle); 4-hydroxyproline (HYP); 3,4-dehydro-L-proline (DHP); aminoheptanoic acid (AHP); (2R,5S)-5-phenyl-pyrrolidine-2-carboxylic acid (2PP); L-a- methylserme (MS); N-methylvaline (MV); 6-aminohexanoic acid (6-AHP); or 7- aminoheptanoic acid (7-AHP). In one specific embodiment, the ENaC inhibitor peptide comprises one or two D-alanines at the amino- and/or carboxyl-terminal ends.

[0103] The disclosure further relates to fusion proteins comprising a peptide of the instant disclosure (a peptide of SEQ ID NO: 1, 2 or 20) and a fusion partner. A "fusion protein'¹ of this disclosure comprises a peptide of SEQ ID NO: 1, 2 or 20, or a functional fragment thereof, that is bonded through a peptide bond to an amino acid sequence that is not bonded to SEQ ID

NO: 1, 2 or 20 in a naturally occurring protein. Illustrative fusion polypeptides include fusions of a peptide of the disclosure (or a fragment thereof) to all or a portion of glutathione-S- transferase, maltose-binding protein, or a reporter protein (e.g., Green Fluorescent Protein, β- glucuronidase, β-galactosidase, luciferase, etc.), hemagglutinin, c-myc, FLAG epitope, immunoglobulin F_c, human serum albumin (HSA) or a fragment thereof, and the like.

[0104] In the fusion proteins, the peptides of the disclosure can be directly bonded to the fusion partner, or indirectly bonded to the fusion partner through a suitable linker. Particularly, the linker is a spacer peptide, which can be of a flexible nature, although other chemical linkages are not excluded, A linker peptide can have a length of from about 1 amino acid to about 40 amino acids, e.g., from about 1 amino acid to about 5 amino acids, from about 5 to about 10 amino acids, from about 10 to about 20 amino acids, from about 20 to about 30 amino acids, or from about 30 to about 40 ammo acids, in length. These linkers can be produced using synthetic, linker-encoding oligonucleotides to couple the proteins. Peptide linkers with a degree of flexibility can be used. The linking peptides may have virtually any amino acid sequence, where in some embodiments the linker peptide will have a sequence that results in a generally flexible peptide. The use of small amino acids, such as glycine and alanine, are of use in creating a flexible peptide. The creation of such sequences is routine to those of skill in the art. Various linkers are commercially available and are considered suitable for use.

[0105] Suitable linkers can be readily selected and can be of any of a suitable of different lengths, such as from 1 amino acid (e.g., Gly) to 40 amino acids, from 2 amino acids to 15 amino acids, from 3 ammo acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0106] Exemplary flexible linker which can be used to join or link a carrier moiety to an ENaC inhibitor peptide or a compound of Formula I, for example, via peptide bonds, include glycine polymers (G)n, (e.g., where n is an integer from 1 to about 20); glycine-serine polymers (including, for example, (GS)n (SEQ ID NO: 35), (GSGGS)n (SEQ ID NO: 36) and (GGGS)n (SEQ ID NO: 37), where n is an integer of between 1 and 10, e.g., I, 2, 3, 4, 5, 6, 7, or more), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are of interest since both of these ammo acids are relatively unstructured, and therefore may serve as a neutral tether between components. Glycine polymers are used in some embodiments. Exemplary flexible linkers include, but are not limited to GG, GGG, GGS, GGSG (SEQ ID NO: 38), GGSGG (SEQ ID NO: 39), GSGSG (SEQ ID NO: 40), GSGGG (SEQ ID NO: 41), GGGSG (SEQ ID NO: 42), GSSSG (SEQ ID NO: 43), and the like.

[0107] In another embodiment, the linker is non-peptide linker. Non-peptide linker moieties can also be used to join or link a carrier moiety to an ENaC inhibitor peptide or a compound of Formula I. The linker molecules are generally about 6-50 atoms long. The linker molecules may also be, for example, aryl acetylene, ethylene glycol oligomers containing 2-10 monomer units, diamines, diacids, amino acids, or combinations thereof.

[0108] In an alternative embodiment, an ENaC inhibitor peptide or a compound of Formula I may be linked to the carrier peptide by a disulfide bond. In some embodiments, the disulfide bond is formed between two cysteines, two cysteine analogs or a cysteine and a cysteine analog. In this embodiment, both the ENaC inhibitor peptide or a compound of Formula I and the carrier peptide contain at least one cysteine or cysteine analog. The cysteine residue or analog may be present as the N-terminal or C-terminal residue of the peptide or as an internal residue of the inhibitor peptide and of the carrier peptide. The disulfide linkage is then formed between the sulfur residues on each of the cysteine residues or analogs. Thus, the disulfide linkage may form between, for example, the N -terminus of the inhibitor peptide and the N-terminus of the carrier peptide, the C-terminus of the inhibitor peptide and the C-terminus of the carrier peptide, the N-terminus of the inhibitor peptide and the C-terminus of the carrier peptide, the C-terminus of the inhibitor peptide and the N-tenninus of the carrier peptide, or any other such combination including at any internal position within the inhibitor peptide and/or the carrier peptide.

[0109] In yet another embodiment, the ENaC inhibitor peptide or a compound of Formula I contains an additional amino acid comprising a reactive side chain, e.g., SH group of cysteine, which may be coupled to a carrier via click chemistry. See Liang et al, J. Angew.Chem., Int. Ed., 48, 965 (2009).

[0110] The disclosure further relates to methods for preparing the EN aC inhibitor peptides or compounds of Formula I. In some embodiments, the ENaC inhibitor peptide can be isolated and purified in accordance with conventional methods of recombinant synthesis. A lysate may^¬ be prepared of the expression host and the lysate purified using high performance liquid chromatography (HPLC), exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. For the most part, the compositions which are used will comprise at least 80% by weight of the desired product, at least about 85% by weight, at least about 95% by weight, or at least about 99.5% by weight, in relation to contaminants related to the method of preparation of the product and its purification. The percentages can be based upon total protein. The compounds of Formula I may be prepared therefrom by adding ! and/or R² groups using techniques known in the art.

[0111] An ENaC inhibitor peptide or a compound of Fonnula I may be prepared by in vitro (e.g., cell-free) synthesis, using conventional methods as known in the art. Various commercial synthetic apparatuses are available, for example, automated synthesizers by Applied Biosystems, Inc., Foster City, CA. By using synthesizers, naturally occurring amino acids may be substituted with unnatural amino acids. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like. [0J 2] In another embodiment, the peptides of the disclosure may be prepared by solid state/solid phase peptide synthesis methods. Such are well known to synthetic chemists and are based on the original Merrifield synthesis methods of building peptides on resins. See, e.g., the disclosure in U.S. Pat. Nos. 4,749,742; 4,466,919; and 4,507,389,

[0113] If desired, various groups may be introduced into the peptide during synthesis or during expression, winch allow for linking to other molecules or to a surface, or provide some other desired property such as increased solubility, increased resistance to proteolysis, increased in vivo half-life, and the like. One or more cysteines can be used to make thioethers, histidines for linking to a metal ion complex, carboxyl groups for forming amides or esters, amino groups for forming amides, and the like.

[0114] An ENaC inhibitor peptide or a compound of Formula I as described herein may be in the form of a pharmaceutically acceptable salt. Pharmaceutically acceptable salts include acid addition salts, such as hydrochloride, hydrobromide, sulfurate, nitrate, phosphorate, acetate, propionate, glycolate, pymvate, oxalate, maiate, malonate, succinate, maleate, fumarate, tartarate, citrate, benzoate, cinnamate, mandelate, methanesulfonate, ethanesulfonate, p- toluene-sulfonate, salicylate and the like, and base addition salts, such as sodium, potassium, calcium, magnesium, lithium, aluminum, zinc, ammonium, ethylenediamine, argmine, piperazine, etc.

[0115] Particularly, it is believed that salts of the peptides of the disclosure having desirable hygroscopic properties will be useful for therapeutic and medical applications. For instance, since the respiratory tract exposes the airborne peptides to 100% relative humidity, which in turn potentially accelerates the rate of aggregation of hygroscopic particles during their transit to the lung. Accordingly salts that are relatively less hygroscopic can be prepared. In addition modifications to reduce hygroscopicity of the peptides may be made, e.g., acetylation of amino acid residues using enzymatic or chemical coupling means. For unmodified peptides, maltodextrins could be used as a carrier.

[0116] In a further aspect, the disclosure provides pharmaceutical formulations and methods of administering the same to achieve any of the therapeutic effects {e.g., modulation of sodium absorption) discussed above. The pharmaceutical formulation may comprise any of the reagents discussed above in a pharmaceutically acceptable carrier, e.g., an ENaC inhibitor}' peptide or a compound of the disclosure or functional fragment thereof.

[0117] By "pharmaceutically acceptable" it is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject without causing any undesirable biological effects such as toxicity . [0J 8] The formulations of the disclosure cars optionally comprise medicinal agents, pharmaceutical agents, carriers, adjuvants, dispersing agents, diluents, and the like. The peptides or compounds of the disclosure can be formulated for administration in a pharmaceutical carrier in accordance with known techniques. See, e.g., Remington, The Science & Practice of Pharmacy (9^th Ed., 1995). In the manufacture of a pharmaceutical formulation according to the disclosure, the peptide or the compound (including tlie physiologically acceptable salts thereof) is typically admixed with, inter alia, an acceptable carrier. The carrier can be a solid or a liquid, or both, and is preferably formulated with the peptide or the compound as a unit-dose formulation, for example, a tablet, which can contain from about 0.01 or 0.5% to about 95% or 99%, particularly from about 1% to about 50%, and especially from about 2% to about 20% by weight of the peptide or the compound. One or more peptides or compounds can be incorporated in tlie formulations of the disclosure, which can be prepared by any of the well-known techniques of pharmacy.

[0119] The formulations of the disclosure include those suitable for oral, rectal, topical, buccal (e.g., sub-lingual), vaginal, parenteral (e.g., subcutaneous, intramuscular including skeletal muscle, cardiac muscle, diaphragm muscle and smooth muscle, intradermal, intravenous, intraperitoneal), topical (i.e., both skin and mucosal surfaces, including airway surfaces), intranasal, transdermal, intraarticular, intrathecal, and inhalation administration, administration via iniraportal delivery, as well as direct organ injection (e.g., into the liver, into the urethra for delivery to the kidneys, etc.). The most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular peptide or the compound which is being used.

[0120] For injection, the carrier will typically be a liquid, such as sterile pyrogen-free water, sterile normal saline, hypertonic saline, pyrogen-free phosphate- buffered saline solution, bacteriostatic water, or CREMOPHOR EL (BASF, Parsippany, NJ). For other methods of administration, the earner can be either solid or liquid.

[0121] For oral administration, the peptide or tlie compound can be administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. Peptides or compounds of the disclosure can be encapsulated in gelatin capsules together with inactive ingredients and powdered carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate and the like. Examples of additional inactive ingredients that can be added to provide desirable color, taste, stability, buffering capacity, dispersion or other known desirable features are red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, edible white ink and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric- coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.

Θ 22] Formulations suitable for buccal (sub-lingual) administration include lozenges comprising the compound in a flavored base, usually sucrose and acacia or tragacanth; and pastilles comprising the compound in an inert base such as gelatin and glycerin or sucrose and acacia.

[0123] Formulations suitable for ocular administration include, e.g., an ointment. In certain instances, ointments are semisolid (e.g., soft solid or thick liquid) formulations that include the peptides or compounds of the disclosure dispersed in an oil-in-water emulsion or a water-in- oil emulsion . The hydrophobic component of an ointment may be derived from an animal (e.g. , lanolin, cod liver oil, and ambergris), plant (e.g., safflower oil, castor oil, coconut oil, cottonseed oil, menhaden oil, palm kernel oil, palm oil, peanut oil, soybean oil, rapeseed oil, linseed oil, rice bran oil, pine oil, sesame oil, or sunflower seed oil), or petroleum (e.g., mineral oil, or petroleum jelly). In certain instances, ointments are semisolid preparations that soften or melt at body temperature (including the temperature of an eye and/or a tissue related thereto). In certain instances, ointments re-hydrate a tissue and are thus useful for ophthalmic disorders characterized by loss of moisture or dryness in the eye. Alternately, the ophthalmic formulation is in the form of a gel or a sol. The ophthalmic formulation may also be in the form of a liquid that gels upon administration to the eye, which prevents tearing and washing away of the peptide or the compound.

[0124] The disclosure further relates to compositions comprising the peptides or the compounds of the disclosure and a nasal carrier. As used herein, the term "nasal carrier" includes solutions, emulsions, suspensions, gels, sols, colloids, and solids, designed for deliver}' of the aforementioned peptide or the compound to the nasal mucosa. The term "solution" refers to a liquid mixture in which the minor component (e.g., the peptide or the compound) is uniformly distributed within the major component (e.g., buffer). "Emulsions" refer to a tine dispersion of minute droplets of one liquid in another in which it is not soluble or miscible (e.g., oil and water). "Suspensions" refer to heterogeneous mixtures in which the solute particles do not dissolve but get suspended throughout the bulk of the medium. "Gels" refer to solid jelly-like material that can have properties ranging from soft and weak to hard and tough and are defined as a substantially dilute cross-linked system, which exhibits no flow. "Sols" refer to colloidal suspensions of very small solid particles in a continuous liquid medium. Preferably, the nasal carrier is a liquid. The liquid nasal carrier includes a diluent suitable for application to the nasal mucosa. Suitable diluents include aqueous or non-aqueous diluents or combination thereof. Examples of aqueous diluents include, but are not limited to, saline, water, dextrose or combinations thereof. Non-aqueous diluents include, but are not limited to, alcohols, particularly polyhydroxy alcohols such as propylene glycol, polyethylene glycol, glycerol, and vegetable and mineral oils. These aqueous and/or non-aqueous diluents can be added in various concentrations and combinations to form solutions, suspensions, oil- in-water emulsions or water-in-oil emulsions. In preferred embodiments, the diluent is saline or water. In some embodiments, the nasal carrier is a saline. The term "saline" refers to substances containing or impregnated with salt, e.g., sodium chloride, potassium chloride, calcium chloride, magnesium chloride, zinc chloride, sodium, bicarbonate, calcium bicarbonate, sodium phosphate, calcium phosphate, etc. The compositions of the present disclosure may include one or more agents that increase viscosity chosen in quantities that preferably do not irritate the nasal mucosa and increase nasal retention time. Preferred agents that increase viscosity include, but are not limited to, methvlceiluiose, carboxymethylcellulose sodium, ethylcellulose, carrageenan, carbopol, and/or combinations thereof. The most preferred agents used to increase viscosity and increase nasal retention time is methyicellulose or carbopol. Formulations suitable for nasal administration include, e.g., aqueous solution as a nasal or pulmonary spray, which may be dispensed in spray form by a variety of methods known to those skilled in the art. Preferred systems for dispensing liquids as a nasal spray are disclosed in U.S. Pat. No. 4,511,069. The formulations may be presented in multi-dose containers, for example in the sealed dispensing system disclosed in U.S. Pat. No. 4,51 ,069. Additional aerosol delivery forms may include, e.g., compressed air-, jet-, ultrasonic-, and piezoelectric nebulizers, which deliver the biologically active agent dissolved or suspended in a pharmaceutical solvent, e.g., water, ethanol, or a mixture thereof. Nasal and pulmonary spray solutions of the present disclosure typically comprise the drug or drug to be delivered, optionally formulated with a surface-active agent, such as a nonionic surfactant (e.g., polysorbate-80), and one or more buffers. In some embodiments of the present disclosure, the nasal spray solution further comprises a propeliant. The pH of the nasal spray solution is optionally between about pH 2.0 and 8, wherein the pH could be adjusted and buffered as desired. Suitable buffers for use within these compositions are as described above or as otherwise known in the art. Other components may be added to enhance or maintain chemical stability, including preservatives, surfactants, dispersants, or gases. Suitable preservatives include, but are not limited to, phenol, methyl paraben, paraben, m-cresol, thiomersal, chlorobutanol, benzyl alkonimum chloride, sodium benzoate, and the like. Suitable surfactants include, but are not limited to, oleic acid, sorbitan trioleate, poiysorbates, lecithin, phosphotidyl cholines, and various long chain diglycerides and phospholipids. Suitable dispersants include, but are not limited to, ethylenediaminetetraacetic acid, and the like. Suitable gases include, but are not limited to, nitrogen, helium, chlorofluorocarbons (CFCs), hydrofluorocarbons (HFCs), carbon dioxide, air, and the like.

[0125] Formulations suitable for vaginal administration include, e.g., emulsions, suspensions, aqueous or anhydrous solutions or dispersions, or alternatively the form of an emulsion or suspension or salve for convenient intravaginal administration. The active compositions and other ingredients may form suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulation agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active compositions and other ingredients may be in powder form, obtained by aseptic isolation of sterile solid or by lyophiiization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use. For intravaginal administration, the therapeutic agents may be formulated as is known in the art for direct application to the vaginal area. Forms chiefly conditioned for vaginal application take the form, for example, of milks, gels, dispersions, microemulsions, lotions thickened to a greater or lesser extent, impregnated pads, ointments, aerosol formulations (e.g., sprays or foams), creams, pastes, jellies, sprays, and aerosols. Alternatively, the composition can be formulated to be part of an adhesive polymer, such as polyacrylate or acrylate/vinyl acetate copolymer. Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents. Liquid sprays are conveniently delivered from pressurized packs, for example, via a specially shaped container or applicator. The active compositions can also be delivered via iontophoresis, e.g., as disclosed m U.S. Pat. Nos. 4, 140, 122; 4,383,529; or 4,051,842.

[0126] Formulations of the present disclosure suitable for parenteral administration comprise sterile aqueous and non-aqueous injection solutions of the peptide or the compound, which preparations are preferably isotonic with the blood of the intended recipient. These preparations can contain anti -oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions can include suspending agents and thickening agents. The forrnulations can be presented in unit/dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for-injection immediately prior to use.

[0127] Ready-to-use injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described. For example, in one aspect of the present disclosure, there is provided an injectable, stable, sterile composition comprising a peptide or a compound of the disclosure, in a unit dosage form in a sealed container. The peptide or a compound or a salt thereof is provided in the form, of a lyophilisate which is capabl e of being reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection thereof into a subject. The unit dosage form typically comprises from about 1 mg to about 10 grams of the peptide or the compound or the salt. When the peptide or the compound or the salt is substantially water-insoluble, a sufficient amount of emulsifying agent which is pharmaceutically acceptable can be employed in sufficient quantity to emulsify the peptide or the compound or the salt in an aqueous carrier. One such useful emulsifying agent is phosphatidyl choline.

[0128] Formulations suitable for rectal administration are preferably presented as unit dose suppositories. These can be prepared by mixing the peptide or the compound with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.

[0129] Formulations suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers which can be used include petroleum jelly, ianoline, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof.

[0130] Formulations suitable for transdermal administration can be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Formulations suitable for transdermal administration can also be delivered by- iontophoresis (Tyle et al, Pharm. Res. 3:318 (1986)} and typically take the form, of an optionally buffered aqueous solution of the peptides or the compounds. Suitable formulations comprise citrate or BIS/TRIS buffer (pH=6) or ethanol/ water and contain from 0.1 to 0.2M of the compound.

[0131] The peptide or the compounds can alternatively be formulated for nasal administration or otherwise administered to the lungs of a subject by any suitable means, e.g., administered by an aerosol suspension of respirabie particles comprising the peptide or the compound, which the subject inhales. The respirable particles can be liquid or solid. The term "aerosol" includes any gas-borne suspended phase, which is capable of being inhaled, e.g., into the lung or nasal passages. Specifically, aerosol includes a gas-borne suspension of droplets, as can be produced in a metered dose inhaler or nebulizer, or in a mist sprayer. Aerosol also includes a dry powder composition suspended in air or other carrier gas, which can be delivered by insufflation from an inhaler device, for example. See Ganderton et al. Drug Delivery to the Respiratory Tract, Ellis Horwood (1987); Gonda (1990) Critical Reviews in Therapeutic Drug Carrier Systems 6:273-313; and Raebura et al, J. Pharmacol Toxicol Meth. 27: 143 (1992). Aerosols of liquid particles comprising the peptide or the compound can be produced by any suitable means, such as with a pressure- driven aerosol nebulizer or an ultrasonic nebulizer, as is known to those of skill in the art. See, e.g., U.S. Pat. No. 4,501,729. Aerosols of solid particles comprising the peptide or the compound can likewise be produced with any solid particulate medicament aerosol generator, e.g., a passive or active dry powder inhaler, by techniques known in the pharmaceutical art.

[0132] Alternatively, one can administer the peptide or the compound in a local rather than systemic manner, for example, in a depot or sustained-release formulation.

[0133] Further, the present disclosure provides liposomal formulations of the peptides or the compounds disclosed herein and salts thereof. The technology for forming liposomal suspensions is well known in the art. When the peptide or the compound or salt thereof is an aqueous- soluble salt, using conventional liposome technology, the same can be incorporated into lipid vesicles. In such an instance, due to the water solubility of the peptide or the compound or salt, the peptide or the compound or salt will be substantially entrained within the hydrophiiic center or core of the liposomes. The lipid layer employed can be of any conventional composition and can either contain cholesterol or can be cholesterol-free. When the peptide or the compound or salt of interest is water-insoluble, again employing conventional liposome formation technology, the salt can be substantially entrained within the hydrophobic lipid bilayer which forms the structure of the liposome. In either instance, the liposomes which are produced can be reduced in size, as through the use of standard sonication and homogenization techniques.

[0134] Hie liposomal formulations containing the peptides or the compounds disclosed herein or salts thereof can be ivophilized to produce a ly ophilizate which can be reconstituted with a pharmaceutically acceptable carrier, such as water, to regenerate a liposomal suspension.

[0135] In the case of water-insoluble peptides or compounds, a pharmaceutical composition can be prepared containing the water-insoluble peptide or compound, such as for example, in an aqueous base emulsion. In such an instance, the composition will contain a sufficient amount of pharmaceutically acceptable emulsifying agent to emulsify the desired amount of the peptide or the compound. Particularly useful emulsifying agents include phosphatidyl cholines and lecithin.

[0136] In particular embodiments, the peptide or the compound is administered to the subject in a therapeutically effective amount, as that term is defined above. Dosages of pharmaceutically active peptides or compounds can be determined by methods known in the art, see, e.g.. Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA). The therapeutically effective dosage of any specific peptide or compound will vary somewhat from peptide to peptide, and patient to patient, and will depend upon the condition of the patient and the route of delivery. As a general proposition, a dosage from about 0.1 to about 50 mg/kg will have therapeutic efficacy, with all weights being calculated based upon the weight of the peptide or the compound, including the cases where a salt is employed. Toxicity concerns at the higher level can restrict intravenous dosages to a lower level such as up to about 10 mg/kg, with all weights being calculated based upon the weight of the peptide or the compound, including the cases where a salt is employed. A dosage from about 10 mg/kg to about 50 mg kg can be employed for oral administration. Typically, a dosage from about 0.5 mg/kg to 5 mg/kg can be employed for intramuscular injection . Particular dosages are about 1 iimol/kg to about 100 umol/kg, and more, particularly to about 20 umol/kg and to 50 umol/kg of the peptide for intravenous or oral administration, respectively.

[0137] In particular embodiments of the disclosure, a therapeutically effective amount of the

ENaC inhibitor peptide or a compound of Formula I is an amount effective to, e.g., (a) inhibit

ENaC internalization by about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to a control (e.g., a vehicle); (b) increase airway hydration by about 20%, 40%, 50%, 75%,

100%, 150%, 250%, or more compared to a control (e.g., a vehicle): (c) increase height of airway lining fluid (ALF) by about 20%, 40%, 50%, 75%, 100%, 150%, 250%, or more compared to a control (e.g., a vehicle); and/or (d) improve a clinical outcome (e.g., survival or time to disease progression) when administered to a subject in vivo. In some embodiments, a therapeutically effective amount of the ENaC inhibitor peptide or the compound of Formula I improves a clinical outcome of a subject qualitatively or quantitatively. Representative types of qualitative improvements include, e.g., improved quality of life as assessed by the four dimensions of the Chronic Respirator}⁷ Questionnaire (CRQ). Representative types of quantitative improvements include, e.g., measurable improvements in survival outcomes, such as, improvement over control subjects by about 20%, 40%, 50%, 75%, 100%, 150%, 250%, 500%, or more, in disease-free survival period (DFS), time to death (TTD), In some embodiments, the improvement in outcome is assessed in subjects by measuring an art- appreciated parameter such as forced expiratory volume in one second (FEVl) or percent predicted FEVl . FEVl is the maximum amount of air that a subject can forcefully blow out of their lungs in one second and is measured using a spirometer, an instrument that measures pulmonary air flow by having a subject blow into a plastic tube. It is used to show lung capacity and helps pulmonologists classify diseased (e.g., asthmatic or COPD) patients into stages. Thus, the lower the FEVl, the more severe the pulmonary disease.

[0138] The results of the FEVl are particularly useful when compared with current standards or expected values based on a healthy person, along with gender, height and race. By comparing a subject's FEVl with the general standard, a doctor can determine the stage of the disease, i.e., how far it has progressed. For instance, an FEVl of 80> of the expected value indicates mild disease; an FEVl between 50-80 percent indicates moderate disease: an FEVl between 30-50 percent indicates severe disease; and an FEVl of <30 percent indicates very severe disease.

[0139] A combination of the parameters, e.g., increase in ALF height and improvement in survival outcome, may also be used to determine therapeutically effective amounts.

[0140] In order to achieve therapeutic effects, more than one administration (e.g., two, three, four, or more administrations) can be employed over a variety of time intervals (e.g., hourly, daily, weekly, monthly, etc.).

[0141] The disclosure relates to ENaC inhibitor peptides or compounds of Formula I or compositions thereof (e.g., pharmaceutical compositions) which are inhibit ENaC. By "inhibit" it is meant to reduce amount, levels, density, turnover, association, dissociation, activity, signaling, or any other feature associated with alpha ENaC, beta ENaC, or gamma ENaC. The

ENaC inhibitor peptides or the compounds of Formula I can inhibit ENaC by promoting or facilitating internalization of the ENaC from cell membrane. The ENaC inhibitor peptide or compound of Formula 1 can increase ENaC internalization by about 20% to about 85%, including, all values in between, e.g., 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80% or more . Particularly, the ENaC inhibitor peptide or a compound of Formula I or a composition thereof can increase alpha ENaC internalization by about 40%, 45%, 50%, 55%, 60%, 70%,

80% or more compared to treatment with a vehicle alone (e.g., a control which is devoid of the

ENaC inhibitor peptide or a compound of Formula I); or the ENaC inhibitor peptide or a compound of Formula I or a composition thereof can increase beta ENaC internalization by about 50%, 55%>, 60%, 65%, 70%, 80%, 90% or more compared to treatment with a vehicle alone (control); or the ENaC inhibitor peptide or a compound of Formula I or a composition thereof can increase gamma ENaC internalization by about 40%, 50%, 55%, 60%, 65%, 70%, 75% or more compared to treatment with a vehicle alone (control).

[0142] The ENaC inhibitor peptides or compound of Formula I or compositions thereof (e.g., pharmaceutical compositions) are preferably physically stable e.g., resistant to degradation by- heat, light, oxidation and the like and more preferably physically stable and enzymatically stable. Herein, "physically stable" relates to retention of a property, e.g., physical property such as size and/or length of the peptide or the compound or a biological property, e.g., ENaC internalization activity, after a given time period, e.g., 7 hours, 14 hours, 21 hours, 28 hours, or more, e.g., 48 hours, 96 hours, 1 week, etc. For example, the peptides or compounds of the disclosure are stable (substantially retain activity) when held at 40°C for at least 7 hours, at least 14 hours, at least 21 hours, or at least 28 hours, or more. Stability can also be assessed by- measuring a loss of a property (LOP) compared to a control, e.g., a random peptide or an S 18 peptide described in US2016/0102121 and WO 16/057795. In these embodiments, the ENaC inhibitor peptides or compounds of Formula I of the disclosure are at least 50% stable (i.e., <50% LOP), particularly at least 60% stable (i.e., <40% LOP), more particularly at least 70% stable (i.e., <30% LOP), and especially at least 80% stable (i.e., <20% LOP) even after a 7, 14, 21 or 28-hour incubation at 40°C.

[0143] The ENaC inhibitor peptides, compounds of Formula I or compositions thereof are also preferably enzymatically stable e.g., resistant to degradation by enzymes such as trypsin or human neutrophil elastase (HNE). Herein, "enzymatically stable" relates to retention of a property, e.g., physical property such as size and/or length of the peptide or the compound or a biological property, e.g., ENaC internalization activity of the peptide or the compound, after coming into contact with an enzyme, e.g., a protease. For example, the peptides and compounds of the disclosure are resistant to protease degradation for at least 30 minutes, particularly for at least 60 minutes, and especially for at least 90 minutes, or more. The ENaC inhibitor peptide or the compound of Formula I of the disclosure can be at least 50% stable (i.e., <50% LOP), particularly at least 60% stable (i.e., <40% LOP), more particularly at least 80% stable (i.e., <20% LOP), and especially at least 90% stable (i.e., <10% LOP) after 1 ,5 hour incubation with trypsin. Alternately, the ENaC inhibitor peptide or the compound of Formula I of the disclosure are at least 80% stable (i.e., <20% LOP), particularly at least 90% stable (i.e., <10% LOP) and especially at least 95% stable (i.e., <5% LOP) after 30, 60 or 90-min incubation with HNE. The results on resistance to FINE compare favorably to that of S18 peptide (>60% LOP after 30, 60 or 90-min incubation with HNE). [0144] The disclosure further relates to ENaC inhibitor peptides or compounds of Formula I that are effective in improving an outcome of a subject suffering from an ENaC -related lung disorder (e.g., cystic fibrosis). In one specific embodiment, the outcome is survival. In another embodiment, the outcome is age at terminal endpoint (ATE), total disease duration (TDD), time to progression of disease (TTP), time-to-death (TTD), and disease-free survival period (DFS), or a combination thereof.

[0145] In some embodiments, the ENaC inhibitor peptides or compounds of Formula I, when administered to a subject suffering from a disease associated with ENaC activity, improves an outcome associated with the disease by about 10% to about 99%, including, all values in between, e.g., 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, or more. More specifically, wherein the outcome is survival of a subject suffering from the disorder, treatment of the subject with the ENaC inhibitor peptides or compounds of Formula I of the disclosure appreciably improves survival outcomes by about at least 30%, 35%, 40%, 45%, 50%, 55%, or more, compared to a control (e.g., a subject receiving a random peptide or an empty vehicle). Alternately, treatment of the subject with the ENaC inhibitor peptides or compounds of Formula I of the disclosure appreciably reduces the subject's mortality by about 30%, 35%, 40%, 45%, 50%, 55%, or more, compared to a control {e.g., a subject receiving a random peptide or an empty vehicle). Still in other embodiments, treatment of the subject with the ENaC inhibitor peptides or compounds of Formula I of the disclosure appreciably increases the number of treated subjects (e.g., survivors) by 1.1-fold, 1.25-fold, 1.5 -fold, 1.75-fold, 2- fold, 2.5-fold, 3-fold, 4-fold, 5-fold, or more, compared to the control subject.

[0146] In another embodiment, the outcome is time to a critical event (e.g., the time point at which 10%, 20%, 30%, 40%, 50% of the subjects in a population die from a disease or a condition). In one specific embodiment, treatment of the subject with the ENaC inhibitor peptides or compounds of Formula I of the disclosure appreciably delays time to the critical event (e.g., time to death of 50% of the subjects) by 4 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 6 months, 9 months, 1 year or more.

[0147] The disclosure further provides ENaC inhibitor peptides or compounds of Formula I that are non-toxic, e.g., at the cellular, tissue, organ, system, or organismal level. In some embodiments, the peptides or compounds are non-toxic at the cellular level at a concentration of about 10 uM, 20 uM, 30 uM, 40 uM, 50 uM, 75 μΜ, 100 μΜ, or more. As exemplified herein, the ENaC inhibitor peptides or compounds of Fonnula I, at a dose of about 100 uM, had no effect on cell viability compared to a control peptide. [0 48] The present disclosure provides nucleic acids, where a subject synthetic nucleic add comprises a nucleotide sequence encoding one or more ENaC inhibitor peptides or compounds of Formula I of the present disclosure. The term "nucleic acid" as used herein, refer to an oligonucleotide, nucleotide, polynucleotide, or any fragment thereof, to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and ma - represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-!ike material In this context, "fragments" refers to those nucleic acid sequences which are greater than about 10 nucleotides in length, and most preferably are at least about 20 nucleotides, at least about 50 nucleotides, or more.

[0149] Included herein are nucleic acids encoding the following ENaC inhibitor peptides of the present disclosure: LPLPLD (SEQ ID NO: 3); LPLPLDQ (SEQ ID NO: 4); LPLPLDQT (SEQ ID NO: 5); LPIPLD (SEQ ID NO: 6); LPIPLDQ (SEQ ID NO: 7); or LPIPLDQT (SEQ ID NO: 8), or the complementary strand thereto, or the RNA equivalent thereof, or a complementary RN A equivalent thereof.

[0 50] Further included herein are nucleic acids encoding tagged ENaC inhibitor peptides, e.g. , histidine-tagged, Flag-tagged, or Myc-tagged ENaC inhibitor peptides, having the sequence LPLPLD (SEQ ID NO: 3): LPLPLDQ (SEQ ID NO: 4); LPLPLDQT (SEQ ID NO: 5); LPIPLD (SEQ ID NO: 6); LPIPLDQ (SEQ ID NO: 7); or LPIPLDQT (SEQ ID NO: 8), or the complementary strand thereto, or the RNA equivalent thereof, or a complementary RNA equivalent thereof.

[0151] In some embodiments, the nucleic acids encoding the aforementioned polypeptides are obtained using computational methods, e.g., Reverse Translate Tool provided via Sequence

Manipulation Suite under Bioinformatics(dot)Org. In another embodiment, the nucleic acids may be designed by ligating a fragment nucleic acid encoding the inhibitor peptide to a plurality of nucleic acids encoding the linker and/or the carrier using routine recombinant DNA technology. Included herein are codon -optimized sequences of the aforementioned nucleic acid sequences and vectors. Codon optimization for expression in a host cell, e.g., bacteria such as

E. coli or insect Hi5 cells, may be routinely performed using Codon Optimization Tool

(CODONOPT), available freely from Integrated DNA Technologies, Inc., Coralville, Iowa.

[0152] Embodiments disclosed herein further relate to variants of the aforementioned polynucleotides. In some embodiments, included herein are variants of aforementioned nucleic acids which comprise, or alternatively consist of, a nucleotide sequence which is at least 80%,

85%, 90%, 95%, 96%, 97%, 98% or 99%, or greater % identity to, for example, the nucleic acids encoding the following peptides: LPLPLD (SEQ ID NO: 3); LPLPLDQ (SEQ ID NO: 4); LPLPLDQT (SEQ ID NO: 5); LPIPLD (SEQ ID NO: 6); LPIPLDQ (SEQ ID NO: 7); or LPIPLDQT (SEQ ID NO: 8), or the complementary strand thereto, or the RNA equivalent thereof, or a complementary RNA equivalent thereof.

[0153] The phrases "percent identity" or "% identity" refer to the percentage of sequence similarity found in a comparison of two or more amino acid or nucleic acid sequences. Percent identity can be determined electronically, e.g., by using the MEGALIGN program (LASERGENE software package, DNASTAR). The MEGALIGN program can create alignments between two or more sequences according to different methods, e.g., the CLUSTAL Method (Higgins el al. Gene 73:237-244 (1988)). The CLUSTAL algorithm groups sequences into clusters by examining the distances between all pairs. The clusters are aligned pairwise and then in groups. The percentage similarity between two amino acid sequences, e.g., sequence A and sequence B, is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of low or of no homology between the two amino acid sequences are not included in determining percentage similarity. Percent identity between nucleic acid sequences can also be calculated by the CLUSTAL method, or by other methods known in the art, such as the Jotun Hein Method, (See, e.g., Hein et al, Methods Enzymol. 183:626-645 (1990)). Identity between sequences can also be determined by other methods known in the art, e.g., by- varying hybridization conditions.

[0154] In another embodiment, included herein are variant polynucleotides which hybridize to one or more nucleic acid molecules under stringent hybridization conditions or lower stringency conditions. "Hybridization," as the term is used herein, refers to any process by winch a strand of nucleic acid bonds with a complementary strand through base pairing. For example, hybridization under high stringency conditions could occur in about 50% formamide at about 37° C to 42° C. Hybridization could occur under reduced stringency conditions in about 35% to 25% formamide at about 30° C. to 35° C. In particular, hybridization could occur under high stringency conditions at 42° C in 50% formamide, 5xSSPE, 0.3% SDS, and 200 ug/ml sheared and denatured salmon sperm DNA. Hybridization could occur under reduced stringency conditions as described above, but formamide at a reduced temperature of

35° C. The temperature range corresponding to a particular level of stringency can be further narrowed by calculating the purine to pyrimidine ratio of the nucleic acid of interest and adjusting the temperature. Variations on the above ranges and conditions are well known in the art. [0155] The term "hybridization complex" as used herein, refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementarv' bases. A hybridization complex may be formed in solution or formed between one nucleic add sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).

[0156] In another embodiment, included herein are variants which are polynucleotide fragments of the aforementioned nucleic acids.

[0157] Also included herein are oligonucleotides, e.g., PCR primers, which hybridize to one or more nucleic acids. The term "oligonucleotide," as used herein, refers to a nucleic acid sequence of at least about 6 nucleotides to 60 nucleotides, preferably about 15 to 30 nucleotides, and most preferably about 20 to 25 nucleotides, which can be used in PCR amplification or in a hybridization assay or microarray. As used herein, the term "oligonucleotide" is substantially equivalent to the terms "amplimers," "primers," "oligomers," and "probes," as these terms are commonly defined in the art.

[0158] Also included herein are modified nucleic acids such as peptide nucleic acid (PNA). P As are described in the art, e.g., Nielsen et a!,. Anticancer Drug Des. 8:53-63, 1993.

[0159] Also included herein are vectors which contain one or more of the aforementioned nucleic acids. In some embodiments, the vector comprises at least one protein encoding nucleic acid, e.g., nucleic acids encoding the polypeptide sequences for LPLPLD (SEQ ID NO: 3); LPLPLDQ (SEQ ID NO: 4); LPLPLDQT (SEQ ID NO: 5); LPIPLD (SEQ ID NO: 6); LPTPLDQ (SEQ ID NO: 7); or LPIPLDQT (SEQ ID NO: 8), in operable linkage with one or more additional sequences. The additional sequences may be synthetic in nature. The terms "operabiy associated" or "operabiy linked," as used herein, refer to functionally related nucleic acid sequences. A promoter is operabiy associated or operabiy linked with a coding sequence if the promoter controls the transcription of the encoded polypeptide. While operabiy associated or operabiy linked nucleic acid sequences can be contiguous and in reading frame, certain genetic elements, e.g., repressor genes, are not contiguously linked to the encoded polypeptide but still bind to operator sequences that control expression of the polypeptide.

[0160] A nucleotide sequence encoding an ENaC inhibitor peptide can be operabiy linked to one or more regulatory elements, such as a promoter and enhancer, that allow expression of the nucleotide sequence in the intended target cells (e.g. , a cell that is genetically modified to synthesize the encoded ENaC inhibitor peptide). In some embodiments, a subject nucleic acid is a recombinant expression vector. [0161] Suitable promoter and enhancer elements are known in the art. For expression in a bacterial cell, suitable promoters include, but are not limited to, lacl, lacZ, T3, T7, gpt, lambda P and trc. For expression in a eukaryotic cell, suitable promoters include, but are not limited to, cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoters present in long terminal repeats from a retrovirus: a metallothionein-1 promoter; and the like.

[0162] In some embodiments, e.g., for expression in a yeast cell, a suitable promoter is a constitutive promoter such as an ADH1 promoter, a PGK1 promoter, an ENO promoter, a PYK1 promoter and the like; or a regulatable promoter such as a GAL1 promoter, a GAL 10 promoter, an ADH2 promoter, a PH05 promoter, a CUP1 promoter, a GAL7 promoter, a MET25 promoter, a MET3 promoter, a CYC 1 promoter, a HIS3 promoter, an ADH1 promoter, a PGK promoter, a GAPDH promoter, an ADC 1 promoter, a TRP1 promoter, a URA3 promoter, a LEU2 promoter, an ENO promoter, a TP I promoter, and AOXl [e.g., for use in Pichia). Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.

[0163] Suitable promoters for use in prokaryotic host cells include, but are not limited to, a bacteriophage T7 K A polymerase promoter; a trp promoter; a iac operon promoter; a hybrid promoter, e.g. , a lac/tac hybrid promoter, a tac/trc hybrid promoter, a trp/Iac promoter, a T7/lae promoter; a trc promoter; a tac promoter, and the like; an araBAD promoter; promoters such as an ssaG promoter or a related promoter (see, e.g., US2004/0131637), a pagC promoter (Pulkkinen et al, J. Bacterial, 1991 : 173(1): 86-93; Alpuche-Aranda et al, PNAS, 1992; 89(21); 10079-83), a nirB promoter (Harborne et al (1992) Mol Micro. 6:2805-2813), and the like (see, e.g., Dunstan et al ( 1999) Infect. Immun. 67:5133-5141; Mc elvie et al (2004) Vaccine 22:3243-3255; and Chatfield et al (1992) Biotechnol 10:888-892); a sigma70 promoter, e.g., a consensus sigma70 promoter (see, e.g., GENBANK Accession Nos. AX798980, AX798961, and AX798183); a stationar ' phase promoter, e.g., a dps promoter, an spy promoter, and the like; a promoter derived from the pathogenicity island SPI-2 (see, e.g., W096/17951); an actA promoter (see, e.g., Shetron-Rama et al (2002) Infect. Immun. 70: 1087-1096); an rpsM promoter (see, e.g., Valdivia and Falkow ( 1996). Mol Microbiol 22:367); a tet promoter; an SP6 promoter (see, e.g.. Melton et al ( 1984) Nucl Acids Res. 12:7035); and the like. Suitable strong promoters for use in prokaryotes such as Escherichia coli include, but are not limited to Trc, Tac, T5, T7, and Ρλ. Non-limiting examples of operators for use in bacterial host cells include a lactose promoter operator (Lacl repressor protein changes conformation when contacted with lactose, thereby preventing the Lad repressor protein from binding to the operator), a tryptophan promoter operator (when complexed with tryptophan, TrpR repressor protein has a conformation that binds the operator; in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind to the operator), and a tac promoter operator (see, for example, deBoer et al. PNAS USA, 80:21 - 25 (1983)).

[0164] A nucleotide sequence encoding an ENaC inhibitor peptide can be present in an expression vector and/or a cloning vector. An expression vector can include a selectable marker, an origin of replication, and other features that provide for replication and/or maintenance of the vector.

[0165] Large numbers of suitable vectors and promoters are known to those of skill in the art; many are commercially available for generating a recombinant construct. Large numbers of suitable vectors and promoters are known to those of skill in the art; many are commercially available for generating a recombinant constructs. The following vectors are provided by way of example. Bacterial: pBs, PHAGESCRIPT, PsiX174, PBLUESCRIPT SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif, USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).

[0166] The present disclosure provides isolated genetically modified host cells (e.g., transformed cells or cell-lines) that are genetically modified with a nucleic acid comprising a nucleic acid sequence which encodes an ENaC inhibitor peptide. In some embodiments, a subject isolated genetically modified host cell can produce an ENaC inhibitor peptide.

[0 67] Suitable host cells include eukaryotic host cells, such as a mammalian cell, an insect host cell, a yeast cell; and prokaryotic cells, such as a bacterial cell. Introduction of a subject nucleic acid into the host cell can be effected, for example by calcium phosphate precipitation,

DEAE dextran mediated transfection, liposome-mediated transfection, electroporation, or other known method. Suitable yeast cells include, but are not limited to, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia op ntiae,

Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis,

Pichia methanolica, Pichia sp., Saccharomyc.es cer visiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium. lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Neurospora crassa,

Chlamydomonas reinhardtii, and the like. Suitable prokaryotic cells include, but are not limited to, any of a variety of laboratory strains of Escherichia coli, Lactobacillus sp., Salmonella sp,, Shigella sp., and the like. See, e.g., Carrier ei a/., J Immunol. 148: 1176-1 181 ( 1992); U.S. Pat. No. 6,447,784; and Sizemore et al. Science 270:299-302 (1995),

[0168] Embodiments disclosed herein further include antibodies which bind specifically to one or more of the aforementioned peptides or fusion proteins thereof.

[0169] In some embodiments, the antibodies bind to polypeptides comprising the following amino acid sequences: LPLPLD (SEQ ID NO: 3); LPLPLDQ (SEQ ID NO: 4); LPLPLDQT (SEQ ID NO: 5); LPIPLD (SEQ ID NO: 6); LPIPLDQ (SEQ ID NO: 7); or LPTPLDQT (SEQ ID NO: 8) or an immunogenic fragment thereof. In another embodiment, the antibodies bind to fragment of these polypeptides. Contemplated herein are antigen-binding fragments of such antibodies, e.g., F(ab) domain, F(ab)i domains, scFv domains, including synthetically generated antibodies (using, e.g., phase display technology). Contemplated herein are antigen- binding fragments of such antibodies, e.g., F(ab) domain, F(ab)?. domains, scFv domains, including synthetically generated antibodies (using, e.g., phase display technology).

[0170] The disclosure further relates to purified biomolecules, e.g., nucleic acids, proteins, peptides, and/or antibody molecules, including, conjugates thereof. The term ''substantially purified," as used herein, refers to nucleic acids, amino acids or antibodies that are removed from their natural environment and are isolated or separated, and are at least about 60% free, preferably about 75% free, most preferably about 90%, especially about 95% free and particularly about 99% free from other components with which they are naturally associated.

[0171] Another aspect of the disclosure relates to a kit comprising the peptide of the disclosure and useful for carrying out the methods of the disclosure. The kit may further comprise additional reagents for carrying out the methods (e.g., buffers, containers, additional therapeutic agents) as well as instructions.

[0172] A further aspect of the disclosure is a method of treating subjects, comprising administering to a subject a therapeutically effective amount of an ENaC inhibitor peptide or a compound of Formula 1 or a pharmaceutical composition comprising the ENaC inhibitor peptide or the compound of Formula I. By the terms "treat," "treating," or "treatment of," it is intended that the severity of the subject's condition is reduced or at least partially improved or modified and that some alleviation, mitigation or decrease in at least one clinical symptom is achieved.

[0173] A "therapeutically effective amount" as used herein is an amount that provides some improvement or benefit to the subject. Alternatively stated, a "therapeutically effective" amount is an amount that will provide some alleviation, mitigation, or decrease in at least one clinical symptom in the subject. Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject. Methods for determining therapeutically effective amount of the ENaC inhibitor peptides or compounds of Formula I of the disclosure, including, compositions comprising such peptides, have been described previously.

[0174] Disorders that are treatable with the methods of the disclosure include, but are not limited to, a lung disorder (e.g., cystic fibrosis, idiopathic pulmonary fibrosis, no -cystic fibrosis bronchiectasis, chronic obstructive pulmonary disease, acute or chronic bronchitis, or asthma), a gastrointestinal disorder (e.g., inflammatory bowel disease), a kidney disorder, or a cardiovascular disorder. The peptides of the disclosure, including compositions thereof, can be used to treat dry- eyes, dry mouth, vaginal dryness, and acute rhinosinusitis.

[0175] Accordingly, in some embodiments, the disclosure relates to a method for treating a lung disorder in a subject in need thereof, comprising administering to the subject, a therapeutically effecti ve amount of a composition comprising the ENaC inhibitor peptide or the compound of Formula I.

[0176] In one specific embodiment, the disclosure relates to a method for treating cystic fibrosis in a subject in need thereof, comprising administering to the subject, a therapeutically effective amount of a composition compri sing the ENaC inhibitor peptide or the compound of Formula I.

[0177] In another specific embodiment, the disclosure relates to a method for treating idiopathic pulmonary fibrosis in a subject in need thereof, comprising administering to the subject, a therapeutically effective amount of a composition comprising the ENaC inhibitor peptide or the compound of Formula I.

[0178] In another specific embodiment, the disclosure relates to a method for treating idiopathic pulmonary fibrosis in a subject in need thereof, comprising administering to the subject, a therapeutically effective amount of a composition comprising the ENaC inhibitor peptide or the compound of Formula I.

[0179] In another specific embodiment, the disclosure relates to a method for treating chronic obstructive pulmonary disease in a subject in need thereof, comprising administering to the subject, a therapeutically effective amount of a composition comprising the ENaC inhibitor peptide or the compound of Formula 1.

[0180] In another specific embodiment, the disclosure relates to a method for treating asthma in a subject in need thereof, comprising administering to the subject, a therapeutically effective amount of a composition comprising the ENaC inhibitor peptide or the compound of Formula I.

[0181] In another specific embodiment, the disclosure relates to a method for treating a gastrointestinal disorder (e.g., inflammatory bowel disease) in a subject in need thereof, comprising administering to the subject, a therapeutically effective amount of a composition comprising the ENaC inhibitor peptide or the compound of Formula I.

[0182] In another specific embodiment, the disclosure relates to a method for treating a kidney disorder in a subject in need thereof, comprismg administering to the subject, a therapeutically effective amount of a composition comprising the ENaC inhibitor peptide or the compound of Fo mula I.

[0183] In another specific embodiment, the disclosure relates to a method for treating dry eyes in a subject in need thereof, comprising administering to the subject, a therapeutically effective amount of a composition comprising the ENaC mhibitor peptide or the compound of Formula I.

[0184] In another specific embodiment, the disclosure relates to a method for treating dry mouth in a subject in need thereof, comprising administering to the subject, a therapeutically effective amount of a composition comprising the ENaC inhibitor peptide or the compound of Fo mula I.

[0185] In another specific embodiment, the disclosure relates to a method for treating vaginal dryness in a subject in need thereof, comprising administering to the subject, a therapeutically effective amount of a composition comprising the EN aC inhibitor peptide or the compound of Formula I.

[0186] In another specific embodiment, the disclosure relates to a method for treating acute rhinosinusitis in a subject in need thereof, comprising administering to the subject, a therapeutically effective amount of a composition comprising the ENaC inhibitor peptide or the compound of Formula I.

[0187] The peptides or compounds of the present disclosure may be administered to a subject, e.g., human subject or an animal, in need thereof by any means known in the art. The present disclosure relates to both veterinary and medical applications. Suitable subjects include both avians and mammals, with mammals being preferred. The term "avian" as used herein includes, but is not limited to, chickens, ducks, geese, quail, turkeys, and pheasants. The term "mammal" as used herein includes, but is not limited to, humans, bovines, ovines, caprines, equines, felines, canines, lagomorphs, etc. Non-limiting examples of suitable subjects may include pigs, cows, horses, goats, sheep, llamas and alpacas; a companion animal such as dogs, cats, rabbits, and birds; a zoo animal such as non-human primates, large cats, wolves, and bears, etc. Human subjects include neonates, infants, juveniles, and adults.

[0188] In one specific embodiment, the disclosure relates to methods of treating a lung disorder, condition, and/or disease by administration of at least one ENaC inhibitor peptide or compound of Formula I by an inhalation composition, kits, and/or systems described herein.

[0189] The term "treatment" and the like, as used herein, encompass any course of medical intervention aimed at a pathologic condition, and includes not only permanent cure of a disease, but prevention of disease, control or even steps taken to mitigate a disease or disease symptoms. For instance, in reference to methods of treating a disorder, such as COPD, idiopathic pulmonary fibrosis (IPF) or cystic fibrosis (CF), the embodiment, generally includes the administration of a compound or composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition (e.g., COPD, IPF or CF) in a subject relative to a subject not receiving the compound or composition. This can include reversing, reducing, or arresting the symptoms, clinical signs, and underlying pathology of a condition in a manner to improve or stabilize a subject's condition (e.g., regression of lung capacity).

[0190] In some embodiments, the therapeutic embodiments are carried out by contacting a tissue of a subject, e.g., epithelial tissue of the airways, with the inhalation delivery system. As defined herein, '"contacting" means that the composition comprising the active ingredient is introduced into a sample containing a target, e.g., cell target, in a test tube, flask, tissue culture, chip, array, plate, microplate, capillary, or the like, and incubated at a temperature and time sufficient to permit binding of the peptide or the compound to the target. In the in vivo therapeutic context, "contacting" means that the peptide or the compound used is introduced into a patient or a subject for the treatment of a lung disorder, e.g., CF, IPF, or COPD, and the peptide or the compound is allowed to come in contact with the patient's target tissue, e.g., epithelial tissue, in vivo.

[0191] In another embodiment, the therapeutic embodiments are carried out by administering the compositions and kits to a subject, e.g., a patient suffering from a lung disorder such as COPD, IPF, or CF. The term "administering" means applying as a remedy, such as by the placement of a drug in a manner in which such drug would be received, e.g., via an inhalation device such as an MDI or DPI or a nebulizer, and be effective in carrying out its intended purpose.

[0192] In some embodiments, the disclosure relates to treatment of chronic obstructive pulmonary disease (COPD) comprising administering, to a subject in need thereof, a therapeutically effective amount of an ENaC inhibitory peptide or a compound of Formula I. COPD is the overall term for a group of chronic conditions that are associated generally with the obstruction of lungs' airways. The disease may be accompanied by pulmonary hypertension (PH) but not necessarily. The disease has two major aspects of pathology, namely chronic bronchitis, characterized by mucus hyper-secretion from the conducting airways, and emphysema, characterized by destructive changes in the alveoli. In clinical practice, COPD is defined by its characteristically low airflow on lung function tests (Nathell et a!,. Respiratory Research 8, 89, 2007),

[0193] In another embodiment, the disclosure relates to treatment of a pathological condition of COPD which is chronic bronchitis, comprising administering, to a subject in need thereof, a therapeutically effective amount of an ENaC inhibitory peptide or a compound of Formula I. Chronic bronchitis is an inflammatory disease that begins in the smaller airways within the lungs and gradually advances to larger airways. It increases mucus production in the airways and increases the occurrence of bacterial infections in the bronchial tree, which, in turn, impedes airflow. This chronic inflammation induces thickening of the walls of the bronchial tree leading to increasing congestion in the lungs that results in dyspnea. By definition, chronic bronchitis refers to a productive cough for at least three months of each of two successive years for which other causes have been ruled out.

[0194] In another embodiment, the disclosure relates to treatment of a pathological condition of COPD which is emphysema, comprising administering, to a subject in need thereof, a therapeutically effective amount of an ENaC inhibitory peptide or a compound of Formula I. Emphysema describes destruction of the lung architecture with enlargement of the airspaces and loss of alveolar surface area. Lung damage is caused by weakening and breaking the air sacs within the lungs. Several adjacent alveoli may rupture, forming one large space instead of many small ones. Larger spaces can combine into an even bigger cavity, called a bulla. As a result, natural elasticity of the lung tissue is lost, leading to overstretching and rapture. There is also less pull on the small bronchial tubes, which can cause them to collapse and obstruct airflow. Air that is not exhaled before the new inhale process gets trapped in the lungs, leading to shortage of breath. The sheer effort it takes to force air out of the lungs when exhaling can be exhausting.

[0195] The most common symptoms of COPD include shortness of breath, chronic coughing, chest tightness, greater effort to breathe, increased mucus production and frequent clearing of the throat. Patients are unable to perform their usual daily activities. Independent development of chronic bronchitis and emphysema is possible, but most people with COPD have a combination of the disorders. Both conditions decrease the lungs' ability to take in oxygen and remove carbon dioxide.

[0196] Worldwide, COPD ranked as the sixth leading cause of death in 1990. It is projected to be the 4^th leading cause of death worldwide by 2030 due to an increase in smoking rates and demographic changes in many countries (Mathers et al, PLoS Med. 3 e442, 2006). Presently, COPD is the 3^rd leading cause of death in the U.S. (Heron et al, Natl Vita! Stat Rep. 65(2): 1- 95, 2013).

[0197] Long-term smoking is the most common cause of COPD, responsible for 80% to 90% of all cases. Other risk factors are heredity, second-hand smoke, air pollution, and a history of frequent childhood respirator}' infections. Cigarette smoking and other inhaled irritants plays a fundamental role in the pathogenesis of COPD, which affects as many as 8% of individuals in industrialized nations.

[0198] The inflammatory response in COPD involves a number of different cell types including mononuclear cells (macrophages), CD4+ and CD8+ T lymphocytes, neutrophils, which can be isolated from the lungs of patients with COPD. When activated, these cells induce mediators of inflammation and cytokines, such as interleukin (IL)-8, tumor necrosis factor-A (TNF-alpha), LTB4 which amplify the inflammatory response and may remodel lung architecture. Moreover, there is excessive activity of proteases, and an imbalance between proteases and endogenous antiproteases. Accordingly, the proteolytic lung tissue microenvironment of COPD patients makes it inherently challenging to use peptide compounds as therapeutic agents.

[0 99] Although there is no cure for COPD, medications that are prescribed for people with

COPD include: fast-acting beta 2-agonists, such as salbutamol which can help to open narrowed airways; anticholinergic bronchodilators, such as ipratropium bromide, and theophylline derivatives, all of which help to open narrowed airways; long-acting bronchodilators, which help relieve constriction of the airways and help to prevent bronchospasm associated with COPD; inhaled or oral corticosteroids, that help reduce inflammation; antibiotics that are often given at the first sign of a respiratory infection to prevent further damage and infection in diseased lungs; expectorants that help loosen and expel mucus secretions from the airways, and may help make breathing easier; lung transplantation is being performed in increasing numbers and may be an option for people who suffer from severe emphysema; lung volume reduction surgery, shows promise and is being performed with increasing frequency; al-antitrypsin (AAT) replacement therapy or gene therapy for AAT deficiency emphysema. Newer developments describe the successful use of vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PA CAP) in the treatment of COPD. See, WO 2003/61680. Hie disclosure therefore further relates to treatment of COPD comprising administering an ENaC inhibitor peptide or a compound of Fonnula I, together with one or more of the aforementioned agents useful for the therapy of COPD.

[0200] In another embodiment, the disclosure relates to treatment of idiopathic pulmonary fibrosis (IPF), comprising administering, to a subject in need thereof, a therapeutically effective amount of an ENaC inhibitor}' peptide or a compound of Formula I. IPF is a restrictive lung disease characterized by progressive interstitial fibrosis of lung parenchyma, affecting approximately 100,000 patients in the United States (Raghu et at. Am JRespir Crit Care Med 174:810-816, 2006). This interstitial fibrosis associated with IPF leads to progressive loss of lung function, resulting in death due to respiratory failure in most patients. The median survival from the time of diagnosis is 2-3 years (Raghu et al.. Am JRespir Crit Care Med 183:788-824, 2011). The prognosis is dire and often lung transplantation is the only resort (Thabut et a!., Annals of Internal Medicine 151 :767-774, 2009). Lung transplantation, however, is associated with considerable morbidity, not all IPF patients are appropriate candidates for it, and there is a relative paucity of suitable donor lungs. Despite numerous attempts, no drug therapies to date have been shown to substantially prolong survival in a randomized, placebo-controlled interventional trial in IPF patients, although some interventions have appeared to slow the rate of lung function decline in some patients (Raghu et at, supra, 2011 ; Richeldi et at, NEJM, 365: 1079-1087, 2011; Rafii et al., J. Thorac. Dis. 5(l):48-73, 2013).

[0201] Common symptoms of IPF include shortness of breath; persistent dry, hacking cough; fatigue; inexplicable weight loss; muscle and/or joint aches muscles; clubbing (widening and rounding of the tips of the fingers or toes).

[0202] In another embodiment, the disclosure relates to the treatment of cystic fibrosis (CF), including, chronic lung disease, in a subject in need thereof, comprising administering a therapeutically effective amount of an ENaC inhibitor peptide or a compound of Formula I. In CF, bacterial colonization of the airways generally occurs within the first year or two after birth. Patients with CF have a predisposition to subsequent chronic colonization and infection with Pseudomonas aeruginosa, an organism whose presence in the CF lung is associated with progressive respirator}' compromise. Infection is associated with an exuberant inflammatory response dominated by neutrophils and the potent inflammatory mediators that are released by activated neutrophils. An inexorable decrease in pulmonary function in the norm, leading eventually to death or to a need for lung tra spla tation . [0203] Symptoms of cystic fibrosis include, for example, abdominal pain, chronic cough (with blood or with phlegm); gastrointestinal issues (diarrhea, fat in stool, heartburn, severe constipation, or bulky stools); respiratory problems (e.g., pulmonary hypertension, shortness of breath, sinusitis, or wheezing); developmental defects in children (e.g., delayed development, delayed puberty, or slow growth); including systemic issues such as fatigue or inability to exercise. Other common symptoms include, e.g., acute bronchitis, deformity of nails, infection, male infertility, nasal polyps, pneumonia, salty sweat, or weight loss.

[0204] Experimental evidence on the function of the CF gene CFTR in regulating epithelial ion transport has provided a compelling account of the pathogenesis of gastrointestinal disease in CF, as well as of the genesis of such CF-associated phenomena as high sodium chloride content in sweat. There is a growing consensus that the CF airway is marked by an aberrant, exaggerated pro-inflammatory propensity that predates infection. In vivo studies using fetal human tracheal xenografts strongly suggest that tins basal pro-inflammatory predisposition of the CF airway leads to the development of mucosal damage after infection, damage that is itself integral to subsequent persistent bacterial colonization of the airway. The airway inflammatory response in CF is persistently neutrophilic, marked by upregulation of neutrophil chemotactic mediators such as interleukm 8 (IL-8) and leukotriene B4 (LTB4); florid accumulation of neutrophils in the airways; and neutrophil activation, with release of toxic products such as neutrophil elasiase. The initial inflammatory response to most bacterial stimuli, in the lung and elsewhere, is "acute", that is, neutrophil dominant. However, in the absence of bacterial clearance there is normally modulation over time to less toxic, "chronic" inflammation, a shift marked by the presence and regulatory activity of monocytes and lymphocytes. An unusual feature of inflammation in the CF airway is that such modulation never takes place.

[0205] Current treatments for cystic fibrosis generally focus on controlling infection through antibiotic therapy and promoting mucus clearance by use of postural drainage and chest percussion. However, even with such treatments, frequent hospitalization is often required as the disease progresses. New therapies designed to increase chloride ion conductance in airway epithelial cells have been proposed, and restoration of the expression of functional cystic fibrosis transmembrane regulator at the cell surface is considered a major therapeutic goal in the treatment of the disease. The disclosure therefore further relates to treatment of CF comprising administering an E aC inhibitor peptide or a compound of Formula I, together with one or more of the aforementioned agents useful for the therapy of CF.

[0206] The disclosure further relates to treatment of dry eyes, dry mouth, vaginal dryness or acute rhinosinusitis comprising administering, to a subject in need thereof, a therapeutically effective amount of an ENaC inhibitor peptide or a compound of Formula I, including the compounds disclosed under Table 1 of U.S. Pub. No. 2017-0226180 (spanning pages 4 to 8 of US2017-0226180), the structures of the compounds, as represented by the amino acid sequences thereof, are incorporated by reference herein.

[0207] In some embodiments, the disclosure relates to treatment of diy eyes, dry mouth, vaginal dryness or acute rhinosmusitis comprising administering, to a subject in need thereof, a therapeutically effective amount of an ENaC inhibitor peptide or a compound of Formula I disclosed herein.

[0208] In another embodiment, the disclosure relates to treatment of dry eyes, diy mouth, vaginal dryness or acute rhinosinusitis comprising administering, to a subject in need thereof, a therapeutically effective amount of a compound disclosed in Table 2 or an N-terminal acetylated derivative thereof. In Table 2, a = D-alanine, Nie = Norleucine, HYP =^;: 4- hydroxyproline, DHP = 3, 4-dehydro-L -proline, Ahp = aminoheptanoic acid, 2PP = (2R,5S)-5- phenyl-pyrrolidine-2-carboxylic acid, MS = L-a-methylserine, and raV

[0209] Table 2: Representative types of peptide compounds

94 NH3 V P V P L D Q T NH2

95 NH3 V P V P L D Q s NH2

96 NH3 L P L P L D Q s NH2

97 NH3 V P L P L D Q s NH2

98 NH3 L P L P L D Q T NH2

HY HY

NH3 V L V E

99 P P Q s NH2

100 NH3 V P L P V E Q s NH2

101 NH3 L P L P L E Q s NH2

102 NH3 L P mV P L D Q s NH2

103 NH3 L P Nle P L D Q T NH2

104 NH3 L P Nle P V E Q s NH2

105 NH3 L P Nle P V E Q s NH2

HY HY

NH3 L Nle L E

106 P P Q s NH2

HY HY

NH3 L Nle V E

107 P P Q s NH2

Ah

NH3 L P P L D T NH2

108 Q

109 NH3 L P F P L D Q T MH2

HY

NH3 a a L Nle P L D a NH2

110 P Q T a

HY

NH3 a a 1, P Nle L D

111 P Q T a a NH2

HY

NH3 L I P L D

1 12 P Q T MH2

1 13 NH3 a L P I P L E Q s a NH2

[0210] Tlie term "dry eye" (also referred to as xerophthalmia) as used herein includes any condition resulting in irritation or a sensation of dryness of the eye, including any type of ocular surface inflammation and it includes any disease or disorder or condition which results in an adverse effect on the quality of the tear film that lubricates the eyes, regardless of etiology. The disease or disorder may be of the eye itself, or of another part of the body, so long as it results in an adverse effect on the quality of the tear film that lubricates the eyes. Dry eye includes ocular inflammatory conditions that include: (i) eyelid inflammations: blepharitis, chronic eyelid edema, meibomitis, ocular rosacea, thyroid eye disease; (ii) conjunctival inflammations: chronic papillary conjunctivitis, chronic follicular conjunctivitis, nonspecific chronic conjunctivitis, giant papillary conjunctivitis, ocular cicatricial pemphigoid, cicatrizing conjunctivitis, allergic conjunctivitis, phlyctenular corneoconjunctivitis; (iii) Corneal inflammations: dry eye syndrome, ocular rosacea, superficial punctate keratitis, infectious keratitis, peripheral ulcerative keratitis, Thygeson's superficial punctate keratitis, corneal graft rejection, disciform keratitis, stromal keratitis: (iv) Uveitis: Anterior uveitis, HLA-B27 uveitis, Behcet's disease, atopic eye disease, juvenile rheumatoid arthritis, pars planitis, sarcoidosis, Vogt-Koyanagi-Harada syndrome, sympathetic ophthalmia, Fuch's heterochromia iridocyclitis, glaucomatocyclitis crisis (Posner-Schlossman syndrome); and (v) Retinal inflammations (not already mentioned): chronic macular edema, central serous choreoretinopathy, white dot syndrome, acute retinal necrosis. Causes include idiopathic, diabetes, congenital alacrima, xerophthalmia, lacrimal gland ablation, and sensory denervation as well as other conditions, factors and phenomena such as prolonged contact lens wear, advanced age, circulating hormones, allergies, ocular surgeries including PRK or LASIK, many medications, environmental conditions, visual tasking such as computer use, ocular fatigue, mechanical influences such as corneal sensitivity, partial lid closure, surface irregularities (e.g., pterygium), and lid irregularities (e.g., ptosis, entropion/ectropion, Pi nguecula), In rare cases, it may be a symptom of collagen vascular diseases, including rheumatoid arthritis, Wegener's granulomatosis, cystic fibrosis, and systemic lupus erythematosus.

[0211] The following are the most common complaints associated with dry eye syndrome (DES): Foreign-body sensation and ocular dryness and grittiness; hyperemia mucoid discharge; ocular irritation; excessive tearing (secondary to reflex secretion); photophobia; and fluctuating or blurry vision. Drugs such as isotretinoin, sedatives, diuretics, tricyclic antidepressants, antihypertensives, oral contraceptives, antihistamines, nasal decongestants, beta-blockers, phenothiazines, atropine, and pain relieving opiates such as morphine can cause or worsen this condition. Infiltration of the lacrimal glands by sarcoidosis or tumors, or postradiation fibrosis of the lacrimal glands, can also cause tins condition. Tests that may be used for diagnosis of dry eye syndrome include the following: impression cytology (e.g. , to monitor the progression of ocular surface changes), measurement of tear breakup time (TBUT), the Schirmer test, and quantification of tear components (e.g., through analysis of tear proteins or tear-film osmo!arity). Additional tests that may be used in workup include the tear stability' analysis system (TSAS), the tear function index (TFI; Liverpool modification), and the tear feraing test (TFT). Criteria for a diagnosis of dry eye syndrome associated with Sjogren syndrome (SS) include an abnormally low Schirmer test result, objective evidence of low salivary flow, biopsy-proven lymphocytic infiltration of the labial salivary glands, and dysfunction of the immune system, as manifested by the presence of serum autoantibodies. Θ212] Lubricating supplements are the medications most commonly used to treat DES . Agents that have been used to treat DES include the following: rebamipide, a mucin secretogogue; artificial tear substitutes; gels and ointments; anti-inflammatory agents; topical cyclosporine, topical corticosteroids; or topical or systemic omega-3 fatty acids. Topical or systemic tetracyclines have also been used. Other treatments include secretagogues such as diquafosol (approved in Japan but not in the United States), autologous or umbilical cord serum, and systemic immunosuppressants. Surgical interventions include use of punctual plugs, sealing of the perforation or descemetocele with corneal cyanoacrylate tissue adhesive; corneal or corneoscleral patching; lateral tarsorrhaphy/temporary tarsorrhaphy; conjunctival flap; surgical occlusion of the lacrimal drainage system; mucous membrane grafting; salivary gland duct transposition; and amniotic membrane transplantation. The disclosure therefore further relates to treatment of dry eyes comprising administering an ENaC inhibitor peptide or a compound of Formula I, together with one or more aforementioned agents useful for the therapy of dr - eyes.

Θ213] In some embodiments, the disclosure relates to treatment of dry mouth in a subject in need thereof, comprising administering an ENaC inhibitor peptide or a compound of Formula

I. The term "dry mouth^"' as used herein (also known as xerostomia or dry mouth syndrome) is the medical term for the subjective symptom of dryness in the mouth, which may be associated with a change in the composition of saliva or reduced salivary flow (hyposalivation) or have no identifiable cause, regardless of the etiology . A result of reduced or no saliva, dry mouth can lead to problems because saliva helps prevent tooth decay by limiting bacterial growth and washing away food particles. Saliva also enhances the ability to taste and makes it easier to swallow. In addition, enzymes in saliva aid in digestion. Dry mouth is very common and is often seen as side effect of many types of medication. It is more common in older people

(mostly because members of this group tend to take several medications) and in people who breathe through their mouths (mouth breathing). Dehydration, radiotherapy involving the salivary glands, and several diseases including inflammatory diseases and autoimmune diseases can cause hyposalivation or a change in saliva consistency leading to dry mouth.

Diseases associated with dr ' eye are also often associated with dry mouth, also called hyposalivation, as the tear ducts in the eye are connected to the mouth and both comprise mucosal cells. Causes of dry mouth include cancer therapy, as chemotherapy drugs can change the nature of saliva and the amount produced. This effect may be temporary, with nonnal salivary flow returning after treatment has been completed. Radiation treatments to the head and neck can damage salivary glands, causing a marked decrease in saliva production. This can be temporary or permanent, depending on the radiation dose and area treated. An injury or surgery that causes nerve damage to the head and neck area can result in dry mouth. Dry mouth can be a consequence of certain health conditions, including the autoimmune disease Sjogren's syndrome or HIV/AIDS. Stroke and Alzheimer's disease may cause a perception of dry mouth, even though the salivary glands are functioning normally. Snoring and breathing with the mouth open also can contribute to dry mouth. Smoking or chewing tobacco can increase dry mouth symptoms. Methamphetaniine use can cause severe dry mouth and damage to teeth, a condition also known as "meth mouth/' More than 400 medicines can cause the salivary glands to make less saliva. Drugs that can cause dryness include antihistamines, decongestants, diuretics, some anti-diarrhea drugs, some anti-psychotic drags, tranquilizers, some antihypertensives (e.g., terazosin, prazosin, clonidine, atenolol, propranolol), antidepressants (tricyclic antidepressants, selective serotonin reuptake inhibitors, lithium), and anti-reflux drugs (proton pump inhibitors, e.g., omeprazole), opioids, cytotoxic drags, retinoids, bupropion, protease inhibitors, didanosine, diuretics, ephedrine, benzodiazepines and IL-2. The disclosure therefore further relates to treatment of dry mouth comprising administering an ENaC inhibitor peptide or a compound of Formula I, together with one or more of the aforementioned agents useful for the therapy of dry mouth.

[0214] The disclosure further relates to the treatment of vaginal dryness in a subject in need thereof comprising administering an ENaC inhibitor peptide or a compound of Formula I. Vaginal dryness is a common problem which can cause physical and emotional distress in women (Key et a!., Nurs. Stand, 5:24-27, 1991). Inadequate lubrication due to vaginal dryness can also result in dyspareunia (characterized by difficult or painful sexual intercourse), a condition thought to affect approximately 40% of women; it has been estimated that over 40 million women will suffer dyspareunia at some time in their lives (Kelly, Clinical Practices and Sexuality 8(8):2, 1992). Vaginal dryness is one of the most common symptoms of menopause. Vaginal dryness is also often seen after childbirth or in women suffering from diseases, such as diabetes mellitus and autoimmune disorders. In other instances, treatments for various conditions, such as chemotherapy or radiotherapy for cancer, can cause vaginal dryness.

[0215] Vaginal dryness can have a significant impact on the quality of life of women by adversely affecting their attitude toward intimate contact, a necessary part of healthy relationships. Current methods for treating vaginal dryness include applying lubricating agents such as lubricating creams, jellies and topical estrogen creams; and hormone replacement therapy. Hormone replacement therapy is also effective for treating vaginal atrophy and dryness, but has several contraindications and unwanted side effects. Water-based lubricating composition are commercially available, for example, ASTROGLIDE® and K-Y JELLY®, as well as silicone-based lubricants. ASTROGLIDE® is typical of the water-based lubricants, which is a clear solution available as either a liquid or a gel . ASTROGLIDE® Liquid contains purified water, glycerin, propylene glycol, polyquatemium 15, methylparaben and propylparaben. ASTROGLIDE® Gel contains purified water, glycerin, hydroxyethyl cellulose, chiorhexidine gluconate, methylparaben, glucono delta lactone, and sodium hydroxide.

[0216] The disclosure further relates to use of an ENaC inhibitor peptide or a compound of the disclosure to treat of rhinosinusitis. As used herein, the term "rhinosinusitis" refers to a condition that has symptoms of both rhinitis and sinusitis. Rhinosinusitis includes acute rhinosinusitis and chronic rhinosinusitis. Acute rhinosinusitis can be caused by an infection, such as a bacterial, viral or fungal infection, or by a chemical irritation. Cigarette-smoke- induced acute rhinosinusitis and chlorine fume-induced chronic rhinosinusitis are examples of acute rhinosinusitis. Chronic sinusitis (CS) and chronic rhinosinusitis (CRS) are conditions that last longer than eight weeks. The underlying causes of acute sinusitis and acute rhinosinusitis may lead to chronic sinusitis or chronic rhinosinusitis if the resulting inflammation persists for more than 8 weeks. Chronic rhinosinusitis includes, e.g., eosinophilic chronic hyperplastic rhinosinusitis. Nasal polyps are most commonly associated with chronic rhinosinusitis (CRS), which is characterized by mucosal inflammation of the nasal cavity and paranasal sinuses with symptoms lasting more than 8 weeks.

[0217] In another embodiment, disclosed herein is a use of an ENaC inhibitor peptide or a compound of Formula I, la, lb, or Ic as described above, for therapy of pulmonary diseases, e.g., COPD, IPF, CF, etc., in a subject in need thereof. Also disclosed herein is a use of an

ENaC inhibitor peptide or a compound of Formula I, la, lb, or Ic as descri bed abov e, for therapy of dry eye, dry mouth, vaginal dryness and/or acute rhinosinusitis in a subject in need thereof.

[0218] In another embodiment, disclosed herein is use of an ENaC inhibitor peptide or a compound of Formula I, la, lb, or Ic as described above, for the manufacture of a medicament for treating pulmonary diseases, e.g., COPD, IPF, CF, etc., in a subject in need thereof. Also disclosed herein is a use of an ENaC inhibitor peptide or a compound of Formula I, la, lb, or

Ic as described above, for the manufacture of a medicament for the therapy of dry eye, dry mouth, vaginal dryness and/or acute rhinosinusitis in a subject in need thereof.

[0219] In another embodiment, disclosed herein are compositions comprising an ENaC inhibitor peptide or a compound of Formula 1, la, lb, or Ic as described abov e, for use in treating pulmonary diseases, e.g., COPD, IPF, CF, etc., in a subject in need thereof. Also disclosed herein are compositions comprising an ENaC inhibitor peptide or a compound of Formula I, la, lb, or Ic as described above, for use in treatment of dry eye, dry mouth, vaginal dryness and/or acute rhinosinusitis in a subject in need thereof.

[0220] The disclosure further relates to a method of regulating salt balance, blood volume, and/or blood pressure in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the ENaC inhibitor peptide or a compound of Formula I, la, lb, or Ic or a functional fragment or homolog thereof.

[0221] The disclosure further relates to a method of inhibiting the activation of a sodium channel, comprising contacting the sodium, channel with an ENaC inhibitor peptide or a compound of Formula ΐ, la, lb, or Ic of the disclosure or a composition comprising the same. The method can be carried out, e.g., on an isolated sodium channel, sodium channel in an artificial membrane, or a sodium channel in a cell (e.g., in vitro). In some embodiments, the sodium channel is present in an isolated cell, e.g., a cultured primary cell or cell line. In another embodiment, the isolated cell is part of an epithelial cell culture, e.g., a natural or artificial epithelial lining, e.g., a. cell culture in a device (such as an Ussing chamber) in which characteristics such as ion flow and/or potential can be measured across lining. In another embodiment, the cell is part of an isolated tissue or a tissue culture. In a further embodiment, the cell can be present in an animal, e.g., an animal that is a disease model or a subject in need of treatment. In some embodiments, the step of contacting (e.g., binding) the sodium channel with an ENaC inhibitor peptide or a compound of the disclosure comprises delivering the ENaC inhibitor peptide or the compound to a cell comprising the sodium channel.

[0222] Another aspect of the disclosure relates to a method of inhibiting sodium absorption through a sodium channel, comprising contacting (e.g., binding) the sodium channel with the ENaC inhibitor peptide or the compound of Formula I of the present disclosure or a functional fragment thereof. Inhibition of sodium absorption can be measured by any technique known in the art or disclosed herein (see, Example 4).

[0223] Another aspect of the disclosure relates to a m ethod of increasing the volume of fluid lining an epithelial mucosal surface, comprising contacting (e.g., binding) a sodium channel present on the epithelial mucosal surface with an effective amount of an ENaC inhibitor peptide or a compound of the disclosure or a functional fragment or homolog thereof. The volume of fluid lining an epithelial mucosal surface can be measured by any technique known in the art or disclosed herein. Θ224] A further aspect of the disclosure relates to a method of reducing the level of a sodium channel present on the surface of a cell, comprising contacting (e.g., binding) the sodium channel with an effective amount of an ENaC inhibitor peptide or a compound of the disclosure or a functional fragment or homolog thereof.

[0225] The peptides or compounds of the present disclosure can optionally be delivered in conjunction with other therapeutic agents. The additional therapeutic agents can be delivered concurrently with the peptides or compounds of the disclosure. As used herein, the word "concurrently" means sufficiently close in time to produce a combined effect (that is, concurrently can be simultaneously, or it can be two or more events occurring within a short time period before or after each other). In some embodiments of the disclosure, the ENaC inhibitor peptide or the compound of the disclosure is delivered to a patient concurrently with a compound that modulates the function of the Cystic fibrosis transmembrane conductance regulator (CFTR) where the combined activity of the ENaC inhibitor peptide or the compound of the disclosure and the CFTR-targeted agent have superior activity to the CFTR-targeted agent alone. In another embodiment of the disclosure, the ENaC inhibitor peptide or the compound of the disclosure is delivered to a patient concurrently with a mucolytic compound where the combined activity of the ENaC inhibitor peptide or the compound of the disclosure and the mucolytic agent have superior activity to the mucolytic agent alone. In yet another embodiment of the disclosure, the ENaC inhibitor peptide or the compound of the disclosure is delivered to a patient concurrently with a long acting B-agonist compound (LABA) where the combined activity of the ENaC inhibitor peptide or the compound of the disclosure and the LABA agent have superior activity to the LABA alone. In yet another embodiment of the disclosure, the ENaC inhibitor peptide or the compound of the disclosure is delivered to a patient concurrently with a glucocorticoid agonist where the combined acti vity of the ENaC inhibitor peptide or the compound of the disclosure and the glucocorticoid agent have superior activity to the glucocorticoid alone.

[0226] Moreover, the peptide compounds of the disclosure are also useful treating diseases that are caused by pathogens, e.g., bacteria, viruses, fungi, protists, etc. The art recognizes an important role of SPLUC1 proteins against pathogenic infections of the airway, e.g., viral infection (Akrarn et al. Mucosal Immunol. 2017 May 17), bacterial infection (Jiang etal., PLoS

One 8(5), 2013), fungal infection (Bartlett et al, Dis Model Mech., 8(5): 501-508, 2015). For instance, analogous to full-length SPLU C 1 proteins, the peptide compounds may play a role in promoting immunity, e.g., enhanced neutrophil innate immune functions, thereby inhibiting the colonization of the epithelial mucosa by microbes. See, Gaily et al, Am J Pathol. 178(5): 2159-2167, 2011); Liu et al. (J Immunol 191 :4259-4268, 2013); Liu et al. {Am J Pathol 182: 1519-1531, 2013); Jiang et al. (PLOS One, 8:ε64689, 2013); and Walton et al. (Biochemistry 55:2979-2991, 2016), Thus, embodiments of the disclosure further relate to use of the peptides or compounds as antimicrobial agents, e.g., inhibiting the growth of pathogenic bacteria (including biofilms comprising bacteria), viruses, fungi, protists, etc. in host systems.

[0227] In some embodiments, the peptides or compounds are useful in inhibiting or disrupting the formation of biofilms. A "biofilm" is a complex organization of pathogens, e.g., bacteria that are anchored to a surface via a matrix containing polysaccharides, which grows into complex architectures such as differentiated towers comprising a plurality of pathogens. The extruded exopolysaccharide matrix, which comprises more than 90% of the biofilm, envelopes the pathogen and provides protection from phagocytosis and oxidative burst mechanisms. This protective effect is observed both in the natural environment and in a host. Pathogens within biofilms are also resistant to the host's humoral defense systems because of a lack of accessibility by immunoglobulins and/or complement system. The attachment of pathogens to a surface triggers the expression of a cassette of genes, which results in the formation of a biofilm. Biofilm bacteria have been demonstrated to be highly resistant to growth in standard planktonic [i.e., free-floating) culture, which is sometimes attributed to differences in gene expression.

[0228] In the in vivo context, the peptides or compounds of the disclosure may disrupt or inhibit biofilm formation or exert anti-microbial effects, for instance, by mediating airway surface liquid (ASL) homeostasis, thereby helping cleanse the airways through the physical removal of pathogens and toxicants on the mucociliary escalator. Accordingly, embodiments of the disclosure further relate to use of peptides or compounds in promoting airway surface liquid (ASL) homeostasis by regulating innate the volume and composition of ASL.

[0229] The present disclosure includes methods to, for example, reduce antimicrobial resistance, reduce inflammation, inhibit microbial growth, bind pathogenic molecules (e.g., LPS) or reduce microbial viability using an effective amount of a peptide or a compound of the present disclosure. The methods may include, for instance, administration of one or more antimicrobial/immune modulatory agents or antibiotics.

[0230] The antimicrobial properties (e.g., bactericidal or viricidal effects) of the peptides disclosed allow them to be included in formulations to inhibit microbial growth and proliferation, and any adverse physiological responses such as inflammation . The purified peptide may be used without further modifications or it may be diluted in a pharmaceutically acceptable carrier. It is contemplated that the peptides and compounds of the disclosure may be administered to humans or animals, included in food preparations, pharmaceutical preparations, medicinal and pharmaceutical products, cosmetic products, hygienic products, cleaning products and cleaning agents, as well as any material to which the peptides or compounds could be sprayed on or adhered to wherein the inhibition of microbial growth on such a material is desired.

[0231] The proper dosage or effective amount of an antimicrobial peptide or compound necessary to prevent microbial growth and proliferation depends upon a number of factors including the types of bacteria that might be present, the environment into which the peptide or compound is being introduced, and the time that the peptide or compound is envisioned to remain in a given area.

[0232] It is further contemplated that the antimicrobial peptides or compounds of the disclosure may be used in combination with or to enhance the activity of other antimicrobial agents or antibiotics. Combinations of the peptide or compounds with other agents may be useful to allow antibiotics to be used at lower doses due to toxicity concerns, to enhance the activity of antibiotics whose efficacy has been reduced or to effectuate a synergism between the components such that the combination is more effective than the sum of the efficacy of either component independently. Antibiotics which may be combined with an antimicrobial peptide in combination therapy include, but are not limited to penicillin, ampicillin, amoxycillin, vancomycin, cycloserine, bacitracin, cephalosporin, imipenem, colistin, methicillin, streptomycin, kanamycin, tobramycin, gentamicin, tetracycline, chiortetracycime, doxycycline, chloramphenicol, lincomycin, clindamycin, erythromycin, oleandomycin, polymyxin nalidixic acid, rifamycin, rifampicin, gantrisin, trimethoprim, isoniazid, paraaminosalicylic acid, and ethainbutol. The present disclosure is thus contemplated for use against all such infections.

[0233] The aforementioned embodiments of the disclosure are further described in view of the following non-limiting examples.

[0234] EXAMPLES

[0235] The structures, materials, compositions, and methods described herein are intended to be representative examples of the disclosure, and it will be understood that the scope of the disclosure is not limited by the scope of the examples. Tliose skilled in the art will recognize that the disclosure may be practiced with variations on the disclosed structures, materials, compositions and methods, and such variations are regarded as within the ambit of the disclosure.

[0236] Example 1 : Assessment of ENaC internalization by the peptide compounds [0237] Human embryonic kidney (HEK293) cells were transfected with alpha, beta, and gamma ENaC for 24 hours. Surface ENaC levels were determined by surface biotinylation. The cells were treated with vehicle (control) or peptide compounds of the present disclosure, in some experiments, unmodified peptide compounds (SPX6, SPX7, and SPX8, having the sequence LPIPLD (SEQ ID NO: 6); LPIPLDQ (SEQ ID NO: 7); and LPIPLDQT (SEQ ID NO: 8), respectively) were employed. In some experiments, peptide compounds that were terminally-modified with two D-alanine residues, one at each N- and C-termini of the template peptide sequence, were employed. The modified peptides are represented with the abbreviation SPX6-D, SPX7-D, and SPX8-D and respectively have the amino acid sequence aLPIPLDa (SEQ ID NO: 14); aLPIPLDQa (SEQ ID NO: 15); and aLPIPLDQTa (SEQ ID NO: 32), wherein a is a D-alanine. In some experiments, peptide compounds that were terminally- modified with a total of four D-alanine residues, two at each N- and C-termini of the template sequence were employed. The plural modified peptides are represented with the abbreviation SPX6-DD, SPX7-DD, SPX8-DD and respectively have the amino acid sequence aaLPIPLDaa (SEQ ID NO: 18); aaLPIPLDQaa (SEQ ID NO: 19); and aaLPTPLDQTaa (SEQ ID NO: 34), wherein a is a D-alanine. The results of the experiments with the alpha subumt of ENaC is shown in the left hand portion of the chart (in orange bars); the results of the experiments with the beta subumt is shown in the middle portion of the chart (in grey bars); and the results of the experiments with the gamma subunit of ENaC is shown in the right hand portion of the chart (in blue bars).

[0238] Results are shown in FIG. 1. The results show that all peptide derivatives internalized α, β and γ ENaC subunits as compared to vehicle control.

[0239] Example 2: Assessment of thermal stability of the peptide compounds

[0240] Peptides were formulated at 50 mg/ml in 0.9% saline and stored at 40°C for up to 28 days. At the indicated times, peptide stability was determined by HPLC and calculated as percent of original peak. The SI 8 peptide, which is employed as a control, is an 18-mer sequence derived from SPLUNC1, whose structure has been described in Tarran et al. (US2016/0102121), The S18 peptide has the amino acid sequence GGLPVPLDQT LPLNVNPA (SEQ ID NO: 1 14) (see, FIG. 1 and the description thereof in Tarran et al (US2016/0102121).

[0241] Results are shown in FIG. 2. The results show that the thermal stability of the 6-mer SPX6 peptide is better than that of the 8-mer SPX8 peptide and the thermal stability of the 7- mer SPX7 peptide is comparable to that of the 8-mer SPX8 peptide.

[0242] Example 3: Assessment of protease resistance of the peptide compounds [Θ243] Peptides were formulated at 50 mg/ml in 0,9% saline and incubated with 0.2% trypsin or 0.1 mg/rnl human neutrophil elastase (HNE) for 90 minutes. Peptide stability was determined by HPLC after denaturing enzymes at 90°C for 5 min.

[0244] Results are shown in FIG, 3. The results on trypsin stability are shown in Panel A and the results on HNE stability are shown in Panel B. The results show that the 6-mer SPX6 polypeptide had the highest enzymatic stability compared to ail the polypeptides tested. In particular, both the modified as well as the unmodified variants of the shorter polypeptide SPX6 displayed greater resistance to trypsin compared to the longer 8-mer polypeptide SPX8. The unmodified SPX7 and/or its modified variant (SPX7-D) also displayed greater resistance to trypsin compared to the unmo ified 8~mer SPX8 polypeptide. The shorter 6-mer, 7~mer and 8- mer polypeptides displayed similar stability profiles against elastase (HNE), which was found to be significantly improved over the parent S 18 polypeptide.

[0245] Example 4: Effect of peptide compounds in the survival of bENaC-Tg mouse model

[Θ246] Transgenic mice over-expressing bENaC (PENaC-Tg) were administered saline or the indicated peptide beginning two days after birth. Dosing consisted of once daily intranasal administration of a 50 mM peptide solution formulated in 0.9% saline. Animal survival was monitored daily until post-natal (PN) day 14.

[0247] Results are shown in FIG. 4. It can be seen that, compared to vehicle (black line) or control peptide (grey line), the peptide compounds of the disclosure promoted survival in the PENaC-Tg mouse model of cystic fibrosis.

Θ248] Example 5: Assessment of cellular toxicity of peptide compounds using a cellular model

[0249] Human embryonic kidney (HEK293T) cells were plated on 96-well plates at a density of about 2,000 cells per well. The next day, media was removed and the cells were incubated with fresh media supplemented with (100 μΜ) or without (control) peptide compounds of the disclosure. Cells were cultured for 72 hours until untreated control reached confluence. Cell viability was measured with PRESTOBLUE cell viability indicator. Cells were stained for 30 minutes at 37°C and fluorescence measurements were made to assess cell viability. Data, which are presented in FIG. 5, shows mean +/- standard deviation (SD) of six technical replicates for each dose.

[0250] The results show that the peptide compounds of the disclosure are non-toxic to the in vitro model human cell-line. [0251] While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and subcombinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions and sub-combinations as are within their true spirit and scope.

[0252] For convenience, certain terms employed in the specification, examples and claims are collected here. Unless defined otherwise, all technical and scientific terms used in this disclosure have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0253] Throughout this disclosure, various patents, patent applications and publications are referenced. The disclosures of these patents, patent applications, accessioned information (e.g., as identified by PUBMED, PUBCHEM, NCBI, UNIPROT, or EBI accession numbers) and publications in their entireties are incorporated into this disclosure by reference in order to more fully describe the state of the art as known to those skilled therein as of the date of this disclosure. This disclosure will govern in the instance that there is any inconsistency between the patents, patent applications and publications cited and this disclosure.

Claims

We claim:

1. A compound of Formula lb and salts thereof,

R'-PEP-R² (lb);

wherein R¹ and R² are, independently, absent, or a blocking or a protecting group; and

PEP is X1-X2-X3-X4-X5-X6-X7 (SEQ ID NO: 2), wherein Xi to X? are each, independently, an amino acid, wherein

Xi is leucine (L), isoleucine (I), methionine (M), phenylalanine (F), or valine (V) or a conservatively substituted amino acid of any one of L, I, M, F, or V;

X2 is proline (P) or arginine (R) or a conservatively substituted amino acid of any one of P or ;

X3 is leucine (L), isoleucine (I), methionine (M), phenylalanine (F), or valine (V) or a conservatively substituted amino acid of any one of L, I, M, F, or V;

X4 is proline (P) or arginine (R) or a conservatively substituted amino acid of any one of P or R4X5 is (L), isoleucine (I), methionine (M), phenylalanine (F), or valine (V) or a conservative^ substituted amino acid of any one of L, I, M, F, or V; Xe is aspartic acid (D), asparagine (N), glutamine (Q), glutamate (E), serine (S), lysine (K), arginine (R), or citra!!ine (Cit), or a consen/atively substituted amino acid of any one of D, N, Q, E, S, , R or Cit; and X? is absent, or glutamine (Q), arginine (R), asparagine (N), aspartate (D), glutamate (E), histidine (H), lysine (K), methionine (M), or serine (S) or a conservatively ammo acid of any one of Q, R, N, D, E, H, K, M, and S.

2, A compound of Formula lb and salts thereof,

R¹ PEP R² (lb);

wherein R^! and R² are, independently, absent, or a blocking or a protecting group; and

PEP is X : X ·· X = X i X? X,·. \- (SEQ ID NO: 2), wherein Xi to X? are each, independently, an amino acid, wherein

Xi is leucine (L), isoleucine (I), methionine (M), phenylalanine (F), or valine (V) or a conservatively substituted amino acid of any one of L, I, M, F, or V; X2 is proline (P) or arginine (R) or a conservatively substituted amino acid of any one of P or R;

X3 is leucine (L), isoleucine (Ϊ), methionine ( ), phenylalanine (F), or valine (V) or a conservatively substituted amino acid of any one of L, I, M, F, or V;X4 is proline (P) or arginine (R) or a conservatively substituted amino acid of any one of P or R;

X5 is (L), isoleucine (I), methionine (M), phenylalanine (F), or valine (V) or a conservatively substituted amino acid of any one of L, I, M, F, or V;

Xe is aspartic acid (D), asparagine (N), glutamine (Q), glutamate (E), or serine (S) or a conservatively substituted amino acid of any one of D, N, Q, E, or S; and

X? is absent, or glutamine (Q), arginine (R), asparagine (N), aspartate (D), glutamate (E), histidine (FT), lysine (K), methionine (M), or serine (S) or a conservatively amino acid of any one of Q, R, N, D, E, H, K, M, and S.

3. The compound of claim 1 or claim 2, wherein X? is glutamine or a conservatively substituted amino acid thereof.

4. The compound of claim 1 or 2, wherein X; is leucine (L) or a conservatively- substituted amino acid thereof.

5. The compound of claim 1 or 2, wherein Xj. is proline (P) or a conservatively- substituted amino acid thereof.

6. The compound of claim 1 or 2, wherein X3 is leucine (L) or a conservatively- substituted ammo acid thereof.

7. The compound of claim 1 or 2, wherein X₄ is proline (P) or a conservatively-substituted amino acid thereof.

8. The compound of claim 1 or 2, wherein Xs is leucine (L) or a conservatively-substituted amino acid thereof.

9. The compound of claim 1 or 2, wherein Xe is aspartic acid (D) or a conservatively- substituted amino acid thereof.

10. The compound of claim 1 or 2, wherein X? is glutamine (Q) or a conservatively- substituted amino acid thereof.

11. The compound of claim 1 or 2, wherein

X = is L, I, M, F, or V;

Xs is L, I, M, F, or V;

Xe is D, N, Q, E, S, K, R, or Cit: and

X? is O. R, N, D, I·.. 1 1. K, M, or S.

12. Tlie compound of claim 1 or 2, wherein

X; is L, I, M, F, or V;

X* is P or R;

Xs is L, I, M, F, or V:

Xe is D, N, Q, E, or S; and

X7 is Q. R, N, D, E, H, K, M, or S.

13. The compound according to any one of claims 1 - 12, wherein PEP is selected from the group consisting of LPLPLDQ (SEQ ID NO: 4) and LPIPLDQ (SEQ ID NO: 7).

14. The compound of claim 1, wherein X? is absent.

15. The compound of claim 14, wherein Xi is leucine (L) or a conservatively-substituted amino acid thereof.

1.6. The compound of claim 14, wherein Xi is proline (P) or a conservatively-substituted amino acid thereof.

17. The compound of claim 14, wherein ¾ is leucine (L) or a conservatively-substituted amino acid thereof.

18. The compound of claim 14, wherein XA is proline (P) or a conservatively-substituted amino acid thereof.

19. The compound of claim 14, wherein Xj is leucine (L) or a conservatively-substituted amino acid thereof.

20. The compound of claim 14, wherein Xe is aspartic acid (D) or a conservatively- substituted amino acid thereof.

21. The compound of claim 14, wherein

X₄ is P or R;

Xe is D, N, Q, E, S, K, R, or Cit.

22. The compound of claim 14, wherein

Xe is D, N, Q, E, or S.

23. The compound of any one of claims 14-22, wherein PEP is selected from the group consisting of LPLPLD (SEQ ID NO: 3) and LPIPLD (SEQ ID NO: 6).

7"7

24. The compound of any one of the preceding claims, wherein R^] and R² are both absent,

25. The compound of any one of claims 1-24, wherein at least one of R^! and R² is a blocking or a protecting group.

26. The compound of claim 25, wherein the compound comprises acetylation at the N- terminus, amidation at the C~terminus, or both acetylation at the N-terminus and amidation at the C-temiinus,

27. The compound of any one of claims 1-2,4, wherein at least one of R¹ and R² is a blocking or protecting group comprising one to about 4 non-proteinogenic amino acids.

28. The compound of claim 27, wherein the non-proteinogenic amino acids are selected from the group consisting of D -amino acids (e.g., D-ala ine), Norleucine (Nle), 4-hydroxyproline (HYP), 3,4-dehydro-L-proline (DHP), aminoheptanoic acid (AHP), (2R,5S)-5-phenyl- pyrrolidine-2-carboxylic acid (2PP), L-a-methylserine (MS), N-methylvaline (MV), 6- aminohexanoic acid (6-AHP), and 7-aminoheptanoic acid (7-AHP).

29. The compound of claim 28, wherein the non-proteinogenic amino acids are D-alanine.

30. The compound of claim 2,7, w herein the compound is selected from the group consisting of:

(a) aLPLPLDa (SEQ ID NO: 12);

(b) aLPLPLDQa (SEQ ID NO: 13);

(c) aLPIPLDa (SEQ ID NO: 14);

(d) aLPIPLDQa (SEQ ID NO: 15);

(e) aaLPLPLDaa (SEQ ID NO: 16);

(1) aaLPLPLDQaa (SEQ ID NO: 17);

(g) aaLPIPLDaa (SEQ ID NO: 18); and

(h) aaLPIPLDQaa (SEQ ID NO: 19);

wherein "a" is a non-proteinogenic amino acid selected from the group consisting of D-amino acids (e.g., D-alanine) (D-Ala); Norleucine (Nle); 4-hydroxyproline (HYP); 3,4-dehydro-L- proline (DHP); aminoheptanoic acid (AHP); (2R,5S)-5-phenyl- pyrrolidine-2-carboxylic acid (2PP); L-a-methylserine (MS); N-methylvaline (MV) 6-aminohexanoic acid (6-AHP); and 7-aminoheptanoic acid (7-AHP).

31. The compound of claim 27, wherein "a" is D-Ala.

32. A peptide consisting essentially of X1-X2-X3-X4-X5-X6-X7 (SEQ ID NO: wherein

Xi is leucine or a conservatively substituted amino acid thereof;

X2 is proline or a conservatively substituted ammo acid thereof;

X3 is leucine or a conservatively substituted amino acid thereof:

X4 is proline or a conservatively substituted amino acid thereof;

XJ is leucine or a conservatively substituted amino acid thereof;

Xe is aspartic acid or a conservatively substituted amino acid; and

X? is absent, or giutamine or a conservatively amino acid thereof; and salts thereof.

33. A method of inhibiting the activation of a sodium channel, comprising contacting a sodium channel with a compound or peptide of any one of claim s 1-32.

34. The method of claim 33, wherein the sodium channel is an epithelial sodium channel (ENaC).

35. The method of claim 33 or 34, wherein the sodium channel is present in an isolated cell

36. The method of claim 35, wherein the isolated cell is part of an epithelial cell culture.

37. The method of claim 33 or 34, wherein the sodium channel is present in a ceil in an animal.

38. The method of claim 37, wherein the animal has a respiratory disease.

39. The method of claim 37 or 38, wherein contacting the sodium channel with the peptide comprises administering the compound or peptide to the animal.

40. The method of claim 33, wherein activation of the sodium channel is inhibited by at least 20%.

41. The method of claim 33, wherein activation of the sodium channel is inhibited by at least 50%.

42. The method of claim 33, wherein activation of the sodium channel is i hibited by at least 90%.

43. A method of inhibiting sodium absorption through a sodium channel, comprising contacting the sodium, channel with a compound or peptide of any one of claims 1 -32.

44. The method of claim 43, wherein the sodium channel is ENaC.

45. A method of increasing the volume of fluid lining an epithelial mucosal surface, comprising contacting a sodium channel present on the epithelial mucosal surface with a compound or peptide of any one of claims 1-32.

46. The method of claim 45, wherein the sodium channel is ENaC,

47. A method of reducing the level of a sodium channel present on the surface of a ceil, comprising contacting the sodium channel with a compound or peptide of any one of claims 1-32.

48. The method of claim 47, wherein the sodium channel is ENaC.

49. A method of treating a disorder responsive to inhibition of sodium absorption across an epithelial mucosal surface in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound or peptide of any one of claims 1-

32,

50. The method of claim 49, wherein the sodium channel is ENaC.

51. The method of claim 49, wherein said disorder is a Sung disorder.

52. The method of claim 51, wherein said lung disorder is cystic fibrosis (CF), idiopathic pulmonary fibrosis (IPF) or chronic obstructive pulmonary disease (COPD).

53. The method of claim 51 , wherein said lung disorder is non-cystic fibrosis bronchiectasis.

54. The method of claim 51, wherein said lung disorder is chronic obstructive pulmonary disease (COPD).

55. The method of claim 51, wherein said lung disorder is acute or chronic bronchitis.

56. The method of claim 51, wherein said lung disorder is asthma.

57. The method of claim 49, wherein said disorder is a gastrointestinal disorder.

58. The method of claim 57, wherein said gastrointestinal disorder is inflammatory bowel disease.

59. The method of claim 49, wherein said disorder is a kidney disorder.

60. A method of regulating salt balance, blood volume, and/or blood pressure in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound or peptide of any one of claims 1-32.

61. The method of claim 60, wherein administering the peptide reduces the level of a sodium channel present on the surface of a cell in the subject.

62. A method for treating a lung disorder, a gastrointestinal disorder, a kidney disorder, or a cardiovascular disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound or peptide of any one of claims 1-32.

63. The method of claim 62, wherein the lung disorder is cystic fibrosis, non-cystic fibrosis bronchiectasis, chronic obstructive pulmonary disease, acute or chronic bronchitis, or asthma.

64. The method of claim 62, wherein the gastrointestinal disorder is inflammatory bowel disease.

65. A method of improving the outcome of a subject suffering from a disease mediated by ENaC activity, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the a compound or peptide of any one of claims 1-

JiZ .

66. The method of claim 65, wherein the disease mediated by ENaC activity is a lung disorder selected from the group consisting of cystic fibrosis, non-cystic fibrosis

bronchiectasis, chronic obstructive pulmonary disease, acute or chronic bronchitis, and asthma.

67. The method of claim 65, wherein the outcome is disease-free survival period (DPS), time to death (TTD), forced expiratory volume in one second (FEV1) or percent predicted FEV1.

68. A method of increasing innate immunity in a subject and/or reducing the susceptibility of the subject to a pathogen, comprising administering to the subject, a therapeutically effective amount of a compound or peptide of any one of claims 1-32.

69. The method of claim 68, wherein the pathogen is selected from bacteria, viruses, fungi, or protists.

70. The method of claim 68, wherein the subject is a human subject.

71. A method for treating dry eye, dry mouth, vaginal dryness, or rhinosinusitis in a subject in need thereof, comprising administering to the subject a therapeutically effecti v e amount of a compound or peptide of any one of claims 1-32 or a pharmaceutical composition comprising the compound or peptide and a carrier.

72. The method of claim 71, wherein a pharmaceutical composition comprising is administered in the form of an ocular formulation, buccal formulation, vaginal formulation, or nasal formulation.

73. A compound or peptide of any one of claims 1 -32 for use in treatment of a lung disorder, a gastrointestinal disorder, a kidney disorder, a cardiovascular disorder, dry eye, dry mouth, vaginal dryness, or rhiiiosinusitis.

74. The compound of claim 73 for use in treatment of a lung disorder selected from the group consisting of COPD, IPF, and CF.

75. Use of compound or peptide of any one of claims 1-32, for the manufacture of a medicament for a lung disorder, a gastrointestinal disorder, a kidney disorder, a

cardiovascular disorder, dry eye, dry mouth, vaginal dryness, or rhinosinusitis.

76. The use of claim 75 for the manufacture of a medicament for treating a lung disorder selected from the group consisting of COPD, IPF, and CF.

77. A kit comprising, in one or separate packages, the compound or peptide of any one of claims I to 32, optionally together with instructions for using the kit.