EP3554527A1

EP3554527A1 - Saline formulations of splunc1 peptides

Info

Publication number: EP3554527A1
Application number: EP17832396.0A
Authority: EP
Inventors: Timothy Morgan CROWDER; David W. Scott; John Clayton TAYLOR
Original assignee: Spyryx Biosciences Inc
Current assignee: Spyryx Biosciences Inc
Priority date: 2016-12-13
Filing date: 2017-12-12
Publication date: 2019-10-23
Also published as: WO2018111903A1; CA3046361A1

Abstract

The present invention relates to pharmaceutical formulations of non-naturally occurring peptides to bind to sodium channels and inhibit activation of the sodium channels. The invention further relates to methods for regulating of sodium absorption and fluid volume and treating disorders responsive to modulating sodium absorption by modulating the binding of specialized non-naturally occurring peptides to sodium channels.

Description

SALINE FORMULATIONS OF SPLUNC1 PEPTIDES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Patent Application No. 62/433,549, filed on December 13, 2016, the entire contents of which are incorporated herein by reference.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on December 12, 2017, is named 102382- 0053_SL.txt and is 61,128 bytes in size.

FIELD OF THE INVENTION

[0003] The present invention relates to optimized peptides that are specialized non-naturally occurring peptides with improved ability to bind to sodium channels and inhibit activation of the sodium channels. The invention further relates to methods for regulating sodium absorption and fluid volume and treating disorders responsive to modulating sodium absorption by activity of sodium channels.

BACKGROUND OF THE INVENTION

[0004] 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⁺- modulated airway surface liquid absorption with Na⁺ passing through the epithelial Na⁺ channel (ENaC) (Hobbs et al, J. Physiol. 591: 4377 (2013), Knowles et al, J. Clin. Invest. 109:571 (2002)). Critically, the mechanism by which ENaC activity is regulated in the airways is poorly understood. 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. Neurobiol. 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., Curr. 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. 111 :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 Us sing 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. Mol. 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 , 2010 Pfleugers Arch, 460: 1-17).

[0005] Recently, it has been shown that the Short Palate Lung and Nasal epithelial Clone (SPLUNC1) 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, C. D., and Craven, C. J. (2002) PLUNC: a novel family of candidate host defense proteins expressed in the upper airways and nasopharynx Hum Mol Genet 11, 937 ; Campos, M. A., et al. (2004) Purification and characterization of PLUNC from human tracheobronchial secretions Am J Respir Cell Mol Biol 30, 184; Lindahl, M., Stahlbom, B., and Tagesson, C. (2001); Identification of a new potential airway irritation marker, palate lung nasal epithelial clone protein, in human nasal lavage fluid with two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-time of flight Electrophoresis 22, 1795). SPLUNC1 appears to be a volume sensing molecule since it can be secreted onto the mucosal surface of the superficial epithelia where ENaC is expressed (Bartlett et al, J. Leukoc. Biol. 83: 1201 (2008); Bingle et al, J. Pathol. 205:491 (2005)). Furthermore, SPLUNC1 has been demonstrated to contain a subdomain that functions as an inhibitor of ENaC through its N-terminal domain. [0006] The present invention discloses novel specialized non-naturally occurring peptides that mimic the properties of SPLUNC1 in regulation of sodium channels by binding to and inhibiting ion transport to regulate sodium absorption and fluid volume and treat disorders responsive to modulating sodium absorption.

SUMMARY OF THE INVENTION

[0007] The present disclosure is based, in part, on the surprising finding that hypertonic formulations containing ENaC inhibitory peptides are more effective in vitro and in vivo compared to corresponding isotonic formulations. For instance, as described and exemplified herein, formulations comprising ENaC inhibitory peptide SPX-101 in hypertonic saline (e.g., 2.7% , 4.2% or 7% saline) significantly improved airway surface liquid (ASL) height in CFTR-mutant human bronchial epithelial cells in comparison to formulations in isotonic saline. In particular, a formulation of SPX- 101 in 3% saline significantly increased ASL height by over 250% compared to the effect obtained using SPX-101 in isotonic saline. The results demonstrate that hypertonic saline and SPX-101 are synergistic in increasing ASL height, and that the formulation produce an effect that was greater than additive compared to the effect conferred by hypertonic saline alone or SPX-101 alone.

[0008] The surprising in vitro effects of formulations containing ENaC inhibitory peptide and hypertonic saline were corroborated in vivo using a sheep model of cystic fibrosis. In particular, sheep were administered a CFTR inhibitor (CFTRinhl72) by nebulization and this treatment resulted in about 50% attenuation of tracheal mucus velocity (TMV) a about four hours post-administration. The attenuation was sustained for more welve hours in vivo. However, administering the ENaC inhibitory peptide SPX-101 (formulated in normal or hypertonic saline) four hours after exposure to CFTRinhl72 restored TMV. All hypertonic saline formulations tested improved TMV in comparison to isotonic formulations, and a dose dependent effect on the restoration of TMV was observed with increasing tonicity of the vehicle. A formulation of SPX-101 in 7.0% saline resulted in complete restoration of TMV in the model, which lasted for the full 12-hour duration of the study. In contrast, isotonic saline (0.9%; control) only restored TMV to 80% of the initial value.. These results further demonstrate synergism of hypertonic saline and SPX-101 in vivo. [0009] The present invention is based, in part, on the design of specialized non-naturally occurring peptides to regulate the activity of sodium channels.

Accordingly, in one aspect the invention relates to a method of inhibiting the activation of a sodium channel, comprising contacting a sodium channel with a specialized non-naturally occurring peptide or a functional fragment thereof. In one embodiment, the sodium channel is an epithelial sodium channel (ENaC). In one embodiment, the specialized non-naturally occurring peptide or a functional fragment thereof binds to the sodium channel.

[00010] Another aspect of the invention relates to a method of inhibiting sodium absorption through a sodium channel, comprising contacting the sodium channel with a specialized non-naturally occurring peptide or a functional fragment thereof. In one embodiment, the specialized non-naturally occurring peptide or a functional fragment thereof binds to the sodium channel.

[0010] A further aspect of the invention relates to 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 specialized non-naturally occurring peptide or a functional fragment thereof. In one embodiment, the specialized non-naturally occurring peptide or a functional fragment thereof binds to the sodium channel.

[0011] Another aspect of the invention relates to a method of reducing the level of a sodium channel present on the surface of a cell, comprising contacting the sodium channel with a specialized non-naturally occurring peptide or a functional fragment thereof. In one embodiment, the specialized non-naturally occurring peptide or a functional fragment thereof binds to the sodium channel.

[0012] A further aspect of the invention relates to a method of treating a disorder responsive to inhibition of sodium absorption across an epithelial mucosal surface in a subject in need thereof, comprising delivering to the subject a therapeutically effective amount of a specialized non-naturally occurring peptide or a functional fragment thereof. In one embodiment, the specialized non-naturally occurring peptide or a functional fragment thereof binds to the sodium channel.

[0013] Another aspect of the invention relates to a method of regulating salt balance, blood volume, and/or blood pressure in a subject in need thereof, comprising delivering to the subject a therapeutically effective amount of a specialized non-naturally occurring peptide or a functional fragment thereof. In one embodiment, the specialized non-naturally occurring peptide or a functional fragment thereof binds to the sodium channel.

[0014] Another aspect of the invention relates to use of a specialized non- naturally occurring peptide or a functional fragment thereof that mimics the sodium channel binding domain of a PLUNC protein and binds to a sodium channel, wherein cleavage of the sodium channel by a protease is inhibited when bound to the peptide, for regulating salt balance, blood volume, and/or blood pressure in a subject in need thereof.

[0015] Another aspect of the disclosure relates to a kit comprising the peptide of the invention.

[0016] Another aspect of the invention relates to the use of a specialized non- naturally occurring peptide or a functional fragment thereof for the preparation of a medicament to treat a disorder responsive to inhibition of sodium absorption in a subject in need thereof.

[0017] These and other aspects of the invention are set forth in more detail in the description of the invention below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 shows the sequence of S18 (SEQ ID NO:l). The residues of subsequence A are essential for ENaC interaction, while the residues of subsequence B were found not to contribute.

[0019] FIGs. 2A-2B show the 1^st half (LPVPLDQT (SEQ ID NO: 113) but not the remainder (DQTLPLNVNP (SEQ ID NO: 114) of S18 is required for inhibition of ENaC and preservation of ASL height.

[0020] FIG. 3A shows that S18 (SEQ ID NO:l) and

aaLPVPLDQTLPLNVNPaa (SEQ ID NO:2) have equal potency and efficacy, despite SEQ ID NO:2 being flanked by D-alanines. Fig 3B shows that SI 8, and

aaLPVPLDQTaa (SEQ ID NO:3) have equal potency and efficacy, despite (SEQ ID NO:3) being flanked by D-alanines. Fig. 3A also shows the relative potency of a sample of peptides, including aaLPNlePLDQTaa (SEQ ID NO:5), which displays increased potency, relative to S18 or SEQ ID NO:4.

[0021] FIG. 4 shows an additional comparison of S18 and SEQ ID NO:5, and increased potency of SEQ ID NO: 5.

[0022] FIGs. 5A-5U show the results of experiments analyzing the effects of various peptides on internalization of alpha-ENaC in HEK293T cells and effects on cell viability when GFP-tagged alpha-ENaC is co-expressed with beta and gamma ENaC. EC50 values are shown at the bottom of several of the figures. In these figures, ADG is a negative control peptide with the sequence: NH3-ADGGLLLLNNPPPPQTVV-NH2 (SEQ ID NO:143). The peptides used in these experiments were S18 (SEQ ID NO:l), SEQ ID NO:2, SEQ ID NO:141, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:128, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID

NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 131, SEQ ID NO: 139; SEQ ID NO: 140, SEQ ID NO: 144 and SEQ ID NO: 127. Also used in these experiments were peptides of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:136, each with an aa cap at both the N- and the C-terminus. Also shown is amiloride, which inhibits ENaC by another mechanism and does not reduce the amount of functional receptor on the surface of cells.

[0023] FIG. 6 shows the results of an experiment analyzing the effects of various peptides on internalization of alpha-ENaC in HEK293T cells when GFP-tagged alpha-ENaC is co-expressed only with gamma ENaC and no beta-ENaC. In this figure, SEQ ID NO: 143 (negative control peptide) and water (vehicle) controls are used along with S18 (SEQ ID NO:l) and SEQ ID NO:128. While SEQ ID NO:l AND SEQ ID NO: 128 are effective in reducing alpha-ENaC when beta- ENaC is co-expressed, no effect is observed in this experiment when beta-ENaC is not present.

[0024] FIG. 7A shows the results of an experiment analyzing the effect on percent survival of ENaC-Tg C57BL:FVB mice after treatment with the S18 peptide (SEQ ID NO:l). S18 (100 mM solution) was administered 3 times per day at 1 yUg of body weight.

[0025] FIG. 7B shows the results of an experiment analyzing the effect on percent survival of ENaC-Tg C57BL:FVB mice after treatment with inhaled SEQ ID NO: 142 or SEQ ID NO: 128.

[0026] FIGs. 7C and 7D show the results of an experiment analyzing the effect on percent survival of ENaC-Tg C57BL:C3H mice after treatment with SEQ ID NO: 129 or SEQ ID NO: 128.

[0027] FIG. 7E shows the results of an experiment analyzing the effect on percent survival (left panel) and weight gain (right panel) of ENaC-Tg C57BL:C3H mice after treatment with a once daily dose of SEQ ID NO: 128. [0028] FIG. 7F shows the results of an experiment analyzing the effect on percent survival of ENaC-Tg C57BL:C3H mice after treatment with SEQ ID NO: 127, SEQ ID NO: 134, or SEQ ID NO: 136.

[0029] FIGs. 8A-8C are graphs showing the results of an experiment analyzing the effect of SEQ ID NO:128 (aaLPIPLDQTaa) on CFTRinhibitor-172 (CFTRinh-172) induced slowing of tracheal mucus velocity (TMV) in sheep. SEQ ID NO: 128 was found to reverse CFTRinh induced slowing of TMV. The reversal was dose dependent. Fig. 8B also shows treatment with amiloride, which inhibits ENaC by another mechanism and does not reduce the amount of functional receptor on the surface of cells.

[0030] FIG. 9 shows that SPX-101 binds selectively to ENaC. (A) The chemical structure of SPX-101. (B) Representative western blot images of biotin- tagged SPX-101 pulldowns from ENaC transfected HEK293T cells. (C) Representative binding of TAMRA-tagged SPX-101 to untransfected HEK293T cells or ones transiently expressing ASIC1, ASIC2, or ENaC. All data are representative of at least three independent experiments. Significance was determined by one-way ANOVA. (p-value = **<0.005).

[0031] FIG. 10 shows that SPX-101 induces internalization of ENaC. (A) HBECs from healthy donors were treated with 1 and 10 μΜ SPX-101, 100 μΜ amiloride, or 10 μΜ control peptide. Surface proteins were labeled with Sulfo-NHS- biotin and enriched by streptavidin pulldown. Enriched lysates and input control were western blotted for α, β, and γ ENaC and total protein in the blots was assessed by Ponceau S staining. HBECs from healthy (B) or CF (C) donors were treated with 10 μΜ SPX-101 for 0-8 hours and proteins were detected as above. Blots are representative and graphs are inclusive of data collected from at least three independent donors.

Significance was determined by Mann- Whitney Test (A) p-value = (*<0.05, **<0.005, ***< 0.0005) (B, C) Significance was determined by two-way ANOVA. p-value = (*<0.05, **<0.005, ***< 0.0005).

[0032] FIG. 11 shows that SPX-101 decreases ENaC current. Amiloride- sensitive current was determined in healthy (A) and CF (B) HBECs two hours after administration of SPX-101. Data are inclusive of five independent experiments for both healthy and CF HBECs. Significance was determined by Wilcoxon Matched Pairs Test, (p-value - **<0.005, ***< 0.0005). [0033] FIG. 12 shows that SPX-101 increases survival of ENaC-Tg mice. (A) Kaplan-Meier survival curve of PENaC-Tg mice treated once-daily with 0.18, 0.9, or 1.8 mg/kg SPX-101, 1.8 mg/kg control peptide, or saline starting two days after birth. (B) Weight gain in mice described in (A). (C) Leukocyte composition in the BALF of mice treated in (A).

[0034] FIG. 13 shows that SPX-101 increases mucus transport in PENaC-Tg mice. Mucus transport was assessed as velocity (A), directionality (B), and the product of those two measurements deemed mucus transport index (MTI) (C). PENaC-Tg mice were treated with a single intranasal dose of 1.8 mg/kg control peptide or 1.8 mg/kg SPX-101 and tracheas were harvested four hours later and bead movement was monitored as described in methods. Wild-type littermate mice were treated with 50 mM saline and served as a healthy control. Data are inclusive of triplicate recording from at least six mice. Significance was determined by Wilcoxon Matched Pairs Test as compared to control peptide, (p-value = **<0.001).

[0035] FIG. 14 shows that SPX-101 restores tracheal mucus velocity in an ovine model of CF. Baseline tracheal mucus velocity (TMV) was obtained (t=0) and immediately thereafter CFTRinh-172 was nebulized to inhibit CFTR. Four hours later SPX-101 (1-4 mg/kg), control peptide (4 mg/kg), or 0.9% isotonic saline was administered via nebulization and TMV measured hourly for eight hours (A).

Additionally, sheep were nebulized with amiloride (0.06 mg/kg) formulated in isotonic (0.9%) or hypertonic saline (4.2%) (B), 1 mg/kg SPX-101 in hypertonic saline (4.2%) (C), and 2 mg/kg SPX-101 in hypertonic saline (D). Data for 0.9% saline is the same in A-D. Data for 4.2% saline is the same in B-D. The data for 1 mg/kg SPX-101 in 0.9% saline is the same in A and C and the data for 2 mg/kg SPX-101 in 0.9% saline is the same in A and D. All dosing was conducted in at least three independent sheep.

Significance was determined by two-way ANOVA analysis at hour 12 time point as compared to vehicle control, p-value = **<0.0001).

[0036] FIG. 15 shows a dose-dependent effect of saline in presence or absence of SPX-101 on ASL height maintained in vitro by CFTR-mutant human bronchial epithelial cells. The error bars indicate SEM and N=8. The data show that treating cells with a formulation of SPX-101 in hypertonic saline solution significantly increased ASL height compared to a control formulation of SPX-lOlin isotonic saline. More importantly, the effect of the formulation of SPX-101 in hypertonic saline solution on ASL height was greater than additive (i.e. , synergistic) compared to the effect achieved via treatment with hypertonic saline alone or SPX-101 alone.

[0037] FIG. 16 shows the in vivo effect of formulations containing SPX-101 (1 mg/kg) in hypertonic saline (2.7%, 4.2% or 7.0% saline) or normal saline over a 12-hour period in restoring tracheal mucus velocity (TMV) in sheep that were treated with a CFTR inhibitor (CFTRinh-172).

DETAILED DESCRIPTION OF THE INVENTION

[0038] The present invention will now be described in more detail with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. This invention 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 invention to those skilled in the art.

[0039] 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 invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. 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.

[0040] Amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either the one-letter code, or the three letter code, both in accordance with 37 C.F.R. §1.822 and established usage.

[0041] As used in the description of the invention 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.

[0042] 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").

[0043] The term "consists essentially of (and grammatical variants), as applied to a peptide sequence of this invention, means a peptide that consists of both the recited sequence (e.g. , SEQ ID NO) and a total of ten or less (e.g. , 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional amino acids on the N-terminal and/or C-terminal ends of the recited sequence such that the function of the peptide is not materially altered. The total of ten or less additional amino acids includes the total number of additional amino acids on both ends added together. The term "materially altered," as applied to peptides of the invention, refers to an increase or decrease in binding activity (e.g. , to a sodium channel or specialized non-naturally occurring peptide) of at least about 50% or more as compared to the activity of a peptide consisting of the recited sequence.

[0044] The term "modulate," "modulates," or "modulation" refers to enhancement (e.g. , an increase) or inhibition (e.g. , a decrease) in the specified level or activity.

[0045] The term "enhance" or "increase" refers to an increase in the specified parameter of at least about 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8- fold, 10-fold, twelve-fold, or even fifteen-fold.

[0046] The term "inhibit" or "reduce" or grammatical variations thereof as used herein refers to a decrease or diminishment in the specified level or activity of at least about 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more. In particular embodiments, the inhibition or reduction results in little or essentially no detectible activity (at most, an insignificant amount, e.g. , less than about 10% or even 5%).

[0047] The term "contact" or grammatical variations thereof as used with respect to a specialized non-naturally occurring peptide and a sodium channel, refers to bringing the specialized non-naturally occurring peptide and the sodium channel in sufficiently close proximity to each other for one to exert a biological effect on the other. In some embodiments, the term contact means binding of the specialized non-naturally occurring peptide to the sodium channel.

[0048] 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.

[0049] 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. [0050] The term "fragment," as applied to a peptide, will be understood to mean an amino acid sequence of reduced length relative to a reference peptide or amino acid sequence and comprising, consisting essentially of, and/or consisting of an amino acid sequence of contiguous amino acids identical to the reference peptide or amino acid sequence. Such a peptide fragment according to the invention may be, where appropriate, included in a larger polypeptide of which it is a constituent. In some embodiments, such fragments can comprise, consist essentially of, and/or consist of peptides having a length of at least about 4, 5, 6, 7, 8, 9, 10, or more consecutive amino acids of a peptide or amino acid sequence according to the invention. In other embodiments, such fragments can comprise, consist essentially of, and/or consist of peptides having a length of less than about 10, 9, 8, 7, 6, 5, 4, or less consecutive amino acids of a peptide or amino acid sequence according to the invention.

[0051] As used herein, the terms "protein" and "polypeptide" are used interchangeably and encompass both peptides and proteins, unless indicated otherwise.

[0052] A "fusion protein" is a polypeptide produced when two heterologous nucleotide sequences or fragments thereof coding for two (or more) different polypeptides not found fused together in nature are fused together in the correct translational reading frame. Illustrative fusion polypeptides include fusions of a peptide of the invention (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, etc.

[0053] As used herein, a "functional" peptide or "functional fragment" is one that 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" peptide or "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, of the biological activity of the native polypeptide (and can even have a higher level of activity 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.

[0054] The term "about," as used herein when referring to a measurable value such as an amount of polypeptide, dose, time, temperature, enzymatic activity or other biological activity and the like, is meant to encompass variations of ± 20%, ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount.

[0055] A first aspect of the invention relates to the ability of specialized non- naturally occurring peptides to bind to a sodium channel and prevent activation of the sodium channel, thereby inhibiting the flow of sodium ions. Thus, one aspect of the present invention relates to a method of inhibiting the activation of a sodium channel, comprising contacting (e.g., binding) a sodium channel with a specialized non-naturally occurring peptide or a functional fragment thereof. In one embodiment, the sodium channel is an epithelial sodium channel (ENaC), e.g., human ENaC, or a non-human mammalian 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). The inhibition of sodium channel activation can be measured by any method known in the art or disclosed herein, including, without limitation, measuring sodium flow or change in potential across a membrane, across a cell, or across a natural or artificial lining. The inhibition can be at least about 20%, e.g. , at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0056] The method of inhibiting the activation of a sodium channel can be carried out, e.g., on an isolated sodium channel, a sodium channel in an artificial membrane, or a sodium channel in a cell. In one embodiment, 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.

[0057] In one embodiment, the step of contacting (e.g. , binding) the sodium channel with a specialized non-naturally occurring peptide comprises delivering the specialized non-naturally occurring peptide or a functional fragment or homolog thereof to a cell comprising the sodium channel.

[0058] As used herein, the term "homolog" is used to refer to a polypeptide which differs from the disclosed specialized non-naturally occurring peptide by modifications to the specialized non-naturally occurring peptide, but which significantly retains a biological activity of the disclosed non-naturally occurring peptide. Minor modifications include, without limitation, changes in one or a few amino acid side chains, changes to one or a few amino acids (including deletions, insertions, and substitutions), changes in stereochemistry of one or a few atoms, and minor

derivatizations, including, without limitation, methylation, glycosylation,

phosphorylation, acetylation, myristoylation, prenylation, palmitoylation, amidation, and addition of glycosylphosphatidyl inositol. The term "substantially retains," as used herein, refers to a fragment, homolog, or other variant of a peptide that retains at least about 20% of the activity of the naturally occurring peptide (e.g. , binding to a sodium channel), e.g. , about 30%, 40%, 50% or more. Other biological activities, depending on the peptide, may include enzyme activity, receptor binding, ligand binding, induction of a growth factor, a cell signal transduction event, etc.

[0059] In one embodiment, the method comprises delivering to a cell comprising a sodium channel an isolated specialized non-naturally occurring peptide. In exemplary embodiments, the specialized non-naturally occurring peptide comprises, consists essentially of, or consists of the disclosed specialized non-naturally occurring peptide or a functional fragment thereof. In another embodiment, the isolated specialized non-naturally occurring peptide comprises, consists essentially of, or consists of an amino acid sequence that is at least 70% identical, e.g. , at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the publicly known amino acid sequence or a functional fragment thereof. In some embodiments, the peptides comprise a portion of the natural amino acid sequence of a PLUNC protein with one or more conservative substitutions with natural or non-natural amino acids and/or one or more additions of non-natural amino acids. Conservative substitutions are described below. In some embodiments, the peptides comprise one or more terminal modifications as described below.

[0060] Non- limiting examples of peptides of the invention are disclosed in Table 1 below. In some embodiments, the peptides of the invention may comprise one or more additional residues at the amino- and/or carboxyl-terminal ends. In some embodiments, the one or more additional residues are D-alanines. For example, a peptide may comprise one or two D-alanines at the amino- and/or carboxyl-terminal ends.

[0061] The disclosure further relates to formulations comprising specialized non-naturally occurring peptides of the disclosure (e.g., SEQ ID NO: 127, SEQ ID NO: 128) and saline. Preferably, the formulation comprises an ENaC inhibitory peptide and hypertonic saline. Hypertonic saline refers to any saline solution with a concentration of sodium chloride (NaCl) higher than physiological concentration (about 0.9 (w/v)). Accordingly, the formulations of the disclosure may include, e.g., about 1%- 10% saline; about 2%-8% saline; about 3%-7% saline, including all sub-ranges in between, e.g. , between l%-8%; l%-7%; l%-6%; l%-5%; l%-4%; l%-3%; l%-2%; 2%-9%; 2%-8%; 2%-7%; 2%-6%; 2%-5%; 2%-4%; 2%-3%; 3%-9%; 3%-8%; 3%-7%; 3%-6%; 3%-5%; 3%-4%; 4%-9%; 4%-8%; 4%-7%; 4%-6%; 4%-5%; 5%-9%; 5%-8%; 5%-7%; 5%-6%; 6%-9%; 6%-8%; 6%-7%; 7%-9%; 7%-8%; and 8%-9% saline. In some embodiments, the formulations of the disclosure may include, e.g., about 1.2%, 1.4%, 1.6%, 1.8%, 2.0%, 2.2%, 2.4%, 2.6%, 2.8%, 3.0%, 3.2%, 3.4%, 3.6%, 3.8%, 4.0%, 3.0%, 4.2%, 4.4%, 4.6%, 4.8%, 5.0%, 5.2%, 5.4%, 5.6%, 5.8%, 6.0%, 6.2%, 6.4%, 6.6%, 6.8%, 7.0%, 7.2%, 7.4%, 7.6%, 7.8%, 8.0%, 8.5%, 9%, or a greater % of NaCl, e.g., about 15% NaCl. If desired, the formulations of the disclosure may comprise two or more ingredients such that the total tonicity of the solution exceeds the physiological concentration of saline (about 0.9% NaCl). For instance, the ingredients may include, e.g., sodium chloride and an osmolality adjusting agent selected from the group consisting of mannitol, xylitol, sorbitol, isomaltol, glucose, lactose, dextrose, sucrose, trehalose, maltose, glycerin, propylene glycol, ethylene glycol, glycerol, glycine, dimethylsulphoxide, calcium chloride, sodium sulfate, magnesium chloride, sodium gluconate, sodium pyruvate, pentosane polysulfate, and a cyclodextrin. Other representative examples of such formulations include, e.g. , a solution comprising 0.9% sodium chloride and 1% sodium phosphate (wherein the total Na content exceeds that of isotonic saline) or a solution of 0.9% sodium chloride and 1% potassium chloride (wherein the total CI content exceeds that of isotonic saline). In contrast, the term "normal saline" means a solution containing 0.9% (w/v) NaCl (also referred herein as isotonic saline).

wherein a = D-alanine, Nle = 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 mV = N-methylvaline

[0062] The specialized non-naturally occurring peptides of the invention also include functional portions or fragments. The length of the fragment is not critical as long as it substantially retains the biological activity of the peptide (e.g. , sodium channel binding activity). Illustrative fragments comprise at least about 4, 5, 6, 7, 8, 9, 10, or more contiguous amino acids of a specialized non-naturally occurring peptide. In other embodiments, the fragment comprises no more than about 10, 9, 8, 7, 6, 5, or 4 contiguous amino acids of a specialized non-naturally occurring peptide.

[0063] Likewise, those skilled in the art will appreciate that the present invention also encompasses fusion polypeptides comprising a specialized non-naturally occurring peptides peptide or a functional fragment thereof. As another alternative, the fusion protein can comprise a reporter molecule. In other embodiments, the fusion protein can comprise a polypeptide that provides a function or activity that is the same as or different from the activity of the peptide, e.g. , a targeting, binding, or enzymatic activity or function.

[0064] Likewise, it will be understood that the peptides specifically disclosed herein will typically tolerate substitutions in the amino acid sequence and substantially retain biological activity. To identify peptides of the invention 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, hydrophilicity, charge, size, and the like.

[0065] In identifying amino acid sequences encoding peptides other than those specifically disclosed herein, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (see, Kyte and Doolittle, /. Mol. Biol. 157: 105 (1982); incorporated herein by reference in its entirety). 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.

[0066] Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, id.), these are: 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); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (- 3.5); lysine (-3.9); and arginine (-4.5). [0067] Accordingly, the hydropathic index of the amino acid (or amino acid sequence) may be considered when modifying the peptides specifically disclosed herein.

[0068] It is also understood in the art that the substitution of amino acids can be made on the basis of hydrophilicity. U.S. Patent No. 4,554,101 (incorporated herein by reference in its entirety) states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.

[0069] As detailed in U.S. Patent No. 4,554,101, the following hydrophilicity values have been assigned to 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).

[0070] Thus, the hydrophilicity of the amino acid (or amino acid sequence) may be considered when identifying additional peptides beyond those specifically disclosed herein.

[0071] Peptides (and fragments thereof) of the invention include peptides that have at least about 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher amino acid sequence identity with the peptide sequences disclosed herein.

[0072] 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 & Waterman, Adv. Appl. Math. 2:482 (1981), by the sequence identity alignment algorithm of Needleman & Wunsch, /. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI), the Best Fit sequence program described by Devereux et al. , Nucl. Acid Res. 72:387 (1984), preferably using the default settings, or by inspection.

[0062] An example of a useful algorithm is PILEUP. PILEUP 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 & Doolittle, /. Mol. Evol. 35:351 (1987); the method is similar to that described by Higgins & Sharp, CABIOS 5:151 (1989). [0063] 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);

blast. wustl/edu/blast/README.html. 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.

[0064] An additional useful algorithm is gapped BLAST as reported by Altschul et al, Nucleic Acids Res. 25:3389 (1997).

[0065] 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-Blast-2 to maximize the alignment score are ignored).

[0066] 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 one embodiment, 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 in the shorter sequence, in one embodiment. In percent identity calculations relative weight is not assigned to various manifestations of sequence variation, such as insertions, deletions, substitutions, etc.

[0067] In one embodiment, 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.

[0068] Peptides and fragments of the invention can be modified for in vivo use by the addition, at the amino- and/or carboxyl-terminal ends, of a blocking agent to facilitate survival of the relevant polypeptide in vivo. This can be useful in those situations in which the peptide termini tend to be degraded by proteases prior to cellular uptake. Such blocking agents can include, without limitation, additional related or unrelated peptide sequences that can be attached to the amino and/or carboxyl terminal residues of the peptide to be administered. This can be done either chemically during the synthesis of the peptide or by recombinant DNA technology by any suitable methods. For example, one or more non-naturally occurring amino acids, such as D-alanine, can be added to the termini. Alternatively, blocking agents such as pyroglutamic acid or other molecules known in the art can be attached to the amino and/or carboxyl terminal residues, or the amino group at the amino terminus 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 noncovalently coupled to pharmaceutically acceptable "carrier" proteins prior to administration.

[0069] In one embodiment, the peptides or fragments thereof of the invention are administered directly to a subject. Generally, the compounds of the invention will be suspended in a pharmaceutically-acceptable carrier (e.g. , physiological saline) and administered orally or by intravenous infusion, or administered subcutaneously, intramuscularly, intrathecally, intraperitoneally, intrarectally, intravaginally, intranasally, intragastrically, intratracheally, or intrapulmonarily. In another embodiment, the intratracheal or intrapulmonary delivery can be accomplished using a standard nebulizer, jet nebulizer, wire mesh nebulizer, dry powder inhaler, or metered dose inhaler. They can be delivered directly to the site of the disease or disorder, such as lungs, kidney, or intestines. The dosage required depends on the choice of the route of administration; the nature of the formulation; the nature of the patient's illness; the subject's size, weight, surface area, age, and sex; other drugs being administered; and the judgment of the attending physician. Suitable dosages are in the range of 0.01 μg/kg-2.0 g/kg. Wide variations in the needed dosage are to be expected in view of the variety of peptides, fragments, and homologs available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by i.v. injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization as is well understood in the art. Administrations can be single or multiple (e.g. , 2-, 3-, 4-, 6-, 8-, 10-; 20-, 50-, 100-, 150-, or more fold). Encapsulation of the peptides, fragments, and homologs in a suitable delivery vehicle (e.g. , polymeric microparticles or implantable devices) may increase the efficiency of delivery, particularly for oral delivery. The methods of the present invention can be practiced with any of the peptides disclosed herein or their functional fragments. In some embodiments, the methods of the invention are practiced with S18 (SEQ ID NO: 1) or a functional fragment thereof. In other embodiments, the methods of the invention are practiced with S8 (SEQ ID NO: 120) or a functional fragment thereof. In certain cases, the methods of the invention are practiced with any of the peptides disclosed in U.S. 2012/0115795, U.S. 2016/0102121 or U.S. 9,127,040 (each of which is incorporated herein by reference in its entirety).

[0070] According to certain embodiments, the peptides, or fragments thereof can be targeted to specific cells or tissues in vivo. Targeting delivery vehicles, including liposomes and targeted systems are known in the art. For example, a liposome can be directed to a particular target cell or tissue by using a targeting agent, such as an antibody, soluble receptor or ligand, incorporated with the liposome, to target a particular cell or tissue to which the targeting molecule can bind. Targeting liposomes are described, for example, in Ho et al, Biochemistry 25:5500 (1986); Ho et al, J. Biol. Chem. 262:13979 (1987); Ho et al, J. Biol. Chem. 262:13973 (1987); and U.S. Pat. No. 4,957,735 to Huang et al, each of which is incorporated herein by reference in its entirety).

[0071] Another aspect of the invention relates to a method of inhibiting sodium absorption through a sodium channel, comprising contacting (e.g., binding) the sodium channel with a specialized non-naturally occurring peptide or fragment thereof. Inhibition of sodium absorption can be measured by any technique known in the art or disclosed herein.

[0072] Another aspect of the invention relates to a method 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 a specialized non-naturally occurring peptide 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.

[0073] A further aspect of the invention 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 a specialized non-naturally occurring peptide or a functional fragment or homolog thereof.

[0074] An additional aspect of the invention relates to a method of treating a disorder responsive to inhibition of sodium absorption across an epithelial mucosal surface in a subject in need thereof, comprising delivering to the subject a therapeutically effective amount of a specialized non-naturally occurring peptide or a functional fragment or homolog thereof. In one embodiment, the invention encompasses a method for treating a symptom of a disorder responsive to inhibition of sodium absorption across an epithelial mucosal surface in a subject in need thereof, comprising administering a peptide comprising a sequence selected from SEQ ID NOS:2-128 to the subject. The disorder in the methods of the invention can be, in non-limiting examples, a lung disorder (e.g. , cystic fibrosis, non-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.

[0075] Another aspect of the invention relates to a method of regulating salt balance, blood volume, and/or blood pressure in a subject in need thereof, comprising delivering to the subject a therapeutically effective amount of a specialized non-naturally occurring peptide or a functional fragment or homolog thereof.

[0076] A third aspect of the invention relates to products that can be used to carry out the methods disclosed herein. Thus, one aspect of the invention relates to a peptide comprising the sequence:

X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO: 116) wherein:

Xi is leucine or a conservative substitution with a natural or non-natural amino acid;

X2 is proline or a conservative substitution with a natural or non-natural amino acid;

X₃ is valine or a conservative substitution with a natural or non-natural amino acid;

X₄ is proline or a conservative substitution with a natural or non-natural amino acid;

X5 is leucine or a conservative substitution with a natural or non-natural amino acid;

X₆ is aspartic acid or a conservative substitution with a natural or non-natural amino acid; X₇ is glutamine or a conservative substitution with a natural or non-natural amino acid; and Xg is threonine or a conservative substitution with a natural or non-natural amino acid; or a functional fragment thereof.

[0077] Another aspect of the invention relates to a peptide comprising the sequence:

X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO: 117) wherein:

Xi is leucine, norleucine, or valine;

X2 is proline, 4-hydroxyproline, (2R,5S)-5-phenyl-pyrrolidine-2-carboxylic acid, or 3,4- dehydro-L-proline;

X₃ is valine, leucine, norleucine, or N-methylvaline;

X₄ is proline, 4-hydroxyproline, (2R,5S)-5-phenyl-pyrrolidine-2-carboxylic acid, or 3,4- dehydro-L-proline;

X5 is leucine, norleucine, or valine;

X₆ is aspartic acid or glutamic acid;

X₇ is glutamine or asparagine; and Xg is threonine, serine, or L-a-methylserine;or a functional fragment thereof.

[0078] A further aspect of the invention relates to a peptide comprising the sequence: Xi-X₂-X₃-X4-X5-X6-X7-X8-X9-Xio-Xii-Xi2-Xi3-Xi4-Xi5(SEQ ID NO: 118) wherein:Xi is leucine or a conservative substitution with a natural or non-natural amino acid; X₂ is proline or a conservative substitution with a natural or non-natural amino acid;

X3 is valine or a conservative substitution with a natural or non-natural amino acid;

X₅ is leucine or a conservative substitution with a natural or non-natural amino acid;

X₆ is aspartic acid or a conservative substitution with a natural or non-natural amino acid; X₇ is glutamine or a conservative substitution with a natural or non-natural amino acid; Xg is threonine or a conservative substitution with a natural or non-natural amino acid; X9 is threonine or a conservative substitution with a natural or non-natural amino acid; X10 is leucine or a conservative substitution with a natural or non-natural amino acid; X11 is proline or a conservative substitution with a natural or non-natural amino acid; X12 is asparagine or a conservative substitution with a natural or non-natural amino acid; Xi3 is valine or a conservative substitution with a natural or non-natural amino acid;

Xi₄ is asparagine or a conservative substitution with a natural or non-natural amino acid; Xi5 is proline or a conservative substitution with a natural or non-natural amino acid; or a functional fragment thereof.

[0079] In some embodiments, the peptide comprises a sequence selected from the group consisting of SEQ ID NOS:4-142 and SEQ ID NO: 144. In one embodiment, the peptide comprises the sequence of SEQ ID NO:5 or SEQ ID NO: 122. In one embodiment, the peptide comprises the sequence of SEQ ID NO:2 (aaLPVPLDQTLPLNVNPaa) or SEQ ID NO: 119. In one embodiment, the peptide comprises the sequence of SEQ ID NO: 127 or SEQ ID NO: 128 (aaLPIPLDQTaa).

[0080] In certain embodiments, the peptides of the invention comprise at least one modified terminus, e.g. , to protect the peptide against degradation. In some embodiments, the N-terminus is acetylated and/or the C-terminus is amidated. In some embodiments, the peptide comprises the sequence of any one of SEQ ID NOS:10-127, 129, 130, 133, 134, or 136-140, further comprising one or two D-alanines at the amino- and/or carboxyl-terminal ends.

[0081] In certain embodiments, the peptides of the invention comprise at least one non- natural amino acid (e.g. , 1, 2, 3, or more) or at least one terminal modification (e.g. , 1 or 2). In some embodiments, the peptide comprises at least one non-natural amino acid and at least one terminal modification. [0082] In certain embodiments, the peptide mimics the sodium channel binding domain of a 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% of the binding activity. In some embodiments, the peptide has at least the same binding activity as the full length PLUNC protein. In one embodiment, 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).

[0083] The peptides of the present invention can optionally be delivered in conjunction with other therapeutic agents. The additional therapeutic agents can be delivered concurrently with the peptides of the invention. 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 one embodiment of the invention, the specialized non-naturally occurring peptide 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 specialized non-naturally occurring peptide and the CFTR-targeted agent have superior activity to the CFTR-targeted agent alone. In another embodiment of the invention, the specialized non- naturally occurring peptide is delivered to a patient concurrently with a mucolytic compound where the combined activity of the specialized non-naturally occurring peptide and the mucolytic agent have superior activity to the mucolytic agent alone. In yet another embodiment of the invention, the specialized non-naturally occurring peptide is delivered to a patient concurrently with a long acting B-agonist compound (LABA) where the combined activity of the specialized non-naturally occurring peptide and the LABA agent have superior activity to the LABA alone. In yet another embodiment of the invention, the specialized non-naturally occurring peptide is delivered to a patient concurrently with a glucocorticoid agonist where the combined activity of the specialized non-naturally occurring peptide and the glucocorticoid agent have superior activity to the glucocorticoid alone.

[0084] Another aspect of the invention relates to a kit comprising the peptide of the invention and useful for carrying out the methods of the invention. The kit may further comprise additional reagents for carrying out the methods (e.g. , buffers, containers, additional therapeutic agents) as well as instructions. [0085] As a further aspect, the invention 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. , a specialized non- naturally occurring peptide or functional fragment thereof.

[0086] 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.

[0087] The formulations of the invention can optionally comprise medicinal agents, pharmaceutical agents, carriers, adjuvants, dispersing agents, diluents, and the like.

[0088] The peptides of the invention can be formulated for administration in a pharmaceutical carrier in accordance with known techniques. See, e.g., Remington, The Science And Practice of Pharmacy (9^th Ed. 1995). In the manufacture of a pharmaceutical formulation according to the invention, the peptide (including the 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 as a unit-dose formulation, for example, a tablet, which can contain from 0.01 or 0.5% to 95% or 99% by weight of the peptide. One or more peptides can be incorporated in the formulations of the invention, which can be prepared by any of the well-known techniques of pharmacy.

[0089] A further aspect of the invention is a method of treating subjects in vivo, comprising administering to a subject a pharmaceutical composition comprising a peptide of the invention in a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered in a therapeutically effective amount. Administration of the peptides of the present invention to a human subject or an animal in need thereof can be by any means known in the art for administering compounds.

[0090] The formulations of the invention 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 to the liver by intraportal delivery, as well as direct organ injection (e.g. , into the liver, into the brain for delivery to the central nervous system, into the pancreas, or into a tumor or the tissue surrounding a tumor). 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 which is being used.

[0091] 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[R] (BASF, Parsippany, N.J.). For other methods of administration, the carrier can be either solid or liquid.

[0092] For oral administration, the peptide 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 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.

[0093] 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.

[0094] Formulations of the present invention suitable for parenteral administration comprise sterile aqueous and non-aqueous injection solutions of the peptide, 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 formulations 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. [0095] Extemporaneous 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 invention, there is provided an injectable, stable, sterile composition comprising a peptide of the invention, in a unit dosage form in a sealed container. The peptide or salt is provided in the form of a lyophilizate which is capable 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 salt. When the peptide or 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 salt in an aqueous carrier. One such useful emulsifying agent is phosphatidyl choline.

[0096] Formulations suitable for rectal administration are preferably presented as unit dose suppositories. These can be prepared by admixing the peptide with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.

[0097] 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, lanoline, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof.

[0098] 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 (see, for example, Tyle, Pharm. Res. 3:318 (1986)) and typically take the form of an optionally buffered aqueous solution of the peptides. Suitable formulations comprise citrate or bis/tris buffer (pH 6) or ethanol/water and contain from 0.1 to 0.2M of the compound.

[0099] The peptide 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 respirable particles comprising the peptide, 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 into the bronchioles 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 & Jones, Drug Delivery to the Respiratory Tract, Ellis Horwood (1987); Gonda (1990) Critical Reviews in Therapeutic Drug Carrier Systems 6:273- 313; and Raeburn et al, J. Pharmacol. Toxicol. Meth. 27:143 (1992). Aerosols of liquid particles comprising the peptide 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. Patent No. 4,501,729. Aerosols of solid particles comprising the peptide can likewise be produced with any solid particulate medicament aerosol generator, by techniques known in the pharmaceutical art.

[0100] Alternatively, one can administer the peptide in a local rather than systemic manner, for example, in a depot or sustained-release formulation.

[0101] Further, the present invention provides liposomal formulations of the peptides disclosed herein and salts thereof. The technology for forming liposomal suspensions is well known in the art. When the peptide 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 salt, the peptide or salt will be substantially entrained within the hydrophilic 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 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.

[0102] The liposomal formulations containing the peptides disclosed herein or salts thereof, can be lyophilized to produce a lyophilizate which can be reconstituted with a pharmaceutically acceptable carrier, such as water, to regenerate a liposomal suspension.

[0103] In the case of water-insoluble peptides, a pharmaceutical composition can be prepared containing the water-insoluble peptide, 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. Particularly useful emulsifying agents include phosphatidyl cholines and lecithin.

[0104] In particular embodiments, the peptide is administered to the subject in a therapeutically effective amount, as that term is defined above. Dosages of pharmaceutically active peptides 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 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 mg/kg to about 2 g/kg will have therapeutic efficacy, with all weights being calculated based upon the weight of the peptide, including the cases where a salt is employed. In certain cases, 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, including the cases where a salt is employed. In specific embodiments, a dosage from about 0.1 mg/kg to about 2 mg/kg, from about 0.5 mg/kg to about 2 g/kg, from about 1 mg/kg to about 2 g/kg, from about 10 mg/kg to about 2 g/kg, from about 100 mg/kg to about 2 g/kg, from about 0.5 g/kg to about 2 g/kg, from about 1 g/kg to about 2 g/kg, from about 0.1 mg/kg to about 1 g/kg, from about 0.5 mg/kg to about 1 g/kg, from about 1 mg/kg to about 1 g/kg, from about 10 mg/kg to about 1 g/kg, from about 100 mg/kg to about 1 g/kg, from about 0.5 g/kg to about 1 g/kg, from 0.1 mg/kg to about 0.5 g/kg, from 0.5 mg/kg to about 0.5 g/kg, from 1 mg/kg to about 0.5 g/kg, from 10 mg/kg to about 0.5 g/kg, from about 100 mg/kg to about 0.5 g/kg, from 0.1 mg/kg to about 50 mg/kg, from 0.5 mg/kg to about 50 mg/kg, from 1 mg/kg to about 50 mg/kg, from about 10 mg/kg to about 50 mg/kg, from 0.1 mg/kg to about 5 mg/kg, from 0.5 mg/kg to about 5 mg/kg, from 1 mg/kg to about 5 mg/kg, or from about 1 mg/kg to about 4 mg/kg can be employed for inhalation, intranasal, oral, intravenous, or intramuscular administration. In one embodiment, a dosage of about 1 mg/kg can be employed for inhalation, intranasal, oral, intravenous, or intramuscular administration. In another embodiment, a dosage of about 2 mg/kg can be employed for inhalation, intranasal, oral, intravenous, or intramuscular administration. In a further embodiment, a dosage of about 4 mg/kg can be employed for inhalation, intranasal, oral, intravenous, or intramuscular administration. In certain embodiments, a dosage from about 0.5 mg/kg to 5 mg/kg can be employed for intramuscular injection. In certain aspects, dosages are about 1 μιηοΐ/kg to 50 μιηοΐ/kg, and more particularly to about 22 μιηοΐ/kg and to 33 μιηοΐ/kg of the peptide for intravenous or oral administration, respectively.

[0105] In particular embodiments of the invention, 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.) to achieve therapeutic effects.

[0106] The present invention finds use in 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. Human subjects include neonates, infants, juveniles, and adults.

[0107] The disclosure relates to the following representative aspects:

[0108] In some aspects, the disclosure provides for a pharmaceutical composition comprising a polypeptide comprising a sodium channel binding domain of a Palate Lung and Nasal epithelial Clone (PLUNC) protein, or a functional fragment, or homolog thereof, wherein the PLUNC protein, or the functional fragment, or homolog thereof binds to a sodium channel, and wherein the PLUNC protein, or the functional fragment, or homolog thereof is formulated in a saline solution containing a salt concentration of at least 1%, e.g. , at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20%. Preferably, the salt concentration in the formulation is between 2% and 10%, particularly between 3% and 8%, and especially between 4% and 7%. Particularly preferably, the salt concentration in the formulation is at least 4.2%, e.g., about 4.2% NaCl.

[0109] In some aspects, the disclosure provides for a pharmaceutical composition comprising a formulation as described above (e.g. , a PLUNC protein or a functional fragment thereof or homolog thereof and saline) and a pharmaceutically acceptable carrier and optionally a pharmaceutically acceptable osmolality adjusting agent. Preferably, the PLUNC protein, or the functional fragment, or homolog thereof is formulated in a saline solution containing a salt concentration of at least 0.9%, e.g. , about 4.2% NaCl. In certain aspects, the osmolality adjusting agent is one or more selected from the group consisting of mannitol, xylitol, sorbitol, isomaltol, glucose, lactose, dextrose, sucrose, trehalose, maltose, glycerin, propylene glycol, ethylene glycol, glycerol, glycine, dimethylsulphoxide, calcium chloride, sodium sulfate, magnesium chloride, sodium gluconate, sodium pyruvate, pentosane polysulfate, and a cyclodextrin.

[0110] In some aspects, the disclosure provides for a pharmaceutical composition comprising at least one polypeptide comprising, consisting essentially of, or consisting of, the amino acid sequence set forth in SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 141, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 128, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 131, SEQ ID NO: 139; SEQ ID NO: 140, SEQ ID NO: 144, SEQ ID NO: 127, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 136, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8, wherein the polypeptide is formulated in a saline solution containing a salt concentration of at least 1%, e.g., at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20%. Preferably, the salt concentration in the formulation is between 2% and 10%, particularly between 3% and 8%, and especially between 4% and 7%. Particularly preferably, the salt concentration in the formulation is at least 4.2%, e.g. , about 4.2% NaCl.

[0111] In some aspects, the disclosure provides for a pharmaceutical composition comprising at least one peptide comprising, consisting essentially of, or consisting of, the sequence set forth in SEQ ID NO: 127 or SEQ ID NO: 128, wherein the peptide is formulated in a saline solution containing a salt concentration of at least 1%, e.g. , at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20%. Preferably, the salt concentration in the formulation is between 2% and 10%, particularly between 3% and 8%, and especially between 4% and 7%. Particularly preferably, the salt concentration in the formulation is at least 4.2%, e.g., about 4.2% NaCl.

[0112] In some aspects, the disclosure provides for a pharmaceutical composition comprising a polypeptide comprising a modified sodium channel binding domain of a PLUNC protein, or a functional fragment, or homolog thereof which is formulated in a saline solution containing a salt concentration of at least 1%. Preferably, the PLUNC protein or the functional fragment or homolog thereof is modified to reduce or eliminate the inactivation of the polypeptide at an acidic pH, e.g. , by the addition of a blocking agent to facilitate survival of the polypeptide in vivo and/or by fusing to a protein that increases the stability of the polypeptide. In some aspects, the modified PLUNC protein or the functional fragment or homolog thereof is modified at the N-terminus via acetylation. In some aspects, the modified PLUNC protein or the functional fragment or homolog thereof comprises at least one non-natural amino acid or at least one terminal modification, e.g. , at least one D-alanine at the N-terminus and/or the C-terminus.

[0113] In some aspects, the disclosure provides for a pharmaceutical composition comprising a polypeptide comprising a modified sodium channel binding domain of a PLUNC protein, or a functional fragment, or homolog thereof which is formulated in a saline solution containing a salt concentration of at least 1%, wherein the formulation is to be administered by inhalation, intranasally, intravenously, intramuscularly, intraocularly, transdermally, or orally, preferably by inhalation, e.g. , via metered dose, inhaler, nebulizer, or in a mist sprayer.

[0114] In some aspects, the disclosure provides for a kit comprising, in one or more packages, a polypeptide comprising a modified sodium channel binding domain of the PLUNC protein, or a functional fragment, or homolog thereof and reagents for formulation of the PLUNC protein or the fragment or homolog thereof in a saline solution containing a salt concentration of at least 1%.

[0115] In some aspects, the disclosure relates to a method of inhibiting activity of a sodium channel, comprising contacting the sodium channel with a pharmaceutical composition comprising a polypeptide comprising a sodium channel binding domain of a PLUNC protein, or a functional fragment, or homolog thereof, wherein the polypeptide, or the functional fragment, or homolog thereof is formulated in a saline solution containing a salt concentration of at least 1%, e.g., at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20%. Preferably, the salt concentration in the formulation is between 2% and 10%, particularly between 3% and 8%, and especially between 4% and 7%. Particularly preferably, the salt concentration in the formulation is at least 4.2%, e.g. , about 4.2% NaCl.

[0116] In some aspects, the disclosure relates to a method of inhibiting activity of a sodium channel, comprising contacting the sodium channel with a pharmaceutical composition comprising at least one polypeptide comprising, consisting essentially of, or consisting of, the amino acid sequence set forth in SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 141, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 128, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 131, SEQ ID NO: 139; SEQ ID NO: 140, SEQ ID NO: 144, SEQ ID NO: 127, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 136, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8, wherein the polypeptide is formulated in a saline solution containing a salt concentration of at least 1%, e.g., at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20%. Preferably, the salt concentration in the formulation is between 2% and 10%, particularly between 3% and 8%, and especially between 4% and 7%. Particularly preferably, the salt concentration in the formulation is at least 4.2%, e.g. , about 4.2% NaCl. Particularly under this aspect, the activity of the sodium channel is inhibited by at least 20%, especially by at least 50%, preferably by at least 60%, and particularly preferably by at least 90%. In some aspects, the inhibition of activity by the pharmaceutical composition of the disclosure is mediated by internalization of the sodium channel, e.g. , internalization of one or more subunits of ENaC, e.g. , a, β, or γ ENaC. [0117] In some aspects, the disclosure relates to a method of inhibiting sodium absorption through a sodium channel, comprising contacting the sodium channel with a pharmaceutical composition comprising a polypeptide comprising a sodium channel binding domain of a PLUNC protein, or a functional fragment, or homolog thereof, wherein the PLUNC protein, or the functional fragment, or homolog thereof is formulated in a saline solution containing a salt concentration of at least 1%, e.g. , at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20%. Preferably, the salt concentration in the formulation is between 2% and 10%, particularly between 3% and 8%, and especially between 4% and 7%. Particularly preferably, the salt concentration in the formulation is at least 4.2%, e.g. , about 4.2% NaCl.

[0118] In some aspects, the disclosure relates to 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 pharmaceutical composition comprising a polypeptide comprising a sodium channel binding domain of a PLUNC protein, or a functional fragment, or homolog thereof, wherein the PLUNC protein, or the functional fragment, or homolog thereof binds to the sodium channel, and wherein the polypeptide, or the functional fragment, or homolog thereof is formulated in a saline solution containing a salt concentration of at least 1%, e.g. , at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20%. Preferably, the salt concentration in the formulation is between 2% and 10%, particularly between 3% and 8%, and especially between 4% and 7%. Particularly preferably, the salt concentration in the formulation is at least 4.2%, e.g. , about 4.2% NaCl. Preferably under this aspect, increasing the volume of fluid lining an epithelial mucosal surface increases the activity of and/or expression level of cystic fibrosis transmembrane conductance regulator (CFTR).

[0119] In some aspects, the disclosure relates to a method of reducing the level of a sodium channel present on the surface of a cell, comprising contacting the sodium channel with a pharmaceutical composition comprising a polypeptide comprising a sodium channel binding domain of a PLUNC protein, or a functional fragment, or homolog thereof, wherein the PLUNC protein, or the functional fragment, or homolog thereof binds to the sodium channel, and wherein the polypeptide, or the functional fragment, or homolog thereof is formulated in a saline solution containing a salt concentration of at least 1%, e.g. , at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20%. Preferably, the salt concentration in the formulation is between 2% and 10%, particularly between 3% and 8%, and especially between 4% and 7%. Particularly preferably, the salt concentration in the formulation is at least 4.2%, e.g., about 4.2% NaCl.

[0120] In some aspects, the disclosure relates to a method of treating a disorder responsive to inhibition of sodium absorption across an epithelial mucosal surface in a subject in need thereof, comprising delivering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a polypeptide comprising a sodium channel binding domain of a PLUNC protein, or a functional fragment, or homolog thereof, wherein the PLUNC protein, or the functional fragment, or homolog thereof binds to a sodium channel, and wherein the polypeptide, or the functional fragment, or homolog thereof is formulated in a saline solution containing a salt concentration of at least 1%, e.g. , at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20%. Preferably, the salt concentration in the formulation is between 2% and 10%, particularly between 3% and 8%, and especially between 4% and 7%. Particularly preferably, the salt concentration in the formulation is at least 4.2%, e.g. , about 4.2% NaCl. In some aspects, the disorder is a lung disorder, e.g. , cystic fibrosis, non-cystic fibrosis bronchiectasis, acute or chronic bronchitis, severe asthma, or chronic obstructive pulmonary disease (COPD). In some aspects, the disorder is a gastrointestinal disorder, e.g., constipation, or inflammatory bowel disease (IBD). In some aspects, the disorder is a kidney disorder.

[0121] In some aspects, the disclosure relates to a method of regulating salt balance and/or fluid volume regulation in a subject in need thereof, comprising delivering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a polypeptide comprising a sodium channel binding domain of a PLUNC protein, or a functional fragment, or homolog thereof, wherein the PLUNC protein, or the functional fragment, or homolog thereof binds to a sodium channel present on the surface of a cell in the subject, and wherein the polypeptide, or the functional fragment, or homolog thereof is formulated in a saline solution containing a salt concentration of at least 1%, e.g. , at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20%. Preferably, the salt concentration in the formulation is between 2% and 10%, particularly between 3% and 8%, and especially between 4% and 7%. Particularly preferably, the salt concentration in the formulation is at least 4.2%, e.g. , about 4.2% NaCl. [0122] In some aspects, the disclosure relates to a method for treating a symptom of 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 pharmaceutical composition comprising a polypeptide comprising a sodium channel binding domain of a PLUNC protein, or a functional fragment, or homolog thereof, wherein the PLUNC protein, or the functional fragment, or homolog thereof binds to a sodium channel present on the surface of a cell in the subject, and wherein the polypeptide, or the functional fragment, or homolog thereof is formulated in a saline solution containing a salt concentration of at least 1%, e.g. , at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20%. Preferably, the salt concentration in the formulation is between 2% and 10%, particularly between 3% and 8%, and especially between 4% and 7%. Particularly preferably, the salt concentration in the formulation is at least 4.2%, e.g. , about 4.2% NaCl. In some aspects, the lung disorder is cystic fibrosis, non- cystic fibrosis bronchiectasis, chronic obstructive pulmonary disease, acute or chronic bronchitis, or asthma. In some aspects, the gastrointestinal disorder includes constipation or inflammatory bowel disease.

[0123] In some aspects, the disclosure relates to a method of inhibiting activity of a sodium channel, comprising contacting the sodium channel with: a pharmaceutical composition comprising a polypeptide comprising a sodium channel binding domain of a PLUNC protein, or a functional fragment, or homolog thereof, wherein the PLUNC protein, or the functional fragment, or homolog thereof binds to the sodium channel; and a hypertonic saline solution, e.g. , a saline solution containing a salt concentration of about 4.2% NaCl. In some aspects, the pharmaceutical composition and the hypertonic saline solution is administered simultaneously. In some aspects, the hypertonic saline solution is administered after the administration of the pharmaceutical composition.

[0124] In some aspects, the disclosure relates to a method of inhibiting sodium absorption through a sodium channel, comprising contacting the sodium channel with: a pharmaceutical composition comprising a polypeptide comprising a sodium channel binding domain of a PLUNC protein, or a functional fragment, or homolog thereof, wherein the PLUNC protein, or the functional fragment, or homolog thereof binds to the sodium channel; and a hypertonic saline solution, e.g. , a saline solution containing a salt concentration of about 4.2% NaCl. [0125] In some aspects, the disclosure relates to 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 pharmaceutical composition comprising a polypeptide comprising a sodium channel binding domain of a PLUNC protein, or a functional fragment, or homolog thereof, wherein the PLUNC protein, or the functional fragment, or homolog thereof binds to the sodium channel; and a hypertonic saline solution, e.g. , a saline solution containing a salt concentration of about 4.2% NaCl.

[0126] In some aspects, the disclosure relates to a method of reducing the level of a sodium channel present on the surface of a cell, comprising contacting the sodium channel with: a pharmaceutical composition comprising a polypeptide comprising a sodium channel binding domain of a PLUNC protein, or a functional fragment, or homolog thereof, wherein the PLUNC protein, or the functional fragment, or homolog thereof binds to the sodium channel; and a hypertonic saline solution, e.g. , a saline solution containing a salt concentration of about 4.2% NaCl.

[0127] In some aspects, the disclosure relates to a method of treating a disorder responsive to inhibition of sodium absorption across an epithelial mucosal surface in a subject in need thereof, comprising delivering to the subject: a therapeutically effective amount of a pharmaceutical composition comprising a polypeptide comprising a sodium channel binding domain of a PLUNC protein, or a functional fragment, or homolog thereof, wherein the PLUNC protein, or the functional fragment, or homolog thereof binds to a sodium channel; and a hypertonic saline solution, e.g. , a saline solution containing a salt concentration of about 4.2% NaCl.

[0128] In some aspects, the disclosure relates to a method of regulating salt balance and/or fluid volume regulation in a subject in need thereof, comprising delivering to the subject a therapeutically effective amount of a pharmaceutical composition comprising: a polypeptide comprising a sodium channel binding domain of a PLUNC protein, or a functional fragment, or homolog thereof, wherein the PLUNC protein, or the functional fragment, or homolog thereof binds to a sodium channel present on the surface of a cell in the subject; and a hypertonic saline solution, e.g. , a saline solution containing a salt concentration of about 4.2% NaCl.

[0129] In some aspects, the disclosure relates to a method for treating a symptom of 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 pharmaceutical composition comprising a polypeptide comprising a sodium channel binding domain of a PLUNC protein, or a functional fragment, or homolog thereof, wherein the PLUNC protein, or the functional fragment, or homolog thereof binds to a sodium channel present on the surface of a cell in the subject; and a hypertonic saline solution, e.g. , a saline solution containing a salt concentration of about 4.2% NaCl.

[0130] In some aspects, the disclosure relates to a method of treating a disorder responsive to inhibition of sodium absorption across an epithelial mucosal surface in a subject in need thereof, comprising delivering to the subject: a therapeutically effective amount of a pharmaceutical composition comprising a polypeptide comprising a sodium channel binding domain of a PLUNC protein, or a functional fragment, or homolog thereof, wherein the PLUNC protein, or the functional fragment, or homolog thereof binds to a sodium channel; and a compound that modulates the function of cystic fibrosis transmembrane conductance regulator (CFTR), e.g. , a compound selected from the group consisting of ivacaftor, lumacaftor, VX-661, tezacaftor (VX-661-111), deuterated ivacaftor (CTP-656), cavosonstat (N91115), acebilustat (CTX-4430), ataluren, riociguat, QR-010, QBW251, FDL169, QBW251, PTI-428, JBT-101, GS-5745, LAU-7b, POL6014, immobilized lipase, liprotamase, gallium nitrate, tobramycin, azithromycin, aztreonam, levofloxacin, amikacin, fosfomycin, vancomycin, and inhaled nitric oxide. In some aspects under the present therapeutic embodiment, the pharmaceutical composition and the compound are delivered to the subject concurrently. In some aspects under the present therapeutic embodiment, the pharmaceutical composition and the compound are delivered to the subject simultaneously.

[0131] In some aspects, the disclosure relates to a method of treating a disorder responsive to inhibition of sodium absorption across an epithelial mucosal surface in a subject in need thereof, comprising delivering to the subject: a therapeutically effective amount of a pharmaceutical composition comprising a polypeptide comprising a sodium channel binding domain of a PLUNC protein, or a functional fragment, or homolog thereof, wherein the PLUNC protein, or the functional fragment, or homolog thereof binds to a sodium channel; and a mucolytic compound, e.g. , a compound selected from the group consisting of acetylcysteine, ambroxol, carbocisteine, erdosteine, mecysteine, dornase alfa, oligoG, VX-371, AZD5634, and inhaled mannitol.

[0132] In some aspects, the disclosure relates to a method of treating a disorder responsive to inhibition of sodium absorption across an epithelial mucosal surface in a subject in need thereof, comprising delivering to the subject: a therapeutically effective amount of a pharmaceutical composition comprising a polypeptide comprising a sodium channel binding domain of a PLUNC protein, or a functional fragment, or homolog thereof, wherein the PLUNC protein, or the functional fragment, or homolog thereof binds to a sodium channel; and a long acting beta-agonist compound (LAB A), e.g. , a LABA compound selected from the group consisting of albuterol, levalbuterol, formoterol, and salmeterol.

[0133] In some aspects, the disclosure provides for a method of treating a disorder responsive to inhibition of sodium absorption across an epithelial mucosal surface in a subject in need thereof, comprising delivering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a polypeptide comprising a sodium channel binding domain of a PLUNC protein, or a functional fragment, or homolog thereof and a

pharmaceutically acceptable osmolality adjusting agent (e.g. , a compound selected from mannitol, xylitol, sorbitol, isomaltol, glucose, lactose, dextrose, sucrose, trehalose, maltose, glycerin, propylene glycol, ethylene glycol, glycerol, glycine, dimethylsulphoxide, calcium chloride, sodium sulfate, magnesium chloride, sodium gluconate, sodium pyruvate, pentosane polysulfate, and a cyclodextrin), wherein the PLUNC protein, or the functional fragment, or homolog thereof binds to a sodium channel.

[0134] The present invention is more particularly described in the following examples that are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art.

EXAMPLE 1

Experimental Methods

[0135] Tissue procurement and cell culture: Cells were harvested by enzymatic digestion from human bronchial tissue as previously described under a protocol approved by the UNC School of Medicine IRB (Tarran et al. , J. Gen. Physiol. 127:591 (2006)). Human excess donor lungs and excised recipient lungs were obtained at the time of lung transplantation from portions of main stem or lumbar bronchi and cells were harvested by enzymatic digestion. All preparations were maintained at an air-liquid interface in a modified bronchial epithelial medium and used 2-5 weeks after seeding on 12 mm T-Clear inserts (Corning Costar) coated with human placental type VI collagen (Sigma). Phosphate buffered saline (PBS) was used for washing human bronchial epithelial culture mucosal surfaces.

[0136] Confocal microscopy: To label airway surface liquid, Ringer containing rhodamine-dextran (2 mg/ml; Invitrogen) was added to human bronchial epithelial culture mucosal surfaces. Perfluorocarbon was added mucosally to prevent evaporation of the airway surface liquid and the culture placed in a chamber containing 100 μΐ Ringer on the stage of a Leica SP8 confocal microscope with a 63x glycerol immersion objective. 10 points per culture were scanned and an average airway surface liquid height determined. For confocal microscopy, human bronchial epithelial cultures were bathed serosally in a modified Ringer solution containing (mM): 116 NaCl, 10 NaHC0₃, 5.1 KC1, 1.2 CaCl₂, 1.2 MgCl₂, 20 TES, 10 glucose, pH 7.4). At all other times, human bronchial epithelial cultures were maintained in a modified BEGM growth medium which contained 24 mM NaHCC>3 gassed with 5% CO2. Perfluorocarbon (FC-77) was obtained from 3M and had no effect on ASL height as previously reported.

[0137] Internalization of alpha-ENaC: HEK293T cells were transfected with gfp- ocENaC where gfp was fused at the N-terminus of ENaC and unlabeled ENaC and yENaC using lipofectamine in 384 well plates as per the manufacturer's instructions and as published previously (see, e.g. , Hobbs et al. Am J Physiol Lung Cell Mol Physiol. 2013

Dec;305(12):L990-L1001. doi: 10.1152/ajplung.00103.2013. Epub 2013 Oct; Garland et al. Proc Natl Acad Sci U S A. 2013 Oct 1;110(40): 15973-8. doi: 10.1073/pnas.l311999110. Epub 2013 Sep 16.; Tan et al. J Physiol. 2014 Dec l;592(Pt 23):5251-68). Twenty-four hours later, peptide or vehicle control were added at t=0 and fluorescence was read using a Tecan Ml 000 plate reader 3 h later. Fluorescence was read at 488 nm (excitation)/ 510 nm (emission).

[0138] Fig. 6 shows the results of an experiment analyzing the effects of various peptides on internalization of alpha-ENaC in HEK293T cells when GFP-tagged alpha-ENaC is co-expressed only with gamma ENaC and no beta- ENaC. In this figure, SEQ ID NO: 143 (negative control peptide) and water (vehicle) controls are used along with S18 (SEQ ID NO: l, and SEQ ID NO: 128. While SEQ ID NO: l AND SEQ ID NO: 128 are effective in reducing alpha-ENaC when beta-ENaC is co-expressed, no effect is observed in this experiment when beta-ENaC is not present.

[0139] Efficacy testing in ENaC mice: Like CF patients, PENaC-Tg mice are disease free at birth, but soon develop obstructive lung disease (Zhou, Z., et al. Am J Respir Crit Care Med 178, 1245-1256 (2008)). C57BL:FVB and C57BL:C3H mixed strains have 90% and 50% mortality, respectively, and the reproducibility of the disease on these backgrounds is sufficiently high that reliable data are produced with n of about 8-10 animals/group.

Furthermore, the PENaC-Tg mouse responds to therapeutic interventions in a fashion similar to CF/COPD in humans, and the development of lung disease can be prevented following inhibition of lung disease at birth (Livraghi, A., et al. J Immunol 182, 4357-4367 (2009)). SI 8- derived peptides or vehicle were dosed one to three times a day by intranasal instillation (Ιμΐ/g body weight) for 14 days in parallel cohorts of mice. Mice were weighed daily and, if a diuretic effect was found, the volume excreted was replaced by sub-cutaneous injections of sterile saline. At the end of the treatment, mice were sacrificed for phenotypic analysis.

[0140] Statistical analyses: All data are presented as the mean + SE for n experiments. Airway cultures derived from three or more separate donors were used for each study and each oocyte study was repeated on three separate occasions. Differences between means were tested for statistical significance using paired or unpaired t tests or their non parametric equivalent as appropriate to the experiment. From such comparisons, differences yielding P < 0.05 were judged to be significant. All binding assays were fitted to the Hill equation.

EXAMPLE 2

Identification of specialized non-naturally occurring peptides

[0141] Based on the ability of normal human bronchial epithelial cultures to regulate airway surface liquid height to 7 μιη, which was paralleled by a decrease in trypsin-sensitive ENaC activity, we previously demonstrated that SPLUNCl derived peptides (namely, SI 8; FIG. 1) can inhibit ENaC with EC₅₀ in the sub micromolar range for up to 24 h following a single dose (Hobbs et al, 2013). While S18 is resistant to proteolysis and heat-stable, we tested whether we could reduce the size of SI 8, increase its stability and/or increase its potency. Any of these actions would increase its utility as a drug.

[0142] To confirm our alanine-scan data, we made a peptide of subsequence A

(LPVPLDQT (SEQ ID NO: 113); FIG. 1). As a control, we made an overlapping 2nd peptide, which contained the charged mid-region (DQT), as well as subsequence B (DQTLPLNVNP (SEQ ID NO: 114)). As shown in FIG. 2, LPVPLDQT (SEQ ID NO: 113) retained similar activity to SI 8, whilst DQTLPLNVNP (SEQ ID NO: 114) was inactive. However, additional experiments, where we examined susceptibility to proteases, demonstrated that whilst

LPVPLDQT (SEQ ID NO: 113) could inhibit ENaC-led fluid absorption, this peptide was more susceptible to proteolytic degradation. Thus, we generated new peptides where we flanked the C- and N- termini with a pair of D-alanines (shown as lower case 'a'). Peptides were added mucosally to HBECs at 30 μΜ and ASL height measured 3 h later by XZ-confocal microscopy. FIG. 2A shows the comparison of the N- terminal region of S18 (LPVPLDQT) (SEQ ID

NO: 113) vs. an overlapping, but C-terminal segment (DQTLPLNVNP) (SEQ ID NO: 114). In FIG. 2B, D-alanines were added to Sub-S18 peptides LPVPLDQT (SEQ ID NO: 113) and LPVPLDQTLPL (SEQ ID NO: 115) to increase the stability of LPVPLDQT (SEQ ID NO: 113). D-alanines are noted as lowercase a. All n = 6-9. Data are shown as mean + SEM. [0143] The 15-mer and 8-mer showed identical activity when compared against S18 (FIGS. 2A-2B). We then generated full dose-responses for S18 vs. the peptide aaLPVPLDQTaa (SEQ ID NO:3). S18 and aaLPVPLDQTaa (SEQ ID NO:3) produced identical dose responses (FIG. 3A), indicating that shortening the peptide and flanking it with unnatural amino acids did not affect its potency or efficacy. Next, we made several new peptides based on the sequence of SEQ ID NO:2. Here, since prolines typically provide kinks in a peptide, they were not altered and instead, we systematically made conservative substitutions of the other amino acids. The dose responses are shown in FIG. 3B. Note, since there are several lines on this graph, the error bars and data symbols were omitted. However, these data were obtained simultaneously using paired airway cultures, so their comparison is valid. Clearly, SEQ ID NO: 5

(aaLPNlePLDQTaa) shows a log-fold increase in potency as compared to SI 8, whilst SEQ ID NO:9 (aaLPVPLDQSaa) and SEQ ID NO:6 (aaLPVPNleDQTaa) show diminished potency and efficacy respectively (FIG. 3B). Other sequences included in FIG. 3B are SEQ ID NO:4 (aaNlePVPLDQTaa), SEQ ID NO:7 (aaLPVPLDNTaa) and SEQ ID NO:8 (aaLPVPLEQTaa).

[0144] Using a fresh batch of airway cells, we performed an additional dose response to SEQ ID NO:5 (FIG. 4). As can be seen, this peptide gave a significant increase in potency, as compared to SI 8, and the IC₅₀ was 30 nM.

[0145] The ability of the peptides to modulate internalization of ocENaC was tested. Results from these experiments are shown in FIGS. 5A-5U. ADG is a negative control peptide with the sequence: NH3 - ADGGLLLLNNPPPPQTVV-NH2 (SEQ ID NO: 143). The peptides used in these experiments were S18 (SEQ ID NO: l), SEQ ID NO:2, SEQ ID NO: 141, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 128, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 131, SEQ ID NO: 139; SEQ ID NO: 140, SEQ ID NO: 144 and SEQ ID NO: 127. Also used in these experiments were peptides of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 136, each with an aa cap at both the N- and the C-terminus. Also shown is amiloride, which inhibits ENaC by another mechanism and does not reduce the amount of functional receptor on the surface of cells. These results demonstrate that the SPLUNC peptides trigger the destruction of the ENaC subunits to inhibit its activity.

[0146] The efficacy of the peptides was tested in ENaC mice. Results from these experiments are shown in Figs. 7A-7F. Fourteen-day treatment with inhaled SEQ ID NO: 128 was more effective than inhaled SEQ ID NO: 142 at increasing percent survival of ENaC-Tg C57BL:FVB mice (Fig. 7B). Fourteen-day treatment with SEQ ID NO: 128 also increased percent survival of ENaC-Tg C57BL:C3H mice (Figs. 7C and 7D). Once daily dosing with SEQ ID NO: 128 increased percent survival of ENaC-Tg C57BL:C3H mice (Fig. 7E, left panel). The right panel of Fig. 7E shows that SEQ ID NO: 128 did not have a diuretic effect. Fig. 7F shows that treatment with SEQ ID NO: 127 increased percent survival of ENaC-Tg C57BL:C3H mice. Sequences of other peptides used in the experiment were SEQ ID NO: 129, SEQ ID NO: 134, and SEQ ID NO: 136.

EXAMPLE 3

Effect of SEP ID NO:128 on CFTRinh-172-Induced Mucociliary Dysfunction in Sheep

[0147] Purpose: To test the ability of SEQ ID NO: 128 (aaLPIPLDQTaa) to reverse the slowing of tracheal mucus velocity (TMV) induced by inhalation of CFTRinhibitor-172 (CFTRinh) in sheep.

[0148] Rationale: We previously reported that sheep challenged with an aerosol of 10 mg CFTRinh- 172 produced a slowing of TMV that persisted for 12 h (Eur. Res. J. S59: PA2054, 2015). We used this model to determine if SEQ ID NO: 128, a compound that inhibits epithelial sodium channels and prevents airway surface liquid hyper absorption in human bronchial epithelial cultures from CF patients for >8 h following a single dose, could reverse this

CFTRinh-induced mucociliary dysfunction.

[0149] Animals Preparation: All procedures used were approved by the Mount Sinai Medical Center Animal Research Committee. The sheep were conscious and intubated for these studies. All instrumentation was performed under local anesthesia. After topical anesthesia of the nasal passages with 2% lidocaine solution, the sheep were nasally intubated, with an endotracheal tube 7.5 cm in diameter (Mallinckrodt Med Inc., St. Louis, MO.) shortened by 6 cm. The cuff of the tube was placed just below the vocal cords (verified by fluoroscopy) to allow for maximal exposure of the tracheal surface area. The inspired air was warmed and humidified using a Bennett Humidifier (Puritan -Bennett, Lenexa, KS). To minimize possible impairment of TMV caused by the inflated cuff, the endotracheal tube cuff remained deflated throughout the study except for the period of drug delivery.

[0150] Tracheal Mucus Velocity: TMV was measured in vivo by a roentgenographic technique. Between 8 and 10 radiopaque Teflon/bismuth trioxide disks, 1-mm diameter, 0.8-mm thick and 1.5-2mg in weight, were insufflated onto the trachea. A modified suction catheter connected to a source of continuous compressed air at a flow of 3-4 L/min was used to introduce the particles via the endotracheal tube. The catheter remained within the endotracheal tube only during particle delivery, so that no contact with the tracheal surface is made. The cephalad-axial velocities of the individual disks were recorded on videotape from a portable image intensifier unit. Individual disk velocities were calculated by measuring the distance traveled by each disk during a l-min observation period. For each run, the mean value of all individual disk velocities was calculated. A collar containing radiopaque reference markers of known length was worn by the sheep, and used as a standard to correct for magnification effects inherent in the fluoroscopy unit.

[0151] Aerosol Delivery Systems: Aerosols were generated using an Airlife medication nebulizer. To control aerosol delivery a dosimetry system activated by a piston respirator (Harvard Apparatus Respiratory Pump, Holliston, MA) was used. The nebulizer was connected to a T-piece with one end attached to the respiratory pump and the other to the animal's tracheal tube. Nebulized aerosols were delivered directly into the trachea only during inspiration at a frequency of 20 breaths / min and at a tidal volume of 500 mL.

[0152] Agents: Ten (10) mg of CFTRinh-172 was dissolved in 0.3 mL of DMSO. The solution was vortexed and then 2.7 mL of ethanol was added bringing the final nebulized solution of CFTRinh to 10 mg/3 mL. The solution was stirred for 10 min before dosing to assure that the CFTRinh was completely dissolved. A new nebulizer was used for each CFTRinh challenge. SEQ ID NO: 128 was dissolved in 3mL 0.9% saline to achieve final doses of 4 mg/kg, 2 mg/kg, and 1 mg/kg and the total amount delivered to the sheep. Some animals were dosed with 3 mM amiloride, which inhibits ENaC by another mechanism and does not reduce the amount of functional receptor on the surface of cells. The mean weight of the sheep used in these studies was 39 kg (range 32-46kg). Cystic fibrosis transmembrane conductance regulator inhibitor (CFTRinh-172, AdooQ Biosciences, Irvine, CA). DMSO and Ethanol, Sigma- Aldrich Inc. (Ronkonkoma, NY). 0.9% saline (Cardinal Health, Westin, FL). Bennett Humidifier (Puritan-Bennett, Lenexa, KS). AirLife ® Brand Misty Max 10 Disposable Nebulizer™

(Cardinal Health, Westin, FL).

[0153] Study Protocol: After a 10 minute period on the humidifier, a baseline TMV was obtained. Immediately following the baseline, the sheep were challenged with an aerosol of 10 mg of CFTRinh. TMV was measured hourly for 4h. Immediately after the 4h TMV measurement, 3 mL of 0.9% normal saline (vehicle) or the different doses of SEQ ID NO: 128 were given by nebulization. TMV was then measured from 5-12h following treatment.

[0154] Results: The results of SEQ ID NO: 128 treatments on the CFTRinh response are shown in Figs. 8A-8C. For statistical comparisons, we compared the average mean responses from 5-12h for each treatment using a one way ANOVA. Post hoc comparisons were made with the Bonferroni test. Values in the figures are mean + se for 3 sheep / group. The average response for the saline treated group between 5-12h was 50 + 0.5%. All treatments significantly improved this response: 73.7 + 0.7% with 1 mg/kg; 88.4 + 0.6% with 2 mg/kg; and 103.0 + 0.6 4 mg/kg. In addition the treatment responses were significantly different from each other (P<0.001).

[0155] Conclusion: These results show that SEQ ID NO: 128 can reverse CFTRinh- induced slowing of TMV. The reversal is dose dependent. Thus, SEQ ID NO: 128 provides useful therapy to correct impaired mucus clearance associated with CFTR dysfunction.

[0156] The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

[0157] Scientific Knowledge on the Subject: In CF lung disease, hyperactivation of the epithelial sodium channel (ENaC) causes an excess of sodium to be absorbed, resulting in an osmotic gradient that draws water away from the airway surface. This airway dehydration leads to the production of thick, static mucus that promotes bacterial colonization and infections, airway obstruction and lung inflammation. Short Palate, Lung, and Nasal Clone 1 (SPLUNCl) is a protein secreted by airway epithelial cells that controls ENaC activity in healthy lungs. SPLUNCl' s regulation of ENaC is pH-sensitive and is lost in the acidic environment of the CF lung. Additional research identified a peptide derived from SPLUNCl' s N- terminus, SI 8, that is responsible for regulating ENaC. Importantly, S18 maintains its function at acid pH.

[0158] What this paper adds to the field: This work describes the actions of SPX-101, an optimized version of S18 allowing for its efficient nebulization into the CF lung. In these studies SPX-101 is shown to internalize all three major ENaC subunits, leading to improved mucus transport in mouse and sheep models of CF lung disease.

[0159] Rationale: Cystic Fibrosis lung disease (CF) is caused by the loss of function of the cystic fibrosis transmembrane conductance regulator (CFTR) combined with hyperactivation of the epithelial sodium channel (ENaC). In the lung, ENaC is responsible for movement of sodium. Hyperactivation of ENaC, which creates an osmotic gradient that pulls fluid out of the airway, contributes to reduced airway hydration causing mucus dehydration, decreased mucociliary clearance, and recurrent, acute bacterial infections. ENaC represents a therapeutic target to treat all CF patients independent of their underlying CFTR mutation.

[0160] Objective: To investigate the in vitro and in vivo efficacy of SPX-101, a peptide mimetic of SPLUNCl' s natural regulation of ENaC activity. [0161] Methods: ENaC internalization by SPX-101 in primary human bronchial epithelial cells from healthy and CF donors was assessed by surface biotinylation and subsequent western blot analysis. SPX-101's in vivo therapeutic effect was assessed by survival of ENaC transgenic mice, mucus transport in these mice, and mucus transport in a sheep model of CF.

[0162] Measurements and Main Results: SPX-101 binds selectively to ENaC and promotes internalization of the α, β, and y subunits. Removing ENaC from the membrane with SPX-101 causes a significant decrease in amiloride-sensitive current. The peptide increases survival of ENaC transgenic mice to >90% with once-daily dosing by inhalation. SPX-101 increased mucus transport in the ENaC mouse model as well as the sheep model of CF.

[0163] Conclusions: These data demonstrate that SPX-101 promotes durable reduction of ENaC membrane concentration leading to significant improvements in mucus transport.

[0164] Cystic fibrosis (CF) is a recessive genetic disease affecting more than 70,000 people globally. The disease is caused by mutations in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which result in lack of expression, or expression of dysfunctional, protein (1). CFTR controls transepithelial secretion of ClTHCCV onto the airway surface. In addition to dysfunctional CFTR, CF is also characterized by hyperactivation of the epithelial sodium channel (ENaC) (2, 3). This hyperactivation causes an excess of sodium to be absorbed via ENaC, resulting in an osmotic gradient that draws water away from the airway surface. As this effect reduces the liquid volume on the airway surface, there is an increase in the viscosity of airway mucus which leads to impaired mucociliary clearance (4-7). This environment of thick, static mucus promotes bacterial colonization and infections, airway obstruction and lung inflammation. Currently, the majority of deaths from CF result from lung infections or related complications (8-10).

[0165] Historically, treatments for CF have focused on the symptoms of the disease without addressing the loss of CFTR function (11-14). More recently, attempts have been made to treat CF by targeting the absent or dysfunctional CFTR protein itself. These efforts have led to the development of drugs such as ivacaftor (VX-770) and lumacaftor (VX-809) which, while beneficial for those carrying specific CFTR mutations, are not therapeutic options for the entire CF population (15, 16). As more than 1900 different mutations can dysregulate CFTR function, finding a single CFTR corrector beneficial to all genotypes remains challenging.

[0166] An alternative therapeutic approach for treating CF, which would work independent of the underlying CFTR mutation, is to block ENaC hyperactivation. Prior

49

63 attempts have been made to target ENaC with inhaled therapeutics, including the use of amiloride or its derivatives, but have proven unsuccessful (17-20). While these compounds are potent ENaC inhibitors, they suffer from two major limitations. First, because of their small size, these compounds cross into systemic circulation, where they affect ENaC in the kidney, leading to diuresis and hyperkalemia (21-23). Second, their actions in the lung are reversible and short-lived, providing no durable changes in mucus hydration or lung function (19, 21, 24, 25).

[0167] Recently, it was discovered that the lung airway's natural regulation of ENaC activity is controlled by Short Palate, Lung, and Nasal Clone 1 (SPLUNC1) (26). In healthy lungs, SPLUNC1 is secreted by airway epithelial cells as an autocrine regulator of ENaC, thereby limiting sodium and water absorption. However, this action is pH-dependent and is lost in the acidic environment of the CF lung due to abnormal folding of SPLUNC1 at low pH (4, 27). Additional research on SPLUNC1 led to the discovery that an 18 amino acid domain (SI 8) on the protein's N-terminus was responsible for controlling ENaC. Unlike the full-length protein, S18 was found to regulate ENaC in a pH-independent manner (27, 28). This discovery underscored the therapeutic potential of using such an ENaC-regulating peptide for the treatment of CF lung disease. We have optimized the natural sequence of S18 for stability and delivery via nebulization into the lung in the form of SPX-101. Herein we report that SPX-101 induces internalization of oc/ /yENaC in normal and CF human bronchial epithelial cells (HBECs), providing a novel mechanism of action for the regulation of water movement in the airway. In vivo, the peptide increases survival of ENaC-Tg mice, increases mucociliary clearance in these mice, and increases tracheal mucus velocity in an ovine model of CF. Taken together, SPX-101 represents a novel peptide promoter of ENaC internalization which we propose has the potential to serve as a new therapeutic for the treatment of all patients with CF.

Methods

Peptides

[0168] All peptides were synthesized by Ambiopharm.

Animal Studies

[0169] All animal studies were approved by the Institutional Animal Care and Use Committees of Spyryx Biosciences and the Mount Sinai Medical Center. The ENaC transgenic ( ENaC-Tg) mouse has been previously described (29). For all studies, hemizygous male C57BL/6N ENaC-Tg mice were crossed with female C3H mice (Charles River Laboratories, Raleigh, NC). All experiments were conducted using Fl generation pups and the ENaC transgene was confirmed by PCR. Pups were treated by once-daily intranasal instillation of SPX-101 (0.18, 0.9, or 1.8 mg/kg), control peptide (1.8 mg/kg), or saline vehicle until two weeks of age. Measurement of tracheal mucus velocity in conscious sheep was previously described (21, 30).

Cell Culture

[0170] HBECs used in these studies were isolated at Spyryx Biosciences and purchased from Lonza (Walkersville, MD). Cells were grown as previously described (31). HEK293T cells were purchased from ATCC (Manassas, VA).

Measurement of amiloride sensitive current

[0171] Transepithelial voltage (Vt) and resistance (R) EVOM measurements were performed on normal and CF HBECs. Current was calculated according to Ohm's law.

Statistical Analysis

[0172] For all experiments a p <0.05 was considered significant.

Results

SPX-101 binds selectively to ENaC

[0173] The structure of SPX-101 is shown in Figure 9A. We first determined the specificity of SPX-101 for binding to ENaC. HEK293T cells were mock-transfected or transfected with ccENaC-GFP, ENaC-FLAG, and yENaC-HA. Twenty-four hours after transfection, cells were lysed and incubated with biotin- tagged SPX-101. Interacting proteins were isolated on streptavidin agarose and detected by western blot. As seen in Figure 9B, all three ENaC subunits were detected in the pulldown. Similar results were previously reported with SPLUNCl and S18 binding to ENaC (26, 28). In additional experiments, TAMRA-tagged SPX-101 binding was significantly increased in the ENaC-expressing cells as compared to those mock transfected or expressing ASIC 1/2, the ion channels most homologous to ENaC, demonstrating specificity of binding (Figure 9C). A final determination of specificity was conducted using a Eurofins Panlabs Safety Assessment Screen, which tests for interactions with 87 common receptors and kinases. SPX-101 did not significantly inhibit or activate any of the proteins tested (data not shown). Together, these data demonstrate that SPX-101 is ENaC- selective.

SPX-101 promotes internalization of ENaC [0174] Previously, it was reported that SPLUNC1 induced internalization of ENaC (32). To determine if SPX-101 had a similar effect, we treated primary HBECs derived from healthy donors with SPX-101, amiloride, or an alphabetized control peptide. As seen in Figure 10A, SPX-101 induced internalization of all three ENaC subunits. SPX-101 did not alter expression of ATP12A demonstrating that these effects were ENaC-specific. As expected, amiloride and the control peptide had no effect on ENaC levels in the plasma membrane. We next determined the duration of this effect in HBECs from healthy and CF donors. SPX-101 induced a rapid and durable internalization of all three ENaC subunits out to eight hours, the longest time point analyzed (Figures lOB/C). These data demonstrate that SPX-101, but not amiloride, promotes internalization of all three ENaC subunits.

SPX-101 attenuates amiloride-sensitive current

[0175] Because SPX-101 internalized ENaC, we hypothesized that the peptide would attenuate amiloride-sensitive current. This was tested experimentally by treating HBECs from healthy and CF donors with SPX-101 for two hours and measuring current before and after administration of amiloride. The average amiloride sensitive current in vehicle treated healthy donor HBECs was 14.7 μΑ/cm² and in vehicle treated CF donor HBEC was 22.8 μΑ/cm² confirming hyperactivation of ENaC in diseased cells, as previously reported (33, 34). SPX-101 inhibited amiloride-sensitive current by 75% in HBECs derived from healthy donors (Figure 11A) and by 61% in CF donor HBECs (Figure 1 IB). These results, combined with the data in Figures lOA-lOC, indicate that SPX-101 induces internalization of ENaC for >8 hours, suggesting a long-term inhibition of the amiloride-sensitive current.

SPX-101 increases survival of ENaC-Tg mice

[0176] We next tested the effects of SPX-101 on growth and survival of ENaC-Tg mice, which develops obstructive pulmonary disease similar to CF (29, 35, 36). Previous studies have shown that when backcrossed to C3H mice, ENaC-Tg mice on a C57BL/6 background produce offspring with 50% survival rate at two weeks of age (37). SPX-101-treated mice demonstrated survival rates of 58%, 79%, and 92% with a once-daily intranasal instillation of 0.18, 0.90, and 1.8 mg/kg, respectively (Figure 12A). In support of increased survival, SPX- 101 treated mice showed a concentration-dependent increase in weight that is indicative of improved health. It has been reported that ENaC-Tg mice have increased proportions of disease-associated neutrophils and eosinophils in their bronchoalveolar lavage fluid (BALF) as compared to healthy wild-type mice (37). SPX-101 treatment decreased the number of neutrophils and eosinophils in the BALF (Figure 12C). Importantly, an alphabetized control peptide did not cause changes in mouse survival or BALF leukocyte composition ruling out an osmotic effect of the peptide as the driving force behind these events. These data demonstrate that SPX-101 prolongs survival and reduces neutrophil and eosinophil infiltration in ENaC-Tg mice.

SPX-101 increases mucociliary clearance in flENaC-Tg mice

[0177] It has previously been reported that ENaC-Tg mice have a significant reduction in mucociliary clearance (MCC) (29). Indeed, our studies indicated that control peptide-treated ENaC-Tg mice had a 50% reduction in MCC, determined by the velocity of fluorescent beads along excised trachea from these mice, as compared to tracheas from wild- type littermates. SPX-101 significantly increased the rate of MCC to 75% of control mice four hours after a single intranasal instillation (Figure 13A). In addition to increasing the velocity of the beads, SPX-101 also increased the directionality of the bead movement along the trachea, fully restoring this defect. In contrast, directionality in control peptide-treated mice remained -25% below wild-type levels (Figure 13B). We then calculated mucus transport index (MTI) as a product of velocity x directionality. Control peptide-treated ENaC-Tg mice exhibited a 59% decrease in MTI compared to wild-type littermate controls. A single dose of SPX-101 doubled the MTI compared to control peptide-treated mice (Figure 13C). These data demonstrate that SPX-101 promotes mucus clearance in ENaC-Tg mice.

SPX-101 increases tracheal mucus velocity in a sheep model of Cystic Fibrosis

[0178] We next assessed the durability of SPX-101 on improving MCC using an ovine model of cystic fibrosis-like disease. In this model, sheep are nebulized with the CFTR small molecule inhibitor, CFTRinhl72, and tracheal mucus velocity (TMV) is measured by the movement of radio-opaque disks. CFTRinhl72 causes a -50% reduction in TMV four hours after administration that is sustained for greater than twelve hours. To determine if SPX-101 could restore TMV, sheep were nebulized with SPX-101 peptide four hours after exposure to CFTRinhl72 and disk movement was then monitored for an additional eight hours. SPX-101 caused a dose-dependent increase in TMV, with the highest dose restoring TMV to 100% for the duration of the experiment (Figure 14A). In contrast, the control peptide had no effect on TMV, demonstrating that osmotic effects were not responsible for the increase in TMV. For purposes of comparison, we also determined the effect of other ENaC-effecting compounds in this model. Amiloride was able to increase TMV to 80% of the initial rate but the effect was poorly sustained (Figure 14B). We then investigated whether hypertonic saline (HS) could increase the effect of SPX-101 on restoring TMV. Dosing sheep with SPX-101 in 4.2% HS showed a significant improvement as compared to the peptide dosed with 0.9% isotonic saline (Figures 14C and 14D). In line with previous reports, amiloride showed no additional benefit when nebulized with HS (19). An area under the curve calculation for each of the above treatments is presented in Table 2. Combined, these data demonstrate that SPX-101, but not amiloride, promotes durable increases in sheep TMV via a differentiated mechanism.

Discussion

[0179] We have shown that SPX-101 binds selectively to ENaC (Figure 1) to induce internalization of α/β/γ ENaC (Figures lOA-lOC), and a decrease in amiloride-sensitive current (Figure 3). SPX-101 increases survival of ENaC-Tg mice while reducing neutrophil and eosinophil infiltration into the lung with a once-daily dosing regimen (Figure 12C). A single intranasal dose of the peptide increases mucociliary clearance velocity and directionality in ENaC-Tg mice (Figures 13A-13C). Finally, the peptide restores tracheal mucus velocity in an ovine model of CF (Figures 14A, 14C, and 14D). The effects described above are specific to SPX-101 and are not achieved by a control peptide or the ENaC pore blocker amiloride. Taken together, SPX-101 is an optimized, first-in-class ENaC-effecting therapeutic capable of restoring durable mucus transport.

[0180] In healthy lungs, SPLUNC1 is secreted by airway epithelia creating an autocrine control mechanism of ENaC membrane concentration (4, 26, 38). This function is lost in CF due to a conformational change in SPLUNC1 that occurs in the acidified CF airway. However, a peptide derived from SPLUNCl's N-terminus, S18, was shown to retain ENaC-regulatory function at acidic pH (27). To create our therapeutic, SPX-101, we optimized S 18 to enhance drug-like properties and deliverability into the lung via nebulization. The reduced size of SPX- 101 increases the dose density and reduces nebulization time, an important consideration for CF patients who spend multiple hours a day receiving therapy.

[0181] The hyperactivation of ENaC in the CF lung has been shown to occur from both an increase in channel number (39), as well as an increase in open probability (Po) of the channel (40, 41). The CF lung contains increased levels of proteases which can activate ENaC (40, 42). Inhibitors such as amiloride decrease ENaC hyperactivation by reducing Po, but the channels and activating proteases are still present in the airway. This scenario ensures that ENaC hyperactivation rapidly resumes when the inhibitor is washed away. In contrast, SPX-101 causes a durable reduction of ENaC hyperactivation by removing channels from the apical membrane of the cell and functionally blocking sodium absorption.

[0182] Herein we have demonstrated in cell and animal models that SPX-101 has a durable effect due to the peptide's ability to internalize ENaC. Highlighting the advantage afforded by this mechanism of action is the ability of SPX-101 to inhibit amiloride-sensitive current. We observed up to a 75% reduction of amiloride-sensitive current two hours after administration of SPX-101. Inhibition of ENaC current occurred even though the peptide had been removed from the cell with repeated washings. The ability of SPX-101 to maintain its reduction in ENaC current occurred even after the peptide was washed away because the number of channels on the surface of the cell has been reduced. In contrast, amiloride potently inhibits ENaC current, but this effect is immediately lost after the apical surface is washed. These findings establish that it is the sustained suppression of ENaC activity, through channel internalization, that provides the clear therapeutic advantage of SPX-101 as compared to small molecule ENaC inhibitors.

[0183] The PENaC-Tg mouse model develops CF-like lung disease that is characterized by mucus plugging of the upper airway and increased neutrophil and eosinophil levels in the BAL fluid (29, 37). In our studies, 45% of PENaC-Tg mice survived through two weeks of age in the absence of therapeutic intervention. After treatment with SPX-101, 58%, 79%, and 92% of mice survived with a once-daily intranasal instillation of 0.18, 0.90, and 1.8 mg/kg, respectively. In comparison, Zhou and colleagues demonstrated that amiloride increased survival of PENaC-Tg mice to -80% but this required thrice daily administration (37). Further, the dose of amiloride used in these studies resulted in a diuretic effect that required supplemental intraperitoneal injections of saline to offset the fluid depletion. SPX-101 did not cause diuresis and no supportive treatments were required, demonstrating the lack of systemic effects afforded by our peptide. This is supported by 28-day preclinical toxicology in rats and dogs, where no shift in blood electrolytes or diuresis was observed (data not shown).

[0184] For any compound to be beneficial in CF lung disease it must increase MCC. It was previously reported that the PENaC-Tg mice have impaired mucus transport (29). We observed an -50% reduction in velocity of fluorescent microspheres along the trachea of PENaC-Tg mice compared to wild-type littermate mice. The mucus velocity in these mice was significantly restored to 77% of wild- type levels by a single dose of SPX-101. In addition to the increase in velocity, SPX-101 also corrected directionality of mucus movement to wild- type levels. This restoration of mucus transport suggests that SPX-101 is likely augmenting mucus hydration in this model. Important future studies will address this effect of SPX-101, as well as its effects on mucus composition and rheology.

[0185] SPX-101 also increased mucus transport in a large animal model. The ovine model based on CFTRinhl72 nebulization provides an opportunity to understand a compound's effects in a complex airway (29). Our data in this model demonstrate that SPX-101 provides a sustained increase in TMV for the duration of the experiment and reached 100% at the highest dose tested. A control peptide did not increase TMV, again ruling out changes in airway osmolality as the driver of the observed effect. We also tested the effects of amiloride in this model and while it was able to increase TMV, the effect was transient and returned to untreated levels by the end of the experiment. This short-lived response is supported by clinical reports where amiloride was unable to provide durable changes in mucus clearance or nasal PD (19, 21, 25).

[0186] It is estimated that >60% of CF patients use HS to enhance mucociliary clearance, therefore we wanted to understand if salt concentration could modulate the effect we observed with SPX-101. At both the 1 and 2 mg/kg dose of SPX-101, 4.2% HS significantly increased TMV above the levels seen with SPX-101 alone. We believe this might be due in part to water absorption across the airway epithelium being decreased due to ENaC internalization. Moreover, the addition of HS should draw water into the airway space and, because sodium absorption has been reduced, the effect on TMV is potentiated. However, when we combined HS with amiloride there was no further enhancement of TMV. This could be explained by amiloride being rapidly washed away due to additional fluid drawn into the airway by the introduction of HS.

[0187] Because CF is caused by the dysregulation of multiple ion channels, it is important to assess a potential CF therapeutic in terms of its effects on total ion transport across the epithelia. Current approved compounds which target CFTR, ivacaftor (VX-770) and lumacaftor (VX-809), underscore the potential of CFTR-based therapeutics. In support of this, Van Goor and colleagues reported on VX-770' s ability to enhance CI^" secretion in

G551D/F508del cells (44). Further, they reported that under these same conditions, VX-770 also reduced ENaC hyperactivation. Unfortunately, these and other CFTR-based therapies are only appropriate for a portion of the CF population due to orientation to specific CF-causing mutations.

[0188] Conversely, SPX-101 has demonstrated in CF HBECs a greater than 60% reduction in amiloride-sensitive current through its mechanism of internalizing ENaC (Figure 3). This reduction restores the amiloride-sensitive current to approximately wild-type levels in HBECs from healthy donors. Additionally, the duration of SPX-101's internalization effect in these cells was shown to exceed 8 hours (Figures 1 OA- IOC). This finding corroborates the results with SPX-101 in the sheep model, which showed full drug response beyond 8 hours (Figures 14A, 14C, and 14D). Fortunately, SPX-101's effect on ENaC is independent of the CF-causing mutations, which means it represents a unique opportunity to treat all CF patients.

[0189] In conclusion, SPX-101 fully differentiates itself from previous ENaC-targeting therapeutics through its mechanism of action and duration of effect. Not simply a channel blocker, SPX-101 replaces the natural ENaC-inhibiting properties of endogenous SPLUNC1 that have been lost in the CF airway. By using the cell's natural means of regulating ENaC, SPX-101 achieves a durable increase in mucus transport in multiple animal models, demonstrating its potential to provide meaningful, clinical benefit to CF patients. Beyond CF, SPX-101 has the potential to also address other diseases associated with MCC defects, such as non-CF bronchiectasis, COPD, and severe asthma.

[0190] Table 2. AUC analysis of sheep TMV data

EXAMPLE 4

Effect of hypertonic saline on the activity of SPX-101

[0191] Studies were conducted to examine the effect of hypertonic saline on the pharmacological activity of SPX-101 in vitro and in vivo.

[0192] For in vitro studies, human bronchial epithelial cells (CFTR status

DF508/DF508) were used. Cells were harvested by enzymatic digestion from human bronchial tissue as previously described. Human excess donor lungs and excised recipient lungs were obtained at the time of lung transplantation from portions of main stem or lumbar bronchi and cells were harvested by enzymatic digestion. All preparations were maintained at an air-liquid interface in a modified bronchial epithelial medium and used 2-5 weeks after seeding on 12 mm T-Clear inserts (Corning Costar) coated with human placental type VI collagen

(Sigma). Phosphate buffered saline (PBS) was used for washing human bronchial epithelial culture mucosal surfaces. To label airway surface liquid, Ringer containing rhodamine-dextran (2 mg/ml; Invitrogen) was added to human bronchial epithelial culture mucosal surfaces. Perfluorocarbon was added mucosally to prevent evaporation of the airway surface liquid and the culture placed in a chamber containing 100 μΐ Ringer on the stage of a Leica SP8 confocal microscope with a 63X glycerol immersion objective. Airway surface liquid (ASL) height was measured six hours after addition of the solutions. 10 points per culture were scanned and an average airway surface liquid height determined. Data are presented as mean + SEM (n=8).

[0193] The results are presented in FIG. 15. The results show a surprising effect when hypertonic saline was used as vehicle for SPX-101 compared to the effect conferred by isotonic saline. In particular, use of a formulation of SPX-101 in 3% saline increased ASL height by over 250% compared to SPX-101 in isotonic saline (about 12 urn with 10 mM SPX-101 in 0.9% saline compared to about 30 μηι with the same dose of SPX-101 in 3.0% saline). Moreover, the combined effects of hypertonic saline and SPX-101 were synergistic, e.g. , greater than additive.

[0194] Assessment of the combined effect of hypertonic saline and SPX-101 in an in vivo sheep model

[0195] The combined effect of SPX-101 and hypertonic saline on improving MCC was further assessed using an ovine model of cystic fibrosis-like disease. In this model, sheep were nebulized with the CFTR small molecule inhibitor, CFTRinhl72, and tracheal mucus velocity (TMV) is measured by the movement of radio-opaque disks. CFTRinhl72 causes a -50% reduction in TMV four hours after administration that is sustained for greater than twelve hours (FIG. 16). To determine if SPX-101 could restore TMV, sheep were nebulized with SPX-101 peptide four hours after exposure to CFTRinhl72 and disk movement was then monitored for an additional eight hours. The study was conducted with three different concentrations of hypertonic saline (2.7%, 4.2%, or 7.0%, as indicated) as vehicle for SPX-101 and compared to isotonic saline vehicle (0.9%; control). A dose dependent effect on the restoration of TMV was observed with increasing salinity of the vehicle, and all hypertonic saline formulations tested improved TMV in comparison to the isotonic saline formulation. Notably, SPX-101 in 7.0% saline confered complete restoration of TMV in this model, which lasted for the full 12-hour duration of the study. In contrast, isotonic saline (0.9%; control) only restored TMV to 80% of the initial value, and the effect waned over time. These results demonstrate that hypertonic saline in combination with SPX-101 synergize in restoring TMV in this sheep model of CF.

[0196] 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 sub-combinations 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.

[0197] 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.

[0198] 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.

[0199] The following references, which have been cited in the instant disclosure, are incorporated by reference herein in their entirety.

[0200] 1. Riordan JR. CFTR function and prospects for therapy. Annu Rev Biochem 2008; 77: 701-726.

[0201] 2. Donaldson SH, Boucher RC. Sodium channels and cystic fibrosis. Chest 2007; 132: 1631-1636.

[0202] 3. Knowles M, Gatzy J, Boucher R. Relative ion permeability of normal and cystic fibrosis nasal epithelium. / Clin Invest 1983; 71: 1410-1417.

[0203] 4. Tarran R, Redinbo MR. Mammalian short palate lung and nasal epithelial clone 1 (SPLUNCl) in pH-dependent airway hydration. Int J Biochem Cell Biol 2014; 52: 130- 135.

[0204] 5. Althaus M. ENaC inhibitors and airway re-hydration in cystic fibrosis: state of the art. Curr Mol Pharmacol 2013; 6: 3-12.

[0205] 6. Mall MA, Graeber SY, Stahl M, Zhou-Suckow Z. Early cystic fibrosis lung disease: Role of airway surface dehydration and lessons from preventive rehydration therapies in mice. Int J Biochem Cell Biol 2014; 52: 174-179. [0206] 7. Haq IJ, Gray MA, Garnett JP, Ward C, Brodlie M. Airway surface liquid homeostasis in cystic fibrosis: pathophysiology and therapeutic targets. Thorax 2016; 71: 284- 287.

[0207] 8. Ciofu O, Hansen CR, Hoiby N. Respiratory bacterial infections in cystic fibrosis. Curr Opin Pulm Med 2013; 19: 251-258.

[0208] 9. Cantin AM, Hartl D, Konstan MW, Chmiel JF. Inflammation in cystic fibrosis lung disease: Pathogenesis and therapy. / Cyst Fibros 2015; 14: 419-430.

[0209] 10. Puchelle E, Bajolet O, Abely M. Airway mucus in cystic fibrosis. Paediatr Respir Rev 2002; 3: 115-119.

[0210] 11. Bennett WD, Wu J, Fuller F, Balcazar JR, Zeman KL, Duckworth H, Donn KH, O'Riordan TG, Boucher RC, Donaldson SH. Duration of action of hypertonic saline on mucociliary clearance in the normal lung. JAppl Physiol (1985) 2015; 118: 1483-1490.

[0211] 12. Yang C, Chilvers M, Montgomery M, Nolan SJ. Dornase alfa for cystic fibrosis. Cochrane Database Syst Rev 2016; 4: CD001127.

[0212] 13. Shteinberg M, Elborn JS. Use of inhaled tobramycin in cystic fibrosis. Adv Ther 2015; 32: 1-9.

[0213] 14. Hurt K, Bilton D. Inhaled interventions in cystic fibrosis: mucoactive and antibiotic therapies. Respiration 2014; 88: 441-448.

[0214] 15. Van Goor F, Hadida S, Grootenhuis PD, Burton B, Cao D, Neuberger T, Turnbull A, Singh A, Joubran J, Hazlewood A, Zhou J, McCartney J, Arumugam V, Decker C, Yang J, Young C, Olson ER, Wine JJ, Frizzell RA, Ashlock M, Negulescu P. Rescue of CF airway epithelial cell function in vitro by a CFTR potentiator, VX-770. Proc Natl Acad Sci U S A 2009; 106: 18825-18830.

[0215] 16. Van Goor F, Hadida S, Grootenhuis PD, Burton B, Stack JH, Straley KS, Decker CJ, Miller M, McCartney J, Olson ER, Wine JJ, Frizzell RA, Ashlock M, Negulescu PA. Correction of the F508del-CFTR protein processing defect in vitro by the investigational drug VX-809. Proc Natl Acad Sci U SA 2011; 108: 18843-18848.

[0216] 17. Graham A, Hasani A, Alton EW, Martin GP, Marriott C, Hodson ME, Clarke SW, Geddes DM. No added benefit from nebulized amiloride in patients with cystic fibrosis. Eur Respir J 1993; 6: 1243-1248. [0217] 18. Hofmann T, Senier I, Bittner P, Huls G, Schwandt HJ, Lindemann H.

Aerosolized amiloride: dose effect on nasal bioelectric properties, pharmacokinetics, and effect on sputum expectoration in patients with cystic fibrosis. J Aerosol Med 1997; 10: 147-158.

[0218] 19. Donaldson SH, Bennett WD, Zeman KL, Knowles MR, Tarran R, Boucher RC. Mucus clearance and lung function in cystic fibrosis with hypertonic saline. N Engl J Med 2006; 354: 241-250.

[0219] 20. Hirsh AJ. Altering airway surface liquid volume: inhalation therapy with amiloride and hyperosmotic agents. Adv Drug Deliv Rev 2002; 54: 1445-1462.

[0220] 21. Hirsh AJ, Sabater JR, Zamurs A, Smith RT, Paradiso AM, Hopkins S, Abraham WM, Boucher RC. Evaluation of second generation amiloride analogs as therapy for cystic fibrosis lung disease. J Pharmacol Exp Ther 2004; 311: 929-938.

[0221] 22. O'Riordan TG, Donn KH, Hodsman P, Ansede JH, Newcomb T, Lewis SA, Flitter WD, White VS, Johnson MR, Montgomery AB, Warnock DG, Boucher RC. Acute hyperkalemia associated with inhalation of a potent ENaC antagonist: Phase 1 trial of GS-9411. J Aerosol Med Pulm Drug Deliv 2014; 27: 200-208.

[0222] 23. App EM, King M, Helfesrieder R, Kohler D, Matthys H. Acute and long- term amiloride inhalation in cystic fibrosis lung disease. A rational approach to cystic fibrosis therapy. Am Rev Respir Dis 1990; 141: 605-612.

[0223] 24. Rodgers HC, Knox AJ. The effect of topical benzamil and amiloride on nasal potential difference in cystic fibrosis. Eur Respir J 1999; 14: 693-696.

[0224] 25. Burrows EF, Southern KW, Noone PG. Sodium channel blockers for cystic fibrosis. Cochrane Database Syst Rev 2014: CD005087.

[0225] 26. Garcia-Caballero A, Rasmussen JE, Gaillard E, Watson MJ, Olsen JC, Donaldson SH, Stutts MJ, Tarran R. SPLUNC1 regulates airway surface liquid volume by protecting ENaC from proteolytic cleavage. Proc Natl Acad Sci U S A 2009; 106: 11412-11417.

[0226] 27. Garland AL, Walton WG, Coakley RD, Tan CD, Gilmore RC, Hobbs CA, Tripathy A, Clunes LA, Bencharit S, Stutts MJ, Betts L, Redinbo MR, Tarran R. Molecular basis for pH-dependent mucosal dehydration in cystic fibrosis airways. Proc Natl Acad Sci U S A 2013; 110: 15973-15978.

[0227] 28. Hobbs CA, Blanchard MG, Alijevic O, Tan CD, Kellenberger S, Bencharit S, Cao R, Kesimer M, Walton WG, Henderson AG, Redinbo MR, Stutts MJ, Tarran R. Identification of the SPLUNCl ENaC-inhibitory domain yields novel strategies to treat sodium hyperabsorption in cystic fibrosis airway epithelial cultures. Am J Physiol Lung Cell Mol Physiol 2013; 305: L990-L1001.

[0228] 29. Mall M, Grubb BR, Harkema JR, O'Neal WK, Boucher RC. Increased airway epithelial Na+ absorption produces cystic fibrosis-like lung disease in mice. Nat Med 2004; 10: 487-493.

[0229] 30. Tarran R, Sabater JR, Clarke TC, Tan CD, Davies CM, Liu J, Yeung A, Garland AL, Stutts MJ, Abraham WM, Phillips G, Baker WR, Wright CD, Wilbert S.

Nonantibiotic macrolides prevent human neutrophil elastase-induced mucus stasis and airway surface liquid volume depletion. Am J Physiol Lung Cell Mol Physiol 2013; 304: L746-756.

[0230] 31. Fulcher ML, Gabriel S, Burns KA, Yankaskas JR, Randell SH. Well- differentiated human airway epithelial cell cultures. Methods Mol Med 2005; 107: 183-206.

[0231] 32. Rollins BM, Garcia-Caballero A, Stutts MJ, Tarran R. SPLUNCl expression reduces surface levels of the epithelial sodium channel (ENaC) in Xenopus laevis oocytes. Channels (Austin) 2010; 4: 255-259.

[0232] 33. Knowles M, Gatzy J, Boucher R. Increased bioelectric potential difference across respiratory epithelia in cystic fibrosis. N Engl J Med 1981; 305: 1489-1495.

[0233] 34. Alton EW, Rogers DF, Logan- Sinclair R, Yacoub M, Barnes PJ, Geddes DM. Bioelectric properties of cystic fibrosis airways obtained at heart-lung transplantation. Thorax 1992; 47: 1010-1014.

[0234] 35. Wielputz MO, Eichinger M, Zhou Z, Leotta K, Hirtz S, Bartling SH, Semmler W, Kauczor HU, Puderbach M, Mall MA. In vivo monitoring of cystic fibrosis-like lung disease in mice by volumetric computed tomography. Eur Respir J 2011; 38: 1060-1070.

[0235] 36. Zhou Z, Duerr J, Johannesson B, Schubert SC, Treis D, Harm M, Graeber SY, Dalpke A, Schultz C, Mall MA. The ENaC-overexpressing mouse as a model of cystic fibrosis lung disease. / Cyst Fibros 2011; 10 Suppl 2: S172-182.

[0236] 37. Zhou Z, Treis D, Schubert SC, Harm M, Schatterny J, Hirtz S, Duerr J, Boucher RC, Mall MA. Preventive but not late amiloride therapy reduces morbidity and mortality of lung disease in betaENaC-overexpressing mice. Am J Respir Crit Care Med 2008; 178: 1245-1256. [0237] 38. Fellner RC, Terryah ST, Tarran R. Inhaled protein/peptide-based therapies for respiratory disease. Mol Cell Pediatr 2016; 3: 16.

[0238] 39. Bangel N, Dahlhoff C, Sobczak K, Weber WM, Kusche-Vihrog K.

Upregulated expression of ENaC in human CF nasal epithelium. / Cyst Fibros 2008; 7: 197- 205.

[0239] 40. Gaillard EA, Kota P, Gentzsch M, Dokholyan NV, Stutts MJ, Tarran R. Regulation of the epithelial Na+ channel and airway surface liquid volume by serine proteases. Pflugers Arch 2010; 460: 1-17.

[0240] 41. Stutts MJ, Canessa CM, Olsen JC, Hamrick M, Cohn JA, Rossier BC, Boucher RC. CFTR as a cAMP-dependent regulator of sodium channels. Science 1995; 269: 847-850.

[0241] 42. Donaldson SH, Hirsh A, Li DC, Holloway G, Chao J, Boucher RC, Gabriel SE. Regulation of the epithelial sodium channel by serine proteases in human airways. J Biol Chem 2002; 277: 8338-8345.

Claims

That which is claimed is:

1. A pharmaceutical composition comprising a polypeptide comprising the sequence of SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 141, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 128, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:131, SEQ ID NO:139; SEQ ID NO: 140, SEQ ID NO: 144, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 136, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8, wherein the polypeptide is formulated in a saline solution containing a salt concentration of at least 1%.

2. The pharmaceutical composition of claim 1, wherein the salt concentration is at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20%.

3. The pharmaceutical composition of claim 2, wherein the salt concentration is at least 4%.

4. The pharmaceutical composition of claim 3, wherein the salt concentration is at least 4.2%.

5. The pharmaceutical composition of claim 4, wherein the salt concentration is 4.2%.

6. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

7. The pharmaceutical composition of claim 1, wherein the salt is sodium chloride.

8. The pharmaceutical composition of any one of claims 1-7, wherein the salt

concentration is between 1%-10%, endpoints inclusive.

9. The pharmaceutical composition of any one of claims 1-7, wherein the salt concentration is between 2%-10%, endpoints inclusive.

10. The pharmaceutical composition of any one of claims 1-7, wherein the salt

concentration is between 2%-7%, endpoints inclusive.

11. The pharmaceutical composition of any one of claims 1-7, wherein the salt

concentration is between 3%-7%, endpoints inclusive.

12. The pharmaceutical composition of any one of claims 1-7, wherein the salt

concentration is between 3%-6%, endpoints inclusive.

13. The pharmaceutical composition of any one of claims 1-7, wherein the salt

concentration is between l%-8%; l%-7%; l%-6%; l%-5%; l%-4%; l%-3%; 1%- 2%; 2%-9%; 2%-8%; 2%-7%; 2%-6%; 2%-5%; 2%-4%; 2%-3%; 3%-9%; 3%-8%; 3%-7%; 3%-6%; 3%-5%; 3%-4%; 4%-9%; 4%-8%; 4%-7%; 4%-6%; 4%-5%; 5%- 9%; 5%-8%; 5%-7%; 5%-6%; 6%-9%; 6%-8%; 6%-7%; 7%-9%; 7%-8%; or 8%-9%, e.g. , about 1.0%, about 1.2%, about 1.4%, about 1.6%, about 1.8%, about 2.0%, about 2.2%, about 2.4%, about 2.6%, about 2.8%, about 3.0%, about 3.2%, about 3.4%, about 3.6%, about 3.8%, about 4.0%, about 3.0%, about 4.2%, about 4.4%, about 4.6%, about 4.8%, about 5.0%, about 5.2%, about 5.4%, about 5.6%, about 5.8%, about 6.0%, about 6.2%, about 6.4%, about 6.6%, about 6.8%, about 7.0%, about 7.2%, about 7.4%, about 7.6%, about 7.8%, about 8.0%, about 8.5%, or about 9%.

14. The pharmaceutical composition of any one of claims 1-13, wherein the polypeptide comprises the sequence of SEQ ID NO: 127 or SEQ ID NO: 128.

15. The pharmaceutical composition of claim 14, wherein the polypeptide comprises the sequence of SEQ ID NO: 127.

16. The pharmaceutical composition of claim 15, wherein the polypeptide consists of the sequence of SEQ ID NO: 127.

17. The pharmaceutical composition of claim 14, wherein the polypeptide comprises the sequence of SEQ ID NO: 128.

18. The pharmaceutical composition of claim 17, wherein the polypeptide consists of the sequence of SEQ ID NO: 128.

19. The pharmaceutical composition of claim 1, wherein the polypeptide is modified to modulate its bioactivity.

20. The pharmaceutical composition of claim 19, wherein the polypeptide is modified to reduce or eliminate the inactivation of the polypeptide at an acidic pH.

21. The pharmaceutical composition of claim 19, wherein the polypeptide is modified by the addition of a blocking agent to facilitate survival of the polypeptide in vivo.

22. The pharmaceutical composition of claim 19, wherein the polypeptide is fused to a protein that increases the stability of the polypeptide.

23. The pharmaceutical composition of claim 19, wherein the N-terminus of the

polypeptide is acetylated.

24. The pharmaceutical composition of claim 19, wherein the polypeptide comprises at least one non-natural amino acid or at least one terminal modification.

25. The pharmaceutical composition of claim 19, wherein the polypeptide further

comprises at least one D-alanine at the N-terminus and/or the C-terminus.

26. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is formulated to be administered by inhalation, intranasally, intravenously, intramuscularly, intraocularly, transdermally, or orally.

27. The pharmaceutical composition of claim 26, wherein pharmaceutical composition is formulated to be administered by inhalation.

28. The pharmaceutical composition of claim 27, wherein pharmaceutical composition is administered by a metered dose, inhaler, nebulizer, or in a mist sprayer.

29. A kit comprising the pharmaceutical composition of claim 28.

30. A method of inhibiting activity of a sodium channel, comprising contacting the

sodium channel with a pharmaceutical composition comprising a polypeptide comprising a sodium channel binding domain of a PLUNC protein, or a functional fragment, or homolog thereof, wherein the polypeptide, or the functional fragment, or homolog thereof binds to the sodium channel, and wherein the polypeptide, or the functional fragment, or homolog thereof is formulated in a saline solution containing a salt concentration of at least 1%.

31. The method of claim 30, wherein the salt concentration is at least 2%, at least 3%, at least 4%, at least 5%, or at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20%.

32. The method of claim 31, wherein the salt concentration is at least 4%.

33. The method of claim 32, wherein the salt concentration is at least 4.2%

34. The method of claim 33, wherein the salt concentration is 4.2%.

35. The method of claim 30, wherein the sodium channel is ENaC.

36. The method of claim 30, wherein the PLUNC protein is a human PLUNC protein.

37. The method of claim 30, wherein the polypeptide is a SPLUNC1 protein.

38. The method of claim 37, wherein the SPLUNC1 protein is a human SPLUNC1

protein.

39. The method of claim 30, wherein the polypeptide comprises the sequence of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 141, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 128, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 131, SEQ ID NO: 139; SEQ ID NO: 140, SEQ ID NO: 144, SEQ ID

NO: 127, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO: 136, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8.

40. The method of claim 39, wherein the polypeptide comprises the sequence of SEQ ID NO: 127.

41. The method of claim 40, wherein the polypeptide comprises the sequence of SEQ ID NO:128.

42. The method of claim 30, wherein the activity of the sodium channel is inhibited by at least 20%.

43. The method of claim 30, wherein the activity of the sodium channel is inhibited by at least 50%.

44. The method of claim 30, wherein the activity of the sodium channel is inhibited by at least 60%.

45. The method of claim 30, wherein the activity of the sodium channel is inhibited by at least 90%.

46. The method of claim 30, wherein the inhibition of activity occurs by internalization of the sodium channel.

47. The method of claim 35, wherein the inhibition of activity occurs by internalization of one or more subunits of ENac.

48. A method of inhibiting sodium absorption through a sodium channel, comprising contacting the sodium channel with a pharmaceutical composition comprising a polypeptide comprising a sodium channel binding domain of a PLUNC protein, or a functional fragment, or homolog thereof, wherein the polypeptide, or the functional fragment, or homolog thereof binds to the sodium channel, and wherein the polypeptide, or the functional fragment, or homolog thereof is formulated in a saline solution containing a salt concentration of at least 1%.

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

50. 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 pharmaceutical composition comprising a polypeptide comprising a sodium channel binding domain of a PLUNC protein, or a functional fragment, or homolog thereof, wherein the polypeptide, or the functional fragment, or homolog thereof binds to the sodium channel, and wherein the polypeptide, or the functional fragment, or homolog thereof is formulated in a saline solution containing a salt concentration of at least 1%.

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

52. The method of claim 50, wherein increasing the volume of fluid lining an epithelial mucosal surface increases the activity of cystic fibrosis transmembrane conductance regulator (CFTR).

53. The method of claim 50, wherein increasing the volume of fluid lining an epithelial mucosal surface increases the expression level of CFTR.

54. A method of reducing the level of a sodium channel present on the surface of a cell, comprising contacting the sodium channel with a pharmaceutical composition comprising a polypeptide comprising a sodium channel binding domain of a PLUNC protein, or a functional fragment, or homolog thereof, wherein the polypeptide, or the functional fragment, or homolog thereof binds to the sodium channel, and wherein the polypeptide, or the functional fragment, or homolog thereof is formulated in a saline solution containing a salt concentration of at least 1%.

55. The method of claim 54, wherein the sodium channel is ENaC.

56. A method of treating a disorder responsive to inhibition of sodium absorption across an epithelial mucosal surface in a subject in need thereof, comprising delivering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a polypeptide comprising a sodium channel binding domain of a PLUNC protein, or a functional fragment, or homolog thereof, wherein the polypeptide, or the functional fragment, or homolog thereof binds to a sodium channel, and wherein the polypeptide, or the functional fragment, or homolog thereof is formulated in a saline solution containing a salt concentration of at least 1%.

57. The method of claim 56, wherein the sodium channel is ENaC.

58. The method of claim 56, wherein the disorder is a lung disorder.

59. The method of claim 58, wherein the wherein the lung disorder is cystic fibrosis.

60. The method of claim 58, wherein the lung disorder is non-cystic fibrosis

bronchiectasis.

61. The method of claim 58, wherein the lung disorder is acute or chronic bronchitis.

62. The method of claim 58, wherein the lung disorder is severe asthma.

63. The method of claim 58, wherein the lung disorder is chronic obstructive pulmonary disease (COPD).

64. The method of claim 56, wherein the disorder is a gastrointestinal disorder.

65. The method of claim 64, wherein the method decreases constipation associated with the gastrointestinal disorder.

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

67. The method of claim 56, wherein the disorder is a kidney disorder.

68. A method of regulating salt balance and/or fluid volume regulation in a subject in need thereof, comprising delivering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a polypeptide comprising a sodium channel binding domain of a PLUNC protein, or a functional fragment, or homolog thereof, wherein the polypeptide, or the functional fragment, or homolog thereof binds to a sodium channel present on the surface of a cell in the subject, and wherein the polypeptide, or the functional fragment, or homolog thereof is formulated in a saline solution containing a salt concentration of at least 1%.

69. The method of claim 68, wherein delivering the pharmaceutical composition reduces the level of the sodium channel.

70. The method of claim 69, wherein delivering the pharmaceutical composition reduces the concentration of the sodium channel.

71. The method of claim 70, wherein delivering the pharmaceutical composition reduces the cell surface concentration of the sodium channel.

72. The method of claim 70, wherein delivering the pharmaceutical composition reduces the intracellular concentration of the sodium channel.

73. The method of claim 69, wherein delivering the pharmaceutical composition reduces the activity of the sodium channel.

74. The method of claim 68, wherein the sodium channel is ENaC.

75. A method for treating a symptom of 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 pharmaceutical composition comprising a polypeptide comprising a sodium channel binding domain of a PLUNC protein, or a functional fragment, or homolog thereof, wherein the polypeptide, or the functional fragment, or homolog thereof binds to a sodium channel present on the surface of a cell in the subject, and wherein the polypeptide, or the functional fragment, or homolog thereof is formulated in a saline solution containing a salt concentration of at least 1%.

76. The method of claim 75, wherein the lung disorder is cystic fibrosis, non-cystic fibrosis bronchiectasis, chronic obstructive pulmonary disease, acute or chronic bronchitis, or asthma.

77. The method of claim 75, wherein the method decreases constipation associated with the gastrointestinal disorder.

78. The method of claim 75, wherein the gastrointestinal disorder is inflammatory bowel disease.

79. A method of inhibiting activity of a sodium channel, comprising contacting the sodium channel with:

(i) a pharmaceutical composition comprising a polypeptide comprising a sodium channel binding domain of a PLUNC protein, or a functional fragment, or homolog thereof, wherein the polypeptide, or the functional fragment, or homolog thereof binds to the sodium channel; and

(ii) a hypertonic saline solution.

80. The method of claim 79, wherein the hypertonic saline solution contains a salt

concentration of 4.2%.

81. The method of claim 79, wherein the pharmaceutical composition and the hypertonic saline solution is administered simultaneously.

82. The method of claim 79, wherein the hypertonic saline solution is administered after the administration of the pharmaceutical composition.

83. The method of claim 79, wherein the polypeptide, or the functional fragment, or homolog thereof is administered by inhalation, intranasally, intravenously, intramuscularly, intraocularly, transdermally, or orally.

84. The method of claim 83, wherein the polypeptide, or the functional fragment, or homolog thereof is administered by inhalation.

85. The method of claim 79, wherein the sodium channel is ENaC.

86. A method of inhibiting sodium absorption through a sodium channel, comprising contacting the sodium channel with:

(ii) a hypertonic saline solution.

87. 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:

(iii) a pharmaceutical composition comprising a polypeptide comprising a sodium channel binding domain of a PLUNC protein, or a functional fragment, or homolog thereof, wherein the polypeptide, or the functional fragment, or homolog thereof binds to the sodium channel; and

(iv) a hypertonic saline solution.

88. A method of reducing the level of a sodium channel present on the surface of a cell, comprising contacting the sodium channel with:

(ii) a hypertonic saline solution.

89. A method of treating a disorder responsive to inhibition of sodium absorption across an epithelial mucosal surface in a subject in need thereof, comprising delivering to the subject:

(i) a therapeutically effective amount of a pharmaceutical composition

comprising a polypeptide comprising a sodium channel binding domain of a PLUNC protein, or a functional fragment, or homolog thereof, wherein the polypeptide, or the functional fragment, or homolog thereof binds to a sodium channel; and

(ii) a hypertonic saline solution.

90. A method of regulating salt balance and/or fluid volume regulation in a subject in need thereof, comprising delivering to the subject a therapeutically effective amount of a pharmaceutical composition comprising:

(i) a polypeptide comprising a sodium channel binding domain of a PLUNC protein, or a functional fragment, or homolog thereof, wherein the polypeptide, or the functional fragment, or homolog thereof binds to a sodium channel present on the surface of a cell in the subject; and

(ii) a hypertonic saline solution.

91. A method for treating a symptom of a lung disorder, a gastrointestinal disorder, a kidney disorder, or a cardiovascular disorder in a subject in need thereof, comprising administering to the subject:

(i) a therapeutically effective amount of a pharmaceutical composition

comprising a polypeptide comprising a sodium channel binding domain of a PLUNC protein, or a functional fragment, or homolog thereof, wherein the polypeptide, or the functional fragment, or homolog thereof binds to a sodium channel present on the surface of a cell in the subject; and

(ii) a hypertonic saline solution.

92. A method of treating a disorder responsive to inhibition of sodium absorption across an epithelial mucosal surface in a subject in need thereof, comprising delivering to the subject:

(i) a therapeutically effective amount of a pharmaceutical composition

(ii) a compound that modulates the function of cystic fibrosis transmembrane conductance regulator (CFTR).

93. The method of claim 92, wherein the compound is selected from the group consisting of ivacaftor, lumacaftor, VX-661, tezacaftor (VX-661-111), deuterated ivacaftor (CTP-656), cavosonstat (N91115), acebilustat (CTX-4430), ataluren, riociguat, QR- 010, QBW251, FDL169, QBW251, PTI-428, JBT-101, GS-5745, LAU-7b, POL6014, immobilized lipase, liprotamase, gallium nitrate, tobramycin, azithromycin, aztreonam, levofloxacin, amikacin, fosfomycin, vancomycin, and inhaled nitric oxide.

94. The method of claim 92, wherein the pharmaceutical composition and the compound are delivered to the subject concurrently.

95. The method of claim 92, wherein the pharmaceutical composition and the compound are delivered to the subject simultaneously.

96. A method of treating a disorder responsive to inhibition of sodium absorption across an epithelial mucosal surface in a subject in need thereof, comprising delivering to the subject:

(i) a therapeutically effective amount of a pharmaceutical composition

(ii) a mucolytic compound.

97. The method of claim 96, wherein the mucolytic compound is selected from the group consisting of acetylcysteine, ambroxol, carbocisteine, erdosteine, mecysteine, dornase alfa, oligoG, VX-371, AZD5634, and inhaled mannitol.

98. A method of treating a disorder responsive to inhibition of sodium absorption across an epithelial mucosal surface in a subject in need thereof, comprising delivering to the subject:

(i) a therapeutically effective amount of a pharmaceutical composition

(ii) a long acting beta-agonist compound (LABA).

99. The method of claim 98, wherein the LABA is selected from the group consisting of albuterol, levalbuterol, formoterol, and salmeterol.

100. A pharmaceutical composition comprising a polypeptide comprising a sodium

channel binding domain of a PLUNC protein, or a functional fragment, or homolog thereof and a pharmaceutically acceptable osmolality adjusting agent, wherein the polypeptide, or the functional fragment, or homolog thereof binds to a sodium channel.

101. The pharmaceutical composition of claim 100, wherein the polypeptide, or the functional fragment, or homolog thereof is formulated in a saline solution containing a salt concentration of at least 0.9%.

102. The pharmaceutical composition of claim 100, wherein the osmolality adjusting agent is one or more selected from the group consisting of mannitol, xylitol, sorbitol, isomaltol, glucose, lactose, dextrose, sucrose, trehalose, maltose, glycerin, propylene glycol, ethylene glycol, glycerol, glycine, dimethylsulphoxide, calcium chloride, sodium sulfate, magnesium chloride, sodium gluconate, sodium pyruvate, pentosane polysulfate, and a cyclodextrin.

103. A method of treating a disorder responsive to inhibition of sodium absorption across an epithelial mucosal surface in a subject in need thereof, comprising delivering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a polypeptide comprising a sodium channel binding domain of a PLUNC protein, or a functional fragment, or homolog thereof and a pharmaceutically acceptable osmolality adjusting agent, wherein the polypeptide, or the functional fragment, or homolog thereof binds to a sodium channel.