WO2016065282A1 - Nasal formulation, nasal kit, and method for enhancing nasal nitric oxide (no) levels - Google Patents

Abstract

A nasal formulation includes a liquid carrier to be introduced into upper airways through a nasal cavity. Present in the liquid carrier are low molecular weight arginine-rich peptides derived from protamine. When introduced into the upper airways, the low molecular weight arginine-rich peptides are to increase nitric oxide (NO) levels in the upper airways. The nasal formulation has a pH ranging from 4 to 10.

Description

NASAL FORMULATION, NASAL KIT, AND

METHOD FOR ENHANCING NASAL NITRIC OXIDE (NO) LEVELS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application S.N. 62/068,155, filed October 24, 2014, which is incorporated by reference herein in its entirety.

BACKGROUND

[0001] Nitric oxide (NO) may be produced by any of several iso forms of the enzyme nitric oxide synthase (NOS). NO is central to the mammalian immune response or defense, and is a cytotoxic agent in the mechanisms used by macrophages to kill L. major, M. bovis, and M. tuberculosis, among numerous other species of bacteria. NO is produced from L-arginine in the airways (e.g., in the upper respiratory tract) by immune cells (macrophages, neutrophils, lymphocytes, etc.) and airway epithelial cells (e.g., conductive and respiratory epithelial cells) primarily through inducible nitric oxide synthase (iNOS). Deficiencies in NO production can lead to decreased immune response and/or biofilm formation. The deficiencies in nasal NO levels have been linked with diseases, such as primary ciliary dyskinesia and chronic rhinosinusitis (CRS).

SUMMARY

[0002] A nasal formulation includes a liquid carrier to be introduced into upper airways through a nasal cavity. Present in the liquid carrier are low molecular weight arginine-rich peptides derived from protamine. When introduced into the upper airways, the low molecular weight arginine-rich peptides are able to increase nitric oxide (NO) levels in the upper airways. The nasal formulation has a pH ranging from 4 to 10.

BRIEF DESCRIPTION OF THE DRAWINGS

[0003] Features of examples of the present disclosure will become apparent by reference to the following detailed description and drawings. [0004] Fig. 1 is a schematic illustration of an example of the nasal formulation being introduced into upper airways through a nasal cavity, where the enlarged illustration shows the production of nitric oxide gas (NO_(g)) from low molecular weight arginine-rich peptides (Arg)_x;

[0005] Fig. 2A illustrates the primary structure of one example of protamine (SEQ ID NO: 1; Grade X from salmon sperm obtained from Sigma Aldrich) and smaller digest fragments (SEQ ID NOS: 4-8) as determined by amino acid analysis;

[0006] Fig. 2B illustrates a flow diagram of a Thermolysin-silica conjugation process;

[0007] Fig. 2C illustrates a Bradford assay calibration curve of cell penetrating peptides (CPP) of molecular weight < 3000 containing at least 6 arginine residues and two tryptophans and one phenylalanine (·) and of low molecular weight arginine-rich peptides (e.g., low molecular weight protamine, LMWP) (■);

[0008] Fig. 2D is a graph illustrating the stability of a Thermolysin-silica column over 14 days of re-use;

[0009] Fig. 2E is a Size Exclusion Chromatogram obtained from a Sephadex G-15 column by gravity elution of 1 mL fractions as measured by UV/Vis spectrophotometry and

conductivity;

[0010] Fig. 3 A is a representative immunoblot of the stimulation of RAW 264.7 cells with cytomix 1 (10 ng/mL IFNg and 20 ng/mL LPS);

[0011] Fig. 3B is a graph illustrating the nitrite concentrations of media in stimulated RAW 264.7 cells compared to unstimulated RAW 264.7 cells (unpaired, one-tailed t-test p-value < 0.001, data are average ± s.d. of four independent experiments for each group of cells);

[0012] Fig. 3C is a representative immunoblot of the stimulation of LA4 cells with cytomix 2 (10 ng/mL IFNg, 20 ng/mL LPS, 10 ng/mL IL-1B, and 2 ng/mL TNFa);

[0013] Fig. 3D is a graph illustrating the nitrite concentrations of media in stimulated LA4 cells compared to unstimulated LA4 cells (unpaired one -tailed t-test p-value < 0.001, data are average ± s.d. of three independent experiments for each group of cells);

[0014] Fig. 3E is a representative immunoblot indicating that the expression of iNOS in LA4 cells is dependent on the L-arginine concentration in the media; [0015] Fig. 3F is a graph illustrating the nitrite concentrations in the media of LA4 cells incubated in L-arginine concentrations from 10 μΜ to 1000 μΜ (data are average ± s.d. for six independent experiments per concentration);

[0016] Fig. 3G is a representative immunoblot indicating that the expression of iNOS in RAW 264.7 cells is dependent on the L-arginine concentration in the media;

[0017] Fig. 3H is a graph illustrating the nitrite concentrations in media of RAW 264.7 cells as L-arginine concentrations are varied from 1 μΜ to 1100 μΜ (data are average ± s.d. for four independent experiments per concentration);

[0018] Fig. 4A is a graph illustrating the NO production (measured as nitrite in the cell media) in RAW 264.7 cells stimulated with 40 μΜ LMWP (400 μΜ total L-arginine) and in control cells (unpaired, two-tailed t-test p < 0.001, data are average ± s.d. for four independent experiments per concentration);

[0019] Fig. 4B is a representative immunoblot illustrating that LMWP does not cause iNOS expression without prior stimulation by cytokines;

[0020] Fig. 4C is a graph illustrating the NO production (measured as nitrite in the cell media) versus the preincubated L-arginine concentration of the media for RAW 264.7 cells preincubated in L-arginine deficient media for 6 hours before stimulation and addition of either additional L-arginine or LMWP (data are average ± s.d. for four independent experiments per experimental condition; using unpaired, two-tailed t-test, *: p = 0.01, **: p = 0.002);

[0021] Fig. 4D is a graph illustrating the NO production (measured as nitrite in the cell media) over a 4 hour period for RAW 264.7 cells after the addition of L-arginine or LMWP, the L-arginine or LMWP having been added to the cells 16 hours after stimulation in media with varying L-arginine concentrations (10 μΜ, 100 μΜ, and 400 μΜ) (each data point corresponds to one independent experiment);

[0022] Fig. 4E is a graph illustrating the nitrite concentrations in media of cells treated with no addition (control), L-arginine, and LMWP, 12 hours, 16 hours, and 24 hours after stimulation and addition (Unpaired, two-tailed t-test, **: p = 0.0021, p < 0.001);

[0023] Fig. 4F is a graph illustrating the increased difference in media nitrite concentration over time for cells treated with LMWP compared to L-arginine (each bar is comprised of four independent experiments); [0024] Fig. 4G is a graph illustrating the densitometry of a nitric oxide synthase (iNOS) immunoblot for cells treated with no addition (control), L-arginine, and LMWP over time (unpaired two-tailed t-test p = 0.0038), each lane of blot included four independent experiments for a total n=12 for each treatment, the error bars are standard deviation of relative density for each protein band to assess for significant differences comparatively;

[0025] Fig. 5A is a graph illustrating the dependence of NO production (measured as nitrite in the cell media) in RAW 264.7 cells on the concentration of the inhibitor s-boronoethyl-L- cysteine (BEC) added to the cell media after 24 hours of incubation (each point represents one experiment);

[0026] Fig. 5B is a graph illustrating that LMWP and BEC together synergistically increase NO production in RAW 264.7 cells over 24 h ($: paired, one-tailed t-test comparing treatment 6 (LMWP+BEC) to the added value of treatments 2 (BEC) and 5 (LMWP) p = 0.0072) and NO production is higher than either L-arginine+BEC or LMWP alone (†,*: paired, two-tailed t-test p-values = 0.0023,0.0026);

[0027] Fig. 5C is a graph illustrating that LMWP is effective in increasing NO production in LA4 cells over 24 hours compared to control cells (*: paired, one-tailed t-test p-value = 0.0037) and those given an equivalent concentration of L-arginine (-: paired, one -tailed t-test p-value = 0.043), LMWP+BEC do not elicit synergistic increases in NO production and are statistically significant compared to treatment with L-arginine alone ($: paired, one-tailed t-test p value = 0.0027) and BEC (†: paired, one-tailed t-test p value = .024);

[0028] Figs. 5D and 5E are representative immunoblots of iNOS expression over 24 hours in stimulated RAW 264.7 and LA4 cells, respectively;

[0029] Fig. 5F is a graph illustrating the time-dependent densitometry analysis of immunoblots of iNOS in stimulated RAW 264.7 cells (LMWP+BEC peaks at 16 hours, which is significant compared to L-arginine [*: paired, two-tailed t-test p-value = 0.0089] and L- arginine+BEC [†: paired, two-tailed t-test p-value = 0.032] but falls after 26 hours; each point is comprised of four independent experiments and error bars are standard deviation of relative density for each protein band to assess for significant differences comparatively);

[0030] Fig. 6 is a graph illustrating the nitrite (in terms of fold increase over control cells having a nitrite level of 0.327+0.085) increase in unstimulated and stimulated human tracheal epithelial cells treated with phosphate buffered saline (PBS), L-arginine, and LMWP (data are average for six replicate experiments; * mean ± standard error of the mean; # = different from all other groups);

[0031] Fig. 7A is a graph illustrating the expression of Interleukin 8 (IL-8) in ng/ml for the unstimulated and stimulated human tracheal epithelial cells treated with phosphate buffered saline (PBS) or PBS and LMWP (data are average for six replicate experiments, mean ± standard error of the mean);

[0032] Fig. 7B is a graph illustrating the expression of lactate dehydrogenase (LDH) in arbitrary units for the unstimulated and stimulated human tracheal epithelial cells treated with phosphate buffered saline (PBS) or PBS and LMWP, and for the cell lysate (data are average for six replicate experiments, mean ± standard error of the mean);

[0033] Fig. 8 is a black and white representation of an originally colored confocal microscope image of human tracheal epithelial cells treated with phosphate buffered saline (PBS) or PBS and LMWP after first being infected for 24 hours with S. Aureus, where the arrows (labeled G) indicate biofilm growth; and

[0034] Fig. 9 is a graph depicting the viable bacteria remaining on the surface of cultured human tracheal epithelial cells treated with phosphate buffered saline (PBS) or PBS and LMWP after first being infected for 24 hours with S. Aureus (data are average for three replicate experiments, mean ± standard error of the mean).

DETAILED DESCRIPTION

[0035] Nitric oxide (NO) deficiencies can either be genetic or polymorphism-related, or induced by pathogens that take advantage of upstream regulation of NO production. L-arginine is the substrate for all isoforms of nitric oxide synthase (NOS, particularly iNOS) and is a major metabolic mediator within the urea cycle. The two isoforms of the enzyme arginase catalyze the conversion of arginine into urea and compete with the action of iNOS during immune response. Induction of iNOS is associated with Ml/Thl activated immune cells, while induction of arginase is associated with the M2/Th2 activation state. Arginase itself is known to be co- induced by immune stimulation in a delayed manner. By upregulating the metabolic usage of arginine and inducing a Th2-like state, a pathogen may be able to drastically reduce NO production and prevent host immune response.

[0036] Many bacteria are able to force the production of arginase-I in murine macrophages via toll-like receptor pathways. On the other hand, arginase-I deficient mice have been shown to display greater host survival rates and a lower lung bacterial load during infection with M.

tuberculosis. Previous research has shown that the only arginase isoform that is constitutively expressed in RAW 264.7 and LA4 cells is arginase-II (model systems for human macrophages and bronchial epithelial cells, respectively). Still, both arginase-I (cytosolic) and arginase-II can be highly induced in a similar manner as iNOS by exposure to TNF-a, and LPS in both macrophages and lung epithelial cells, showing tight regulation of NO production even during the Thl macrophage activation state. In addition, lung macrophages in allergen-sensitized and allergen-challenged mice have displayed an altered activation profile that causes substantial arginase-I upregulation in response to rhinovirus infection.

[0037] Due in part to its connection with Thl activation, the present inventors believe that arginase-I plays an important role in inflammatory response at normal levels, but either increased activity of arginase or decreased activity of iNOS may result in immune deficiency within the respiratory system. In other words, reduced levels of NO may be due to the effects of decreased iNOS expression and/or enhanced arginase expression in the sinus epithelium due to increased Th2 cytokine expression and increased activity of bacteria-derived arginase that competes for L- arginine consumption, decreasing NO production. It is further believed that a therapeutic that allows for the augmentation of iNOS activity and/or the reduction of extracellular arginase activity would counteract these deficiencies. In the examples disclosed herein, a nasal formulation (either pre-formulated or to be formulated by a user) allows for the augmentation of iNOS activity and/or the reduction of arginase activity, and thus an increase in NO production within epithelial cells and immune cells typically present in sinonasal tissue. This nasal formulation may be beneficial for treating or even preventing upper airway infections, including CRS.

[0038] The nasal formulation disclosed herein increases NO production within epithelial cells and immune cells, which can also help control ciliary beat frequency. NO is a potent antibacterial agent, and increased amounts of NO can increase ciliary beat frequency. The ciliary beat frequency is directly correlated with mucociliary function, which is one of the primary innate immune defense mechanisms in the airway epithelium. As such, the nasal formulation described herein may help to restore/enhance mucociliary function, which may increase the defense against chronically colonized pathogens and reduce, or even prevent, disease

perpetuation.

[0039] In some examples disclosed herein, the nasal formulation is a pre-formulated nasal spray or sinus rinse. In these examples, the nasal formulation includes a liquid carrier and low molecular weight arginine-rich peptides present in the liquid carrier. The nasal formulation has a pH ranging from 4 to 10.

[0040] The liquid carrier may be any suitable liquid that is capable of being introduced into the upper airways of a human being through the nasal cavity. The liquid carrier may be water, a saline solution, a saline solution with sodium bicarbonate (NaHCOs), a phosphate buffered saline solution (PBS), an aqueous solution with calcium chloride (CaCl₂), or an aqueous solution with a sodium phosphate buffer or a potassium phosphate buffer.

[0041] The nasal formulation includes the low molecular weight arginine-rich peptides dissolved or dispersed in the liquid carrier. The concentration of the low molecular weight arginine-rich peptides may vary, depending upon the type of peptide and the liquid carrier. In an example of the nasal formulation, the liquid carrier consists of water, and the concentration of the low molecular weight arginine-rich peptides ranges from about 0.01 mM (10 μΜ) to about 5 mM (5000 μΜ). In other examples, the upper limit of the concentration of the low molecular weight arginine-rich peptides in the nasal formulation is 1 mM (1000 μΜ).

[0042] The low molecular weight arginine-rich peptides have a molecular weight less than 2 KDa. In an example, the molecular weight ranges from about 0.5 KDa to 2 KDa. The low molecular weight arginine-rich peptides are also derived from a protamine source via treatment with the enzyme thermolysin. The protamine source may be any protamine-containing substance from which arginine-rich peptides having at least 8 amino acids are obtained after treatment with thermolysin. It is believed that the protamine source may be obtained from any species within the Oncorhynchus genus. As examples, the protamine source may be salmon sperm (e.g., chum salmon sperm) or rainbow trout sperm or any other source containing protamine from which arginine-rich peptides may be derived. An example of the primary protamine sequence derived from salmon sperm is shown in Fig. 2A as PRRRRSSSRPVRRRRRPRVSRRRRRGGRRRRR, SEQ ID NO: 1 (i.e., Pro Arg Arg Arg Arg Ser Ser Ser Arg Pro Val Arg Arg Arg Arg Arg Pro Arg Val Ser Arg Arg Arg Arg Arg Gly Gly Arg Arg Arg Arg Arg). Other examples of the primary protamine sequence include MPRRRRASRRVRRRRRPRVSRRRRRGGRRRR, SEQ ID NO: 2 (i.e., Met Pro Arg Arg Arg Arg Ala Ser Arg Arg Val Arg Arg Arg Arg Arg Pro Arg Val Ser Arg Arg Arg Arg Arg Gly Gly Arg Arg Arg Arg), which is from rainbow trout; and MPRRRRSSSRPVRRRRRPRVSRRRRRRGGRRRR, SEQ ID NO: 3 (i.e., Met Pro Arg Arg Arg Arg Ser Ser Ser Arg Pro Val Arg Arg Arg Arg Arg Pro Arg Val Ser Arg Arg Arg Arg Arg Arg Gly Gly Arg Arg Arg Arg), which is from chum, salmon. It is to be understood that the amino acid sequence of arginine-rich peptides derived from other protamine sources may vary.

[0043] In an example, a stable, immobilized thermolysin- silica column is used to produce the low molecular weight arginine-rich peptides. More particularly, protamine obtained from the protamine source is weighed out, and is mixed (and ultimately dissolved) in a suitable salt solution. An example salt solution includes NaCl, CaCl₂ and Tris-HCl, has a pH 8.0, and is buffered to a final concentration of 10 mg/mL. The mixture may be heated in order to facilitate dissolution of the protamine. The solution is passed via a peristaltic pump into a thermolysin- bound silica column submerged in a 70° C water bath. The protamine solution may be cycled through the immobilized enzyme column for a predetermined time period (e.g., 3 hours) to ensure complete digestion. The column may also be completely dehydrated by filtration through a suitable filter. The filtrate, including the low molecular weight arginine-rich peptides, is collected.

[0044] The filtrate may then be subjected to purification in order to de-salt and isolate desirable arginine-rich peptide fragments through size-exclusion chromatography. In the examples disclosed herein, size-exclusion chromatography is used to isolate those arginine-rich peptides with at least 8 amino acids. Some examples of the arginine-rich peptide fragments obtained from the primary protamine structure are shown in Fig. 2A and are labeled as TDSP1 through TDSP5. TDSP1 is PRRRR (or Pro Arg Arg Arg Arg, SEQ ID NO: 4); TDSP2 is PRRRRSSSRP (or Pro Arg Arg Arg Arg Ser Ser Ser Arg Pro, SEQ ID NO: 5); and TDSP3 is RPVRRRRRPR (or Arg Pro Val Arg Arg Arg Arg Arg Pro Arg, SEQ ID NO: 6). [0045] As mentioned above, the low molecular weight arginine-rich peptides have a structure with at least 8 amino acids. Some suitable examples include TDSP4 shown in Fig. 2A,

VSRRRRRGGRRRR or Val Ser Arg Arg Arg Arg Arg Gly Gly Arg Arg Arg Arg (SEQ ID NO: 7) and TDSP5 shown in Fig. 2A, VSRRRRRGGRRRRR or Val Ser Arg Arg Arg Arg Arg Gly Gly Arg Arg Arg Arg Arg (SEQ ID NO: 8). In the nasal formulation, these low molecular weight arginine-rich peptides may be used alone or in combination.

[0046] In another example, the protamine (e.g., 1 mM) may be incubated with immobilized thermolysin on silica particles. The incubation may be performed at a temperature of about 80°C for about 8 hours. Centrifugation may be used to separate protamine fragments from the solid phase thermolysin. The supernatant may be subjected to size fraction column chromatography or cation exchange chromatography on an immobilized heparin column to isolate the desirable arginine-rich peptide fragments.

[0047] In an example, the nasal formulation excludes pure L-arginine monomer. In another example, pure L-arginine monomer may be added along with the low molecular weight arginine- rich peptides. In this other example, it is to be understood that the rate of uptake of pure L- arginine monomer by cells is much slower than the low molecular weight arginine-rich peptides derived from protamine.

[0048] The nasal formulation may also include an inhibitor of the L-arginase enzyme. The inhibitor is a cell-permeable arginase inhibitor. The inhibitor acts by reversibly binding to the active site of arginase due to similarities in electronic geometry between itself and arginine. In an example, the trigonal planar boronic acid moiety of s-boronoethyl-L-cysteine (BEC) readily binds to the active site of arginase and reacts to form a tetrahedral boronate anion as a transition- state analog of arginase. The inhibitor is an effective agent for increasing NO production in murine macrophages and endothelial cells by decreasing consumption of arginine, the substrate for iNOS. In the examples disclosed herein, the inhibitor may be selected from the group consisting of: s-boronoethyl-L-cysteine (BEC), 2(S)-amino-6-(borono) hexanoic acid), N5- (benzyloxycarbonyl)-N2-(tert-butoxycarbonyl)-L-thiocitrulline tert-butyl ester, N5-[N- (benzyloxycarbonyl)-N'-(methoxycarbonylmethoxy)amidino]-N2-(tert-btoxycarbonyl)-L- ornithine tert-butyl ester), 6-(dihydroxyboranyl)-2-(3-phenoxypropyl)norleucine hydrochloride, 6-(dihydroxyboranyl)-2-(hydroxymethyl)-L-norleucine, 2-amino-6-(dihydroxyboryl)-2- [3 -(4- piperidinyl)propyl]hexanoic acid, 2-[4-(dihydroxyboranyl)butyl]lysine, 6-(dihydroxyboranyl)-2- [2-(piperidin-l-yl)ethyl]-L-norleucine dihydrochloride, (3R,4S)-3-amino-l-[3-(4- carboxyphenyl)propyl]-4-[3-(dihydroxyboranyl)propyl]pyrrolidine-3-carboxylic acid), (2S)-2- amino-6-(dihydroxyboryl)-2- [cis-3 - [[(4-fluoro- 1 naphthyl)methyl]amino] cyclobutyl]hexanoic acid), and (2S)-2-amino-2-[cz^'s-3 - [ [(4 ' -chloro-3 -fluorobiphenyl-4-yl)methyl] amino] cyclobutyl] - 6-(dihydroxyboryl)hexanoic acid.

[0049] In other examples, the low molecular weight arginine-rich peptides disclosed herein may be part of a nasal rinse kit. In this example, the low molecular weight arginine-rich peptides are part of a powder composition that also includes the inhibitor of the L-arginase enzyme. This powder composition may also include a salt selected from the group consisting of sodium chloride, sodium bicarbonate, calcium chloride, a sodium phosphate buffer, a potassium phosphate buffer, and combinations thereof. Similar to the nasal formulation, the powder composition may also exclude pure L-arginine monomer, or may include pure L-arginine monomer in addition to the low molecular weight arginine-rich peptides.

[0050] The nasal rinse kit includes a bottle (of any type) for dissolving the powder composition in water. The powder composition is formulated so that when water is added, the concentration includes from about 10 μΜ to about 5000 μΜ of the arginine-rich peptides and from about 10 μΜ to about 200 μΜ of the L-arginase enzyme inhibitor.

[0051] In the examples disclosed herein, the nasal formulation or the kit may be used in a method for enhancing nasal nitric oxide (NO) levels. An example of this is shown in Fig. 1. When the nasal formulation (shown as a nasal spray including the arginine-rich peptides in a saline solution with a pH of 7.4) is introduced into the nasal cavity, the low molecular weight arginine-rich peptides derived from protamine (shown as (Arg)_x in Fig. 1) are also introduced into the nasal cavity. Upon spraying the nasal formulation into a nostril, a significant volume of the nasal formulation is retained as a layer of solution within pockets along the surface of the sinus/nasal mucosal tissue that line the inner walls of sinus cavities. The low molecular weight arginine-rich peptides are not degraded by extracellular arginase, but rather enter innate immune cells and sinonasal airway epithelial cells rapidly (almost immediately) by a non-disruptive phenomenon. The active site of iNOS within the nasal cavity is highly specific to L-arginine (Arg), and thus the low molecular weight arginine-rich peptides themselves are not used by iNOS to generate NO. Rather, within the wall of the sinus cavity, the low molecular weight arginine-rich peptides may be digested/cleaved by intracellular proteases (e.g., peptidases), which convert the low molecular weight arginine-rich peptides to L-arginine. This increases the intracellular L-arginine substrate levels, which can then used for NO biosynthesis. Once cleaved, the L-arginine that is generated would enhance iNOS expression (NO Synthase in Fig. 1), which further helps to increase NO levels.

[0052] As previously described, the low molecular weight arginine-rich peptides enter the cells, and increase intracellular L-arginine levels quickly, leading to upregulation of iNOS levels and increased production of NO by these cells. Increased levels of NO may be sufficient to disrupt bacterial biofilm formation and/or disperse antibiotic resistant biofilms, kill bacteria, improve mucociliary function, and reduce inflammation. Elevated NO production may be observed nearly immediately and for an extended period (e.g., up to about 16 hours). The nasal formulation disclosed herein may be used to fight an infection. Furthermore, daily use of the nasal formulation disclosed herein may help maintain NO levels equivalent to those of healthy individuals, which, in turn, may prevent formation of bacterial biofilm and CRS disease perpetuation.

[0053] The risk of producing too much NO (toxic levels) is unlikely, since host mucosal cells can upregulate expression of intracellular arginase when NO levels become too high, thereby self-regulating the upper levels of NO that can be produced.

[0054] In addition, the inhibitor may further protect the delivered intracellular L-arginine and enhance NO production by both increasing iNOS expression and increasing substrate

availability. Together, the low molecular weight arginine-rich peptides disclosed herein and the inhibitor act to alleviate arginine limitation and prevent inhibition of iNOS expression. By simultaneously delivering these agents to NO-generating cells within the sinonasal cavity, NO generation as a result of inflammatory signaling can be significantly augmented. It may even be possible to restore NO concentrations to levels above their antimicrobial threshold.

[0055] By providing the target cells with the polycationic peptide(s), it may be possible to both protect the supplemented arginine from extracellular arginase activities and circumvent the regulation of the transport of L-arginine across the cell membranes. The delivery of protamine- derived peptides may be achieved through endocytotic mechanisms which are enhanced during immune activation and display rapid internalization.

[0056] To further illustrate the present disclosure, examples are given herein. It is to be understood that these examples are provided for illustrative purposes and are not to be construed as limiting the scope of the present disclosure.

EXAMPLE 1

[0057] In this example, it is demonstrated that low molecular weight arginine-rich peptides (such as low molecular weight protamine, LMWP) derived from the FDA-approved protamine (obtained from salmon sperm) can effectively raise NO production in both model systems of immune (i.e., murine macrophages, RAW 264.7) and bronchial epithelial (LA4) cells.

[0058] Materials

[0059] Protamine (Grade X, from salmon sperm), thermolysin (from Thermoproteolytikus Rokko), lipopolysaccharide from E. Coli, Sephadex G-15 (Medium), N-hydroxysuccinimide, 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide, Bradford Reagent, L-arginine free base, and L- lysine free-base, SILAC RPMI 1640, and SILAC Ham's 12 media were purchased from Sigma- Aldrich (St. Louis, MO). Murine recombinant interferon-gamma, human recombinant

Interleukin-lbeta, and murine recombinant Tumor Necrosis Factor-alpha were purchased from Peprotech (Rocky Hill, NJ). S-boronoethyl-l-cysteine (BEC) and polyclonal rabbit anti-mouse iNOS igG were purchased from Cayman Chemical (Ann Arbor, MI). 4x Laemmli buffer, goat anti-mouse and goat anti-rabbit horseradish peroxidase-conjugated IgG were obtained from Bio- Rad (Hercules, CA). A SuperSignal West Pico chemiluminescence substrate was obtained from Thermo Scientific (Rockford, IL). RAW 264.7 and LA4 cells were obtained as frozen stocks from ATCC Cell Lines (Bethesda, MD). RPMI 1640 culture media, Cell-Dissociation Buffer, and Ham's F12 media were obtained from Life Technologies, and FBS was obtained from Gibco.

[0060] Preparation of LMWPs Derived from Protamine

[0061] Protamine was weighed out and dissolved in 20 mM NaCl, 10 mM CaCl₂, 10 mM Tris-HCl, pH 8.0, buffer to a final concentration of 10 mg/rnL. The mixture was heated with a water bath at 70° C in order to facilitate dissolution of protamine. The solution was then passed via a peristaltic pump into a thermolysin-bound silica column submerged in a 70° C water bath. Conjugation efficiency (expressed as a mean ± standard error of measurement (SEM) of three conjugation experiments) was measured by adding 100 μΐ, of conjugation filtrate, passed through a sterile 0.2 μιη syringe filter (Millipore, Billerica, MA), to 900 of Bradford reagent. The absorbance at 595 nm measured on a Perkin-Elmer 25 UV7VIS Spectrophotometer (Perkin- Elmer, Waltham, MA) after 20 min was compared to a calibration curve obtained by a wide range of concentrations of unbound thermolysin. The total calculated mass of thermolysin in the conjugation filtrate was compared to the total weighed mass of thermolysin to generate a w/w conjugation percentage.

[0062] The protamine solution was cycled through this immobilized enzyme column for 3 hours to ensure complete digestion, and the column was completely dehydrated by filtration through a sterile 0.2 um syringe filter.

[0063] The collected filtrate was analyzed by the Bradford assay by adding 100 μΐ, to 900 μΐ, of Bradford reagent and incubating for 20 min before measuring absorbance at 595 nm. The digestion efficiency was calculated by dividing the enzyme column filtrate absorbance at 595 nm by the absorbance at 595 nm of a 100 of 10 mg/mL of protamine mixes with 900 μΐ_^ of Bradford reagent.

[0064] Purification and Storage of LMWP

[0065] After spectrophotometric analysis, the filtrate obtained from the immobilized thermolysin column was subjected to purification in order to de-salt and isolate the two largest molecular weight arginine-rich peptide fragments through size-exclusion chromatography. A 15 cm length Sephadex G-15 column (average particle diameter = 100 μιη; diameter of the column = 1.5 cm) was prepared. The column was conditioned by passing 3 column volumes of 10 mM Tris-HCl (pH 8.0) buffer through the column before sample loading. The sample of peptide fragments was loaded at <30% of the column volume for peptide separation. Fractions of 1 mL volume were collected by gravity elution. Fractions were quantitatively assayed for peptide content via absorbance at 210 nm by UV7VIS Spectrophotometer measurements, and for salt content by conductivity measurement in order to generate a chromatogram. The initially eluted fractions containing the target fragments VSRRR RGGRRR (SEQ ID NO: 7) and VSRRRRRGGRRRRR (SEQ ID NO: 8) were isolated and subjected to lyophilization and stored at -20° C.

[0066] Cell Culture

[0067] RAW 264.7 and LA4 cells were cultured in RPMI 1640 and Ham's F12 medium, respectively. Each medium contained 10% fetal bovine serum (FBS) and 100 μg/ml penicillin and 100 U/ml streptomycin. For experiments involving the addition of L-arginine or LMWP, cells were cultured in SILAC RPMI 1640 or SILAC Ham's F12 media partially constituted with L-arginine (depending on the experiment) and L-lysine (400 μΜ) and supplemented with 10% FBS. Unless otherwise indicated, RAW 264.7 cells were plated at a density of 5 x 10⁵ cells per well in 24 well plates and LA4 cells were plated at a density of 2 x 10⁵ cells per well in 6 well plates. After cell adhesion to the culture dishes, LA4 cells were stimulated with a mixture of mouse recombinant IFN-γ (10 ng/ml), human recombinant IL-Ιβ (10 ng/ml) and mouse recombinant TNF-a (2 ng/ml). RAW 264.7 cells were stimulated with combination of LPS (20 ng/ml) and mouse recombinant IFN-γ (10 ng/ml). Cells were then treated with L-arginine (10 mM stock) or LMWP (1 mM stock) to a final concentration of 400 μΜ total L-arginine. In some experiments cells were pretreated with 90 μΜ BEC. Cell culture supernatant was collected at indicated times and stored at 4°C for nitrite analysis as an indicator of NO production. Cell lysates were prepared and stored at -20°C for Western blot analyses.

[0068] Nitrite Analysis

[0069] Media samples collected from cell culture experiments were analyzed for nitrite concentrations using a Sievers Nitric Oxide Analyzer (GE Healthcare). A reaction cell was prepared with a N₂-purged solution of 0.5 M H₂S0₄ and 0.5 M KI in order to reduce nitrite in the sample to NO. The generated NO was collected by a N₂ sweep gas and delivered to the instrument for reaction with ozone to produce excited nitrogen dioxide. The quantitative release of photons from the intermediate was measured by a photomultiplier tube to generate an NO concentration as ppb. The data was integrated over time in order to determine total nitrite content of the samples. NO generation was allowed to return to baseline before addition of successive samples. [0070] Western Blot

[0071] Cells were washed three times in PBS and lysed for 30 minutes on ice in 10 mM Tris buffer pH 8.0 containing 0.1% SDS, l%Triton X-100, 0.1% sodium deoxycholate, and complete protease inhibitors (Roche, Indianapolis, IN). Cell lysates were centrifuged at 13,000 x g for 20 minutes at 4°C, and protein concentrations of the cleared cellular lysates were determined by the Lowry assay. The samples were mixed 3 : 1 with 4x Laemmli sample buffer, boiled for 5 minutes, and 30 μg aliquots were separated on an 8% SDS-PAGE gel. The proteins were transferred to a 0.2 μιη pore nitrocellulose membrane (Millipore), which was then blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.05% Tween 20 for 1 h at 25° C. The membrane was incubated with a 1 :5,000 dilution of polyclonal rabbit anti-iNOS antibody or 1 : 10,000 monoclonal mouse anti-P-actin antibody overnight at 4°C and subsequently incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG for 1 hour at 25°C. The membrane was then incubated with chemiluminescence substrate. Finally, blots were exposed on X-ray film for 30 seconds and developed using a Kodak X-Omat Automatic

Processor (Kodak, Rochester, NY). The images were scanned and the band intensities were quantified by Image J and expressed as fold change over β-actin.

[0072] Results

[0073] Preparation and Purification of Low Molecular Weight Protamine

[0074] Protamine obtained from salmon sperm displays a significant molecular weight distribution but contains characteristic repeats of up to six arginine residues. One primary structure of protamine obtained from salmon sperm is shown in Figure 2A. The lower molecular weight fragments of protamine containing these arginine repeats have been shown to neutralize heparin due to their high charge density at neutral pH, while significantly minimizing

immunogenicity and toxicity. These protamine fragments are labeled as TDSP1 through TDSP5 in Fig. 2A. Of these fragments, TDSP4 and TDSP5 present the greatest potential for cellular uptake while limiting immunogenicity and toxicity. These peptides are highly hygroscopic and completely insoluble in solvents such as methanol, ethanol, and isopropanol, leading to numerous isolation and purification challenges. As a result, these fragments were targeted for an optimized, high-throughput, and low cost method for synthesis, purification, and quality assurance. [0075] Themolysin can be immobilized using amine-functionalized silica sol-gel microparticles with a glutaraldehyde cross-linker to exposed primary amines on the exterior of thermolysin. Using this approach, the present inventors facilitated the continuous production of LMWP and stabilized the enzyme for repeated use. However, they found that the use of a glutaraldehyde crosslinker presented significant stability hurdles and lower enzyme conjugation efficiencies. In this example, the immobilization procedure was adapted with fumed silica particles functionalized with 3-aminopropyltriethoxysilane followed by conjugation to a N- hydroxysuccinimidyl ester of thermolysin. As such, the process does not require use of a glutaraldehyde crosslinker. A flow diagram of the process to generate these functionalized silica particles is shown in Fig. 2B.

[0076] As shown in Fig. 2B, silanol groups were generated on the surface of fumed silica particles. This was accomplished by submerging the fumed silica particles in a well-stirred 1M HC1 solution overnight at room temperature. The silanol-modified fumed silica particles were dried. More particularly, the silanol-modified fumed silica particle solution was added to a buchner funnel with a piece of filter paper attached to a large filter flask, and vacuum was applied for hours until the silica particles were fully dried. The exact time for fully drying depends, in part, on the volume of the solution and the size of funnel. Other drying techniques may be used. 0.5 wt% of (3-ammopropyl)triethoxysilane (APTES) in anhydrous toluene was added to the dried particies. The mixture was incubated in an N? environment overnight (to form silica-APTES particles), and dried. For drying, the silica- APTES particles were poured into a large buchner funnel with a piece of filter paper attached to a large filter flask, and vacuum was applied until the particles were fully dried. The time required for fully drying was longer than 30 minutes. The dried silica-APTES particles were washed twice successively with toluene, ethanol, and w^rater. After washing, the particies (APTES-silica) were again dried, this time at 120° C in a vacuum oven for about 1 hour. Thermolysin was incubated in 10 niM N- hydroxysuccinimide (NHS) and 40 mM l-Ethyf-3-(3-dimethylaminopropyl)carbodiimide (EDC) for about 10 minutes. The thermolysin (enzyme) was mixed with the APTES-silica particies for about 30 minutes, dried, and then washed twice successively with ethanol and water. The thermolysin functionalized silica particles (referred to below as the active gel) were stored at 4°C. [0077] Conjugation was measured by dehydrating the thermolysin-conjugated column and performing the Bradford assay against a known calibration curve. Using this approach, the present inventors observed conjugation efficiencies of 92.5 ± 2.7 wt% thermolysin enzyme to the silica substrate. The active gel was mechanically ground before being washed twice successively with ethanol and water and dried again before being packed into a column and stored at 4°C.

[0078] The Bradford reagent displayed very little to no color change for protamine digest as opposed to other cell penetrating peptides (Fig. 2C) and even protamine itself. This important distinction allows for a quantitative measure of the efficiency of digestion by comparing the binding of the Coomassie Brilliant Blue G-250 dye to a 10 mg/mL solution of protamine vs. the binding of the dye to the collected filtrate from the enzyme column. As shown in Fig. 2D, the enzyme column was stable over multiple uses and 3 hours of substrate cycling through the column resulted in digestion efficiencies of 93.1% on first use with minimum digestion efficiencies of 81.0% following further column use.

[0079] To isolate the LMWP fragments and exchange the buffer composition to a composition with a low salt concentration, a size-exclusion chromatography column was employed. Sephadex G- 15 is a suitable column material for peptide separation due to minimal interaction with hydrophobic, charged, or polar samples, and due to its exclusion cutoff of about 1.5 kDa. While the putative molecular weights of TDSP1 (SEQ ID NO: 4) through TDSP3 (SEQ ID NO: 6) lie below this cutoff, the molecular weight of LMWP lies near 2 kDa, and as a result, should elute in a low-salt buffer (Tris 10 mM, pH 8.0) immediately after the void volume and before the low molecular weight constituents. The results of the column purification are shown in Fig. 2E, using absorbance at 220 nm to detect eluted peptide content and conductivity for measurement of salt concentration. The chromatogram displayed six clear peaks in order of molecular weight corresponding to partially digested protamine, TDSP5 (SEQ ID NO: 8), TDSP4 (SEQ ID NO: 7), TDSP3 (SEQ ID NO: 6), TDSP2 (SEQ ID NO: 5), and TDSP1 (SEQ ID NO: 4). As expected, the conductivity of the column effluent rose significantly as the lower molecular weight constituents were eluted from the column, corresponding to the higher salt content of the digestion buffer. The first 11 fractions were pooled and subjected to lyophilization and the resulting powder was weighed to determine yield. [0080] Inducible Nitric Oxide Synthase Activity and Expression in RA W 264.7 and LA4 Cells is Dependent on Extracellular Arginine Levels

[0081] The L-arginine K_m for iNOS has been determined to lie in the low μΜ range (~5 μΜ), yet previous literature has demonstrated that NO synthesis can increase significantly when RAW 264.7 cells are activated in media containing L-arginine concentrations from 10 μΜ to 1.6 mM. This dependence of NO synthesis on L-arginine concentration has been attributed to increased iNOS expression and implies that diminished L-arginine availability can significantly attenuate NO production. As shown in Fig. 3A, RAW 264.7 cells that are co-stimulated (+) with LPS and IFNy (cytomix 1) express iNOS 26 hours after stimulation, while unstimulated cells (-) do not express detectable levels of iNOS. The nitrite content in the media of stimulated and unstimulated cells 24 hours post-stimulation, shown in Fig. 3B, corroborates the analysis of iNOS expression in RAW 264.7 cells, as it increased from 0.54 ± 0.11 μΜ in unstimulated to 56.99 ± 0.48 μΜ in stimulated cells. The nitrite content in the stimulated cells was about 100 fold higher than the unstimulated cells.

[0082] LA4 cells, when stimulated (+) by a mixture of IFNy, IL-Ιβ, and TNFa (cytomix 2), also expressed iNOS, while unstimulated cells (-) displayed no detectable iNOS expression (Fig. 3C). The nitrite content of the collected media (Fig. 3D) in each case again confirmed the activity of iNOS as it increased from 0.27 ± 0.22 μΜ in unstimulated cells to 7.84 ± 0.19 μΜ in stimulated cells. This was about a 23 fold increase.

[0083] When plated in media of varying L-arginine concentration, it was found that RAW 264.7 cells (Figs. 3G and 3H) displayed L-arginine-dependent NO synthesis during a 24 hour period over a range of 10 μΜ to 1100 μΜ L-arginine. The nitrite levels increased from 0.42 ± 0.04 μΜ to 11.08 ± 0.59 μΜ at 100 μΜ L-arginine to 56.99 ± 0.48 μΜ at 1100 μΜ L-arginine. Fig. 3G is a representative immunoblot indicating that the expression of iNOS in RAW 264.7 cells is dependent on media L-arginine concentration, as the expression is shown to increase as the concentration is modulated from 10 μΜ to 1000 μΜ.

[0084] LA4 cells were plated in media of varying L-arginine concentrations to determine if this same L-arginine dependence could be observed in these cells (Figs. 3E and 3F). iNOS expression in LA4 cells was similarly dependent on extracellular L-arginine concentration. Total NO production appeared not to be substrate-limited, with L-arginine concentrations approaching 1000 μΜ with nitrite levels peaking at roughly 9 μΜ. Nitrite levels were seen to increase from 0.29 ± 0.07 μΜ at 10 μΜ L-arginine to 28.6 ± 3.5 fold higher at 400 μΜ L-arginine to 28.6 ± 3.1 fold higher (8.34 μΜ) at 1000 μΜ. Arginine-induced iNOS expression may protect against substrate limitation due to arginase induction by pro-inflammatory stimuli (all differences were statistically significant with p < 0.001). Fig. 3E shows a representative immunoblot indicating that the expression of iNOS in LA4 cells is dependent on the L-arginine concentration media as expression increases as L-arginine concentration increases from 100 - 1000 μΜ.

[0085] Low Molecular Weight Protamine Treatment Yields Increased NO Production in RA W 264.7 and LA4 Cells Compared to L-Arginine Treatment

[0086] Although iNOS expression is L-arginine dependent, its transmembrane transport is slow and can be inhibited by other cationic amino acids such as lysine and ornithine. The present inventors have shown that the large, arginine-rich LMWP species (TDSP4 and TDSP5) are rapidly taken up by both RAW 264.7 cells and LA4 cells and subsequently enhance iNOS expression and NO production compared to equivalent total concentrations of monomeric L- arginine.

[0087] As shown in Fig. 4A, supplementation of RAW 264.7 cells with 40 μΜ LMWP caused a significant increase in NO production over 20 hours compared to control cells, and LMWP itself did not cause induction of iNOS. The increased NO production indicates successful entry into target cells and arginine availability. Four independent experiments were conducted per experimental condition (cell density of 5 x 10⁴ cells/well) and media was collected 20 hours after stimulation.

[0088] In addition, as shown in Fig. 4B, iNOS expression in RAW 264.7 cells was significantly enhanced upon treatment with LMWP compared to control cells (1.28 ± 0.08 vs. 0.70 ± 0.14 iNOS/p-actin). Four independent experiments were conducted per experimental condition (cell density of 5 x 10⁴ cells/well) and media was collected 20 h after stimulation. Treatment of RAW 264.7 cells with 40 μΜ LMWP resulted in a significant increase in NO production compared to cells treated with 400 μΜ L-arginine over 20 hours in cultures pre- incubated with 10 μΜ (39.5 ± 2.9 vs. 31.5 ± 1.1 μΜ), 100 μΜ (41.4 ± 2.0 vs. 32.2 ± 1.0 μΜ), and 400 μΜ L-arginine (46.9 ± 1.6 vs. 38.6 ± 1.1 μΜ) (Fig. 4C). Since RAW cells pre-cultured in a low-arginine environment for 18 hours (as low as 10 μΜ) showed increased NO generation with LMWP treatment, these data show that the presence of L-arginine is not necessary for the beneficial action of LMWP species.

[0089] In order to determine if LMWP could more rapidly provide a substrate for iNOS compared to L-arginine, RAW 264.7 cells were stimulated in various arginine-deficient conditions (10, 100, and 400 μΜ L-arginine) prior to addition of either LMWP or L-arginine, and media nitrite content was measured at 1, 2, and 4 hours after treatment. As shown in Fig. 4D, treatment with LMWP or L-arginine led to nearly identical NO production over the first four hours for each level of iNOS expression. Each data point in Fig. 4D corresponds to one independent experiment.

[0090] Over the 3 incubation concentrations of L-arginine and over the first three hours, LMWP on average was only 1.01 ± 0.06 times more effective than L-arginine. This data, coupled with the low L-arginine K_m for iNOS, implies that the effect of added L-arginine or added LMWP lies in the increased expression of iNOS in stimulated cells. Indeed, as shown in Fig. 4E, a significant difference between NO synthesis as a result of L-arginine (5.91 ± 0.44 μΜ) or LMWP addition (8.52 ± 0.38 μΜ) when added concomitantly with cytomix 1 can be observed as early as 12 hour post-treatment. In addition, the additional NO generated by LMWP treatment compared to L-arginine treatment continues to increase from 5.07 ± 0.75 μΜ after 16 h to 8.3 ± 2.0 μΜ after 24 hours (Fig. 4F). As such, the LMWP is effective in increasing NO production as compared to L-arginine from 12-24 hours after stimulation and addition.

[0091] These findings were mirrored in the expression of iNOS (Fig. 4G), with significantly higher expression seen with LMWP treatment (1.28 ± 0.08 iNOS/p-actin), compared to control cells (0.70 ± 0.14 iNOS/p-actin) or those given additional L-arginine (0.88 ± 0.06 iNOS/p-actin), that is gradually increasing, even up to 26 hours after stimulation.

[0092] Treatment with Low Molecular Weight Protamine and s-Boronoethyl-l-cysteine can Synergistically Increase NO Production in RA W 264.7 Cells but not in LA4 cells

[0093] Arginase can be present in the extracellular environment during infection or chronic inflammation due to secretion by leukocytes or as a pathogen defense mechanism. Once LMWP has been digested by intracellular proteases, the present inventors hypothesize that BEC should further protect the delivered intracellular L-arginine and enhance NO production by both increasing iNOS expression and increasing substrate availability. As shown in Fig. 5A, BEC treatment (over a range of 26 μΜ to 155 μΜ) to stimulated RAW 264.7 cells in 400 μΜ L- arginine yielded increasing NO production, with an optimum concentration of about 100 μΜ BEC. When treated with LMWP and BEC together, a synergistically higher NO synthesis over a 24 hour period in RAW cells was observed as NO levels were significantly higher (1.55 ± 0.03 fold increase over control cells) than the sum of levels due to LMWP and BEC alone (1.42 ± 0.04 fold increase over control cells).

[0094] This synergistic effect was not observed with treatment of L-arginine and BEC (Fig. 5B) as combined treatment of L-arginine and BEC yielded a 1.14 ± 0.02 fold increase over control cells, but the sum of the L-arginine and BEC treatments resulted in 1.20 ± 0.04 fold increase over control cells.

[0095] The treatment of LA4 cells with either LMWP or L-arginine and BEC, however, did not result in synergistically higher NO synthesis (1.26 ± 0.05 fold increase for L-arginine+BEC vs. 1.28 ± 0.04 fold increase over control cells for sum of L-arginine and BEC) (Fig. 5C). The data collected from RAW cells was unexpectedly not mirrored in the expression of iNOS over the same 24 hour period (Fig. 5E). It was found that the expression of iNOS for cells treated with either LMWP/BEC or L-arginine/BEC increased to peaks of 1.12 ± 0.13 and 0.78 ± 0.13 iNOS/p-actin 16 hours after treatment, but decreased after 26 hours to 1.00 ± 0.18 and 0.58 ± 0.09, respectively. Treatment with LMWP, L-arginine, or BEC alone led to continuously rising expression (Fig. 5F). This data suggests that the large amount of NO produced from RAW cells treated with LMWP or L-arginine and BEC may have triggered an NO-dependent negative feedback mechanism for oxidative stress. Similarly, the expression of iNOS in LA4 cells was diminished for treatment with LMWP and LMWP+BEC whose nitrite levels were near 9 μΜ (Fig. 5D). As shown in Fig. 5E, treatment of stimulated LA4 cells with BEC yielded a greater change in iNOS expression from control cells as opposed to RAW 264.7 cells (1.18 ± 0.04 vs. 1.09 ± 0.02 fold increase), which may correspond to higher levels of induced arginase in these cells. If this is the case, it is likely that the oxidative stress limit for these cells (corresponding to cells that comprise a significant portion of total pulmonary nitric oxide) is lower than in RAW cells.

[0096] The augmentation of endogenous NO production in respiratory tract is an attractive therapeutic target due to NO's role as a potent antimicrobial agent. In this example, the effect of LMWP on augmentation of NO synthesis in respiratory cells was examined. LMWP derived from the FDA-approved protamine is significantly less toxic than regular protamine (which is used routinely as antidote for heparin induced anticoagulation). As shown herein, these peptides can be synthesized using a cost-effective enzyme-conjugated column and separated with the Sephadex G-15 resin. Digestion efficiencies can be monitored by the Bradford assay as LMWP shows very little binding affinity towards Coomassie Brilliant Blue G-250. This may indicate that stabilization of the sulfonic acid moiety of Coomassie Brilliant Blue G-250 requires the presence of hydrophobic residues in addition to basic residues. LMWP is extremely hydrophilic due to the high number of arginine residues, and as a result, may not be able to interact with the hydrophobic core of Coomassie Brilliant Blue G-250.

[0097] The results set forth herein have shown that LMWP is effective in increasing NO synthesis in two murine cell lines relevant to respiratory disease in humans - RAW 264.7 cells and LA4 cells. The activity of iNOS is highly regulated by the presence of L-arginine via substrate availability and by transcriptional regulation and therefore additional L-arginine supplementation can be efficacious in increasing iNOS expression and activity. The presence of pathogens and a chronic inflammatory environment, however, can significantly attenuate the benefit of L-arginine supplementation due to the high levels of arginase in the environment. In contrast, the arginine -rich LMWP species, which are not substrates for arginase and have the capacity to enter the target cells rapidly, may be more efficacious than L-arginine in infection and inflammatory environment. In these results, it has been demonstrated that LMWP can be successfully transported into the target cells and be used, after intracellular protease cleavage, for NO biosynthesis. As the active site of iNOS is highly specific to L-arginine, LMWP itself cannot be used by iNOS to generate NO; it must be first cleaved by proteases before enhancing iNOS expression and being converted to NO. The results indicate that LMWP can be efficacious in increasing iNOS expression even in conditions with L-arginine concentrations as low as 10 μΜ, resulting in enhanced NO synthesis. Interestingly, although endocytosis is known to be faster than receptor-mediated transport, the rate of additional NO synthesis after addition of either L-arginine or LMWP is nearly identical. This implies that LMWP itself does not present a larger substrate reservoir for target cells than L-arginine. Yet, accumulated nitrite levels over 12, 16, and 24 hours show a clear statistically significant advantage of LMWP over an equivalent total concentration of L-arginine, with the gap widening over time.

[0098] In addition, the arginase inhibitor BEC further bolsters the efficacy of LMWP by protecting intracellular and extracellular L-arginine from metabolism. The results indicate moderate efficacy of the inhibitor BEC alone, and that arginase inhibition was more effective in LA4 rather than RAW 264.7 cells possibly due to higher arginase expression in this cell type. Surprisingly, the accumulated nitrite content in the media of those cells treated with LMWP and BEC was greater than all other experimental treatments, but the expression of iNOS in these cells after 24 hours was lower than treatment with LMWP alone. This suggests that the majority of the NO synthesis responsible for the larger nitrite accumulation had occurred prior to the 24 hour time point. Indeed, the expression of iNOS reached a peak at around 16 hours after stimulation in RAW 264.7 cells given LMWP and BEC, followed by a decrease. In addition to L-arginine consumption by arginase-I and -II, iNOS is regulated by nitrosylation of NF-κΒ and self- nitrosylation, explaining why this greatly increased iNOS expression over 16 hours may have led to a reduction of iNOS expression.

[0099] In addition, it is believed that LA4 cells also display an oxidative stress limit as evidenced by limitation of NO synthesis at L-arginine concentrations exceeding 400 μΜ (with nitrite concentrations consistently peaking at about 9 μΜ). It is likely that a similar mechanism underlies decreased iNOS expression, due to exogenous NO treatment of cells generating large amounts of endogenous NO. In fact, the digestion of LMWP by intracellular proteases may prolong the expression of iNOS by limiting oxidative stress. It is important to note that the production of NO and ROS species is very tightly regulated and even a 10-20% increase in NO production can have a significant antimicrobial effect. However, it is not anticipated that LMWP -induced NO production (approaching double that of L-arginine over 24 hours) is due to the regulation of oxidative stress. Instead, it is anticipated that the expression of iNOS should continue to increase past the 24 hour period compared to control cells, further widening the gap in efficacy between LMWP and L-arginine.

[0100] It is believed that similar results may be seen in vivo.

[0101] As illustrated in this example, in RAW 264.7 cells, LMWP substantially increased iNOS expression and total NO production (measured as nitrite in the cell media) compared to cells given L-arginine at an equivalent total arginine concentration and control cells at the 12 to 24+ hour post-treatment time points. LMWP did not significantly enhance iNOS expression in LA4 cells, but did moderately enhance NO production when compared to an equivalent concentration of L-arginine over 24 hours. Furthermore, the arginase inhibitor BEC in combination with LMWP resulted in synergistically higher NO in RAW 264.7 cells over 24 hours.

EXAMPLE 2

[0102] The results of this example suggest that low molecular weight arginine-rich peptides (such as low molecular weight protamine, LMWP as described in Example 1) derived from the FDA-approved protamine (obtained from salmon sperm) may stimulate epithelial cell innate immune function by increasing NO generation, which provides the antimicrobial environment to reduce viable S. aureus counts on the surface of the epithelial cells.

[0103] NO Production in Human Tracheal Epithelial (HTE) Cells

[0104] In this example, primary basal epithelial (HTE) cells were isolated from trachea of normal subjects (under approved IRB protocol) and cultured at air/liquid interface to promote mucoliary-differentiated tracheal epithelial cells. The HTE cells were stimulated basolaterally with interleukin 13 (IL-13) (50 ng/ml). After 2 hours, the cell cultures were treated apically with 10 μΐ PBS (negative control), or with 10 μΐ PBS containing 50 nmoles of L-arginine or the LMWP (with an equivalent total of L-arg) and incubated for another 16 hours.

[0105] Cellullar iNOS mRNA levels were determined by qPCR. The cells stimulated with IL-13 exhibited a 44±5.2 fold increase in iNOS mRNA levels over unstimulated cells, which did not change following treatment with L-arginine or the LMWP. In contrast, the LMWP, but not L-arginine, significantly increased nitrite levels in the apical secretions of IL-13 -stimulated cells (see Fig. 6). Since NO produced by the cells is converted almost immediately to nitrite, the nitrite levels in the apical washes was measured. Nitrite was detected in the wash solutions by the chemiluminescence method, in which the samples of wash solution were treated with acid and also iodide to reduce nitrite to NO that is purged from the solution and measured by chemiluminescence NO analyzer. As such, NO production within the cells was assessed by measuring the nitrite levels. The control cells, which were not treated with LMWP, showed nitrite levels of 0.327 ± 0.085 μΜ. [0106] At the same time, protein (i.e., Interleukin 8 (IL-8) or lactate dehydrogenase (LDH)) expression within the cultured human cells was determined by Western blot analysis. For LDH assays, supernatants from cell lysates were used as assay positive controls. The LMWP did not stimulate pro-inflammatory response or cause cytotoxicity as determined by the lack of further expression of Interleukin 8 (IL-8) or lactate dehydrogenase (LDH), respectively, as shown in Figs. 7A and 7B.

[0107] Together, the results in Figs. 6 and 7A and 7B suggest that IL-13 may stimulate arginase in addition to iNOS. Since arginase competes with iNOS for L-arginine, the LMWP may increase NO generation by providing an additional substrate for iNOS.

[0108] Reduced Biofilm Bacterial Density

[0109] HTE cells were infected apically with S. aureus at 0.01 multiplicity of infection and incubated for 8 hours. The apical surface was washed to remove unbound bacteria and L- arginine or LMWP (50 nmoles total L-arg each, in μΐ PBS) was added to the apical surface and further incubated for 24 hours.

[0110] L-arginine treated HTE cells completely disintegrated due to bacterial overgrowth, because L-arg is a good nutrient for the bacteria. In contrast, cells infected with bacteria alone or along with LMWP were intact. Some of these cultures were fixed and immunostained with an antibody to S. aureus and zona occludin-1 and observed under confocal microscope to assess biofilm formation. HTE cells infected with bacteria alone (Fig. 8, left side) showed several large microcolonies resembling biofilm embedded in the mucus layer on the HTE cell surface, whereas cells treated with LMWP (Fig. 8, right side) showed much smaller microcolonies in the mucus layer (see Fig. 8). The microcolonies resembling biofilm were green in the original colored images and some of them are labeled G in Fig. 8. In the original colored confocal images, the nuclei were blue and some of them are labeled B in Fig. 8, and the tight junction protein zona occludin-1 was red and some of them are labeled R in Fig. 8.

[0111] The bacteria remaining on the surface of the human cells in each of the several wells were cultured to estimate the number of viable bacterial cells. The results are reported in Fig. 9 as the Log of colony forming units (CFU) per well. The estimation of viable bacteria by plating cell lysates indicated one log unit less bacteria in LMWP treated cells than in the controls (i.e., the PBS treated cells) (Fig. 9). [0112] In addition, there was a lack of further expression of IL-8 or LDH following LMWP treatment, indicating that there was no excessive release of toxins from dying bacteria (data not shown).

[0113] Similar reduction in bacterial counts in LMWP treated cultures were observed when both S. aureus and P. aeruginosa strains were used to generate biofilm on the surface of the HTE cultures (data not shown). These data indicate that LMWP reduce biofilm density and also kill bacteria. To determine whether LMWP reduce biofilm density or kill bacteria in the absence of epithelial cells, bacterial biofilm formed on the pegs of MBEC plates was exposed to LMWP for 1 hour and biofilm density was determined by crystal violet assay. From an identical experiment, bacterial viability was also determined. LMWP neither reduced biofilm density nor killed biofilm bacteria (data not shown). Together these results suggest that LMWP stimulates epithelial cell innate immune function by increasing NO generation, and it is this enhanced localized NO level that provides the antimicrobial environment to reduce viable S. aureus counts on the surface of the epithelial cells.

[0114] Reference throughout the specification to "one example", "another example", "an example", and so forth, means that a particular element (e.g., feature, structure, and/or characteristic) described in connection with the example is included in at least one example described herein, and may or may not be present in other examples. In addition, it is to be understood that the described elements for any example may be combined in any suitable manner in the various examples unless the context clearly dictates otherwise.

[0115] It is to be understood that the ranges provided herein include the stated range and any value or sub-range within the stated range. For example, a range from about 10 μΜ to about 200 μΜ should be interpreted to include not only the explicitly recited limits of about 10 μΜ to about 200 μΜ, but also to include individual values, such as 25 μΜ, 100 μΜ, 112.5 μΜ, etc., and subranges, such as from about 15 μΜ to about 175 μΜ, from about 50 μΜ to about 150 μΜ, etc. Furthermore, when "about" is utilized to describe a value, this is meant to encompass minor variations (up to +/- 10%) from the stated value.

[0116] In describing and claiming the examples disclosed herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. [0117] While several examples have been described in detail, it will be apparent to those skilled in the art that the disclosed examples may be modified. Therefore, the foregoing description is to be considered non-limiting.

Claims

What is claimed is:

1. A nasal formulation, comprising:

a liquid carrier to be introduced into upper airways through a nasal cavity; and low molecular weight arginine-rich peptides derived from protamine present in the liquid carrier to increase nitric oxide (NO) levels in the upper airways when introduced thereto;

wherein the nasal formulation has a pH ranging from 4 to 10.

2. The nasal formulation as defined in claim 1 wherein the low molecular weight arginine-rich peptides are derived from a protamine source via treatment with an immobilized thermolysin enzyme.

3. The nasal formulation as defined in claim 1 wherein the low molecular weight arginine-rich peptides have a structure with at least 8 amino acids.

4. The nasal formulation as defined in claim 3 wherein the low molecular weight arginine-rich peptides are selected from the group consisting of VSRRRRRGGRRRR (SEQ ID NO: 7), VSRRRRRGGRRRRR (SEQ ID NO: 8), and combinations thereof.

5. The nasal formulation as defined in claim 1 , excluding pure L-arginine monomer.

6. The nasal formulation as defined in claim 1 , further comprising an inhibitor of a L- arginase enzyme, the inhibitor selected from the group consisting of: s-boronoethyl-L-cysteine, 2(S)-amino-6-(borono) hexanoic acid), N5-(benzyloxycarbonyl)-N2-(tert-butoxycarbonyl)-L- thiocitrulline tert-butyl ester, N5-[N-(benzyloxycarbonyl)-N'-

(methoxycarbonylmethoxy)amidino]-N2-(tert-btoxycarbonyl)-L-ornithine tert-butyl ester), 6- (dihydroxyboranyl)-2-(3-phenoxypropyl)norleucine hydrochloride, 6-(dihydroxyboranyl)-2- (hydroxymethyl)-L-norleucine, 2-amino-6-(dihydroxyboryl)-2-[3-(4-piperidinyl)propyl]hexanoic acid, 2-[4-(dihydroxyboranyl)butyl]lysine, 6-(dihydroxyboranyl)-2-[2-(piperidin-l-yl)ethyl]-L- norleucine dihydrochloride, (3R,4S)-3-amino- 1 -[3-(4-carboxyphenyl)propyl]-4-[3- (dihydroxyboranyl)propyl]pyrrolidine-3-carboxylic acid), (2S)-2-amino-6-(dihydroxyboryl)-2- [cz5-3-[[(4-fluoro-lnaphthyl)methyl]amino]cyclobutyl]hexanoic acid), and (2S)-2-amino-2-[c 5-

3-[[(4'-chloro-3-nuorobiphenyl-4-yl)methyl]amino]cyclobutyl]-6-(dihydroxyboryl)hexanoic acid.

7. The nasal formulation as defined in claim 1 wherein the liquid carrier is selected from the group consisting of water, a saline solution, a saline solution with sodium bicarbonate (NaHC0₃), a phosphate buffered saline solution (PBS), an aqueous solution with calcium chloride (CaCl₂), and an aqueous solution with a sodium phosphate buffer or a potassium phosphate buffer.

8. The nasal formulation as defined in claim 7 wherein the liquid carrier consists of water, and wherein a concentration of the low molecular weight arginine-rich peptides in the water ranges from about 0.01 mM to about 5 mM.

9. A nasal kit, comprising:

a powder composition including low molecular weight arginine-rich peptides derived from protamine and an inhibitor of a L-arginase enzyme; and

a bottle for dissolving the powder composition in water to a concentration ranging from about 10 μΜ to about 5000 μΜ of peptides and from about 10 μΜ to about 200 μΜ of the L- arginase enzyme inhibitor.

10. The nasal kit as defined in claim 9 wherein the powder composition further comprises a salt, wherein the salt is selected from the group consisting of sodium chloride, sodium bicarbonate, calcium chloride, a sodium phosphate buffer, a potassium phosphate buffer, and combinations thereof.

11. The nasal kit as defined in claim 9 wherein the low molecular weight arginine-rich peptides derived from protamine have a structure with at least 8 amino acids.

12. The nasal kit as defined in claim 1 1 wherein the low molecular weight arginine-rich peptides are selected from the group consisting of VSRRRRRGGRRRR (SEQ ID NO: 7), VSRRRRRGGRRRRR (SEQ ID NO: 8), and combinations thereof.

13. The nasal kit as defined in claim 9 wherein the powder composition excludes pure L- arginine monomer.

14. A method for enhancing nasal nitric oxide (NO) levels, the method comprising introducing, via a nasal rinse or spray, low molecular weight arginine-rich peptides derived from protamine into a nasal cavity.