Compositions having thioredoxin activity and related methods

Abstract

To provide a method to decrease viscoelasticity of mucus or sputum in a patient that has excessively viscous or cohesive mucus or sputum.SOLUTION: Provided is a method including contacting the mucus or sputum of the patient with a composition comprising a protein or peptide comprising a thioredoxin monocysteinic active site in a reduced state, where the protein or peptide does not contain any cysteine residues except for a single Cys at the N-terminal position of the thioredoxin monocysteinic active site.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 956,994, filed January 3, 2020, the disclosure of which is incorporated herein by reference.

[0002] Sequence Listing Reference This application contains a sequence listing that has been submitted electronically as a text file via EFS-Web. The text file, named "7579-2-PROV_sequence_listing_v2_ST25.txt," has a size of 21 KB and was recorded on January 2, 2020. The information contained in the text file is incorporated herein by reference in its entirety in accordance with 37 CFR § 1.52(e)(5).

[0003] The present invention relates generally to the preparation, formulation, and use of thioredoxin proteins or peptides containing the thioredoxin active site in a reduced state to treat diseases and / or conditions, for example, to reduce mucus or sputum viscoelasticity, inflammation, and hypertension. [Background technology]

[0004] Thioredoxin (Trx) is an essential intracellular human gene product secreted by mucosal epithelia of the lungs, upper and lower GI tract, eyes, and reproductive system, where, together with the small tripeptide glutathione (GSH), it constitutes the majority of extracellular biological reducing power. In contrast to intracellular proteins, where most cysteine ​​(Cys) residues remain reduced due to the local reducing environment, oxygen exposure causes most Cys residues in extracellular proteins to form covalent disulfide bonds. Biological reducing agents act to reverse this disulfide bond nonselectively in the case of GSH and other small thiols, but selectively in the case of thioredoxin oxidoreductase, whose structural and chemical characteristics confer specificity for only certain disulfide conformations. Secreted thioredoxin has evolved to fulfill various homeostatic functions through a unique and efficient thiol-disulfide exchange mechanism that targets protein disulfides with specific conformations, including allosteric binding configurations associated with reversible regulatory control by thioredoxin-family oxidoreductases, as well as vicinal and other highly constrained disulfides, such as those formed intramolecularly in oxidized mucus proteins (mucins), and reduction by thioredoxin results in significant viscoelastic normalization of sputum in CF patients.Secreted thioredoxin regulates mediator release and inhibits neutrophil chemotaxis to inflammatory sites (Tian, ​​H., Matsuo, Y., Fukunaga, A., Ono, R., Nishigori, C., and Yodoi, J., 2013, Thioredoxin ameliorates cutaneous inflammation by regulating the epithelial production and release of pro-inflammatory cytokines. Frontiers in Immunology 4: 1-12) and inhibits inflammatory protease activity (Lee, RL, Rancourt, RC, del Val, G., Pack, K., Pardee, C., Accurso, FJ, and White, CW, 2005, Thioredoxin and dihydrolipoic acid inhibit elastase activity in cystic fibrosis sputum. Am J Physiol Lung Cell Mol Physiol 289: Thioredoxin exerts various anti-inflammatory effects through its thioredoxin-mediated reduction (L875-882). Thioredoxin has also been identified as a specific extracellular activator of a class of constitutively expressed endogenous antibacterial proteins (defensins) that are secreted onto mucosal surfaces and exhibit significantly enhanced potency and target pathogen range upon thioredoxin-mediated reduction of their central disulfide bond (Jaeger et al., 2013, Cell-mediated reduction of human β-defensin 1: a major role for mucosal thioredoxin. Mucosal Immunology 6, 1179-90). Thioredoxin is also classically considered an antioxidant protein due to its ability to selectively activate peroxidases and directly donate electrons to certain oxidized substrates.

[0005] There is a significant unmet medical need for safe, well-tolerated, and effective drugs for the treatment of patients with diseases characterized by thickened pathological mucus, chronic infection, and chronic inflammation. One such disease is cystic fibrosis (CF), a genetic disorder caused by mutations in the gene encoding cystic fibrosis transmembrane regulator (CFTR), a critical transmembrane channel involved in maintaining normal epithelial transport of chloride and bicarbonate. Defects in CFTR expression, accumulation, or function, resulting from approximately 2,000 cftr gene mutations, reduce cAMP-mediated release of chloride and bicarbonate transport, leading to dehydration and increased viscoelasticity of airway mucus, reduced periciliary layer depth, and impaired mucociliary transport (MCT). The resulting accumulation of inadequately cleared pathological mucus in the airways is central to the development of chronic endobronchial bacterial infection and persistent neutrophilic inflammation characteristic of CF (Fahy, JV, and Dickey, BF, 2010, Airway Mucus Function and Dysfunction. NEJM 363: 2233-2247). CF remains the most common inherited fatal disease, primarily in populations of Northern European descent, affecting more than 30,000 individuals in the United States and over 80,000 worldwide. Chronic cough, excessive sputum production, and respiratory complications are major causes of morbidity and reduced quality of life. Although life expectancy for CF patients continues to increase from 18 years before 1980 to over 40 years today, there remains a pressing need for improved therapies to further extend life expectancy and enhance quality of life. Therapies independent of CF genotype are particularly desirable.

[0006] Mucus is a continuously secreted supramolecular polymer gel that forms a protective barrier on epithelial surfaces and is responsible for transporting inhaled debris and bacteria out of the lungs via ciliary action and coughing. Therefore, proper viscoelasticity and hydration of the mucus layer, which allows efficient cilia-driven transport, are crucial for mucus function and the prevention of infection and inflammation. Normal mucus consists mostly of water (97%), with the remaining solid matter comprising mucin proteins, non-mucin proteins, salts, lipids, and cellular debris. The polymeric mucin glycoproteins MUC5AC and MUC5B are primarily responsible for the viscoelastic properties of the respiratory mucus gel. O-linked glycan hydroxyl groups contribute to water binding, while the mucins themselves form a tangled network that also participates in covalent and non-covalent inter- and intra-chain linkages. High molecular weight mucins are hypersecreted in response to disease stress and inflammation and are notable for their extremely high cysteine ​​content—294 and 273 Cys per mature monomer for MUC5AC (UniProt accession P98088) and MUC5B (UniProt accession Q9HC84), respectively. These abundant mucin Cys have the potential to form numerous intrachain disulfides upon exposure to O2 in the airways, and a nearly seven-fold increase in mucin disulfide bonds has been observed in CF patients relative to normal individuals (Yuan et al., 2015, Oxidation increases mucin polymer cross-links to stiffen airway mucus gels, Science Translational Medicine 7, 276ra227).

[0007] Similar to the shortening of a tightly wound rubber band, the increase in intrachain disulfide bonds in pathological mucus gels contracts mucin filaments and compacts the polymeric mucus gel structure. This disulfide-mediated tightening of the CF mucin mesh may provide a mechanism for the observed increase in mucus concentration and osmotic coefficient present in individuals with CF, which is responsible for causing dehydration of the periciliary layer (PCL) and loss of mucus transport. The fundamental importance of increased mucus viscoelasticity, rather than dehydration per se, in CF pathophysiology is supported by direct measurements of PCL hydration and MCT at the epithelial surface of living airways using recently developed high-resolution non-invasive imaging techniques (Birket, SE, et al., 2014, A functional anatomical defect of the cystic fibrosis airway, American Journal of Respiratory and Critical Care Medicine 190, 421-432; Chu, KK, et al., 2016, In vivo imaging of airway cilia and mucus clearance with micro-optical coherence tomography, Biomed Opt Express 7, 2494-2505).

[0008] Naturally secreted GSH and thioredoxin are likely compounds involved in preventing excess mucin disulfide bond formation in extracellular mucus. Net mucosal surface disulfide bond increases result from either reducing agent deficiency or elevated mucin protein levels, given the balance between oxidation-driven disulfide formation and reducing agent-driven disulfide destruction. Mucus is known to be hypersecreted in CF, and it has been observed that CF patients have an approximately 70% decrease in both reduced and oxidized forms of glutathione compared with normal subjects (Wetmore, DR, et al., 2010, Metabolomic profiling reveals biochemical pathways and biomarkers associated with pathogenesis in cystic fibrosis cells. JBC 285: 30516-22). This decrease in GSH is consistent across multiple published studies examining extracellular lung fluid in CF patients, suggesting a possibly indirect role for functional CFTR in maintaining normal rates of airway GSH efflux.

[0009] Even more strikingly, impaired CFTR-mediated bicarbonate efflux in CF epithelia is associated with an abnormally acidic airway pH that drops from 7.2 in normal individuals to less than 6.5 in CF patients (Garland AL, Walton WG, Coakley RD, et al., 2013, Molecular basis for pH-dependent mucosal dehydration in cystic fibrosis airways, Proceedings of the National Academy of Sciences 110:15973-8).

[0010] Due to the inherently high acid dissociation constant (pKa) of small molecule thiol agents, a low pH environment greatly attenuates the ability of endogenous or exogenous GSH (pKa 9.1) to form the deprotonated, reactive free thiolate anion required for nucleophilic disulfide bond attack. It has been calculated that at CF airway pH, only 0.25% of the natural GSH pool is in the active thiolate form. Thus, in CF, not only is there an increase in secreted mucus (and thus, mucin Cys capable of forming disulfide bonds), but there is also a decrease in GSH secretion, and the remaining GSH is functionally impaired due to the reduced pH of the CF airway.

[0011] Similarly, related thiol compounds used as investigational or approved mucolytic drugs, including NAC, cysteamine, and mesna (pKa values ​​of 9.5, 8.3, and 9.2, respectively), similarly lack the potential for significant disulfide-reducing activity in diseased airways. Furthermore, these agents derive their mechanism from simple reduced thiols that indiscriminately target any oxidized substrate, without the selectivity characteristic of enzyme therapeutics.

[0012] In contrast, thioredoxin, a large molecule with an unusually acidic pKa of 6.2 due to hydrogen bonding in its highly conserved enzyme active site, is significantly less sensitive to acidic pH. Recent proteomic studies have shown that thioredoxin comprises a significant proportion of secreted submucosal gland proteins along with newly formed airway mucus (Joo, NS, Evans, IA, Cho, HJ, Park, IH, Engelhardt, JF, and Wine, JJ 2015. Proteomic analysis of pure human airway gland mucus reveals a large component of protective proteins. PLoS One 10, e0116756), suggesting a more important functional role for this oxidoreductase in airway disulfide bond homeostasis than previously thought.

[0013] Treatment of Pathological Mucus: Therapeutically, clearance of mucus from obstructed airways is a key aspect of alleviating ongoing chronic infection and inflammation in obstructive / inflammatory diseases such as CF. Physical therapy, mechanical percussion devices, and inhaled mucus-liquefying (mucolytic) medications are all components of current treatment regimens for sputum clearance in CF patients. However, existing treatments are largely symptomatic and have not been shown to be effective in alleviating the underlying mucus defects that mechanistically lead to inadequate clearance.

[0014] The most commonly used mucolytic compound in CF is recombinant human DNase I (DNase; Dornase Alfa), trademarked by Genentech as Pulmozyme®. DNase improves lung function by hydrolyzing sticky, accumulated neutrophil-derived nucleic acids, but recent studies have shown that excess disulfide bonds in mucus proteins, rather than extracellular DNA accumulation, may play a major role in disease pathogenesis.

[0015] Despite its widespread use, DNase has numerous drawbacks. DNase is a disulfide-bonded, glycosylated, human enzyme with a moderately large monomeric size that requires mammalian cell culture for production, making it one of the more expensive drugs to produce. The target of DNase, excess free nucleic acids, may be present as a result of severe chronic infection or may not be found at appreciable levels in early / less severe CF (although some patients with early-stage disease report benefit), and DNase has not demonstrated clinical benefit in other obstructive pulmonary diseases. Clinical deterioration of lung function with DNase treatment is seen in 6–30% of pediatric patients. DNase may also exacerbate inflammation by promoting the activity of neutrophil elastase, a proteolytic enzyme that is inhibited by the presence of nucleic acids targeted by DNase.

[0016] Despite aggressive use of DNase, response is low, and CF disease usually progresses to bronchiectasis, respiratory failure, and death or transplant in children or early adulthood. Although airway hydration / cough induction therapies, such as mannitol or hypertonic saline inhalation, have shown promise in clinical trials and some are currently used in patient care, there remains a significant lack of effective mucus treatments, especially those that directly target pathological mucus.

[0017] Unfortunately, the results of various thiol-containing small molecules that have been evaluated as mucus drugs have been disappointing. These agents include NAC and nasisterone (NAC; NAC + L-lysine), as well as reduced GSH and cysteamine. While mostly safe, to date, these small molecule agents have not demonstrated clear clinical benefit in either oral or inhaled form.

[0018] Part of this poor efficacy may be the result of loss of potency caused by autoxidation upon inhalation delivery as well as the potential for pulmonary enzymes to rapidly convert GSH to an inactive form, but as noted above, it is more likely to involve the inherently low activity of nonenzymatic thiol agents at acidic CF airway pH caused by their highly basic thiol pKa.

[0019] Like mucus overproduction, defective bicarbonate secretion and airway acidification may underlie other obstructive pulmonary diseases that are not limited to CF and affect large populations. Therefore, truly effective, mechanistically directed mucus-modulating treatments could offer widespread medical benefit. It would be highly desirable to improve thiol agents by combining disulfide targeting with the superior potency, stability, and specificity of biological drugs like thioredoxin. Summary of the Invention [Problem to be solved by the invention]

[0020] Unfortunately, development of formulation strategies for thiol-based therapeutics that can safely stabilize proteins in their reduced form has been limited. Previous attempts (PCT WO2006 / 090127) required painstaking screening of numerous excipients to find a combination of sugar and chemical stabilizer components that would allow both components to remain reduced during long-term storage in solid form and when reconstituted into a liquid solution for delivery. The reduced sugar-based formulations resulting from this strategy proved to be significantly inflammatory and therefore unsuitable for use in inhalation delivery. Native thioredoxin reconstituted in saline using this sucrose formulation resulted in high levels of neutrophil influx and proinflammatory cytokine release when delivered to rats via intratracheal administration (Rancourt, RC, et al., 2007, Reduced thioredoxin increases proinflammatory cytokines and neutrophil influx in rat airways: modulation by airway mucus. Free Radic Biol Med 42, 1441-53). Therefore, there remains a need for safe and effective formulation approaches for thiol-based protein therapeutics. [Means for solving the problem]

[0021] One aspect of the present invention is a method for reducing the viscoelasticity of mucus or sputum in a patient having excessively viscous or sticky mucus or sputum, the method comprising contacting the patient's mucus or sputum with a composition comprising a protein or peptide comprising a reduced thioredoxin monocysteine ​​active site, wherein the protein or peptide does not contain any cysteine ​​residues except for a single Cys at the N-terminal position of the thioredoxin monocysteine ​​active site.

[0022] In another aspect of the present invention, a pharmaceutical composition is provided, the composition comprising a protein or peptide having a reduced thioredoxin monocysteine ​​active site and containing no cysteine ​​residues except for a single Cys at the N-terminal position of the thioredoxin monocysteine ​​active site, and a pharmaceutically acceptable excipient.

[0023] A further aspect of the invention is a composition comprising a protein or peptide having a thioredoxin active site in a reduced state and an aqueous solvent having a vapor pressure of at least about 3 mmHg.

[0024] In yet another aspect of the present invention, there is provided a pharmaceutical composition consisting essentially of a protein or peptide containing a reduced state of a thioredoxin active site, water, and sodium chloride.

[0025] Another aspect of the invention is a method of preparing a dry composition comprising providing an aqueous composition comprising a protein or peptide comprising a reduced thioredoxin active site and an aqueous solvent having a vapor pressure of at least about 3 mmHg, the method further comprising volatilizing the aqueous solvent to produce a dry composition comprising the protein or peptide.

[0026] A still further aspect of the invention is a composition consisting essentially of, or consisting of, a protein or peptide containing a reduced state of a thioredoxin active site and normal saline.

[0027] Another aspect of the invention is a composition consisting essentially of a protein or peptide containing a reduced state of a thioredoxin active site, wherein the composition is a dry powder.

[0028] Another method of the invention is a method of treating inflammation in a subject, the method comprising administering to the subject a pharmaceutical composition comprising a protein or peptide comprising a reduced thioredoxin monocysteine ​​active site, wherein the subject has or is at risk of developing inflammation.

[0029] A still further aspect of the invention is a method of treating a bacterial infection in a subject, wherein the subject has or is at risk of developing a bacterial infection by administering a pharmaceutical composition comprising a protein or peptide having a reduced thioredoxin monocysteine ​​active site.

[0030] A further aspect of the present invention is a composition comprising a thioredoxin monocysteine ​​active site operable to activate one or more endogenous antimicrobial peptides, wherein the activation results in a therapeutically effective reagent for treating or preventing infectious disease.

[0031] A further method of the invention is a method of modulating the microbiome composition of a subject by topically administering to a mucosal surface of the subject a composition comprising a protein or peptide having a reduced thioredoxin monocysteine ​​active site.

[0032] A still further method is for determining the disulfide bond reducing activity of a protein or peptide containing a monocysteine ​​thioredoxin active site by selecting a protein or peptide containing a monocysteine ​​thioredoxin active site that does not contain any cysteine ​​residues except for a single Cys in the thioredoxin monocysteine ​​active site, and measuring the overall cysteine ​​thiol reduction state of the protein or peptide.

[0033] Yet another aspect of the invention is a method of treating a viral respiratory disease in a subject having or at risk of developing a viral respiratory disease by administering to the subject a composition comprising a protein or peptide comprising a reduced state of a thioredoxin monocysteine ​​active site.

[0034] The present invention further includes a method of reducing pulmonary inflammation associated with a viral infection in a subject in need thereof by administering to the subject in need thereof a pharmaceutical composition comprising a protein or peptide comprising a reduced state of a thioredoxin monocysteine ​​active site.

[0035] Another aspect of the invention is a composition comprising a protein or peptide having a thioredoxin active site, wherein the composition does not contain a thioredoxin protein fraction having a UV absorbance greater than about 400 nm wavelength.

[0036] A further aspect of the invention is a method of producing a composition comprising a protein or peptide that comprises a thioredoxin active-site by providing a lysate containing a protein or peptide that comprises a thioredoxin active-site, concentrating the protein or peptide in the lysate, and removing thioredoxin peptide or protein fractions having an absorbance greater than about 400 nm to produce the composition.

[0037] In various embodiments of the invention, a thioredoxin active site comprising an amino acid sequence selected from the group consisting of CXXS (SEQ ID NO:24), CXXX (SEQ ID NO:17), XCXXXX (SEQ ID NO:19), XCGPXX (SEQ ID NO:21), WCGPXK (SEQ ID NO:23), XCXXSX (SEQ ID NO:25), XCGPSX (SEQ ID NO:26), and WCGPSK (SEQ ID NO:27) is a thioredoxin monocysteine ​​active site, wherein the X residue is any amino acid residue other than cysteine. In other embodiments, the protein or peptide comprises a sequence at least about 80% identical to SEQ ID NO:28 or SEQ ID NO:29, and the thioredoxin active site is a thioredoxin monocysteine ​​active site at a position corresponding to positions 32-35 of SEQ ID NO:28 or SEQ ID NO:29. In still further embodiments, the protein or peptide comprises the sequence of SEQ ID NO:28 or SEQ ID NO:29. In other embodiments, the protein comprises human thioredoxin.

[0038] In some embodiments of the present invention, the patient has a pulmonary disease in which abnormal or excessive viscosity or stickiness of mucus or sputum is a symptom or cause of the disease. The patient may also have a pulmonary disease in which abnormal or excessive viscosity or stickiness of mucus or sputum is associated with a lack of biological reducing agent activity. In other embodiments, the patient has a disease selected from the group consisting of cystic fibrosis, chronic obstructive pulmonary disease, bronchiectasis, asthma, sinusitis, idiopathic pulmonary fibrosis, pulmonary hypertension, dry eye disease, and gastrointestinal disease. In another embodiment, the patient has cystic fibrosis. In some embodiments, the patient is human.

[0039] In embodiments of the method for reducing the viscoelasticity of excessively viscous or sticky mucus or sputum in a patient, the step of contacting the patient's mucus or sputum with the composition is carried out by introducing the composition into the patient by a route selected from the group consisting of nasal, intratracheal, bronchial, direct pulmonary placement, inhalation, oral, and ocular. In other embodiments, the mucus or sputum to be contacted is in the patient's respiratory tract, and in other embodiments, after the step of contacting the patient's mucus or sputum with the composition, the patient has an increase in forced expiratory volume (FEV) of at least about 2.5% compared to before the contacting step.

[0040] In a further embodiment, a protein or peptide containing a thioredoxin monocysteine ​​active site is covalently attached to a cysteine ​​residue in a mucus protein, such as when the mucus protein is a mucin, e.g., a respiratory mucus protein.

[0041] In embodiments of the invention, the composition may include a pharmaceutically acceptable carrier, and in such pharmaceutical compositions, the protein or peptide may comprise the thioredoxin monocysteine ​​active site sequence of SEQ ID NO: 1. The pharmaceutical compositions of the invention may be formulated for administration to a patient by a route selected from oral, rectal, nasal, inhalation, intratracheal, bronchial, direct instillation, topical, and ocular.

[0042] In embodiments of the present invention having an aqueous solvent with a vapor pressure of at least about 3 mmHg, the aqueous solvent may be selected from ammonium acetate, ammonium bicarbonate, ammonium formate, triethylammonium acetate, and triethylammonium bicarbonate. The aqueous solvent may be ammonium acetate. In other embodiments, the aqueous solvent may be at a concentration of between about 1 mM and about 50 mM and / or have a pH of between about 4 and about 7.

[0043] In some embodiments, the compositions of the present invention do not contain sugars or sugar derivatives, hi other embodiments, the aqueous compositions do not contain any compounds other than proteins or peptides that have a vapor pressure of less than about 3 mmHg.

[0044] In other embodiments of the invention, the protein or peptide contains no cysteine ​​residues except for one or two Cys in the thioredoxin active site. In still other embodiments, the thioredoxin active site may comprise an amino acid sequence selected from CXXC (SEQ ID NO: 16), XCXXCX (SEQ ID NO: 20), XCGPCX (SEQ ID NO: 22), WCGPCK (SEQ ID NO: 3), where the X residue is any amino acid residue other than cysteine. In further embodiments, the thioredoxin active site is a monocysteine ​​thioredoxin active site, and the protein or peptide may contain no cysteine ​​residues except for a single Cys at the N-terminus of the thioredoxin monocysteine ​​active site.

[0045] In embodiments of the present invention that include sodium chloride, the sodium chloride may be present at about 9 grams of sodium chloride per liter of water.

[0046] In embodiments that include volatilizing the aqueous solvent, the volatilizing step can include subjecting the composition to conditions selected from the group consisting of reduced pressure, elevated temperature, and combinations thereof. In such embodiments, the volatilizing step can be performed under a non-oxidizing atmosphere, such as a nitrogen atmosphere. In other embodiments, the volatilizing step can include lyophilization.

[0047] In embodiments of the invention that involve forming a dry pharmaceutical composition, the method may include solubilizing the dry composition in a diluent, for example, a saline solution having a pH between about 4 and about 7. Such solubilized pharmaceutical compositions may be at least 80% stable in the reduced form for at least about 1 day at a temperature of about 25° C., or may be at least 80% stable in the reduced form for at least about 1 week at a temperature of about 25° C. In other such embodiments, the thioredoxin may comprise a monocysteine ​​thioredoxin active site, or more particularly, a thioredoxin monocysteine ​​active site in a reduced state, and the protein or peptide does not contain any cysteine ​​residues except for a single cysteine ​​residue N-terminal to the thioredoxin monocysteine ​​active site.

[0048] In the methods of the present invention for treating inflammation, the release of the protein or peptide may inhibit the release of an inflammatory cytokine, for example, an inflammatory cytokine selected from IL-8, IL-1β, IL-6 and TNFα.

[0049] In the methods of the present invention for treating bacterial infections, the compositions may include crude or purified extracts of microbial cells that express the protein or peptide.

[0050] In embodiments of the present invention that include compositions that activate one or more endogenous antimicrobial peptides, the antimicrobial peptides may be defensins.

[0051] In embodiments for modulating the microbiome composition of a subject, the mucosal surface may be a pulmonary surface, a nasopharyngeal surface, or a gastrointestinal surface.

[0052] In embodiments for determining the disulfide bond reducing activity of a protein or peptide, the cysteine ​​thiol reduction state can be measured using a method selected from a chromogenic assay, a fluorometric assay, and a turbidometric assay. For example, the chromogenic assay can be a DTNB assay.

[0053] In embodiments relating to treating viral respiratory diseases, the disease may be selected from acute respiratory distress syndrome (ARDS), severe acute respiratory distress syndrome (SARS), Middle East respiratory syndrome (MERS), SARS-coronavirus-2 (SARS-CoV-19 or COVID-19), influenza, asthma, pneumonia, bronchitis, tuberculosis, reactive airway disease syndrome, and viral infections associated with interstitial lung disease. In such embodiments, the viral respiratory disease may be caused by a virus selected from coronavirus, influenza virus, respiratory syncytial virus (RSV), parainfluenza virus, and respiratory adenovirus.

[0054] In various embodiments of the present invention, the compositions may be administered in a nebulized or aerosolized form, or in the form of a dry powder for inhalation.

[0055] In compositions of the present invention having a protein or peptide having a thioredoxin active site and not including a thioredoxin protein fraction having a UV absorbance greater than about 400 nm wavelength, such compositions may further include an aqueous solvent having a vapor pressure of at least about 3 mmHg. Alternatively, the compositions may consist essentially of a protein or peptide having a reduced thioredoxin active site, water, and sodium chloride. Furthermore, such compositions may be dried to a water content of less than about 3.0% by weight.

[0056] In methods of the invention for producing a composition having a protein or peptide with a thioredoxin active site and removing a thioredoxin peptide or protein fraction having an absorbance at greater than about 400 nm to produce the composition, the composition can be further characterized as having a peptide or protein fraction having an absorbance at a UV light wavelength of less than about 300 nm. In such embodiments, the removing step can include hydrophobic interaction chromatography and / or drying the composition to a water content of less than about 3.0% by weight. [Brief explanation of the drawings]

[0057] [Figure 1] FIG. 1 shows the mechanism of mutation of cysteine ​​35 to serine (C35S TRX). [Figure 2] FIG. 2 shows the pH dependence of the thiol agent reducing activity against thioredoxin. [Figure 3] FIG. 3 shows the stability of the lyophilized solid form and solution of monothiol C35S thioredoxin. [Figure 4] FIG. 4 shows the UV spectra of the main thioredoxin fraction (top) and the red fraction with absorbance >400 nm (bottom) isolated by hydrophobic interaction chromatography. [Figure 5] Figure 5 shows the reduction in A. elastic modulus (G'), B. viscous modulus (G"), and C. mucin molecular weight (GPC-MALLS) of 4% solids dry weight mucus reduced with DTT (1 mM) and ORP100S (0.01, 0.1, and 1.0 mM concentrations) at 37°C for 1 hour. [Figure 6] FIG. 6 shows μOCT analysis of ORP100S (“Theradux”) in primary HBE from a CF patient donor. [Figure 7] FIG. 7 shows μOCT analysis of ORP100S (“Theradux”) in CF patient sputum. [Figure 8] FIG. 8 shows the effect of thioredoxin and C35S thioredoxin on the levels of IL-6 or TNFalpha induced after 24 hours in the basolateral ALI medium of primary HBE cultures derived from nasal epithelium of non-CF and CF donors. [Figure 9] FIG. 9 shows nebulized aerosol delivery of ORP-100 and ORP100S in rats and attenuation of formulation-induced neutrophil influx in vivo. DETAILED DESCRIPTION OF THE INVENTION

[0058] The present invention relates generally to the use of thioredoxin proteins or peptides containing a reduced thioredoxin active site to treat mucosal diseases characterized by symptoms including one or more of abnormal mucus, inflammation, infection, or high blood pressure. More specifically, the inventors have discovered that proteins or peptides having a thioredoxin active site, including thioredoxin proteins or peptides containing a monocysteine ​​active site, are effective agents for reducing the viscoelasticity and / or adhesiveness of inflamed or abnormal sputum or mucus, thereby normalizing sputum or mucus.

[0059] Thus, proteins or peptides containing the reduced thioredoxin active site, or nucleic acid molecules encoding such proteins, can be used alone or in compositions to treat various conditions or diseases associated with unwanted mucus or thick, viscous sputum, as well as inflammation, hypertension, and / or infection. For example, respiratory diseases, such as cystic fibrosis, chronic obstructive pulmonary disease, bronchiectasis, sinusitis, idiopathic pulmonary fibrosis, pulmonary hypertension, and asthma, including status asthmaticus, are particularly suitable for treatment using the products and processes of the present invention. Gastrointestinal diseases associated with thickened or sticky mucus, such as coccidiosis, are also particularly suitable for treatment using the products and processes of the present invention. Similar diseases of other mucosal surfaces characterized by abnormally thickened mucus secretion, inflammation, and / or infection, such as dry eye disease, are also suitable for treatment, as are eye diseases involving oxidative stress and inflammation, including macular degeneration, diabetic retinopathy, glaucoma, and cataracts.

[0060] Thus, the present invention relates to the use of proteins or peptides containing a reduced thioredoxin active site, including those containing a monocysteine ​​active site, to reduce the viscoelasticity of mucus or sputum, particularly mucus or sputum that is abnormally or excessively viscous and / or sticky. The proteins can be administered to patients suffering from or affected by such abnormal or excessive mucus or sputum in a manner and amount effective to reduce the viscoelasticity of the mucus or sputum, preferably to provide a therapeutic benefit to the patient. Furthermore, the proteins can be administered to patients suffering from inflammation or infection, including those with or at risk of a runaway inflammatory response, e.g., cytokine release syndrome (CRS), and related acute lung injury, e.g., acute respiratory distress syndrome (ARDS).

[0061] Proteins and peptides containing the thioredoxin active site Thioredoxin-1 (Trx) is a small (12 kDa), naturally occurring redox protein with protein disulfides as its preferred substrate. Trx is an essential human protein that plays a key biological role in regulating protein and enzyme activity through potent, specific disulfide bond reduction.

[0062] Trx has a redox-active dithiol in its highly conserved Cys-Gly-Pro-Cys (SEQ ID NO: 1) active site, which is reduced from the oxidized form by the flavoenzyme thioredoxin reductase (TrxR) and the cofactor NADPH. Together, these three components form the thioredoxin system, whose reducing potential is many times more potent than that of small molecule reducing agents.

[0063] Due to its role in reversible disulfide bond regulation, mammalian Trxs are involved in numerous intracellular and extracellular redox signaling activities, including serving as cofactors for methionine sulfoxide reductase, modifying the DNA-binding activity of receptors and transcription factors, and participating in protein folding. Furthermore, Trxs can scavenge free radicals and protect cells from oxidative stress, and secreted Trxs are required at mucosal surfaces for the activation (by disulfide bond reduction) of the important secreted antibacterial human β-defensin-1, hBD-1.

[0064] Thioredoxin proteins or peptides as disclosed herein have advantages over other reducing agents for use in treating conditions such as cystic fibrosis. For example, unlike other reducing agents, such as N-acetylcysteine ​​(NAC), nacystelyn (NAL), dithiothreitol (DTT), or reduced glutathione (GSH), the mutant thioredoxins disclosed herein are less susceptible to inactivation by enzymatic or autooxidative mechanisms, including reactions that generate superoxide, hydrogen peroxide, hydroxyl radicals, and other toxic oxygen metabolites. Furthermore, native or wild-type thioredoxin is a naturally occurring compound that is normally secreted extracellularly onto the airway surface; therefore, introduction of thioredoxin into the airways should be non-irritating and unlikely to induce an inappropriate immune response. Thioredoxin is also not glycosylated, and as such, is more easily manufactured, and administration of the native or recombinant form of the protein should not induce an innate immune response. Perhaps more importantly, reduced thioredoxin, in contrast to other reducing agents, restores treated mucus or sputum to normal viscosity levels more rapidly and potently, and this normalization lasts for a longer period of time. NAC, NAL, DTT, and GSH, for example, become "consumed" or oxidized over time, at which point normalized sputum or mucus may return to an abnormally viscous state. In contrast, the reduction in viscosity or viscoelasticity brought about by thioredoxin lasts longer, likely due to periodic re-reduction by its reduction system. Furthermore, the monocysteine ​​active site thioredoxin disclosed herein remains covalently bound to mucin Cys residues, thereby creating a more potent and longer-lasting reduction in viscosity compared to native thioredoxin. Finally, thioredoxin is both more potent and more specific for disulfide bond reduction than other reducing agents, and therefore can be used at significantly lower doses than other agents to achieve beneficial effects.

[0065] As discussed above, thioredoxin (Trx) is a protein disulfide reductase that catalyzes numerous thiol-dependent cellular reduction processes. Native thioredoxins contain two redox-active cysteines that are highly conserved across species. In their oxidized form, these cysteines form disulfide bridges that protrude from the three-dimensional structure of the protein. Protein disulfides are the preferred substrates for Trx-mediated reduction. Modification of one of the two Trx active site cysteines to a residue other than cysteine ​​results in a monocysteine ​​active site.

[0066] The present invention generally relates to the use of thioredoxin proteins or peptides containing a reduced thioredoxin active site. Reference to a "thioredoxin active site" includes either a thioredoxin monocysteine ​​(i.e., monothiol) active site comprising the amino acid sequence CXXX (SEQ ID NO: 17) or a native (or wild-type) thioredoxin dithiol active site containing two redox-active cysteines (an N-terminal cysteine ​​and a C-terminal cysteine) comprising the amino acid sequence CXXC having SEQ ID NO: 16. As used herein, an amino acid residue designated "C" is a cysteine ​​residue, and an amino acid residue designated "X" can be any amino acid residue other than a cysteine ​​residue, particularly any of the remaining 20 standard amino acid residues or synthetic, non-natural, or modified amino acids. The identity of an X residue is independent of other X residues; that is, the identity of any X residue can be the same or different from other X residues.

[0067] A thioredoxin active site of the present invention may comprise the amino acid sequence CGPX (SEQ ID NO: 18), with the native or wild-type sequence comprising the amino acid sequence CGPC (SEQ ID NO: 1). A thioredoxin active site may further comprise the amino acid sequence XCXXXX (SEQ ID NO: 19), with the native or wild-type sequence comprising the amino acid sequence XCXXCX (SEQ ID NO: 20). Additionally, a thioredoxin active site of the present invention comprises the amino acid sequence XCGPXX (SEQ ID NO: 21), with such amino acid residues designated "G" being glycine residues and such amino acid residues designated "P" being proline residues, with the native or wild-type sequence comprising the amino acid sequence XCGPCX (SEQ ID NO: 22). Another thioredoxin active site of the present invention comprises the amino acid sequence WCGPXK (SEQ ID NO: 23), with such amino acid residues designated "W" being tryptophan residues and such amino acid residues designated "K" being lysine residues, with the native sequence comprising the amino acid sequence WCGPCK (SEQ ID NO: 3). A thioredoxin active site may comprise the amino acid sequence CXXS (SEQ ID NO: 24). Such thioredoxin active sites of the present invention preferably comprise the amino acid sequence CGPS ​​(SEQ ID NO: 1). Thioredoxin active sites may further comprise the amino acid sequence XCXXSX (SEQ ID NO: 25), XCGPSX (SEQ ID NO: 26), or WCGPSK (SEQ ID NO: 27), where the amino acid residue represented by "X" can be any amino acid residue except a cysteine ​​residue. Monocysteine ​​thioredoxin active sites can be altered from the corresponding native sequence by substituting the C-terminal cysteine ​​of the native active site, as described above. Additionally, thioredoxin active sites can be altered by deleting the C-terminal cysteine ​​of the native active site.

[0068] Further variants of thioredoxin A thioredoxin protein or peptide containing a thioredoxin active site may further comprise one or more cysteine ​​deletions, substitutions, or a combination thereof at non-active site cysteine ​​residues outside the thioredoxin active site. In one embodiment, one or more cysteines outside the thioredoxin active site are substituted with any amino acid residue other than a cysteine ​​residue. In one embodiment, one or more cysteines outside the thioredoxin active site are substituted with any amino acid residue other than a cysteine ​​or alanine residue. In one embodiment, one or more cysteines outside the thioredoxin active site are substituted with a serine residue. In a further embodiment, all of the non-active cysteines outside the thioredoxin active site in the thioredoxin protein or peptide are deleted, substituted with a serine residue, or a combination thereof. In still further embodiments, all of the non-active cysteines outside the thioredoxin active site in the thioredoxin protein or peptide are deleted and / or substituted with serine residues, or a combination thereof, and the C-terminal cysteine ​​in the thioredoxin active site is also substituted with a serine residue.

[0069] Types of thioredoxin In one aspect of the present invention, the thioredoxin protein containing the thioredoxin active site is a full-length thioredoxin protein or any fragment thereof containing the thioredoxin active site as structurally and functionally described above. Preferred thioredoxin proteins having an active site include prokaryotic thioredoxin, yeast thioredoxin, plant thioredoxin, and animal thioredoxin, with mammalian and human thioredoxin being further embodiments of animal thioredoxin. Nucleic acid and amino acid sequences of thioredoxin proteins from various organisms are known in the art and are intended to be encompassed by the present invention. For example, SEQ ID NOs: 4 to 15 represent the amino acid sequences of thioredoxins from Pseudomonas syringae (SEQ ID NO: 4), Porphyromonas gingivalis (SEQ ID NO: 5), Listeria monocytogenes (SEQ ID NO: 6), Saccharomyces cerevisiae (SEQ ID NO: 7), Gallus (SEQ ID NO: 8), Mus musculus (SEQ ID NO: 9), Rattus norvegicus (SEQ ID NO: 10), Bos taurus (SEQ ID NO: 11), Homo sapiens (SEQ ID NO: 12), Arabidopsis thaliana (SEQ ID NO: 13), Zea mays (SEQ ID NO: 14), and Oryza sativa (SEQ ID NO: 15). Referring to each of these sequences, the CXXC motif having SEQ ID NO:16 can be found as follows: SEQ ID NO:4 (positions 34-37), SEQ ID NO:5 (positions 29-32), SEQ ID NO:6 (positions 28-31), SEQ ID NO:7 (positions 30-33), SEQ ID NO:8 (positions 32-35), SEQ ID NO:9 (positions 32-35), SEQ ID NO:10 (positions 32-35), SEQ ID NO:11 (positions 32-35), SEQ ID NO:12 (positions 32-35), SEQ ID NO:13 (positions 60-63), SEQ ID NO:14 (positions 89-92) and SEQ ID NO:15 (positions 95-98).

[0070] Modifications outside the active site With reference to SEQ ID NO: 12, inactive cysteine ​​residues outside the thioredoxin active site that can be deleted and / or substituted can be found at positions 62, 69, and 73 of the human thioredoxin-1 sequence. Again referring to SEQ ID NO: 12, active site Cys are found at positions 32 and 35, with the cysteine ​​at position 32 referred to as the N-terminal cysteine ​​and the cysteine ​​at position 35 referred to as the C-terminal cysteine. In one embodiment, the cysteines at positions 62, 69, and 73 of SEQ ID NO: 12 or corresponding positions in other thioredoxins are deleted, substituted with any amino acid residue other than a cysteine ​​residue, or a combination thereof. In yet another embodiment, the cysteines at positions 62, 69, and 73 of SEQ ID NO: 12 or corresponding positions in other thioredoxins are substituted with any amino acid residue other than a cysteine ​​or alanine residue. In a further embodiment, the cysteines at positions 62, 69, and 73 of SEQ ID NO: 12, or the cysteines at corresponding positions in other thioredoxins, are substituted with serine. In yet another further embodiment, the cysteines at positions 35, 62, 69, and 73 of SEQ ID NO: 12, or the cysteines at corresponding positions in other thioredoxins, are substituted with serine. In one embodiment, the cysteines at positions 62, 69, and 73 of SEQ ID NO: 12, or the cysteines at corresponding positions in other thioredoxins, are deleted, and the cysteine ​​at position 35 of SEQ ID NO: 12, or the cysteine ​​at the corresponding position in other thioredoxins, is substituted with any amino acid residue other than a cysteine ​​residue, preferably with a serine residue. In yet another embodiment, the cysteines at positions 62, 69, and 73 of SEQ ID NO: 12 or the cysteines at corresponding positions in other thioredoxins are deleted and / or substituted with any amino acid residue other than a cysteine ​​residue, and / or a combination thereof, and the cysteine ​​at position 35 of SEQ ID NO: 12 or the cysteine ​​at corresponding positions in other thioredoxins is substituted with any amino acid residue other than a cysteine ​​residue, preferably with a serine residue.

[0071] Single cysteine ​​thioredoxin In certain embodiments of the invention, the thioredoxin protein is a protein or peptide comprising a reduced thioredoxin monocysteine ​​active site, where the protein or peptide does not contain any cysteine ​​residues except for a single cysteine ​​residue in the thioredoxin monocysteine ​​active site, e.g., the thioredoxin proteins are set forth as SEQ ID NOs:28 and 29, which are fully monocysteine ​​variants of SEQ ID NO:12, with the only cysteine ​​at position 32 (or more generally, at the N-terminal position of the thioredoxin active site). This embodiment of the invention also includes variants of SEQ ID NOs:28 and 29 that have amino acids substituted and / or deleted and are still fully monocysteine ​​and have the only cysteine ​​at position 32 (or more generally, at the N-terminal position of the thioredoxin active site). Such variants may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 such substitutions and / or deletions relative to the sequence of SEQ ID NO:28 or SEQ ID NO:29. In alternative embodiments, variants may be characterized as having at least about 80% identity to SEQ ID NO:28 or SEQ ID NO:29, at least about 85% identity to SEQ ID NO:28 or SEQ ID NO:29, at least about 90% identity to SEQ ID NO:28 or SEQ ID NO:29, at least about 95% identity to SEQ ID NO:28 or SEQ ID NO:29, at least about 99% identity to SEQ ID NO:28 or SEQ ID NO:29 or any integer percent identity between 80% and 99%.

[0072] The three-dimensional structures of several thioredoxin proteins, including human and bacterial thioredoxins, have been elucidated. Thus, the structures and active sites of thioredoxins from multiple organisms are known in the art, and those skilled in the art will be able to readily identify and generate fragments or homologs of full-length thioredoxins that can be used in the present invention, including thioredoxins with monocysteine ​​active sites in combination with deletions, substitutions, or combinations thereof of inactive cysteine ​​residues outside the thioredoxin active site. An example of a thioredoxin protein is ORP-100, a thioredoxin protein with a monothiol active site in which the second active site cysteine ​​at position 35 (i.e., the C-terminal cysteine) is replaced with a serine residue. ORP100S is a thioredoxin protein (SEQ ID NO: 29) with a monothiol active site in which the second active site cysteine ​​at position 35 (i.e., the C-terminal cysteine) has been replaced with serine, and all of the non-active cysteine ​​residues outside the thioredoxin active site (found at positions 62, 69, and 73) have been replaced with serine residues.

[0073] Reduced cysteine The phrase "in a reduced state" specifically describes the state of cysteine ​​residues in the active site of a protein or peptide of the invention. In the reduced state, adjacent cysteine ​​residues form a dithiol (i.e., two free sulfhydryl groups, -SH). In contrast, in the oxidized form, such cysteine ​​residues form an intramolecular disulfide bridge, and such molecules are sometimes referred to as cystine. In the reduced state, the thioredoxin active site can participate in redox reactions via reversible oxidation of its active site thiol to a disulfide, catalyzing a thiol-disulfide exchange reaction that results in covalent linkage to one of the target disulfide cysteines. For proteins or peptides of the invention that contain a thioredoxin monothiol active site and further include deletion, substitution with any amino acid residue other than cysteine, or a combination thereof, of one or more cysteine ​​residues outside the thioredoxin active site, the N-terminal cysteine ​​in the active site is in a reduced state as a monothiol and therefore can form a stable mixed disulfide with a cysteine ​​on the target protein.

[0074] Protein or peptide size As used herein, a protein or peptide of the invention containing a thioredoxin active site can be the thioredoxin active site itself or a thioredoxin active site conjugated to another amino acid by a glycosidic bond. Thus, the minimum size of a protein or peptide of the invention is about 4 to about 6 amino acids in length, with preferred sizes varying depending on whether a full-length, fusion, multivalent, or simply functional portion of such a protein is desired. Preferably, the length of a protein or peptide of the invention ranges from about 4 to about 100 amino acid residues or more, with peptides of any intermediate length, including integers (i.e., 4, 5, 6, 7...99, 100, 101...), specifically contemplated. Short thioredoxin mimetic peptides interrupted at the N- and C-termini, such as those described by Bachnoff et al., Free Radical Biol Med 50:1355-67, 2011, may also be used.

[0075] homologue In a more preferred embodiment, the protein of the present invention may be a full-length protein or any homolog of such a protein. As used herein, the term "homologue" refers to a protein or peptide that differs from a naturally occurring protein or peptide (i.e., an "original" or "wild-type" protein) by modifications to the naturally occurring protein or peptide, but maintains the basic protein and side chain structure of the naturally occurring form and / or the basic three-dimensional structure of at least a biologically active portion of the naturally occurring protein (e.g., the thioredoxin active site). Such modifications include, but are not limited to, changes in one or several amino acid side chains, deletions (e.g., truncated versions of proteins or peptides), insertions, and / or substitutions, changes in the stereochemistry of one or several atoms, and / or minor derivatizations, including, but not limited to, methylation, glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitoylation, amidation, and / or the addition of glycosylphosphatidylinositol. According to the present invention, any protein or peptide useful in the present invention, including homologs of native thioredoxin proteins, has a thioredoxin monothiol active site such that, in a reduced state, the protein or peptide is capable of participating in a redox reaction via oxidation of its active site thiol to a disulfide and / or of decreasing the viscoelasticity or stickiness of mucus or sputum or increasing the liquefaction of mucus or sputum.

[0076] As used herein, a protein or peptide containing a thioredoxin active site and further comprising deletion and / or substitution of one or more cysteine ​​residues outside the thioredoxin active site with any amino acid residue other than cysteine ​​and / or a combination thereof may have characteristics similar to thioredoxin, and is preferably a thioredoxin selected from the group of prokaryotic thioredoxin, fungal thioredoxin (including yeast), plant thioredoxin, animal thioredoxin, or mammalian thioredoxin. In a particularly preferred embodiment, the protein is human thioredoxin.

[0077] Homologues can be the result of natural allelic variation or natural mutation. A naturally occurring allelic variant of a nucleic acid encoding a protein is a gene that occurs at essentially the same locus(s) in the genome as the gene encoding such a protein, but has a similar but non-identical sequence due to natural variations caused, for example, by mutation or recombination. An allelic variant usually encodes a protein with similar activity to that of the protein encoded by the gene being compared. One class of allelic variants may encode the same protein but have a different nucleic acid sequence due to the degeneracy of the genetic code. An allelic variant may also contain changes in the 5' or 3' untranslated region of the gene (e.g., in a regulatory control region). Allelic variants are known to those skilled in the art.

[0078] Homologues can be produced using techniques known in the art for producing proteins, including, but not limited to, direct modifications to an isolated naturally occurring protein, direct protein synthesis, or modifications to a nucleic acid sequence encoding the protein, for example, using classical or recombinant DNA techniques to achieve random or targeted mutagenesis.

[0079] Modifications in a homolog compared to a wild-type protein either agonize, antagonize, or do not substantially alter the basic biological activity of the homolog compared to the naturally occurring protein. Generally, the biological activity or biological effect of a protein refers to any function(s) exhibited or exerted by the protein due to the naturally occurring form of the protein, measured or observed in vivo (i.e., in the protein's natural physiological environment) or in vitro (i.e., under laboratory conditions). Protein modifications, such as those in homologs or mimetics (discussed below), can result in proteins with the same biological activity as the naturally occurring protein or proteins with reduced or increased biological activity compared to the naturally occurring protein. Modifications that result in reduced protein expression or reduced protein activity can be referred to as inactivation (complete or partial), downregulation, or reduced protein action. Similarly, modifications that result in increased protein expression or increased protein activity can be referred to as amplification, overproduction, activation, enhancement, upregulation, or increased protein action.

[0080] Preparation of Thioredoxin Compositions Due to the structural stability and physical robustness characteristic of thioredoxin, a primary formulation development goal was maintaining the stored protein in a fully reduced, active form. As a first approach, the reduced protein was exchanged into PBS using DTT, degassed with nitrogen to remove oxygen, and frozen at -80°C in single-use aliquots to avoid continuous freeze-thaw cycles. This strategy required considerable care during storage and use and was suboptimal, as the protein rapidly oxidized in aqueous solution, even when flash-frozen. Based on extensive work conducted at Syngenta Corp. and Octoplus (formulation development specialists), which utilized various complex combinations of sugars and chemical excipients in an attempt to stabilize the reduced state of native Trx in dry storage formulations, other formulations were evaluated. Only one of these formulations (9% sucrose, 1.7 mM EDTA, pH 5.2) was found to provide adequate redox stability for thioredoxin after lyophilization, resulting in almost complete retention of starting activity even during accelerated storage at 40°C for 6 months. However, this complex formulation raised concerns about potential irritation when inhaled, and the high concentration of sucrose undesirably increased solution viscosity, making isotonic reconstitution in a suitable buffer difficult at the protein concentrations required for drug delivery. Despite extensive experimentation, no benign formulation capable of adequately maintaining thioredoxin in the reduced state without oxidation, dimerization, or multimerization was reported.

[0081] The inventors have made the groundbreaking realization that a formulation approach utilizing a volatile solvent, e.g., 20 mM ammonium acetate (pH 5.5), could enable the thioredoxin proteins or peptides of the invention to be frozen and lyophilized in reduced form, during which process the solvent would completely evaporate, leaving only the pure protein in the lyophilizate. Surprisingly, this opposite approach was found to confer beneficial redox-stabilizing properties to the reduced thioredoxin, even superior to Syngenta's complex sucrose formulations, and lacked the pro-inflammatory effects of sucrose and EDTA. Furthermore, by eliminating the residual excipients in the lyophilized material, the stable thioredoxin could be reconstituted in any desired buffer without regard for osmolality, allowing stable solution concentrations in excess of 5-10 mM.

[0082] A further embodiment of the present invention is a method for preparing a composition useful for the storage and transport of thioredoxin proteins and peptides of the present invention. Such compositions are useful for preparing pharmaceutical compositions comprising the thioredoxin proteins and peptides of the present invention for administration to patients. In particular, the thioredoxin proteins and peptides of the present invention can be stably stored and transported in a reduced state in a minimal formulation without complex stabilizers or other formulation requirements. As a result, the reconstituted thioredoxin proteins and peptides of the present invention prepared from such compositions can be in a minimal, naked formulation without complex formulation requirements or the need for any excipients. The inventors have found the surprising and unexpected result that this method allows for retention of protein and redox stability that is as good as or better than that obtained using complex, non-volatile excipients, which required considerable experimentation to derive, including, for example, sucrose-EDTA formulations as described in WO 2006 / 090127.

[0083] The method includes providing a composition comprising a protein or peptide containing a thioredoxin active site in a reduced state and an aqueous solvent having a vapor pressure of at least about 3 mmHg. The method then includes volatilizing the aqueous solvent to produce a dry composition containing the protein or peptide. Such compositions and the resulting pharmaceutical compositions are substantially free of contaminants, such as diluents, solvents, other solutions or liquids, buffers, salts, surfactants, and other chemicals. Such compositions can consist of, or consist essentially of, the protein or peptide. Basic and novel properties of such compositions include one or more of the following characteristics: the thioredoxin active site of the protein or peptide is in a reduced state and lacks significant capacity to undergo spontaneous oxidation or lacks significant capacity to undergo spontaneous dimerization, and / or the protein or peptide is active in its reduced form (i.e., can form a stable disulfide bond with a target) once reconstituted in isotonic saline. In the case of monocysteine ​​active site thioredoxins, they can further be covalently bound to Cys residues of target protein disulfides, which reduces the ability of thioredoxins to be internalized in an active form.

[0084] The term "solvent," as used herein, refers to a liquid or solution in which a protein or peptide of the invention is suspended and / or dissolved prior to removal of the solvent by volatilization, e.g., lyophilization. The term "diluent," as used herein, refers to a liquid or solution in which a protein or peptide of the invention is reconstituted after removal of the solvent by volatilization. Such reconstitution may result in the protein or peptide of the invention being suspended or dissolved in the diluent.

[0085] Preparation of a composition according to this method for producing a protein or peptide of the invention can be accomplished by starting with a protein or peptide of the invention suspended or dissolved in a solvent. The solvent can have a vapor pressure suitable for lyophilization of thioredoxin, e.g., at least about 3 mmHg. The vapor pressure can also be at least about 1 mmHg, 2 mmHg, 3 mmHg, 4 mmHg, 5 mmHg, 6 mmHg, or 7 mmHg, or any decimal value between about 1 mmHg and 7 mmHg. In other embodiments, the vapor pressure of the aqueous solvent can be within the range defined by any two values ​​between 1 mmHg and 10 mmHg. Volatilization can then be carried out according to a suitable method, e.g., lyophilization according to standard protocols, to produce a reduced-state protein or peptide of the invention free of solvent or other liquids. Such resulting protein or peptide can be substantially pure (e.g., at least about 95, 96, 97, 98, 99, 99.5, 99.9%, or 100% pure).

[0086] In some embodiments, the aqueous solvent may be selected from the group consisting of ammonium acetate, ammonium bicarbonate, ammonium formate, triethylammonium acetate, and triethylammonium bicarbonate. The aqueous solvent may have a concentration of about 1 mM to about 50 mM, or any integer range between 1 mM and about 50 mM, and / or have a pH of about 4 to about 7, or any decimal value between about 4 and about 7. In some embodiments, a composition having a protein or peptide comprising a reduced thioredoxin active site and an aqueous solvent having a vapor pressure of at least about 3 mmHg does not contain a saccharide or saccharide derivative, and in some embodiments, does not contain any compounds other than the protein or peptide having a vapor pressure of less than about 3 mmHg.

[0087] The mixture of solvent and protein or peptide of the present invention provided according to this method can consist essentially of a single solvent and protein or peptide of the present invention. The mixture can also consist of a protein or peptide of the present invention and multiple solvents (i.e., a mixed solvent) that collectively meet the vapor pressure limitations of this method. The mixture can also contain two or more proteins or peptides of the present invention, for example, two or more of the thioredoxin variants and / or other proteins or peptides disclosed herein.

[0088] Alternatively, the mixture of the protein or peptide of the invention and solvent can consist essentially of the protein or peptide of the invention and solvent. A basic and novel feature of such an embodiment is that the components of the mixture other than the protein or peptide of the invention can be volatilized while the protein or peptide of the invention remains in a reduced state. In this manner, the protein or peptide of the invention can be stably stored in a reduced state, lyophilized to a state that is stable for storage, and easily reconstituted in a reduced state for pharmaceutical and / or non-pharmaceutical use.

[0089] An important aspect of the lyophilization procedure is its ability to produce a protein or peptide of the invention that is stable in its reduced state. When the starting material for lyophilization is reduced thioredoxin, the process described herein can preserve the thioredoxin active site cysteine ​​in its reduced state, resulting in a thioredoxin having a substantially pure active site cysteine ​​in its reduced state. Furthermore, the thioredoxin active site cysteine ​​can be preserved in its reduced state, regardless of whether other cysteines are present in the thioredoxin.

[0090] Proteins or peptides of the invention having a substantially pure active site cysteine ​​in a reduced state are then suitable for storage and are more amenable to storage at various temperatures and for various periods of time than protein compositions prepared by other methods. For example, proteins or peptides of the invention prepared by this method can be stable (e.g., having an active site cysteine ​​in a reduced state), e.g., having greater than about 50% activity, greater than about 60% activity, greater than about 70% activity, greater than about 80% activity, greater than about 90% activity, or greater than about 95% activity. Such activity levels can be achieved for at least about 3 hours, at least about 6 hours, at least about 12 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 2 weeks, or at least about 1 month. Thus, the resulting compositions can be stored at temperatures between -80°C and 40°C and retain thioredoxin reduction activity.

[0091] Compositions produced by this method may be substantially free of salt, e.g., having less than about 0.01, 0.1, 0.5, 1, 2, 3, 4, 5, or 10 mM salt. Moreover, the lyophilized compositions may be further characterized as comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% by weight of a protein or peptide of the invention.

[0092] The proteins or peptides of the present invention prepared as described above can then be reconstituted for various uses, including pharmaceutical and non-pharmaceutical uses. Reconstitution can be achieved by resuspending or dissolving the proteins or peptides of the present invention in a suitable diluent. For example, the proteins or peptides of the present invention can be reconstituted using sterile water, isotonic saline, hypertonic saline, phosphate-buffered saline (PBS), combinations thereof, or other diluents suitable for reconstitution. In some embodiments, the diluent is suitable for administration to patients, e.g., human patients, but also other animals, including non-human mammals, birds, fish, reptiles, and amphibians.

[0093] Alternatively, the proteins or peptides of the invention prepared as described above can be used without reconstitution. For example, the proteins or peptides of the invention can be administered as a powder or as a dry component in food or tablets. Other uses of unreconstituted thioredoxin are also possible.

[0094] The preparation methods described herein can be used for any of the thioredoxin variants, fragments, active sites, homologs or other versions of thioredoxin described herein.

[0095] The method and resulting formulation developed by the inventors utilize volatile solvents and result in a pure, dry protein after lyophilization. This process allows for reconstitution with components such as isotonic buffered saline (PBS), significantly simplifying the formulation and delivery process. Notably, the novel lyophilized protein possesses comparable redox stability to sucrose-based formulations while eliminating the potential irritation risk of inhaled sucrose / EDTA. This novel formulation allows for the elimination of all excipients in the final lyophilized material, facilitating reconstitution with a simple isotonic saline buffer for delivery via devices such as an electrically vibrating mesh nebulizer. The resulting formulation has the compelling advantage of being simple and salt / excipient-free, addressing the potential safety and delivery / efficiency challenges of sucrose-containing inhaled drug products. Preliminary stability data indicates that the formulation may be comparable to or even superior to the original sucrose formulation.

[0096] In further embodiments, the present invention includes compositions comprising a protein or peptide containing a thioredoxin active site, the compositions being free of thioredoxin protein fractions having ultraviolet (UV) absorbance at wavelengths of light greater than about 400 nm, and methods for making the same. Surprisingly, it has been found that, after production of the proteins of the invention, purification of the protein fraction having thioredoxin activity, including removal of protein fractions having absorbance at wavelengths of light greater than about 400 nm, increases the stability of the peptide or protein in the composition, whether in dried (lyophilized) form or reconstituted from a dried form, e.g., with a saline buffer. It has also been found that such peptide or protein compositions can be dried to lower water contents, e.g., less than about 5.0%, 4.0%, 3.0%, or any 0.1% increment between about 5.0% and 1.0% by weight.

[0097] In some embodiments, the remaining peptide or protein fraction in the composition has an absorbance at a light wavelength of less than about 400 nm, less than about 390 nm, less than about 380 nm, less than about 370 nm, less than about 360 nm, less than about 350 nm, less than about 340 nm, less than about 330 nm, less than about 320 nm, less than about 310 nm, less than about 300 nm, less than about 290 nm, or less than about 280 nm.

[0098] Embodiments of the present invention, including compositions comprising a protein or peptide comprising a thioredoxin active site, but not a fraction having an absorbance greater than about 400 nm, can be in various formats, as described elsewhere herein. For example, such compositions can be in a form suitable for lyophilization, in which the protein or peptide comprising a thioredoxin active site is in a reduced state and the composition further comprises an aqueous solvent having a vapor pressure of at least about 3 mmHg. Alternatively, such compositions can be in a dry state with a low water content, as described above. Furthermore, such compositions can be reconstituted, making them suitable for administration, for example, in a composition consisting essentially of a protein or peptide comprising a thioredoxin active site in a reduced state, water, and sodium chloride.

[0099] Other embodiments include methods for producing a composition containing a protein or peptide comprising a thioredoxin active site, wherein the composition does not contain a fraction having an absorbance greater than about 400 nm. Such methods include providing a lysate containing a protein or peptide comprising a thioredoxin active site, concentrating the protein or peptide in the composition, and removing the peptide or protein fraction having an absorbance greater than about 400 nm. For example, but not by way of limitation, the proteins of the present invention can be produced by recombinant production in host cells. The cells can be lysed and clarified. The resulting clarified composition can be subjected to further purification, such as ion exchange chromatography. It has been found that the fraction having an absorbance greater than about 400 nm is not separated from the remaining major protein fraction having thioredoxin activity and an absorbance at wavelengths less than about 400 nm, e.g., about 280 nm, by an ion exchange chromatography step. However, subjecting this fraction to hydrophobic interaction chromatography results in the separation of the major protein fraction from the fraction having an absorbance greater than about 400 nm. The resulting major protein fraction has the beneficial properties described above of having low water content when dried and increased stability (e.g., as measured by free SH groups and percent monomer).

[0100] Pharmaceutical Composition The present invention also relates to pharmaceutical compositions comprising a solution of a protein or peptide containing a reduced thioredoxin active site. In one embodiment, the protein or peptide contains no cysteine ​​residues except for a single cysteine ​​residue in the thioredoxin monocysteine ​​active site. Such pharmaceutical compositions also include a pharmaceutically acceptable excipient. In various embodiments, the excipient may be selected from ammonium acetate buffer, formic acid, acetic acid with ammonium, acetic acid without ammonium, and combinations thereof.

[0101] In some embodiments, the thioredoxin monocysteine ​​active site comprises an amino acid sequence selected from the group consisting of CXXS (SEQ ID NO:24), CXXX (SEQ ID NO:17), XCXXXX (SEQ ID NO:19), XCGPXX (SEQ ID NO:21), WCGPXK (SEQ ID NO:23), XCXXSX (SEQ ID NO:25), XCGPSX (SEQ ID NO:26), and WCGPSK (SEQ ID NO:27), where the X residue is any amino acid residue other than cysteine. In other embodiments, the protein or peptide comprises the thioredoxin monocysteine ​​active site sequence of SEQ ID NO:1. In further embodiments, the protein or peptide comprises a sequence selected from SEQ ID NO:28 and a sequence having at least about 80% identity to SEQ ID NO:28, and the thioredoxin monocysteine ​​active site is at a position corresponding to positions 32-35 of SEQ ID NO:28. In a further embodiment, the protein or peptide comprises a sequence selected from SEQ ID NO:29 and a sequence having at least about 80% identity to SEQ ID NO:29, wherein the thioredoxin monocysteine ​​active site is at a position corresponding to positions 32-35 of SEQ ID NO:29.

[0102] Such pharmaceutical compositions may be formulated for administration to a patient by a route selected from oral, rectal, nasal, intratracheal, bronchial, direct placement in the lungs, inhalation, oral, topical and ocular.

[0103] Administration Furthermore, compositions, including pharmaceutical compositions of the present invention, can be administered to patients in pharmaceutically acceptable carriers. As used herein, a pharmaceutically acceptable carrier refers to any substance suitable for delivering therapeutic proteins, nucleic acids, or other compounds useful in the methods of the present invention to a suitable in vivo or ex vivo site. A preferred pharmaceutically acceptable carrier is one that, when the protein, nucleic acid molecule, or compound arrives at the desired site (e.g., the site where the mucus or sputum to be treated is secreted or excreted), can maintain the protein, nucleic acid molecule, or compound in a form that allows it to contact the mucus or sputum (in the case of a protein or compound) or enter cells, be expressed and secreted by cells (in the case of a nucleic acid molecule), and thereby allow the expressed protein in a reduced state to contact the mucus or sputum.

[0104] A suitable or effective amount of a thioredoxin protein or peptide containing a thioredoxin active site as disclosed herein for administration to a patient is an amount that is capable of participating in a redox reaction via reversible oxidation of its active site thiol to a disulfide, catalyzing a thiol-disulfide exchange reaction, and in particular, decreasing the viscoelasticity or viscosity of mucus or sputum and / or increasing the liquefaction of mucus or sputum in the patient sufficiently to provide a therapeutic benefit to the patient. The decrease in viscoelasticity or viscosity or the increase in liquefaction of mucus or sputum can be measured, detected, or determined as previously described herein or by any suitable method known to one of skill in the art. As discussed above, such measurements include determining and comparing the percentage of free thiols in mucus or sputum samples obtained from the patient before and after contact with a suitable or effective amount of a protein or peptide containing a thioredoxin monocysteine ​​active site, and determining and comparing the patient's FEV levels before and after contact with a suitable or effective amount of a protein or peptide containing a reduced thioredoxin monocysteine ​​active site.

[0105] Thioredoxin proteins or peptides having a reduced thioredoxin active site as disclosed herein, in addition to reducing the viscoelasticity of mucus or sputum, also have therapeutic uses, e.g., treatment of inflammation, hypertension, oxidative stress, or infection, where the thioredoxin proteins or peptides are administered topically by inhalation, direct application, instillation, or orally, or by infusion, injection, or other administration routes suitable for systemic extracellular treatment.

[0106] Methods for determining the activity of a reduced state thioredoxin protein formulated in a pharmaceutically acceptable solution include determining the redox state of a cysteine ​​in the thioredoxin protein by an assay, e.g., a fluorometric and / or colorimetric assay, e.g., a DTNB assay (using 5,5'-dithiobis-(2-nitrobenzoic acid). In one embodiment, the thioredoxin protein contains a single cysteine ​​amino acid. In one embodiment, the redox state of the N-terminal cysteine ​​in the thioredoxin active site is determined by a fluorometric and / or colorimetric assay, e.g., a DTNB assay.

[0107] In one embodiment, a suitable or effective amount of a thioredoxin protein or peptide containing a thioredoxin active site as disclosed herein to be administered to a patient is about 10 μmol / kg, 15 μmol / kg, 20 μmol / kg, 25 μmol / kg, 30 μmol / kg, 35 μmol / kg, 40 μmol / kg, 45 μmol / kg, 50 μmol / kg, 55 μmol / kg, 60 μmol / kg, 65 μmol / kg, 70 μmol / kg, 75 μmol / kg, 80 μmol / kg, 90 μmol / kg, 100 μmol / kg, 110 μmol / kg, 120 μmol / kg, 130 μmol / kg, 140 μmol / kg, 150 μmol / kg, 160 μmol / kg, 170 μmol / kg, 180 μmol / kg, 190 μmol / kg, 200 μmol / kg, 210 μmol / kg, 220 μmol / kg, 230 μmol / kg, 240 μmol / kg, 250 μmol / kg, 260 μmol / kg, 270 μmol / kg, 280 μmol / kg, 290 μmol / kg, 300 μmol / kg, 310 μmol / kg, 320 μmol / kg, 330 μmol / kg, 340 μmol / kg, 350 μmol / kg, 360 μmol / kg, 370 μmol / kg, 380 μmol / kg, 390 μmol / kg, 400 μmol / kg, 410 μmol / kg, 420 μmol / kg, 430 μmol / kg, 440 μmol / kg, 450 μmol / kg, 460 μmol / kg, 470 μmol / kg, 4 mol / kg, 85 μmol / kg, 90 μmol / kg, 95 μmol / kg, 100 μmol / kg, 105 μmol / kg, 110 μmol / kg, 115 μmol / kg, 120 μmol / kg, 125 μmol / kg, 130 μmol / kg, 135 μmol / kg, 140 μmol / kg, 145 μmol / kg, 150 μmol / kg, 175 μmol / kg, 200 μmol / kg, 225 μmol / kg, 250 μmol / kg, 275 μmol / kg, 300 μmol / kg, 325 μmol / kg, 350 μmol / kg kg, 375 μmol / kg, 400 μmol / kg, 425 μmol / kg, 450 μmol / kg, 475 μmol / kg, 500 μmol / kg, 525 μmol / kg, 550 μmol / kg, 575 μmol / kg, 600 μmol / kg, 625 μmol / kg, 650 μmol / kg, 675 μmol / kg, 700 μmol / kg, 725 μmol / kg, 750 μmol / kg, 775 μmol / kg, 800 μmol / kg, 825 μmol / kg, 850 μmol / kg, 875 μmol / kg, 900 μmol The range of concentrations of 1000 μmol / kg, 925 μmol / kg, 950 μmol / kg, 975 μmol / kg, 1000 μmol / kg, 1100 μmol / kg, 1200 μmol / kg, 1300 μmol / kg, 1400 μmol / kg, 1500 μmol / kg, 1600 μmol / kg, 1700 μmol / kg, 1800 μmol / kg, 1900 μmol / kg, 2000 μmol / kg, 2100 μmol / kg, 2200 μmol / kg, 2300 μmol / kg, 2400 μmol / kg or between about 2500 μmol / kg.

[0108] In another embodiment, when the route of delivery is aerosol delivery to the lungs or a similar route, the amount of a thioredoxin protein or peptide containing a thioredoxin active site as disclosed herein to be administered to a patient comprises between about 0.25 mg per dosage unit (e.g., a dosage unit for humans is typically about 2-3 ml) and about 100 mg per dosage unit to achieve an effective concentration of at least 100 μM at the target site. Preferably, the amount of a thioredoxin protein or peptide containing a thioredoxin active site as disclosed herein to be administered to a patient comprises about 0.25 mg, 0.50 mg, 1.0 mg, 5.0 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, or about 100 mg per dosage unit. Depending on the device used for aerosol delivery, only about 10% of the volume in the aerosol can actually be delivered to the lungs with some aerosol delivery devices. However, when the delivery device is a vibrating mesh nebulizer, about 90% of the volume in the aerosol can be delivered. Electric vibrating mesh nebulizers are smaller, more portable devices that can deliver drugs much more quickly, making them highly preferred by CF patients (Geller, DE, Pediatric Pulmonology, 43(S9):S5-S17, 2008). Vibrating mesh nebulizers are also more efficient at delivering drugs and have a lower residual dose compared to air-jet nebulizers. This is particularly important for reducing treatment costs, as fewer doses are needed to achieve therapeutic benefit. Devices such as these do not result in a reduction in the biological activity of proteins (Kesser, KC, et al. Resp Care, 54(6):754-768, 2009; Scherer, T., et al. J Pharm Sci, 100(1):98-109, 2011).Thus, it is readily apparent that for other administrations where the volume of composition delivered to the site is greater, smaller doses of the protein or peptide containing the thioredoxin active site may be used.

[0109] The optimal amount of the protein of the present invention to be administered to an animal varies depending on the route of administration. For example, if the protein is administered by inhalation (aerosol) route, the optimal amount to be administered may be different from the optimal amount to be administered by intratracheal microspray. Varying the amount depending on such administration route is within the ability of those skilled in the art. It is important to note that the appropriate amount of the protein of the present invention is an amount that is not toxic to animals and has the desired function. Other administration routes include, but are not limited to, oral administration, particularly for the treatment of digestive mucus, or topical administration for the treatment of reproductive mucus.

[0110] In one embodiment of the present invention, compositions, including pharmaceutical compositions of the present invention, containing a thioredoxin protein comprising a thioredoxin active site as disclosed herein are further formulated for delivery with one or more agents that maintain the thioredoxin active site in a reduced state after initial reduction using a reducing agent. Such reducing agents for use in the present invention include, but are not limited to, dithiothreitol (DTT), lipoic acid, NADH- or NADPH-dependent thioredoxin reductase, ethylenediaminetetraacetic acid (EDTA), reduced glutathione, dithioglycolic acid, 2-mercaptoethanol, tris-(2-carboxyethyl)phoshene, N-acetylcysteine, NADPH, NADH, and other biological or chemical reducing agents. As described herein, preferred lyophilized storage formulations of reduced thioredoxin have surprisingly been found to require no formulation excipients, although specific delivery formulations for certain mucosal or epithelial targets may be beneficial.

[0111] Therapeutic Aspects The thioredoxin proteins or peptides disclosed herein have several advantages and benefits over native or wild-type thioredoxins. Active site modifications substituting the C-terminal cysteine ​​with any amino acid residue other than cysteine, along with deletion and / or substitution of one or more cysteine ​​residues outside the thioredoxin active site with any amino acid residue other than cysteine, or a combination thereof, are designed to minimize potential side effects of thioredoxin associated with intracellular signaling or systemic exposure, such as those described by Rancourt et al. (Free Radical Biol & Med 42:1441-43, 2007). These modifications prevent nucleophilic attack on the mixed disulfide formed between thioredoxin and target protein disulfides, catalyzed by the N-terminal thioredoxin active site cysteine ​​(e.g., located at position 32 in human thioredoxin, SEQ ID NO: 12).

[0112] Surprisingly, the inventors have determined that such thioredoxins are more potent than wild-type thioredoxins at reducing (inclining) and normalizing the viscoelasticity of diseased human mucus. The inventors have found that thioredoxins containing a monothiol active site, together with deletion of one or more cysteine ​​residues outside the thioredoxin active site and / or substitution with any amino acid residue other than cysteine, or a combination thereof, not only exhibit no impairment in activity compared to wild-type thioredoxin, but also exhibit greater stability and quantitative ability to reduce human CF mucus viscosity, particularly in rheological assays, compared to thioredoxins that still retain the three non-active site Cys residues at positions 62, 69, and 73.

[0113] Mucus obstruction of the airways can cause significant morbidity and mortality in patients with CF. The inventors have demonstrated that the viscoelastic properties that promote the persistence of these secretions in the airways are significantly reduced by the thioredoxin proteins or peptides disclosed herein, and that even dosages as high as 40 mg / kg in rats cause no adverse effects.

[0114] Thus, one embodiment of the present invention relates to a method for normalizing and reducing the viscoelasticity of mucus or sputum in a patient having excessively viscous or sticky mucus or sputum, comprising contacting the patient's mucus or sputum with a composition comprising a thioredoxin protein or peptide having a thioredoxin active site in a reduced state effective to reduce the viscoelasticity of the mucus or sputum compared to before the contacting step, wherein the thioredoxin protein or peptide comprises deletion and / or substitution of one or more cysteine ​​residues outside the thioredoxin active site with any amino acid residue other than cysteine ​​and / or a combination thereof, with modification of all non-active site cysteines to other non-cysteine ​​amino acids being preferred.

[0115] According to the present invention, the term "mucus" generally refers to a normally clear, viscous fluid secreted by mucous membranes in various tissues of the body, including the respiratory, gastrointestinal, and reproductive systems. Mucus moistens, lubricates, and protects the tissues into which it is secreted. Its gel-forming components include mucin macromolecules (including mucus proteins, nucleic acids, and carbohydrates). Mucus proteins include, but are not limited to, respiratory mucus proteins, gastrointestinal mucus proteins, reproductive mucus proteins, and ocular mucus proteins. The viscoelastic properties of normal mucus vary depending on the concentration, molecular weight, and degree of intertwining of mucin polymers. The term "sputum" generally refers to a mixture of secretions from the respiratory tract, including saliva and mucus. Sputum is typically an expectorated mixture of saliva and mucus (and other secretions from respiratory tissues). Thus, mucus is the primary component of sputum, and as such, the presence of excessively viscoelastic mucus can result in sputum that is itself excessively viscoelastic. The present invention relates to reducing the viscosity and / or hardness of abnormally viscoelastic mucus or sputum.

[0116] The term "liquefaction" refers to the action of becoming more liquid. Thus, increasing the liquefaction of mucus or sputum refers to an increase in the liquid phase or state of mucus or sputum compared to the more solid or viscous phase. In cases of abnormally viscous or excessive mucus associated with disease, the goal is to restore normal levels of mucus viscosity. Thus, liquefaction (or "normalization") can also be thought of as a reduction in mucus viscosity. Excessive liquefaction is harmful in itself, so liquefying agents that naturally limit their activity so that mucus is normalized rather than completely liquefied are particularly desirable.

[0117] It is understood that normal mucus function is achieved by having an appropriate ratio of biological reducing agent to oxidizable cysteine. Thus, a deficiency in biological reducing agent activity is caused by either an excess of oxidized cysteine ​​or a lack of biological reducing agent. Therefore, restoring an appropriate level of biological reducing activity is a means of ensuring the correct balance between oxidation (disulfide bond formation) and reduction (disulfide bond cleavage) when either oxidative stress increases and / or mucus levels rise, or natural reducing agent activity levels decrease.

[0118] The general function of mucus and sputum in the body requires that mucus (and therefore the mucus components of sputum) have viscoelastic properties. In individuals with normal mucus and sputum (i.e., healthy individuals, or more specifically, individuals not suffering from symptoms or conditions caused or exacerbated by the viscosity or stickiness of mucus or sputum), viscoelasticity varies depending on the concentration, molecular weight, and intertwining of mucin polymers (Verdugo et al., Biorheology 20:223-230, 1983). In CF specifically, when mucins in mucus interact with DNA and f-actin released from dying inflammatory cells, mucus (and therefore sputum) can become even more densely viscous. The inability to clear abnormally thickened mucus by coughing or mucociliary clearance facilitates colonization of the lungs by opportunistic pathogens.

[0119] Thus, abnormally or excessively viscous and / or sticky mucus is characterized as mucus that is measurably or detectably more viscous or sticky than mucus obtained from a normal or healthy patient (preferably an age- and sex-matched patient), and / or as mucus that, due to its level of viscosity and / or stickiness, causes discomfort or pain to the patient, or causes or exacerbates a condition or disease, or causes or contributes to at least one symptom in the patient. In other words, abnormally or excessively viscous and / or sticky sputum is a deviation from normal mucus or sputum, and it is desirable to treat the patient to provide some relief from a condition or other therapeutic benefit. Abnormal mucus can be mobile secretory mucus, as in the case of the respiratory tract surface, or stationary secretory mucus, as in the gastrointestinal tract, buccal and nasopharyngeal cavities, reproductive system, or eye.

[0120] The methods and compositions of the present invention can be used to treat any patient in whom it is desirable to reduce the viscoelasticity of mucus or sputum, as well as for the treatment of inflammation, hypertension, fibrosis, oxidative stress, or infection, and more preferably, the thioredoxin protein or peptide is administered by infusion or injection. Patients with certain pulmonary, sinus, nasal, ocular, digestive or gastrointestinal, or reproductive diseases or conditions can benefit from treatment using the methods and compositions of the present invention.

[0121] The present invention is most useful for ameliorating or reducing at least one symptom of a condition or disease caused or exacerbated by abnormal or excessive viscoelasticity and / or viscosity of mucus or sputum, which may of course include lung-related diseases such as cystic fibrosis and gastrointestinal diseases such as coccidiosis or inflammatory bowel disease in which abnormally viscoelastic mucus may be combined with inflammation and impaired response to pathogens.

[0122] Other diseases may, at least sometimes, be associated with abnormal or excessive viscoelasticity and / or stickiness of mucus or sputum, and when such symptoms occur, the methods of the present invention can be used to reduce the viscoelasticity of the mucus or sputum and provide at least some relief or therapeutic benefit to the patient. Examples of such diseases include, but are not limited to, cystic fibrosis, chronic or acute bronchitis, bronchiectasis (non-CF and CF bronchiectasis), COPD / emphysema, acute tracheitis (caused by bacterial, viral, mycoplasmal or other organisms), acute or chronic sinusitis, atelectasis (lung or lobar collapse) due to acute or chronic mucus obstruction of the airways (occasionally seen in various diseases such as asthma, including status asthmaticus), bronchiolitis (viral or otherwise), acute, subacute or chronic intestinal obstruction due to dried-up mucus including, but not limited to, meconium ileus or its equivalent in CF or similar disorders, other gastrointestinal diseases and infertility due to, but not limited to, obstruction of the cervix, vas deferens or other vital reproductive structures, and dry eye disease in which abnormally thickened mucus secretions promote a vicious cycle of inflammation and further abnormal secretions. Furthermore, because impaired mucociliary clearance is associated with the clearance of bacteria and other pathogens from the lungs, the compositions and methods of the present invention may be useful for reducing symptoms associated with excessive viscoelasticity and / or stickiness of mucus or sputum in patients with various respiratory infections, including both viral and bacterial infections.

[0123] Thioredoxin plays a role in modulating runaway inflammatory responses and acute lung injury. Extracellular thioredoxin acts broadly to reduce inflammation in animals exposed to ongoing inflammatory processes. This was observed in a mouse model of COPD, in which neutrophil inflammation was inhibited by thioredoxin, and in a model of acute lung injury induced by influenza A virus infection, in which exogenous delivery or transgenic overexpression of thioredoxin prevented viral pneumonia in mice. Thioredoxin was found to suppress the induction of the pro-inflammatory mediators TNF-α and CXCL1 in lavage fluid and lung tissue in mice in vivo and in mouse lung epithelial cells in vitro. In mice, thioredoxin inhibited lipopolysaccharide-induced neutrophil chemotaxis and LPS-induced IL-1b expression in human macrophages. The anti-inflammatory and immunomodulatory effects of thioredoxin have been proposed to involve the management of cytokine mediator release, suppression of intercellular adhesion molecule-1 (ICAM-1) expression, and inhibition of inflammasome activity. Importantly, thioredoxin also acts to protect airway AT2 stem cells from inflammatory damage. These effects are likely exerted through allosteric regulatory mechanisms as well as by direct activity against inflammatory targets. However, rapid clearance and poor pharmacology have been found to be significant functional limitations for the therapeutic use of exogenous natural thioredoxin. Furthermore, high concentrations of thioredoxin in the cell nucleus paradoxically lead to proinflammatory cytokine release in response to stimuli.

[0124] Thus, one embodiment of the present invention relates to a method for treating pulmonary inflammation, runaway inflammatory responses, and acute lung injury, such as those associated with viral respiratory diseases. Such methods involve administering a composition containing a protein or peptide containing a reduced thioredoxin monocysteine ​​active site to a subject suffering from or at risk of developing such conditions and / or viral respiratory diseases. The protein or peptide containing a monocysteine ​​active site can be any of the inhaled, topical anti-inflammatory, mucus-normalizing therapeutic agents described herein. Such therapeutic compositions are believed to provide compartmentalization of activity to prevent intracellular / nuclear reductive stress and improve pharmacokinetics compared to native thioredoxin.

[0125] In this embodiment, the pulmonary inflammation, runaway inflammatory response, and acute lung injury may be associated with a viral respiratory disease, which is a disease caused by a virus and affects the airways. Such viral respiratory diseases may include acute respiratory distress syndrome (ARDS), severe acute respiratory distress syndrome (SARS), Middle East respiratory syndrome (MERS), SARS-coronavirus-2 (SARS-CoV-19 or COVID-19), influenza, asthma, pneumonia, bronchitis, tuberculosis, reactive airway disease syndrome, and viral infections associated with interstitial lung disease. Viruses included those capable of causing one or more viral respiratory diseases, including coronaviruses, influenza viruses, respiratory syncytial viruses (RSV), parainfluenza viruses, and respiratory adenoviruses.

[0126] Emerging evidence strongly implicates SARS-CoV-2 infection of respiratory epithelia in initiating a cascade of events that can lead to severe COVID-19 disease. In these severely affected individuals, dysregulated airway cytokine release after infection can result in cytokine release syndrome (CRS), in which immune system hyperresponsiveness triggers a runaway response to infection, causing more damage than the pathogen itself. Alveolar epithelial type II (AT2) cells, stem cells in the adult lung and also cells that produce airway surfactant, respond to pathogens and alveolar damage by secreting cytokines, signaling macrophages to recruit and initiate their activation, and defending the alveoli. When this response becomes abnormally activated, it can lead to CRS, which, in severely affected patients, can become systemic and result in overwhelming, fatal pathology. However, loss of AT2 cells due to the direct cytotoxic effects of viral infection may also lead to impaired respiratory function, as the accumulation of dead cells prevents efficient mucociliary clearance, enhancing pulmonary fluid accumulation and pneumonia, leading to a vicious cycle of an ever-increasing inflammatory response.

[0127] ARDS, one of the most devastating complications of COVID-19 and severe influenza, is associated with widespread inflammation in the lungs. The underlying mechanisms of ARDS include diffuse injury to cells that form the barrier of the lung's microscopic air sacs (alveoli), surfactant dysfunction, and immune system activation. The fluid accumulation in the lungs associated with ARDS is partially explained by inflammation-induced vascular leakage. A key aspect of infection-triggered ARDS is the initial release of chemical signals and other inflammatory mediators secreted by lung epithelial and endothelial cells. Neutrophils and some T-lymphocytes migrate into inflamed lung tissue and contribute to the amplification / exacerbation of ARDS. Decreased production of lipid mediators of inflammation (prostaglandins) may impair the resolution of inflammation associated with ARDS (Fukunaga, et. al., Cyclooxygenase 2 Plays a Pivotal Role in the Resolution of Acute Lung Injury. Journal of Immunology 2005; 174:5033-5039.; Gao et al J Immunol 2017; 199:2043-2054).

[0128] Additional diseases or conditions for which the methods of the present invention can be used include treating inflammation, hypertension, oxidative stress, infection, or fibrosis. Thus, in some embodiments, the present invention includes a method for treating a bacterial or other infectious disease in a subject. In this embodiment, the method may include a composition formulated for administration to a patient by a route selected from the group consisting of oral, rectal, nasal, inhalation, intratracheal, bronchial, direct placement, topical, and ocular, including ocular injection. In some embodiments for treating infectious diseases, the composition may be a purified pharmaceutical composition, a dietary supplement, or a crude or purified extract of microbial cells expressing a protein or peptide. Such extracts are useful, for example, for use in animal feed compositions. In some embodiments, the present invention includes a composition comprising a thioredoxin monocysteine ​​active site operable to activate an antimicrobial peptide, where activation results in a therapeutically effective reagent for treating or preventing infectious disease. Such an antimicrobial peptide may be a defensin.

[0129] Another embodiment of the present invention includes a method for modulating the microbiome composition of a subject, comprising topically administering a composition comprising a protein or peptide having a reduced thioredoxin monocysteine ​​active site to a mucosal surface of the subject. Such a mucosal surface may be a pulmonary surface, a nasopharyngeal surface, or a gastrointestinal surface. In such an embodiment, microbiome modulation may be achieved by a protein or peptide of the present invention that activates one or more antimicrobial peptides.

[0130] Therapeutic benefit does not necessarily mean a cure for a particular disease or condition, but rather preferably encompasses results that most usually include alleviation of the disease or condition, elimination of the disease or condition, reduction or elimination of symptoms associated with the disease or condition, prevention or alleviation of secondary diseases or conditions resulting from the occurrence of a primary disease or condition (e.g., infectious diseases caused by opportunistic pathogenic microorganisms that take advantage of excessively viscous mucus in the respiratory tract) and / or prevention of the underlying disease or condition or symptoms associated with the disease or condition.

[0131] As used herein, the phrase "protected from disease" refers to reducing the symptoms of a disease, treating symptoms (alleviating or mitigating the symptoms of a disease without achieving a cure), reducing the occurrence and / or severity of a disease, or alleviating at least one symptom, sign, or cause of a disease, disorder, or condition. Preventing refers to the ability of a composition of the invention to prevent a disease from occurring when administered to a patient. Curing (or disease-modifying) refers to the ability of a composition of the invention to cure a disease when administered to a patient. Protecting a patient from disease includes treating a patient with a disease (therapeutic treatment). Preventing a disease / condition includes preventing disease occurrence (prophylactic treatment). In particular, protecting a patient from disease (or preventing disease) is achieved by increasing (normalizing) the liquefaction of abnormally viscous mucus or sputum in a patient by contacting the mucus or sputum with a thioredoxin protein or peptide as disclosed herein that contains a reduced thioredoxin active site, such that a beneficial effect is achieved. The beneficial effects can be readily assessed by one of ordinary skill in the art and / or by a skilled clinician treating the patient.

[0132] The term "disease" refers to any deviation from a patient's normal health, and includes conditions in which disease symptoms are present as well as conditions in which a deviation (e.g., infection, genetic mutation, genetic defect, etc.) has occurred but symptoms have not yet become apparent.

[0133] Contacting a patient's mucus and / or sputum with a reduced state thioredoxin protein or peptide as disclosed herein (or a composition comprising such a protein) is intended to result in a decrease in viscoelasticity / increased liquefaction of the mucus or sputum compared to before contact with the composition. According to the present invention, normalization of mucus or sputum can be any measurable or detectable increase in the level of liquefaction of the mucus or sputum compared to the previous level of liquefaction, and preferably is a statistically significant increase (i.e., the difference in the measurable level of liquefaction between the patient sample and the baseline control is statistically significant with a confidence level of at least p<0.05).

[0134] Because normal, healthy individuals generally cannot produce sufficient sputum to serve as a control, the "baseline control" is usually a patient sample prior to administration of treatment, although sputum obtained from normal, healthy individuals is not excluded as a baseline control. Furthermore, reduced viscosity leads to improved lung function. This improvement can be determined by various means, including patient-reported outcomes, mean time between exacerbations and hospitalization, and / or an increase in forced expiratory volume (FEV1).

[0135] In one embodiment of the invention, an increase in FEV is described as an increase of at least about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, about 9.0%, and 9.5% and about 10% compared to a sample obtained from the patient prior to contact with a composition or protein of the invention. Preferably, contacting a composition or protein of the invention with mucus or sputum from a patient sample results in an increase of about 2.5% compared to a sample obtained from the patient prior to contact with a composition or protein of the invention.

[0136] Liquefaction and / or reduction in viscoelasticity of mucus or sputum can be measured using any suitable technique known in the art, including but not limited to compression assays such as those described in the Examples section, which measure the amount of mucus or sputum in the solid phase (gel) versus the aqueous phase (liquid).

[0137] In other embodiments of the invention, the relative viscosity or stickiness of mucus or sputum can be measured using other parameters or indicators, including, but not limited to, viscoelasticity (e.g., measured by rheometry or magnetic microrheometry), glycoprotein content, or DNA content. In another embodiment of the invention, changes in mucus protein disulfide bonds can be estimated by using reagents such as NEM (N-ethylmaleimide), which preferentially reacts with unbound (free) Cys residue thiol groups created by disulfide bond disruption (Rancourt, R. et al., Free Radic Biol Med, 42(9):1441-1453, 2007).

[0138] In one aspect of the present invention, the level of liquefaction is described as the amount of a given mucus or sputum sample that is in the aqueous (liquid) phase as a percentage of the total volume of the mucus or sputum sample. In patients with cystic fibrosis, for example, the level of liquefaction of mucus or sputum can be as low as less than 10% or even less than 5% of the total volume. Preferably, contact of a protein or composition of the invention with mucus or sputum results in a change in the liquefaction of the mucus or sputum such that at least about 15% of the total volume is in the liquid phase, more preferably at least about 20% is in the liquid phase, more preferably at least about 25% of the total volume is in the liquid phase, more preferably at least about 30% of the total volume is in the liquid phase, more preferably at least about 35% of the total volume is in the liquid phase, more preferably at least about 40% of the total volume is in the liquid phase, more preferably at least about 45% of the total volume is in the liquid phase, more preferably at least about 50% of the total volume is in the liquid phase, or until the functional blockage or inhibition caused by the mucus is resolved (e.g., until the patient's airway is sufficiently cleared to begin expectorating fluid). An increase of more than 80 or 90% is generally undesirable, as complete liquefaction, resulting in mucin depolymerization, destroys the beneficial viscoelasticity necessary for mucus transport by ciliary action. Excessive liquefaction of mucus or sputum can also be harmful to the patient (e.g., liquefied sputum may reflux before it can be cleared by the patient, flooding small airways with potentially infectious, dilute fluid). In this regard, target-selective natural reducing agents, such as thioredoxin, are highly preferred because they favor highly structured disulfide bonds over planar disulfides that form the intermolecular bonds necessary to create the polymeric structure of mucus (e.g., as described in Passam, FJ, and Chiu, J., Allosteric disulphide bonds as reversible mechano-sensitive switches that control protein functions in the vasculature, Biophys Rev 11, 419-430, 2019).Small molecule reducing agents lack target preference and therefore may result in adverse effects due to excessive liquefaction. In some embodiments, contact of a protein or composition of the invention with mucus or sputum results in a change in the liquefaction of the mucus or sputum such that between about 15% and about 90% of the total volume, or any integer range between 15% and 90%, is in the liquid phase.

[0139] Therefore, in general, phlegm or mucus liquefaction is preferably increased in small, stepwise increments until the airways or passages (e.g., in the gastrointestinal or reproductive system) are cleared without excessive phlegm liquefaction. Preferably, contact of the proteins, peptides, or compositions of the present invention with the mucus or phlegm results in an increase in mucus or phlegm volume of liquefaction of at least about 1%, more preferably at least about 2%, etc., compared to before treatment, until the patient's airways or other obstructed passages are cleared. Once such clearance is achieved, for example, by removal of the so-called "mucous plug," improving drug access to the small airways and alveoli, lower doses of maintenance therapy can be administered to maintain newly secreted mucin proteins in a normal disulfide-bonded state. Thioredoxins, particularly target-binding monothiol thioredoxins, are much less likely to cause excessive liquefaction than non-selective reducing agents, greatly increasing the therapeutic window between effective and toxic doses.

[0140] In one embodiment, therapy is performed in conjunction with a method for removing the reduced concentration material from the patient's affected tissues (respiratory tract, digestive tract, reproductive system). For example, in the case of the respiratory system, the method of the present invention can be used in conjunction with postural drainage, huffing coughs and other respiratory movements, or any suitable method for expectorating liquefied mucus or phlegm.

[0141] According to the present invention, mucus or sputum from a patient to be treated is contacted with a thioredoxin protein (or a composition containing the protein) disclosed herein, which contains a substitution of one or more cysteine ​​residues outside the thioredoxin active site with any amino acid residue other than cysteine. The protein is effective in reducing the viscoelasticity and stickiness of the sputum or mucus and / or increasing the liquefaction of the sputum or mucus compared to before the contacting step. As previously described, thioredoxin is a protein disulfide reductase found in most organisms and participates in multiple thiol-dependent cellular reduction processes. In humans, thioredoxin is also called adult T-cell leukemia-derived factor (ADF). Within cells, most of this ubiquitous low-molecular-weight (11,700) protein remains reduced. Reduced or oxidized thioredoxin can enter intact cells or absorb into the cell membrane, where small amounts are gradually internalized over time. Native thioredoxin has two adjacent cysteine ​​residues in the active site, which, in the oxidized form, form a disulfide bridge located in a protrusion from the protein's three-dimensional structure. The flavoprotein thioredoxin reductase catalyzes the NADPH-dependent reduction of this disulfide. Furthermore, engineered versions of thioredoxin reductase modified for altered cofactor specificity may utilize NADH instead of or in addition to NADPH, as described in U.S. Patent No. 7,071,307, incorporated herein by reference. A small increase in thioredoxin can cause a significant change in the sulfhydryl-disulfide redox state in a protein.Oxidized thioredoxin, particularly the secreted form, can also be reduced by the action of glutathione along with the secreted enzyme glutaredoxin (Du, Y., Zhang, H., Lu, J., and Holmgren, A., Glutathione and glutaredoxin act as a backup of human thioredoxin reductase 1 to reduce thioredoxin 1 preventing cell death by aurothioglucose, Journal of Biological Chemistry 287, 38210-38219, 2012). Both GSH and glutaredoxin are abundant in the airways.

[0142] In addition to its ability to effect the reduction of cellular proteins, thioredoxin can act directly as an antioxidant (e.g., by preventing the oxidation of oxidizable substrates by scavenging reactive oxygen species) as well as through the activity of the peroxidase enzyme, although it is recognized that thioredoxin, unlike other thiols, does not generally contribute to oxidative stress in cells through autoxidation (e.g., autoxidation to generate superoxide radicals). White et al., U.S. Pat. No. 5,985,261, supra, showed that thioredoxin directly induces the production of MnSOD and that such induction is achieved by reduced thioredoxin.

[0143] Further therapeutic variants In one embodiment, thioredoxin proteins or peptides containing a thioredoxin active site as disclosed herein may be the product of drug design or selection and can be produced using various methods known in the art. Such proteins or peptides are sometimes referred to as mimetics. A mimetic refers to any peptide or non-peptide compound that can mimic the biological action of a naturally occurring peptide, often because it has a basic structure that mimics the basic structure of a naturally occurring peptide and / or possesses the silent biological properties of a naturally occurring peptide. Mimetics can include, but are not limited to, peptides with substantial modifications from their original form, such as lacking side chain similarity to naturally occurring peptides (such modifications may, for example, reduce their susceptibility to degradation), anti-idiotypic and / or catalytic antibodies or fragments thereof, non-proteinaceous portions of isolated proteins (e.g., carbohydrate structures), or synthetic or natural organic molecules, including, for example, nucleic acids and drugs identified by combinatorial chemistry.

[0144] Such mimetics can be designed, selected, and / or otherwise identified using various methods known in the art. Various methods of drug design useful for designing or selecting mimetics or other therapeutic compounds useful in the present invention are disclosed in Maulik et al., 1997, Molecular Biotechnology: Therapeutic Applications and Strategies, Wiley-Liss, Inc., which is incorporated herein by reference in its entirety. Thioredoxin mimetic peptides capable of potent, selective redox activity are described by Bachnoff et al., Free Radical Biol Med 50:1355-67 (2011), which is incorporated herein by reference in its entirety. Mimetics can be obtained, for example, from molecular diversity strategies (combinations of related strategies that allow the rapid construction of large, chemically diverse molecular libraries), libraries of natural or synthetic compounds, particularly chemical or combinatorial libraries (i.e., libraries of compounds that differ in sequence or size but have similar building blocks), or by rational, directed, or random drug design. See, for example, Maulik et al., supra.

[0145] In molecular diversity strategies, large compound libraries are synthesized using biological, enzymatic, and / or chemical approaches, for example, from peptides, oligonucleotides, carbohydrates, and / or synthetic organic molecules. Critical parameters in the development of molecular diversity strategies include subunit diversity, molecular size, and library diversity. The general goal of screening such libraries is to obtain high-affinity ligands for desired targets using sequential applications of combinatorial selection, and then optimize the lead molecules by either random or directed design strategies. Molecular diversity methods are described in detail in Maulik, et al., ibid.

[0146] Maulik et al. also disclose methods such as directed design, in which a user directs the process of creating novel molecules from a fragment library of appropriately selected fragments; random design, in which a user uses a genetic or other algorithm to randomly mutate fragments and their combinations while simultaneously applying selection criteria to evaluate the fitness of candidate ligands; and grid-based approaches, in which a user calculates interaction energies between three-dimensional receptor structures and small fragment probes and then links convenient probe sites together.

[0147] Diversity generation methods such as those described above can be combined with other techniques designed to improve the function or pharmacology of molecules of reduced size, such as active site mimetics. For example, one approach that has shown promise in early-stage research is hydrocarbon-stapled α-helical peptides, a novel class of synthetic miniproteins locked into their bioactive α-helical fold by the site-specific introduction of chemically braced, all-hydrocarbon staples. Stapling can significantly improve the pharmacological performance of peptides, increasing their target affinity and proteolytic resistance, while creating smaller peptide versions of larger proteins / enzymes that are amenable to chemical synthesis (Verdine, GL and Hilinsky, GJ, Methods Enzymol, 503:3-33, 2012).

[0148] In one embodiment of the present invention, a thioredoxin protein suitable for use in the present invention has an amino acid sequence comprising, consisting essentially of, or consisting of the full-length sequence of a thioredoxin protein or any fragment thereof having a thioredoxin active site as described herein. For example, any one of the native sequences of SEQ ID NOS: 4-15, or fragments or other homologs thereof, containing a thioredoxin active site as described herein, is encompassed by the present invention. Such homologs can include proteins having an amino acid sequence that is at least about 10% identical to the amino acid sequence of a full-length thioredoxin protein, or at least 20% identical, or at least 30% identical, or at least 40% identical, or at least 50% identical, or at least 60% identical, or at least 70% identical, or at least 80% identical, or at least 90% identical, or greater than 95% identical to the amino acid sequence of a full-length thioredoxin protein, including any percentage between 10% and 100% (10%, 11%, 12%, ... 98%, 99%, 100%).

[0149] As used herein, unless otherwise specified, references to percent (%) identity refer to assessment of homology performed using (1) BLAST 2.0 Basic BLAST homology searches (as described in Altschul, S.F., Madden, T.L., Schaeaeffer, A.A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D.J. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs." Nucleic Acids Res. 25:3389-3402, which is incorporated herein by reference in its entirety), using blastp for amino acid searches and blastn for nucleic acid searches with standard default parameters, with the query sequence filtered for low complexity regions by default; (2) BLAST 2 alignment (using parameters described below); (3) and / or PSI-BLAST (position-specific iterative BLAST) with standard default parameters. Due to some differences in standard parameters between BLAST and BLAST2, it is known that two particular sequences may be recognized as having significant homology using the BLAST2 program, but a search performed in BLAST2.0 Basic BLAST using one of the sequences as the query sequence may not identify the second sequence as a top match. Furthermore, PSI-BLAST provides an automated, easy-to-use version of the "profile" search, which is a sensitive method for finding sequence homologs. The program first performs a gapped BLAST database search. The PSI-BLAST program uses information from any significant alignments returned to construct a position-specific score matrix, which then replaces the query sequence in the next round of database searching. Therefore, it should be understood that percent identity can be determined using any one of these programs.

[0150] Two specific sequences can be aligned to each other using BLAST2 sequences, as described in Tatusova and Madden, (1999), "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences," FEMS Microbiol Lett. 174:247-250, which is incorporated herein by reference in its entirety. BLAST2 sequence alignments are performed in blastp or blastn using the BLAST2.0 algorithm, which performs a gapped BLAST search (BLAST2.0) between two sequences, allowing for the introduction of gaps (deletions and insertions) in the resulting alignment. For clarity herein, BLAST2 sequence alignments are performed using standard default parameters as follows: In blastn, use the BLOSUM62 matrix: Match Reward = 1 Mismatch penalty = -2 Open gap (5) and extended gap (2) penalties Gap x_Dropoff(50) Expected(10) WordSize(11) Filter(On) In blastp, using the BLOSUM62 matrix: Open gap (11) and extended gap (1) penalties gap_dropoff(50) expectation(10) wordsize(3) filter(on).

[0151] Proteins useful in the present invention can also include thioredoxin proteins having an amino acid sequence containing at least 10 contiguous amino acid residues of any full-length thioredoxin protein containing the active site (i.e., 10 contiguous amino acid residues that are 100% identical to 10 contiguous amino acids of a reference sequence, such as a native sequence represented by SEQ ID NOS: 4-15), with deletions and / or substitutions of inactive cysteine ​​residues outside the active site. In other embodiments, homologs of thioredoxin proteins include those containing at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 contiguous amino acid residues of the amino acid sequence of a naturally occurring thioredoxin protein, up to the full length of the protein, including any intervening integers (10, 11, 12, etc.), and including the amino acid sequence containing the active site.

[0152] According to the present invention, the term "contiguous" or "consecutive" refers to a sequence described herein that is connected in an uninterrupted sequence. For example, a first sequence comprising 30 consecutive (or consecutive) amino acids of a second sequence means that the first sequence comprises an uninterrupted sequence of 30 amino acid residues that is 100% identical to the uninterrupted sequence of 30 amino acid residues in the second sequence. Similarly, a first sequence having "100% identity" with a second sequence means that the first sequence exactly matches the second sequence, with no gaps between nucleotides or amino acids.

[0153] In another embodiment, proteins useful in the present invention include thioredoxin proteins having an amino acid sequence sufficiently similar to a native thioredoxin amino acid sequence that a nucleic acid sequence encoding the homologue is capable of hybridizing to (i.e., with) a nucleic acid molecule encoding the native thioredoxin protein (i.e., with the complement of a nucleic acid strand encoding the native thioredoxin amino acid sequence) under moderate, high, or very high stringency conditions (described below). Such hybridization conditions are described in detail below.

[0154] A nucleic acid sequence complement of a nucleic acid sequence encoding a thioredoxin protein of the invention refers to the nucleic acid sequence of the nucleic acid strand that is complementary to the strand encoding thioredoxin. It will be recognized that double-stranded DNA encoding a given amino acid sequence includes a single-stranded DNA and its complementary strand having a sequence that is the complement of the single-stranded DNA. As such, the nucleic acid molecules of the invention can be either double-stranded or single-stranded and include nucleic acid molecules that form stable hybrids under stringent hybridization conditions with a nucleic acid sequence encoding the amino acid sequence of a thioredoxin protein and / or with the complement of a nucleic acid sequence encoding such an amino acid sequence. Methods for deducing complementary sequences are known to those of skill in the art.

[0155] As used herein, hybridization conditions refer to the standard hybridization conditions that nucleic acid molecules are used to identify similar nucleic acid molecules.Such standard conditions are disclosed in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, 1989.Sambrook et al., ibid., and are incorporated herein by reference in their entirety (see, in particular, pages 9.31-9.62).In addition, the formula for calculating the appropriate hybridization and washing conditions for achieving hybridization that allows various degrees of nucleotide mismatch is disclosed in, for example, Meinkoth et al., 1984, Anal. Biochem. 138, 267-284; Meinkoth et al., ibid., and are incorporated herein by reference in their entirety.

[0156] More specifically, as referred to herein, the medium stringency hybridization and washing conditions refer to the conditions that allow the isolation of nucleic acid molecules that have at least about 70% nucleic acid sequence identity with the nucleic acid molecule used to search in hybridization reaction (i.e., the conditions that allow about 30% or less mismatch of nucleotides).As referred to herein, the high stringency hybridization and washing conditions refer to the conditions that allow the isolation of nucleic acid molecules that have at least about 80% nucleic acid sequence identity with the nucleic acid molecule used to search in hybridization reaction (i.e., the conditions that allow about 20% or less mismatch of nucleotides).As referred to herein, the very high stringency hybridization and washing conditions refer to the conditions that allow the isolation of nucleic acid molecules that have at least about 90% nucleic acid sequence identity with the nucleic acid molecule used to search in hybridization reaction (i.e., the conditions that allow about 10% or less mismatch of nucleotides).

[0157] As discussed above, one skilled in the art can use the formulas in Meinkoth et al., ibid., to calculate appropriate hybridization and washing conditions to achieve these particular levels of nucleotide mismatch. Such conditions vary depending on whether DNA:RNA or DNA:DNA hybrids are formed. The calculated melting temperature for DNA:DNA hybrids is 10°C lower than for DNA:RNA hybrids.

[0158] In certain embodiments, stringent hybridization conditions for DNA:DNA hybrids are 6×SSC (0.9 M Na + ) at a temperature of between about 20°C and about 35°C (lower stringency), more preferably between about 28°C and about 40°C (more stringent), and even more preferably between about 35°C and about 45°C (even more stringent), along with appropriate wash conditions. In certain embodiments, stringent hybridization conditions for DNA:RNA hybrids include 6x SSC (0.9M NaCl), 1 ... + ) at temperatures between about 30°C and about 45°C, more preferably between about 38°C and about 50°C, and even more preferably between about 45°C and about 55°C, along with similarly stringent washing conditions. These values ​​are based on calculations of melting temperatures for molecules of greater than about 100 nucleotides, 0% formamide, and a G+C content of about 40%. Alternatively, T m can be empirically calculated as set forth in Sambrook et al., supra, pp. 9.31-9.62. In general, wash conditions should be as stringent as possible and should be appropriate for the chosen hybridization conditions. For example, hybridization conditions should be determined by combining salts and the calculated T m The wash conditions can include a combination of temperature conditions approximately 20-25°C lower than the calculated T of the particular hybrid. mAn example of hybridization conditions suitable for use with DNA:DNA hybrids includes a combination of temperature conditions approximately 12-20° C. lower than the reference temperature. One example of hybridization conditions suitable for use with DNA:DNA hybrids includes hybridization in 6×SSC (50% formamide) at about 42° C. for 2-24 hours, followed by a wash step including one or more washes in about 2×SSC at room temperature, followed by additional washes at a higher temperature and lower ionic strength (e.g., at least one wash in about 0.1×-0.5×SSC at about 37° C., followed by at least one wash in about 0.1×-0.5×SSC at about 68° C.).

[0159] Fusion of thioredoxin with various sequences. The thioredoxin proteins of the present invention may also be fusion proteins comprising a segment containing a thioredoxin active-site and a fusion segment that may have various functions. For example, such a fusion segment may function as a tool to simplify the purification of the proteins of the present invention, e.g., to enable purification of the resulting fusion protein using affinity chromatography. A suitable fusion segment may be a domain of any size that has a desired function (e.g., imparting increased stability to the protein, imparting increased immunogenicity to the protein, and / or simplifying protein purification). It is within the scope of the present invention to use one or more fusion segments. The fusion segment may be joined to the amino- and / or carboxyl-terminus of the thioredoxin active-site-containing segment. The link between the fusion protein segment and the thioredoxin active-site-containing domain may be susceptible to cleavage to allow direct recovery of the thioredoxin active-site-containing domain of such a protein. Fusion proteins are preferably produced by culturing recombinant cells transformed with a fusion nucleic acid molecule encoding a protein comprising a fusion segment attached to either the carboxyl- and / or amino-terminus of the thioredoxin active-site-containing domain.

[0160] In one embodiment of the present invention, any of the amino acid sequences described herein, e.g., the amino acid sequence of a naturally occurring thioredoxin protein or thioredoxin-containing active site, can be generated with at least one and up to about 20 additional heterologous amino acids flanking each of the C- and / or N-terminal ends of the specified amino acid sequence. The resulting protein or polypeptide is sometimes referred to as "essentially consisting of" the specified amino acid sequence. According to the present invention, heterologous amino acids are sequences of amino acids that are not naturally found (i.e., not found naturally in vivo) adjacent to the specified amino acid sequence, or that are not associated with the function of the specified amino acid sequence, or that are not encoded by nucleotides adjacent to a naturally occurring nucleic acid sequence encoding the specified amino acid sequence as it occurs in a gene when such nucleotides in the naturally occurring sequence are translated using the standard codon usage of the organism from which the given amino acid sequence is derived. Similarly, the phrase "essentially consisting of," when used in reference to a nucleic acid sequence herein, refers to a nucleic acid sequence encoding the specified amino acid sequence that may be flanked by multiple additional heterologous nucleotides, at least one and up to about 60, at each of the 5' and / or 3' ends of the nucleic acid sequence encoding the specified amino acid sequence. Heterologous nucleotides are not naturally found (i.e., not found in nature, in vivo) adjacent to a nucleic acid sequence encoding a particular amino acid sequence as occurs in a native gene, or encode a protein that confers any additional function to the protein or alters the function of a protein having the particular amino acid sequence.

[0161] Sources of thioredoxin In one embodiment, thioredoxin proteins or peptides as disclosed herein containing a thioredoxin active-site suitable for use with the methods of the invention include proteins or peptides containing a thioredoxin active-site derived from a substantially similar species of animal to which the protein is administered. In another embodiment, any thioredoxin protein or peptide as disclosed herein containing a thioredoxin active-site can be used in a given patient, including those derived from a variety of sources, e.g., microorganisms, plants, and fungi.

[0162] In another embodiment, thioredoxin proteins or peptides as disclosed herein containing a thioredoxin active site suitable for use with the methods of the invention include isolated or biologically pure proteins. As such, "isolated" and "biologically pure" do not necessarily reflect the extent to which the protein has been purified. Isolated proteins of the invention can be obtained, for example, from natural sources, produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning), or chemically synthesized.

[0163] In yet another embodiment, the chemically synthesized thioredoxin proteins or peptides containing a thioredoxin active site of the present invention may also refer to stabilized versions, such as those containing a structurally constrained active site, e.g., by stapled peptide technology, by cyclization, or by N- or C-terminal constraints. Preferably, the thioredoxin proteins containing a thioredoxin active site to be used in the methods of the present invention have an in vivo half-life sufficient to cause a measurable or detectable increase in liquefaction (or a decrease in viscosity or stickiness) of mucus or sputum in a patient and / or to provide a measurable, detectable, or perceptible therapeutic benefit to the patient associated with mucus and sputum in the patient. Such a half-life can be achieved by the method of delivery of such proteins. Proteins of the present invention preferably have a half-life of greater than about 5 minutes in animals, more preferably greater than about 4 hours in animals, and even more preferably greater than about 16 hours in animals. In a preferred embodiment, the protein of the present invention has a half-life of between about 5 minutes and about 24 hours in an animal, preferably between about 2 hours and about 16 hours in an animal, more preferably between about 4 hours and about 12 hours in an animal.

[0164] Thioredoxin and related nucleic acid molecules Further embodiments of the present invention include nucleic acid molecules encoding thioredoxin proteins or peptides as disclosed herein that contain a thioredoxin active site. Such nucleic acid molecules can be used to generate proteins useful in the methods of the present invention in vitro or in vivo. Nucleic acid molecules of the present invention include nucleic acid molecules comprising, consisting essentially of, or consisting of a nucleic acid sequence encoding any of the proteins previously described herein. According to the present invention, an isolated nucleic acid molecule is a nucleic acid molecule (polynucleotide) that has been removed from its natural environment (i.e., has been subject to human manipulation) and may include DNA, RNA, or derivatives of either DNA or RNA, including cDNA. As such, "isolated" does not reflect the extent to which the nucleic acid molecule has been purified. The phrase "nucleic acid molecule" primarily refers to the physical nucleic acid molecule, while the phrase "nucleic acid sequence" primarily refers to the sequence of nucleotides in a nucleic acid molecule; the two terms can be used interchangeably, particularly with respect to nucleic acid molecules or nucleic acid sequences capable of encoding proteins.

[0165] The isolated nucleic acid molecules of the present invention can be isolated from their natural sources or produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis. Isolated nucleic acid molecules can include, for example, genes, natural allelic variants of genes, coding regions or portions thereof, and coding and / or regulatory regions modified by nucleotide insertions, deletions, substitutions, and / or inversions in a manner such that the modifications do not substantially interfere with the ability of the nucleic acid molecule to encode the desired protein of the present invention or to form stable hybrids with natural gene isolates under stringent conditions. Isolated nucleic acid molecules can contain degeneracy. As used herein, nucleotide degeneracy refers to the phenomenon in which one amino acid can be coded for by different nucleotide codons. Thus, the nucleic acid sequence of a nucleic acid molecule encoding a given protein useful in the present invention may vary due to degeneracy.

[0166] According to the present invention, reference to a gene includes all nucleic acid sequences associated with a native (i.e., wild-type) gene as well as those associated with the thioredoxin monocysteine ​​active site, such as regulatory regions (including, but not limited to, transcriptional, translational, or post-translational control regions) that control the production of the protein encoded by the gene, as well as the coding region itself. In another embodiment, a gene may be a naturally occurring allelic variant that contains a similar, but not identical, sequence to the nucleic acid sequence encoding a given protein. Allelic variants have been previously described above. The terms "nucleic acid molecule" and "gene" can be used interchangeably when the nucleic acid molecule comprises the above-mentioned gene.

[0167] Preferably, the isolated nucleic acid molecules of the present invention are produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis. Isolated nucleic acid molecules include naturally occurring nucleic acid molecules and their homologs, including, but not limited to, naturally occurring allelic variants and modified nucleic acid molecules in which nucleotides have been inserted, deleted, substituted, and / or inverted in such a way that such modifications provide a desired effect on protein biological activity. Allelic variants and protein homologs (e.g., proteins encoded by nucleic acid homologs) are discussed in detail above.

[0168] Nucleic acid molecule homologues can be generated using several methods known to those skilled in the art (e.g., as described in Sambrook et al., ibid.). For example, nucleic acid molecules can be modified using a variety of techniques, including, but not limited to, classical mutagenesis and recombinant DNA techniques (including, but not limited to, site-directed mutagenesis, chemical treatment, restriction enzyme digestion, ligation of nucleic acid fragments and / or PCR amplification), or by in vitro or in vivo recombination of mixtures of molecules to "construct" a reassembled library of nucleic acid molecules containing various combinations thereof by the process of synthesis of oligonucleotide mixtures and chemical ligation or gene shuffling (i.e., molecular breeding; see, e.g., U.S. Patent No. 5,605,793 to Stemmer; Minshull and Stemmer, Curr. Opin. Chem. Biol. 3:284-290, 1999; Stemmer, PNAS USA 91:10747-10751, 1994, all of which are incorporated herein by reference in their entirety). These and other similar techniques known to those skilled in the art can be used to efficiently introduce multiple simultaneous changes into a protein. Nucleic acid molecule homologs can then be selected by hybridization with a given gene or can be screened directly by expression for the function and biological activity of the protein encoded by such nucleic acid molecules.

[0169] One embodiment of the present invention relates to a recombinant nucleic acid molecule comprising the above-described isolated nucleic acid molecule operably linked to at least one transcriptional control sequence. More specifically, according to the present invention, a recombinant nucleic acid molecule typically includes a recombinant vector and an isolated nucleic acid molecule as described herein. According to the present invention, a recombinant vector is an engineered (i.e., artificially generated) nucleic acid molecule used as a tool for manipulating a selected nucleic acid sequence and / or introducing such a nucleic acid sequence into a host cell. Thus, a recombinant vector is suitable for use in cloning, sequencing, and / or otherwise manipulating a selected nucleic acid sequence, for example, by expressing the selected nucleic acid sequence and / or delivering it into a host cell to form a recombinant cell. Such vectors typically contain heterologous nucleic acid sequences, i.e., nucleic acid sequences not naturally found adjacent to the nucleic acid sequence to be cloned or delivered, but the vector also contains regulatory nucleic acid sequences (e.g., promoters, untranslated regions) that are naturally found adjacent to the nucleic acid sequence of the present invention or that are useful for expression of the nucleic acid molecule of the present invention (discussed in detail below). Vectors can be either RNA or DNA, either prokaryotic or eukaryotic, and are usually plasmids. Vectors can be maintained as extrachromosomal elements (e.g., replicating plasmids) or can be integrated into the chromosome of the recombinant host cell, although for most applications of the present invention, it is preferable for the vector to remain separated from the genome. The entire vector can remain in place within the host cell, or under certain conditions, the plasmid DNA can be deleted, leaving behind the nucleic acid molecule of the present invention. The integrated nucleic acid molecule can be under the control of a chromosomal promoter, a native or plasmid promoter, or a combination of promoters. Single or multiple copies of the nucleic acid molecule can be integrated into the chromosome. The recombinant vector of the present invention can contain at least one selectable marker.

[0170] In one embodiment, the recombinant vector used in the recombinant nucleic acid molecule of the present invention is an expression vector. As used herein, the phrase "expression vector" refers to a vector suitable for producing a product (e.g., a protein of interest) to be encoded. In this embodiment, a nucleic acid sequence encoding the product to be produced (e.g., a protein containing a thioredoxin monocysteine ​​active site) is inserted into the recombinant vector to produce a recombinant nucleic acid molecule. The nucleic acid sequence encoding the protein to be produced is inserted into the vector in a manner that allows the nucleic acid sequence to be operably linked to the regulatory sequence in the vector, thereby enabling the transcription and translation of the nucleic acid sequence in the recombinant host cell.

[0171] In another embodiment of the present invention, the recombinant nucleic acid molecule comprises a viral vector. The viral vector comprises the isolated nucleic acid molecule of the present invention integrated into a viral genome or a portion thereof, in which the nucleic acid molecule is packaged in a viral coat, which allows the DNA to enter a cell. Several viral vectors can be used, including, but not limited to, those based on alphaviruses, poxviruses, adenoviruses, herpesviruses, lentiviruses, adeno-associated viruses, and retroviruses.

[0172] Typically, a recombinant nucleic acid molecule comprises at least one nucleic acid molecule of the present invention operably linked to one or more expression control sequences. As used herein, the phrase "recombinant molecule" or "recombinant nucleic acid molecule" primarily refers to a nucleic acid molecule or nucleic acid sequence operably linked to an expression control sequence, but can be used synonymously with the phrase "nucleic acid molecule" when such a nucleic acid molecule is a recombinant molecule as discussed herein. According to the present invention, the phrase "operably linked" refers to linking a nucleic acid molecule to an expression control sequence in such a way that the molecule can be expressed when transfected (i.e., transformed, transduced, transfected, conjugated, or introduced) into a host cell.

[0173] Transcription control sequences are expression control sequences that control the initiation, elongation, or termination of transcription. Particularly important transcription control sequences are those that control transcription initiation, such as promoter, enhancer, operator, and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in the host cell or organism into which the recombinant nucleic acid molecule is to be introduced. The recombinant nucleic acid molecule of the present invention may also contain additional regulatory sequences, such as translational regulatory sequences, origins of replication, and other regulatory sequences compatible with recombinant cells.

[0174] In one embodiment, recombinant molecules of the invention, including those that integrate into a host cell chromosome, also contain a secretion signal (i.e., a signal segment or signal sequence nucleic acid sequence) that enables the expressed protein to be secreted from the cell that produces the protein. Suitable signal segments include signal segments naturally associated with the protein to be expressed or any heterologous signal segment capable of directing the secretion of a protein according to the invention.

[0175] In another embodiment, a recombinant molecule of the present invention comprises a leader sequence that enables the expressed protein to be delivered to and inserted into the membrane of a host cell. Other signal sequences include those that can direct periplasmic or extracellular secretion or retention in a desired compartment. Suitable leader sequences include the leader sequence naturally associated with the protein or any heterologous leader sequence capable of directing delivery and insertion of the protein into the membrane of a cell.

[0176] According to the present invention, the term "transfection" refers to any method by which an exogenous nucleic acid molecule (i.e., a recombinant nucleic acid molecule) can be inserted into a cell. The term "transformation" can be used synonymously with the term "transfection" when such terms are used to refer to the introduction of a nucleic acid molecule into a microbial cell or a plant. In microbial systems, the term "transformation" is used to describe a heritable change due to the acquisition of an exogenous nucleic acid by a microorganism and is essentially synonymous with the term "transfection." However, in animal cells, transformation has acquired a second meaning that may refer, for example, to a change in the growth characteristics of cells in culture after they become cancerous (see above). Therefore, to avoid confusion, the term "transfection" is preferably used in reference to the introduction of an exogenous nucleic acid into an animal cell, and is generally used herein to encompass the transfection of animal cells and the transformation of plant and microbial cells, insofar as these terms relate to the introduction of an exogenous nucleic acid into a cell. Thus, transfection techniques include, but are not limited to, transformation, particle bombardment, electroporation, microinjection, lipofection, adsorption, infection, and protoplast fusion.

[0177] Administration to humans and non-human vertebrates In the methods of the present invention, compositions, including pharmaceutical compositions, can be administered to a patient that is any member of the class of vertebrates, including, but not limited to, primates, rodents, livestock, chickens, turkeys, and domestic pets, companion animals, or racehorses.

[0178] As discussed above, compositions, including pharmaceutical compositions of the invention, are administered to a patient in a manner effective to deliver the composition, particularly a thioredoxin protein and / or any other compound as disclosed herein that comprises a thioredoxin active site therein, to a target site (e.g., the mucus or sputum to be treated for proteins and compounds, or the target host cell that becomes the mucus or sputum to be treated or is in the environment of the mucus or sputum to be treated for recombinant nucleic acid molecules). Suitable administration protocols include any in vivo or ex vivo administration protocol.

[0179] According to the present invention, an effective administration protocol (i.e., administering a composition of the present invention in an effective manner) includes suitable dose parameters and modes of administration that result in contact of the thioredoxin protein and / or other compounds disclosed herein containing a thioredoxin active site in the composition with the mucus or sputum to be treated, preferably such that the patient derives some measurable, observable, or perceptible benefit from such administration. Alternatively, effective dose parameters can be determined by experimental methods using in vitro samples, in vivo animal models, and ultimately, if the patient is human, clinical trials. Effective dose parameters can be determined using methods standard in the art for a particular disease or condition. Such methods include, for example, determining survival rates, side effects (i.e., toxicity), and disease progression or regression, as well as related physiological parameters, such as forced expiratory volume in 1 second (FEV, FEV1).

[0180] According to the present invention, suitable methods for administering the compositions of the present invention to a patient include any in vivo administration route suitable for delivering the composition to the desired site in or on the patient. Preferred administration routes will be apparent to those skilled in the art, depending on whether the compound is a protein or other compound (e.g., a drug), the part of the body to which the composition is to be administered, and the disease or condition the patient is experiencing. In general, suitable methods for in vivo administration of thioredoxin proteins or peptides as disclosed herein include, but are not limited to, transdermal delivery, intratracheal administration, inhalation (e.g., aerosol), nasal, oral, pulmonary administration, and catheter instillation. Ear delivery may include ear drops, intranasal delivery may include nasal drops or intranasal injection, and intraocular delivery may include eye drops or the use of a suitable device for passing the drug across the sclera and / or to the back of the eye. Aerosol (inhalation) delivery can also be performed using standard methods in the art (see, e.g., Stribling et al., Proc. Natl. Acad. Sci. USA 189:11277-11281, 1992, incorporated herein by reference in its entirety). Oral delivery can include solids or liquids that can be ingested by mouth, for example, as tablets or capsules, and incorporated into food and beverage products or animal feed or feed pellets. Other routes of administration useful for mucosal tissues include bronchial, intranasal, other inhalation, rectal, topical, transdermal, transvaginal, transcervical, perisectorial, and urethral routes. Additionally, administration protocols can include pretreatment devices, such as application of proteins, peptides, or compositions in a pessary (e.g., to the cervix), and surgically assisted local administration, such as injection into the paranasal sinuses, for use in applications such as infertility.

[0181] In a preferred embodiment of the invention, when a protein or composition of the invention is administered to treat excessively or abnormally viscous or sticky sputum or mucus in the respiratory tract (airways), the protein or peptide (or composition) or other compound containing a thioredoxin monocysteine ​​active site is administered by routes including, but not limited to, inhalation (i.e., by inhaling an aerosol, for example, in or with a surfactant), direct placement into the lungs via a bronchoscope, an endotracheal tube, and / or any artificial ventilation device, nasal administration (intranasal or transnasal), or intrabronchial or tracheal administration (i.e., by injection directly into the trachea or by tracheotomy), either directly or via lipid encapsulation or surfactant. Any conceivable method of introducing the composition or protein into the airways so that it can come into contact with the mucus or sputum is encompassed by the invention.

[0182] feed Another embodiment of the present invention relates to an animal feed composition comprising a thioredoxin protein or peptide as disclosed herein that contains a thioredoxin active site in a reduced state.

[0183] Animal feed is used to meet the nutritional needs of any type of domesticated animal. Animal feed includes both forage and forage. For example, the thioredoxin protein or peptide disclosed herein can be used in or on forage and / or forage by mixing in or with, applying or incorporating into or on the forage and / or forage by any means. Examples of animal feed include, but are not limited to, hay, straw, silaged grass, compressed, pelleted feed, oil and mixed feed, germinated grain, legumes, crop residues, grain, cereal crop and corn.

[0184] Animal feed includes feed for companion animals, livestock, and other types of animals that are desired to meet their nutritional needs. Companion animals include, but are not limited to, dogs, cats, other animals, birds, reptiles, amphibians, fish, and other companion animals. Livestock include, but are not limited to, cattle, horses, buffalo, sheep, goats, pigs, other ungulates, chickens, turkeys, ducks, other birds, salmon, trout, carp, tilapia, catfish, other fish, or other types of livestock. The thermostability of monothiol thioredoxin makes it particularly suitable for incorporation into pelleted feed, which must withstand heating above 80°C for several minutes.

[0185] Each of the publications and other references discussed and cited herein is incorporated herein by reference in its entirety.

[0186] While various embodiments of the present invention have been described in detail, it is apparent that modifications and adaptations of those embodiments will occur to those skilled in the art, but it is to be expressly understood that such modifications and adaptations are within the scope of the present invention, as set forth in the following claims. [Example]

[0187] For purposes of the following examples, "ORP100S" is provided as an exemplary thioredoxin protein, and any of the thioredoxin proteins disclosed herein can be substituted for ORP100S. [Example 1]

[0188] ORP100S expression and characterization This example demonstrates the structure, construction, expression, and evaluation of ORP100S. Compared to the C35S monocysteine ​​active site thioredoxin ORP-100, ORP100S further incorporates mutations of the three remaining non-active site thioredoxin-1 Cys residues to Ser, resulting in an intact monocysteine ​​thioredoxin with improved stability, activity, and more robust assays.

[0189] Monothiol C35S active site thioredoxins ORP-100 and ORP100S The active site of native Trx enzymes contains two redox-active Cys residues that are highly conserved across species. In their inactive, oxidized form, these Cys contribute to a disulfide bridge that protrudes from the protein's three-dimensional structure (Holmgren A., 1985, Thioredoxin, Annu Rev Biochem 54:237-71). Reduction of this active center (by TrxR enzymes, GSH / glutarodoxin, or via synthetic activation with chemical reducing agents) allows Trx to function as an electron carrier capable of dithiol / disulfide exchange. Protein disulfides are the preferred substrates for Trx-mediated reduction activity. First, after nucleophilic attack by the Cys32 thiolate anion, a transient mixed disulfide is formed between the N-terminal Cys32 of the thioredoxin active site and a Cys of a compatible target disulfide (Holmgren A., 1995, Thioredoxin structure and mechanism: conformational changes on oxidation of the active-site sulfhydryls to a disulfide. Structure 3:239-43). In native Trx, the C-terminal active site cysteine ​​(Cys35) then becomes active due to a conformational change in the active site, which stabilizes the Cys35 thiolate anion, lowers its pKa, and allows it to attack the intramolecular mixed disulfide bond, resulting in the release of oxidized Trx and the now fully reduced target (Wynn R, Cocco MJ, Richards FM., 1995, Mixed disulfide intermediates during the reduction of disulfides by Escherichia coli thioredoxin. Biochemistry 34:11807-13).

[0190] ORP-100 and ORP100S are modified versions of Trx engineered by mutation of the active site Cys35 to Ser (C35S Trx). As illustrated in Figure 1, this eliminates the second step of Trx-disulfide reduction by preventing degradation of the mixed disulfide intermediate formed by the primary Cys32 reaction, resulting in stable covalent linkage of this Cys to the protein target. In the case of human CF mucus, treatment with reduced C35S Trx disrupts disulfide bonds in excess condensed mucin proteins, normalizing mucus viscosity while blocking the ability of Trx-mucin adducts to reform new mucin Cys disulfides. Furthermore, covalent linkage to mucus anchors the C35S Trx enzyme extracellularly, preventing its cellular uptake. This unique blocking mechanism allows the modified Trx to act appropriately on mucosal surfaces while reducing or eliminating the chance of activating inflammatory pathways or other off-target effects that may be induced by Trx signaling intracellularly. Importantly, this monothiol active site C35S Trx strategy allows for the first time to replace or supplement the activity of secreted Trx without significantly affecting intracellular Trx activity.

[0191] ORP100S vs ORP-100 Compared to ORP-100, ORP100S is further modified by Cys-to-Ser mutations of the remaining non-active site Trx Cys residues located at positions 62, 69, and 73. The rationale for this was two-fold: 1) to eliminate reactive Cys that could mediate protein:protein interactions and homodimerization / multimerization, which could result in reduced availability of functional protein and increased instability of fully reduced monomeric C35S thioredoxin, and 2) to enable the use of Cys redox state quantification as a robust, simple in-process assay to monitor overall protein reduction levels and catalytic activity potential. The three non-active site Cys residues serve no structural function in native Trx and are thought to primarily play a regulatory role through the formation of intermolecular linkages, including homodimerization at Cys73, which attenuates disulfide bond reduction activity (Weichsel, A., Gasdaska, JR, Powis, G., and Montfort, WR, 1996, Crystal structures of reduced, oxidized, and mutated human thioredoxins: evidence for a regulatory homodimer. Structure 4, 735-51). The non-active site Trx Cys residues have also been shown to be the site of S-nitrosylation by GSNOR and potentially other post-translational modifications (Wu C, Liu T, Chen W, et al. Redox regulatory mechanism of transnitrosylation by thioredoxin, 2010, Molecular & Cellular Proteomics 9:2262-75). The oxidative stability of ORP100S, a fully monocysteine ​​Trx, is also increased by eliminating the possibility of multimerization, since ORP100S can only dimerize at Cys32, as opposed to the possibility of multiple dimeric and highly multimeric forms in the C35S Trx mutant, which also retains one or more non-active site Cys.

[0192] Although any reduced thiol of Trx Cys can reduce a chromogenic substrate such as DTNB (5,5'-dithiobis-(2-nitrobenzoic acid)) to induce a quantifiable absorbance change, only Cys32 can form mixed disulfides with appropriate protein disulfide substrates such as insulin. Therefore, removal of all Cys except Cys32 also means that the reduced state of total Cys in ORP100S is identical to the reduced state of Cys32. Therefore, the activity of ORP100S to reduce DTNB is identical to its ability to reduce protein disulfide bonds. This allows spectrophotometric monitoring of DTNB reduction to be used as a direct measure of ORP100S protein activity, rather than the more complex and time-consuming determination of insulin reduction state using reverse-phase high-performance liquid chromatography (RP-HPLC).

[0193] ORP100S Design and Construction The ORP100S sequence was codon-optimized for expression in Escherichia coli (E. coli) using a custom algorithm based on the amino acid sequence of human thioredoxin-1. This was hypothesized to increase expression levels and prevent amino acid misincorporation due to RNA depletion, which is less common in bacteria than in humans, both of which are significant challenges for the recombinant expression of native eukaryotic thioredoxin gene sequences in E. coli (Harris et al., 2012, Determination and control of low-level amino acid misincorporation in human thioredoxin protein produced in a recombinant Escherichia coli production system. Biotechnology and Bioengineering 109, 1987-95).

[0194] ORP100S was synthesized as a DNA fragment flanked by AflII and HindIII restriction sites for convenient manipulation, cloned into the expression vector pD861 (DNA2.0 / Atum) under the control of a rhamnose-inducible promoter, and transformed into BL21 E. coli. In some strains of E. coli, the rhaB gene was deleted to enhance rhamnose induction. Rhamnose-inducible expression was verified by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) after small-scale growth in 2-ml culture blocks.

[0195] ORP100S expression, purification and analysis The initial strategy for benchtop-scale production of ORP100S was as follows: cells were grown under fed-batch conditions in a 1.5 L fermentor (Dasgip, Eppendorf). The cell paste was harvested by centrifugation, then disrupted and primary recovered by ultrafiltration. ORP100S protein was purified to >95% purity by anion exchange chromatography, followed by size-exclusion fast protein liquid chromatography (SEC-FPLC) and ultrafiltration / diafiltration. For activation (reduction), ORP100S was treated with 10 mM dithiothreitol (DTT) and then exchanged into lyophilization buffer using an endotoxin clearance step to remove DTT and endotoxin. The reduced protein was frozen at -80°C and lyophilized for 24–36 h (Virtis). After size confirmation and purity determination by SDS-PAGE and analytical SEC-HPLC, sequence identity and homogeneity were verified by MALDI-TOF and ESI mass spectrometry. The color was slightly yellow, resulting in an off-white lyophilizate.

[0196] Functional assays 1. The reduced state of ORP100S was quantified using DTNB, which reacts with free SH groups and produces a yellow color change at 412 nm. A 96-well plate was charged with 50 microliters of 2.5 mM rhTrx, followed by 175 microliters of sample buffer and 25 microliters of 6 mM DTNB. After initiating the reaction with the addition of DTNB, the kinetic absorbance change at 412 nm due to DTNB reduction was monitored spectrophotometrically at 30°C. ORP100S concentration was determined using the extinction coefficient of human Trx-1 (7,000) as the A 280 The actual concentration of free sulfhydryl groups was determined by Nanodrop. The actual concentration of free sulfhydryl groups was calculated based on the absorbance at 412 nm and the extinction coefficient of DTNB (14,150) to determine the reduction state as a percentage of free sulfhydryls. For ORP100S, this represents 100% protein disulfide reduction capacity, and for ORP-100, where only one of the four Cys is target-reducible, it represents 25%.

[0197] 2. The percentage of free monomeric ORP100S in solution was determined using SEC. Samples were analyzed on a BioBasic S-300 250 × 4.6 column (Thermo Scientific) run on an Agilent 1100 HPLC system. The SEC-HPLC mobile phase buffer consisted of 40 mM ammonium acetate (pH 5.5), 2 mM EDTA, and 450 mM NaCl. The low pH minimized dimerization, and the 450 mM NaCl concentration improved separation. The flow rate was 0.35 mL / min, and absorbance was monitored at 280 nm. The length of each run was 20 min. The percentage of ORP100S monomer was determined by integrating the area under the peak of the monomer fraction divided by the total area under the curve of the chromatogram.

[0198] 3. The disulfide bond reduction activity of ORP100S was quantified by assaying the reduced state of a small protein (human insulin), which, in its heterodimeric form, contains two inter- and one intramolecular disulfides, all three of which are known to be suitable thioredoxin substrates. Insulin reduction has classically been used to quantify thioredoxin activity by the change in absorbance after the addition of NADPH and TrxR (Holmgren A., 1979, Thioredoxin catalyzes the reduction of insulin disulfides by dithiothreitol and dihydrolipoamide, J Biol Chem 254:9627-32). Such an approach is not suitable for the monothiol C35S thioredoxins ORP-100 and ORP100S due to 1) the lack of cycling resulting from stoichiometric covalent linkage to disulfide-linked substrates and 2) the inability of NADPH and TrxR to reduce Cys32 in the oxidized monothiol Trx active site. As a result, a novel assay based on the use of reverse-phase (RP) HPLC was developed to monitor the rate of conversion of the disulfide-linked insulin heterodimer to its monomeric form. The two chains of human insulin contain a total of six Cys residues, which form three disulfide bonds in its mature structure. When incubated with dimeric insulin, the reduced form of ORP100S reacts with and disrupts these disulfide bonds, simultaneously forming a covalent linkage to ORP100S Cys32. This resulted in a shift in the mobility of the intact insulin heterodimer, which could be detected using RP-HPLC separation, allowing quantification of the change in heterodimer peak area over time as a measure of protein disulfide reduction activity. ORP100S samples were incubated with 10 mg / mL insulin for various time points (0–90 min), and the reaction was stopped by the addition of iodacetic acid (IA) and trifluoroacetic acid (TFA).Relative activity at each time point was determined from the change in area under the insulin heterodimer peak after separation on an RP-HPLC (Agilent 1100) using a WP-RP 50x3 (Imtakt) column. Buffer A was 0.1% TFA, and buffer B was 0.1% TFA in acetonitrile. The gradient was 0-3% B in 5 min, followed by 30-60% B in 45 min, and then 60-80% B for an additional 5 min. The flow rate was 0.2 mL / min, and absorbance was measured at 280 nm. To assess changes in intact insulin molecules, a time 0 baseline was first established using 1 M IA and 0.1% TFA without the addition of ORP100S. The area under the intact insulin heterodimer peak was then determined and set as equivalent to 100%. After reaction with ORP100S, the area of ​​the intact insulin peak (corresponding to the retention time) was measured. The percent reduction of intact insulin was then calculated from the decrease in area after reduction divided by the area at time 0 and multiplied by 100. [Example 2]

[0199] pH dependence of reducing activity This example illustrates that molecules of the invention that contain a thioredoxin active site have significantly greater activity at physiologically relevant pH than conventional thiol reducing agents due to their lower pKa values.

[0200] Human CF airway surface fluid is approximately 0.8 pH units more acidic than that of unaffected individuals due to the loss of bicarbonate-mediated buffering of proton secretion (Garland AL, Walton WG, Coakley RD, et al., 2013, Molecular basis for pH-dependent mucosal dehydration in cystic fibrosis airways. PNAS 110:15973-8; Shah VS, Meyerholz DK, Tang XX, et al., 2016, Airway acidification initiates host defense abnormalities in cystic fibrosis mice, Science 351:503-7). This has led to the clinical use of reducing agents to treat condensed, abnormal mucus. The approved investigational thiol agents N-acetylcysteine ​​(NAC), glutathione (GSH), cysteamine, and 2-mercaptoethanesulfonate Na (MesNa) all exhibit low levels of disulfide reduction activity at CF airway pH due to the highly basic equilibrium point (pKa) between the inactivated (protonated) and active (deprotonated) forms. Cys thiol pKa values ​​range from pH 8.5 (cysteamine) to pH 9.5 (NAC). In stark contrast to these classical thiols, the structurally stabilized pKa of the Trx active site Cys32 is 2–3 logs lower (pH 6.1–6.3), allowing for high activity even at acidic CF airway pH (Figure 2). We experimentally verified that ORP-100 and ORP100S share the same pKa as native Trx (data not shown) and demonstrated that the monocysteine ​​active site modification does not interfere with the unique hydrogen bond that stabilizes the deprotonated thiolate anion at Trx Cys32.

[0201] Figure 2A shows the percent of thiols calculated to be in the deprotonated, active form for thioredoxin (and ORP-100 / ORP100S) over the pH range 6-9 for four representative small molecule thiol agents. Figure 2B shows RP-HPLC traces of a representative insulin reduction experiment showing the conversion of the insulin heterodimer peak using 1.25 and 12.5 mM NAC at pH 6 (left, top) and pH 8 (left, bottom). Panel B, top right, shows the trace obtained for 0.025 mM ORP-100 at pH 6. Overlaying the time 0 and 60 min traces demonstrates the absence of reduction over the 60 min incubation period: 1.25 mM NAC is inactive for insulin reduction at pH 6 or 8, and NAC can reduce insulin only at pH 8, but not at pH 6, as indicated by the shaded peak area indicating the conversion of the insulin heterodimer to monomer at 60 min. In contrast, even at an acidic pH of 6, ORP-100 can significantly reduce the insulin heterodimer peak at a concentration 500 times lower than that of NAC (Figure 2B, right panel, top). Table: Relative activity of NAC vs. ORP-100 at pH 6-9. Similar results were obtained using native Trx and ORP100S vs. NAC or GSH. These results demonstrate that the monothiol active site C35S thioredoxin (ORP-100, ORP100S) retains the remarkable and potent disulfide-reducing activity of thioredoxin across the entire physiological pH range predicted for the human airway, including neutral to acidic pH levels where exogenous and endogenous (e.g., GSH) small thiols are virtually inactive. [Example 3]

[0202] Correlation between ORP100S reduction state and activity This example demonstrates that fully monothiol ORP100S exhibits a strong correlation between global protein reduction state and disulfide bond reducing activity.

[0203] ORP100S was reduced with 100 mM DTT, and residual reducing agent was removed from the sample using a SEPHADEX™ G-25 column (e.g., a GE Healthcare NAP-5 column) for exchange into 20 mM ammonium acetate, pH 5.5. Naturally reduced ORP100S ("Red") was mixed with various ratios of oxidized ORP100S, treated with iodoacetamide (IA), and exchanged into ammonium acetate buffer to remove unreacted iodoacetamide ("Ox"). Results are shown in Table 1. ORP100S solutions containing various ratios of reduced:oxidized protein were analyzed by DTNB, SEC, and RP-HPLC insulin reduction assays as described in Example 1. The maximum insulin heterodimer reaction using fully reduced material was 45% for the time and conditions used. Insulin reduction values ​​are expressed as relative reduction to maximum. Although it is clear from the 100% IA treatment:0% reduction treatment that there is residual activity in the "fully oxidized" sample, the results nevertheless confirm that the monothiol Trx ORP100S, unlike native Trx with five reducible Cys residues or ORP-100 with four (not shown), exhibits an extremely good linear correlation between overall reduction state and disulfide bond reduction activity. For example, ORP-100, with a total of four Cys residues, can be reduced by as much as 75% but still has 0% insulin-reducing activity when the active site Cys32 is fully oxidized by dimerization. We verified that oxidation proceeds primarily via intermolecular disulfide formation, generating ORP100S homodimers (as well as higher-order multimers in the case of Trx or ORP-100).

[0204] [Table 1] [Example 4]

[0205] Stability in solution versus lyophilized form This example demonstrates that the monothiol active site thioredoxins ORP-100 and ORP100S produced on a laboratory scale using the original manufacturing process described in Example 1 are significantly more stable, as measured by free SH groups and percent monomer, when lyophilized as pure proteins from volatile solvents than when stored as solutions of the compounds. Stability was comparable to that obtained using complex sucrose and EDTA formulations, which were previously the only formulations capable of maintaining thioredoxins in the reduced form during long-term storage.

[0206] Previous studies, such as WO 2006 / 090127, teach compositions for maintaining thioredoxin in a reduced state. Deriving compositions capable of maintaining thioredoxin in the reduced form during storage required considerable experimentation, and these compositions were complex, requiring sugar derivatives and EDTA as excipients. Therefore, our discovery that eliminating all volatile solvents that sublimate upon lyophilization by solubilizing reduced thioredoxin in an aqueous solution incorporating them resulted in comparable stability was unexpected. The results of a stability analysis using this formulation strategy are shown in Figure 3. ORP-100 and ORP100S proteins were reduced with 100 mM DTT and exchanged into a volatile 20 mM ammonium acetate buffer at pH 5.5 on a NAP-5 column to remove residual reducing agent. Half of the material was frozen at -80°C and then lyophilized, while the remainder was maintained in solution at either 5°C or 40°C for various time points ("PH 5.5"). The lyophilized protein was reconstituted back into 20 mM ammonium acetate, pH 5.5, and immediately evaluated at each time point ("lyophilized"). Stability was assessed by measuring both free SH groups (DTNB chromogenic assay) and percent monomer (SEC-HPLC). As shown in Figure 3, excellent storage stability was obtained in the lyophilized form, even after 6 months under accelerated stability conditions at 40°C. While nearly identical to ORP-100, ORP100S exhibited slightly better monomer stability by SEC-HPLC, particularly during the first week of storage in liquid formulations. Based on our previous results, the higher percent monomer, DTNB relative to ORP-100, does not contribute to activity and reflects reduction of a non-active site Cys that is not present in ORP100S (see Example 3).

[0207] For Figure 3: pH 5.5: ORP-100 or ORP100S protein maintained in a liquid formulation at 20 mM ammonium acetate, pH 5.5 for 0, 3, 7, 14, 21, 28, 90, or 180 days. Lyophilized: ORP-100 or ORP100S protein stored and assayed in lyophilized form at 5°C or 40°C for 0, 3, 7, 14, 21, 28, 90, or 180 days after reconstitution with 20 mM ammonium acetate, pH 5.5. Top panel: Percent free sulfhydryls (reduced state) at 5°C. Second panel: Percent monomer determined by SEC assay at 5°C. Third panel: Percent free sulfhydryls at 40°C. Fourth panel: Percent monomer determined by SEC assay at 40°C. [Example 5]

[0208] Large-scale production and removal of thioredoxin protein fractions with high UV absorbance This example demonstrates the production of a thioredoxin protein composition of the invention in which the fraction of thioredoxin protein having a UV absorbance greater than 400 nm has been removed, and describes the stability and water absorption characteristics of the resulting composition.

[0209] A composition containing ORP100S was prepared by culturing recombinantly engineered rhaB E. coli to express the protein in a 150 L fermenter and inducing expression using rhamnose supplementation. The resulting fermentation broth was harvested 48 hours post-induction, homogenized to lyse the cells, and then frozen for further processing. The final titer of ORP100S in the 105 L fermentation broth was 16 g / L. The frozen lysate was thawed and clarified by standard techniques. The protein composition in the clarified lysate was subjected to a first anion exchange chromatography step in bind-and-elute mode (Capto Q resin 15 L - 5 L x 3, catalog 117531604, GE Healthcare) and a second anion exchange chromatography step in flow-through mode (Sartobind STIC PA chromatography - Sartorius) for endotoxin removal. The resulting protein composition was subjected to hydrophobic interaction chromatography (Capto Phenyl ImpRes 10L-5L x 2, catalog 17548404, GE Healthcare) in a bind-and-elute mode. The HIC-purified composition contained a major thioredoxin protein fraction with a single UV absorbance peak at approximately 280 nm, a second thioredoxin protein fraction with a more prominent UV absorbance peak at approximately 423 nm (corresponding to approximately 10% of the thioredoxin amount), and a minor peak in the 500-600 nm range (Figure 4). The minor fraction was yellowish-pink in color (the "red fraction"), while the major fraction was virtually transparent. All purification steps were performed in the presence of DTT to maintain complete reduction.

[0210] Both the main and red fractions were individually exchanged into ammonium acetate buffer pH 5.5 by ultrafiltration / diafiltration and frozen in 500 ml bottles at -80°C. Complete removal of DTT was verified by HPLC-MS. The composition was dried by lyophilization as follows: The frozen product was thawed by transferring it from -80°C to a refrigerator at 2-8°C for 60 hours. The thawed product was filtered using a 0.2 μM PES filter and a 500 ml sterile filter / bottle combination. The filtered product was transferred to Gore LyoGuard lyophilization trays (1.5 L of product / tray, equivalent to 3 x 500 ml product bottles), and the filled trays were transferred to the lyophilizer shelves with a temperature probe placed on top of the tray. The lyophilization cycle began after purging with nitrogen gas.

[0211] Five batches of product were freeze-dried according to the following freeze-drying cycle program:

[0212] [Table 2]

[0213] After lyophilization, the trays were purged with nitrogen and the lyophilized cakes were broken down into powder, which was packaged in sterile storage bottles for storage at -20°C (final recovery of 88.7%). The products were then assayed for moisture content, which ranged from 0.81% to 2.18% by weight for the main fractions across the five batches. In contrast, the red thioredoxin fraction could only be dried to a minimum moisture content of approximately 6.0% by weight.

[0214] Characterization: After drying and reconstitution with saline, the main fraction exhibited comparable disulfide-reducing activity by DTNB and RP-HPLC to thioredoxin compositions purified without removing the red fraction during the HIC purification step. However, the main fraction composition lacking the red fraction with UV absorbance >400 nm demonstrated significantly greater stability when reconstituted in saline and maintained at room temperature compared to thioredoxin compositions purified by methods insufficient to remove the colored fraction. The stability of ORP100S without the red fraction was assessed at room temperature (25°C) by SEC analysis as described in previous examples. Protein solutions were prepared in PBS (saline) at concentrations of 70, 80, 90, 100, and 110 mg / ml, corresponding to concentrations of 6.0, 6.8, 7.7, 8.5, and 9.4 mM. These were incubated at room temperature for 0, 20, 44, 68, 140, 188, and 232 hours. At each time point, tubes were centrifuged at 1000 RFC to check for precipitation, and the percent monomeric ORP100S was determined by SEC. Additionally, at the 68-hour time point, a DTNB assay was performed to assess the degree of reduction. Virtually no change in percent monomer was observed across all concentration levels, which decreased by only 2% from time 0 to 232 hours (see table below). By comparison, an ORP100S composition without red fraction removal reconstituted in PBS (physiological saline) at an equivalent concentration of 5 mM (59 mg / ml) had a starting percent monomeric fraction of 95% at time 0, which decreased to 55% at 72 hours and 26% at 168 hours when incubated at 25°C.

[0215] [Table 3]

[0216] Overall, material purified as described, in which the red fraction was removed, was of greater purity, was more uniform in appearance, and had significantly lower endotoxin levels.

[0217] [Table 4] [Example 6]

[0218] ORP100S mucus rheology and mucin molecular weight reduction This example demonstrates the efficacy of monocysteine ​​human thioredoxin-1 ORP100S for reducing the viscoelastic properties of human CF mucus and the molecular weight (MW) of mucin glycoproteins. The ability of ORP100S to reduce the viscosity and elastic modulus of 4% CF mucus cultured in vitro from primary human bronchial epithelium (HBE) was assessed, as well as the effect of ORP100S treatment on mucin polymer size using gel permeation chromatography (GPC) / multi-angle light scattering (MALLS). Collectively, these results demonstrate the potent ability of ORP100S to normalize the viscoelastic properties and transportability of CF mucus and sputum, suggesting that ORP100S may be a promising CF treatment optimized for activity across a wide range of airway pH microenvironments.

[0219] method: Mucus preparation: Sterile mucus was collected from over 100 individual HBE cultures from 20 different CF donors and prepared to 4 percent (4%) solids by weight, a concentration representative of chronic obstructive pulmonary disease (COPD) and mild CF (Hill, DB et al., 2014, A biophysical basis for mucus solids concentration as a candidate biomarker for airways disease, PloS one 9, e87681; Anderson, WH et al., 2015, The relationship of mucus concentration (hydration) to mucus osmotic pressure and transport in chronic bronchitis., Am J Resp Crit Care Med 192: 182-90).

[0220] Rheology: HBE cell culture mucus was treated with DTT (1 mM) and several concentrations of ORP100S (0.01, 0.1, and 1.0 mM) for 1 hour at 37°C according to previously established methods (Hill, D.B., and Button, B., 2012, Establishment of respiratory air-liquid interface cultures and their use in studying mucin production, secretion, and function. In Mucins: Methods and Protocols, D.J. Thornton, ed., pp. 245-58; Youngren-Ortiz, S. et al., 2017, Development of optimized inhalable gemcitabine-loaded gelatin nanocarriers for lung cancer, J Aerosol Med Pulm Drug Delivery 30:299-321; Seagrave, J., et al., 2012, Effects of guaifenesin, N-acetylcysteine, and ambroxol on MUC5AC and mucociliary transport in primary differentiated human tracheal l-bronchial cells, Respir Res 13: 98). Concentration and time course assays were performed using a TA Instruments DHR3 rheometer to assess the bulk macroscopic biophysical effects of test articles and controls on HBE mucus properties. Briefly, the linear regime of 1 Hz amplitude (stress) sweeps was identified for each treatment condition.A frequency of 1 Hz was chosen because it lies between the frequencies associated with tidal breathing (approximately 0.25 Hz) and mucociliary clearance (10–15 Hz) and has been shown to correlate with mucociliary clearance (Tomkiewicz, R. et al., 1994, Mucolytic treatment with N-acetylcysteine ​​L-lysinate metered-dose inhaler in dogs: airway epithelial function changes. Eur Resp J 7: 81–87). Creep-recovery experiments were performed in which known stresses (between 0.05 and approximately 100 Pa) were applied to treated or control mucus for 10 seconds, and the rheological recovery of the fluid was recorded for an additional 50 seconds. In successive runs, the applied stress was increased logarithmically until the fluid's yield stress (i.e., the stress at which the fluid's viscosity suddenly and dramatically decreases) was reached. From the measured parameters, the viscosity and elasticity of the fluid were determined as a function of the applied stress. Frequency sweeps were performed at both constant stress and strain and used to determine the baseline physical properties of mucus and its elastic and viscous components (G' and G" respectively).

[0221] Mucin Molecular Weight Determination: Molecular weight reduction after test article treatment was determined using gel permeation chromatography combined with multi-angle laser light scattering on a Wyatt Heleos MALLS system. MALLS is a rapid and accurate means of determining the molecular size and mass of high-MW biomolecules in a nondestructive manner without the need for reference standards. Briefly, 4% HBE-treated samples were diluted 100-fold with 0.9% NaCl containing 10 mM EDTA and 0.01% sodium azide. 0.2 mL of the diluted sample was eluted through a Sepharose CL2B column to separate high-MW mucins from other mucus proteins, and the mucin fraction was loaded onto the MALLS system. Mucin MW was determined by fitting the Berry model to light scattering from 11 different angles using Wyatt Astra software.

[0222] result Viscoelasticity: ORP100S demonstrated a concentration-dependent ability to reduce both the elastic (storage, G') and viscous (loss, G") moduli of 4% HBE mucus (Figure 5, top). At the lowest tested ORP100S concentration (10 μM), no significant reduction in G' was evident, and a nearly two-fold reduction in G" was observed. At 100 μM ORP100S, G' decreased from a baseline value of 0.28 Pa to 0.19 Pa, and G" was reduced by a similar amount to that observed with 10 μM. The degree of rheological reduction achieved by this concentration of ORP100S was similar to that observed with 1 mM DTT, a potent dithiol reducing agent with potent mucolytic properties. Remarkably, 1 mM ORP100S exhibited significantly greater rheological reducing properties than DTT, reducing both G' and G" by nearly three-fold.

[0223] Figure 5, A and B (top): Reduction of elastic (G') and viscous (G") moduli of a 4% solids dry-weight mucus reduced with DTT (1 mM) and ORP100S (0.01, 0.1, and 1.0 mM concentrations) for 1 hour at 37 °C. Results show that 0.1 mM ORP100S reduces G' as efficiently as a 10-fold higher concentration of DTT. All data were collected by examining frequency sweeps of the mucus conducted in the linear regime and analyzed at 1 rad / s on a TA DHR3 rheometer.

[0224] Mucin Size: Unlike 1 mM DTT, which demonstrated a moderate increase in mucin molecular weight (from 180 MDa to 210 MDa), all three concentrations of ORP100S equivalently reduced mucin molecular weight to approximately 150 MDa (Figure 5, C bottom). All compounds reduced the concentration of mucin present in the refractometry system (data not shown). The mild increase in mucin average molecular weight with 1 mM DTT may be a sign of a compound opening reactive cysteine ​​residues, which may allow the mucin macromolecule to interact with itself as well as with other mucus proteins. Monothiol reducing agents such as ORP100S, which cap free Cys thiols, are not expected to react in this manner, nor are higher concentrations of DTT expected to completely reduce mucin macromolecules to monomers.

[0225] Figure 5C. Mucin molecular weight reduction (GPC-MALLS) of 4% solids dry heavy mucus reduced with DTT (1 mM) and ORP100S (0.01, 0.1, and 1.0 mM concentrations) at 37 °C for 1 hour.

[0226] Conclusion: ORP100S demonstrated a significantly greater ability to reduce the rheology of unusually viscoelastic CF mucus, mole per mole, than DTT. Importantly, the potent viscoelasticity-modulating effect of ORP100S did not result in complete reduction and polymer degradation of the mucin, suggesting a degree of enzymatic selectivity for intramolecular mucin disulfides that increase polymer density over intermolecular disulfides that link mucin monomers into functional gels. This mucus normalization, in contrast to mucolysis, is consistent with the predicted behavior of a natural airway mucus disulfide homeostasis mechanism based on the thioredoxin, glutathione, and glutaredoxin redox cycles, all three of which are present in airway surface liquid in vivo (Du, Y., Zhang, H., Lu, J. & Holmgren, A., 2012, Glutathione and glutaredoxin act as a backup of human thioredoxin reductase 1 to reduce thioredoxin 1 preventing cell death by aurothioglucose, J Biol Chem 287, 38210-19; Bartlett, JA et al., 2013, Protein composition of bronchoalveolar lavage fluid and airway surface liquid from newborn pigs, Am J Physiol - Lung Cell Mol Physiol 305:L256-66). [Example 7]

[0227] CF mucus and sputum transport potential after treatment with ORP100S This example demonstrates that ORP100S increases CF mucus and sputum transport potential in vitro in cultured primary human bronchial epithelial cells and in situ in isolated adult rat tracheas.

[0228] method Primary human bronchial epithelial studies Primary human bronchial epithelial (HBE) cells were derived from lung explants from healthy failed donors and CF patients homozygous for F508del CFTR. Cells were expanded and grown to confluency, seeded onto 6.5 mm diameter permeable supports (0.5 × 106 cells / filter, Corning) coated with NIH 3T3 fibroblast unconditioned medium, and grown in differentiation medium for at least 6–8 weeks until terminal differentiation (Birket SE, Chu KK, Houser GH, Liu L, Fernandez CM, Solomon GM, Lin V, Shastry S, Mazur M, Sloane P, et al., 2016, Combination therapy with Cystic Fibrosis Transmembrane Conductance Regulator modulators augment the airway functional microanatomy, Am J Physiol Lung Cell Mol Physiol. ajplung 00395, Birket SE, Chu KK, Liu L, Houser GH, Diephuis BJ, Wilsterman EJ, Dierksen G, Mazur M, Shastry S, Li Y, et al. (2014, A functional anatomical defect of the cystic fibrosis airway, Am J Respir Crit Care Med. 190(4):421-32). Cells were washed with PBS and grown for 48 hours to reestablish a fresh mucus layer, followed by apical treatment (to mimic aerosol deposition) with ORP100S (1–3 mM), vehicle control (PBS, -MG++, -Ca++), or positive control DTT (1.6 mM, Sigma-Aldrich, St. Louis, MO). Micro-optical coherence tomography (μOCT) images were acquired at baseline and 3 hours post-treatment in four regions of interest per monolayer, three to four monolayers per condition. Only first- or second-passage cells were used.

[0229] Effect of ORP100S on sputum transit ex vivo To determine the effect of ORP100S on CF sputum transportability, spontaneously expectorated sputum samples were collected from four CF patients hospitalized for pulmonary exacerbations, stored at 4°C, and then divided into 200 μL aliquots and treated with ORP100S (3 mM), PBS, DTT (1.6 mM), or DNase (10 or 25 μg / ml) on the day of collection or the day after collection. Upon treatment, sputum aliquots were placed in a 37°C water bath for 2 hours and then applied (3 μl per sample) to the distal end of excised tracheas from adult non-CF rats for μOCT imaging. Tracheas were washed twice with 500 μl of PBS before sputum addition. Sample conditions were applied in triplicate in random order at distinct anatomical locations—with two washes using 500 μl of PBS between each sample addition—and at least three images were collected from each region of interest. Confirmation of tracheal viability was obtained by imaging with PBS upon completion of the experiment.

[0230] μOCT imaging Using 1 micron resolution spectral domain μOCT, measurements were obtained of mucociliary transport (MCT) velocity and ciliary beat frequency (CBF) in HBE monolayers, as well as of MCT velocity in sputum ex vivo. This first-of-its-kind, high-speed (40 frames per second, 512 lines per frame) microscopic reflectance imaging modality allows simultaneous anatomical imaging in cell cultures and intact tissues that readily distinguish CF characteristics compared with normal epithelium. (Liu L, Chu KK, Houser GH, Diephuis BJ, Li Y, Wilsterman EJ, Shastry S, Dierksen G, Birket SE, Mazur M, et al., 2013, Method for quantitative study of airway functional microanatomy using micro-optical coherence tomography, PLoS One 8(1):e54473; Tuggle KL, Birket SE, Cui X, Hong J, Warren J, Reid L, Chambers A, Ji D, Gamber K, Chu KK, et al., 2014, Characterization of defects in ion transport and tissue development in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-knockout rats. PLoS One 9(3):e91253). In addition to MCT velocity and CBF, μOCT also has the ability to assess the physical characteristics of airway surface liquid. Images were captured and MCT velocity and CBF were calculated as described in previous references.

[0231] statistical analysis Inferential statistics (mean, SD, SE) were calculated using ANOVA with Tukey's post-hoc test for multiple comparisons where appropriate. Statistics are presented as mean ± SE, and a P value <0.05 was considered significant. All statistical analyses were performed using GraphPad Prism version 7.0a (La Jolla, CA).

[0232] result ORP100S enhances MCT rates in non-CF and CF primary HBE cells To determine whether ORP100S alters mucus transport, we evaluated its effects on MCT velocity and CBF in primary HBE cells derived from healthy non-CF donors and CF donors homozygous for F508del CFTR. For these studies, we used μOCT imaging, which allows measurement of these and other parameters of airway functional microanatomy without the use of exogenous particles or dyes. Results demonstrate that ORP100S ("Theradux")-treated (1-3 mM) non-CF cells exhibited significantly higher MCT velocity (2.18 ± 0.3 mm / min, P < 0.01) relative to PBS (0.05 ± 0.007 mm / min) at 3 hours post-treatment, exceeding the effect of DTT (1.6 mM, 1.41 ± 0.1 mm / min) (Figure 6A). This effect was replicated in CF cells, which showed significantly higher MCT rates at 3 hours with ORP100S (54.73 ± 15.3 mm / min, P < 0.05) compared with PBS (7.30 ± 2.9 mm / min) or DTT (33.33 ± 12.9 mm / min), increased from baseline (15.4 ± 15.3 mm / min) (Figure 6C and D). ORP100S did not induce significant differences in CBF in either non-CF or CF cells, supporting its disulfide-reducing properties as a mechanism for promoting MCT improvement (Figure 6B, E, and F).

[0233] Figure 6. μOCT analysis of ORP100S (Theradux) in primary HBE cells (non-CF and CF). (A) Raw mucociliary transport (MCT) velocity and (B) ciliary beat frequency (CBF) at 3 hours post-treatment for non-CF cells. For CF cells, (C) raw MCT velocity at 3 hours and (D) change in MCT velocity relative to baseline at 3 hours post-treatment were also measured. (E) Raw CBF and (F) change in CBF relative to baseline were also measured. N = 3-4 monolayers per condition across one non-CF and one CF donor. Each data point represents the average treatment effect per monolayer. N = 3-4 monolayers per condition. * P<0.05 ** P<0.01

[0234] ORP100S improves mucus clearance in the intact trachea The effect of ORP100S on mucus clearance was further evaluated using intact rat tracheas, which contain airway surface complexities such as fully differentiated mucosal surface glandular expression. Spontaneously expectorated sputum was collected from four CF patients (mean age = 31 years, mean FEV1 = 1.62 L) with the genotypes F508del / F508del, F508del / S589N (N = 2), and F508del / 1973_1985del13InsAGAAA. Sputum was then applied to the surface of live wild-type rat tracheas and imaged using μOCT under physiological conditions. Figure 7A-D shows representative re-slice μOCT images depicting mucus transport for each treatment condition. MCT was measured by projecting a cross-sectional line through the mucus over time, with the gradient of the particle trajectory indicating velocity. As summarized in Figure 7E and F, ORP100S-treated cells exhibited a higher MCT velocity (4.68 ± 0.9 mm / min, P < 0.0001) relative to PBS (0.97 ± 0.17 mm / min), which significantly exceeded the effects of standard treatments DNase (2.30 ± 0.28 mm / min) and DTT (2.31 ± 0.34 mm / min). The change in MCT velocity normalized to the effect of PBS was 3.80 ± 0.35 mm / min (P < 0.0001), again exceeding that observed with DNase (2.95 ± 0.40 mm / min, P < 0.01) or DTT (3.01 ± 0.61 mm / min, P < 0.01). As seen in representative μOCT images of ORP100S vs. PBS-treated sputum (Fig. 7G and H), ORP100S was effective in reducing sputum density, consistent with previously observed viscoelastic data.

[0235] Figure 7. μOCT analysis of ORP100S ("Theradux") in CF sputum. (A-D) Representative re-slice μOCT images of each treatment condition depicting mucus transport. MCT was measured by projecting a cross-sectional line through the mucus over time. The slope of the particle trajectory indicated velocity. (E) Raw mucociliary transport (MCT) velocity and (F) change in MCT velocity normalized to PBS across samples from N=4 CF donors. Representative images depicting ORP100S effect on (H) PBS and (G) mucus density (mucus (mu), epithelium (ep). Mean + / - SEM, ** P<0.01, **** P<0.0001.

[0236] conclusion ORP100S-treated primary non-CF and CF HBE cells showed an increase in MCT beyond the effect of DTT. In expectorated CF sputum, ORP100S-enhanced mucus transport potential relative to the positive control was also observed, accompanied by a decrease in sputum density. Collectively, these results demonstrate that disulfide bond reduction by ORP100S increases mucus transport potential and suggests that ORP100S may be a promising CF treatment optimized for activity across a wide range of airway pH microenvironments. [Example 8]

[0237] The reduced form of ORP100S is non-inflammatory in vitro In vitro studies in normal and CF HBEs (HBE-ALI) cultured at the air-liquid interface were performed to evaluate the potential of monothiol thioredoxin to induce proinflammatory cytokine release after microspray application to cell monolayers.

[0238] Nasal airway epithelial cells from normal, healthy volunteers or CF subjects were cultured in serum-free medium at the air-liquid interface with mucociliary differentiation, based on a method adapted from the approach of Schlegel and colleagues (Suprynowicz, FA, et al., Proc Natl Acad Sci USA, 2012, 109(49): pp. 20035-40; Becker, MN, et al., AM J Respir Crit Care Med, 2004, 169(5): pp. 645-53). Using this technique, several unique CF and healthy control cells were collected, expanded, and cryopreserved. From each of three unique normal or CF donors (homozygous for F508del), 30 wells were cultured for 30 days until differentiation at the ALI. Triplicate cultures were exposed to PBS, native Trx, or ORP-100 at the apical surface, and both apical and basolateral media samples were collected at 4 and 24 hours postloading. Apical collection was performed by placing 200 μL of sterile PBS on the apical surface and withdrawing it after a 15-minute incubation. IL-6 ELISA was performed in duplicate for each sample, while others were measured using a multiplexed assay. Media was centrifuged to remove debris and stored at -80°C until ELISA analysis.

[0239] Representative data for changes in the proinflammatory cytokines IL-6 (left) and TNF-alpha (right) upon addition of saline (PBS) in the presence or absence of ORP-100 or thioredoxin are shown in Figure 8 for HBE-ALI derived from healthy and CF donors. These data demonstrate that the reduced form of ORP-100 is non-inflammatory and exhibits significant anti-inflammatory effects at concentrations above 100 μM for the drug formulated in a defined concentration of (isotonic) PBS relative to application of PBS vehicle alone, as observed in CF HBE-ALI (but not those derived from healthy donors), where application of isotonic or hypertonic saline was sufficient to induce both TNF-alpha and IL-6. The reduced, but not oxidized, ORP-100 form suppressed the inflammatory effects of saline. These results were reproduced in vivo in an acute intratracheal instillation study in normal rats.

[0240] Figure 8: Induced IL-6 or TNFα levels after 24 hours in the basolateral ALI medium of primary HBE cultures from nasal epithelia of non-CF (left series of bars) and CF donors (right series of bars). Delivery: 15-minute apical surface bolus application of a 200 μL volume of control or test article solution. PBS: 0.9% phosphate-buffered saline, negative vehicle control (black bars); ORP100-1000: 1 mM ORP-100 in PBS (dark filled bars); ORP100-1000: 100 μM ORP-100 in PBS (light filled bars); Trx-1000: 1 mM native thioredoxin-1 in PBS (dark hatched bars); Trx-100: 100 μM native thioredoxin-1 in PBS (light hatched bars). All concentrations reflect the volume delivered to the HBE apical surface. [Example 9]

[0241] The reduced form of ORP100S is anti-inflammatory in vivo This example evaluated and compared the in vivo inflammatory potential among three forms of C35S monothiol Trx (oxidized ORP-100, reduced ORP-100, and reduced ORP100S) formulated in a sucrose / EDTA formulation composition described in PCT WO2006 / 090127 and delivered as a nebulized aerosol at doses of 4 and 40 mg / kg to normal rats.

[0242] All three forms of ORP-100 were lyophilized after reconstitution with DTT and formulated in sucrose buffer (9% sucrose, 1.17 mM EDTA, pH 5.2). All test articles were evaluated at low (10 mg / kg) and high (40 mg / kg) target delivery doses. Test articles were administered as aerosols to Charles River Sprague-Dawley rats via inhalation. Aerosols of each test article were generated using a commercially available vibrating mesh nebulizer, the output of which was attached to a nose-only exposure system.

[0243] The target dose was achieved by modulating the aerosol exposure time while maintaining a constant aerosol concentration. Animals in the low-dose group received a single dose of test article for a 20-minute exposure to achieve a target delivered dose of 10 mg / kg and a deposited dose of 1 mg / kg. Animals in the high-dose group received a single dose of test article for a 75-minute exposure to achieve a target delivered dose of 40 mg / kg and a deposited dose of 4 mg / kg.

[0244] The concentrations of total ORP-100 in the aerosols were determined by BCA analysis and ranged from 601.1 to 868.0 μg / L for the low-dose group and 708.7 to 798.0 μg / L for the high-dose group. Aerosol particle sizes were less than 3.0 microns MMAD, as determined by cascade impactor analysis (Figure 9, left). The achieved deposition doses, assuming a 10% deposition fraction, were 0.9, 1.2, and 1.2 mg / kg for the low-dose oxidized and reduced ORP-100 and ORP100S, respectively, and 3.8, 3.8, and 4.2 mg / kg for the high-dose group. For all dose groups, the actual deposited dose was within 25% of the target.

[0245] Animals in each study group were evaluated for potential toxicity by measuring various parameters, including clinical signs, body weight, clinical pathology (BALF cell counts, LDH, and albumin measurements), lung tissue cytokines, lung weight, and gross and microscopic pathology of respiratory target tissues. Additionally, three animals in each low-dose group had lung tissue harvested for immunohistochemistry (Alizee Pathology, Thurmont, MD). Three (3) animals in each low-dose group were euthanized at 1 and 4 hours post-exposure for blood and tissue collection. Four (4) animals in each high-dose group were euthanized at 4 and 20 hours post-exposure for blood and tissue collection.

[0246] [Table 5]

[0247] When reduced ORP-100 and ORP100S were compared with the oxidized ORP-100 control group, differences in BALF LDH, albumin, cell counts, and differential white blood cell counts were modest, as were measurements of various lung tissue cytokines. LDH levels, although slightly higher in the low-dose group, varied among test article and dose groups with no clear trend. Albumin levels were highest at 1 hour for all dose groups but were similar among all test article and dose groups at 4 and 20 hours post-exposure.

[0248] BALF total cells were generally similar among the three test articles, regardless of dose level or evaluation time. Macrophages were the most common cell type observed in BALF, but no clear trends were observed among the three test articles. Most groups had low lymphocyte, neutrophil, and eosinophil counts at all time points, although slightly higher cell counts were observed in all high-dose groups sacrificed at 20 hours. The % macrophages in BALF were similar among all low-dose animals at 1 and 4 hours, as well as in the high-dose animals at 4 hours. The % macrophages in BALF was slightly lower at 20 hours compared to the other sampling times.

[0249] The decrease in macrophage percentage in all high-dose groups compared with the low-dose group was primarily explained by an increase in neutrophil percentage. However, the changes were similar to those observed in the oxidized ORP-100 control group. Differences in lymphocytes and eosinophils varied between TA and dose groups, with no clear trends.

[0250] All animals survived to necropsy. Gross pathology findings were generally few and minimal in severity, consisting primarily of minimal red discoloration of the lungs. Common microscopic findings consisted of rare to few mononuclear cells and scattered infiltrates of eosinophils. These findings were observed in animals in all groups and were considered background, interpreted as occurring before test article exposure, or were artifacts of the sacrifice procedure.

[0251] Overall, there was no evidence of adverse test article effects in clinical observations, BALF chemistry, cell counts, cell fractions, lung tissue cytokines, macroscopic findings, or microscopic lung changes in Sprague-Dawley rats exposed to oxidized ORP-100 and reduced ORP-100S test articles at target delivery doses of 10 mg / kg and 40 mg / kg and examined 1, 4, or 20 hours post-exposure. However, this study utilized a sucrose / EDTA formulation developed to provide the greatest degree of redox stability to thioredoxin when lyophilized in its reduced form (PCT WO2006 / 090127), and as shown in Figure 9, right, the sucrose formulation effect was evident. In the oxidized ORP-100 control (lacking thioredoxin activity), neutrophil influx, indicative of inflammation, was induced. Reduced ORP-100 and reduced ORP100S were able to partially alleviate the formulation effect, but almost completely inhibited neutrophil influx.

[0252] Because our study design did not include a vehicle-only group, it was not possible to isolate the effect of the sucrose / EDTA formulation itself on BALF cell counts and cytokines. However, we did show that an oxidized thioredoxin inactive protein control formulated in normal saline had identical BALF cell count and cytokine results to normal saline alone. Considered in the context of intratracheal delivery (Rancourt, RC, et al., 2007, Reduced thioredoxin increases proinflammatory cytokines and neutrophil influx in rat airways: modulation by airway mucus, Free Radic Biol Med 42, 1441-53), our results (showing the highest response from the oxidized ORP100 inactive protein control group) are consistent with the sucrose / EDTA formulation contributing to the observed dose-dependent inflammatory response suppressed by reduced ORP100S.

[0253] The invention illustratively disclosed herein may suitably be practiced in the absence of any element not specifically disclosed herein. However, it will be apparent to those skilled in the art that numerous variations, variations, modifications, other uses and applications of the invention are possible, and that variations, variations, modifications, other uses and applications that do not depart from the spirit and scope of the invention are deemed to be covered by the invention, which is limited only by the claims that follow.

[0254] The foregoing discussion of the invention has been presented for purposes of illustration and description. The foregoing is not intended to limit the invention to the form(s) disclosed herein. In the foregoing Detailed Description of the Invention, for example, various features of the invention are grouped together in one or more embodiments for the purpose of streamlining the disclosure. Features of the embodiments of the invention may be combined into alternative embodiments other than those discussed above. This method of disclosure is not to be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment. Thus, the following claims are hereby incorporated into this Detailed Description of the Invention, with each claim standing on its own as a separate preferred embodiment of the invention.

[0255] Furthermore, while the description of the invention includes a description of one or more embodiments and certain variations and modifications, other variations, combinations, and modifications are also within the scope of the invention, for example, as may be within the knowledge of one of ordinary skill in the art after understanding the present disclosure. No right is intended to include, to the extent permitted by law, alternative embodiments, including alternative, interchangeable, and / or equivalent structures, functions, ranges, or steps to those claimed, whether or not such alternative, interchangeable, and / or equivalent structures, functions, ranges, or steps are disclosed herein, without intending to dedicate any patentable subject matter to the public.

Claims

1. 1. A method for reducing the viscoelasticity of mucus or sputum in a patient having excessively viscous or sticky mucus or sputum, the method comprising contacting the patient's mucus or sputum with a composition comprising a protein or peptide comprising a reduced thioredoxin monocysteine ​​active site, wherein the protein or peptide does not contain any cysteine ​​residues except for a single cysteine ​​residue at the N-terminal position of the thioredoxin monocysteine ​​active site.

2. a protein or peptide comprising a reduced thioredoxin monocysteine ​​active site and containing no cysteine ​​residues except for a single cysteine ​​residue at the N-terminal position of the thioredoxin monocysteine ​​active site; b. a pharmaceutically acceptable excipient; A pharmaceutical composition comprising:

3. a. a protein or peptide containing a reduced thioredoxin active site; b. an aqueous solvent having a vapor pressure of at least about 3 mmHg; A composition comprising:

4. a. a protein or peptide containing a reduced thioredoxin active site; b. Water; c. Sodium chloride and 1. A pharmaceutical composition consisting essentially of:

5. 1. A method for preparing a dry composition, comprising: a. providing an aqueous composition comprising a protein or peptide comprising a reduced thioredoxin active site and an aqueous solvent having a vapor pressure of at least about 3 mmHg; b. volatilizing the aqueous solvent to produce a dry composition comprising the protein or peptide; A method comprising:

6. A composition consisting essentially of a protein or peptide containing a reduced thioredoxin active site and normal saline.

7. A composition consisting essentially of a protein or peptide containing a reduced thioredoxin active site, said composition being a dry powder.

8. A method for treating inflammation in a subject, comprising administering to a subject having or at risk of developing inflammation a pharmaceutical composition comprising a protein or peptide comprising a reduced thioredoxin monocysteine ​​active site.

9. A method for treating a bacterial infection in a subject, comprising administering to a subject having or at risk of developing a bacterial infection a pharmaceutical composition comprising a protein or peptide comprising a reduced thioredoxin monocysteine ​​active site.

10. A composition comprising a thioredoxin monocysteine ​​active site operable to activate one or more endogenous antimicrobial peptides, wherein the activation results in a therapeutically effective reagent for treating or preventing infectious disease.

11. 1. A method for modulating the microbiome composition of a subject, comprising topically administering to a mucosal surface of the subject a composition comprising a protein or peptide comprising a reduced thioredoxin monocysteine ​​active site.

12. A method for determining the disulfide bond reducing activity of a protein or peptide containing a monocysteine ​​thioredoxin active site, comprising selecting a protein or peptide containing a monocysteine ​​thioredoxin active site that does not contain any cysteine ​​residues except for a single cysteine ​​residue in the thioredoxin monocysteine ​​active site, and measuring the overall cysteine ​​thiol reduction status of the protein or peptide.

13. A method for treating a viral respiratory disease, comprising administering to a subject having or at risk of developing the viral respiratory disease a composition comprising a protein or peptide that comprises a reduced thioredoxin monocysteine ​​active site.

14. 1. A method for reducing pulmonary inflammation associated with a viral infection in a subject in need thereof, comprising administering to the subject in need thereof a pharmaceutical composition comprising a protein or peptide comprising a reduced thioredoxin monocysteine ​​active site.

15. A composition comprising a protein or peptide containing a thioredoxin active site, the composition not including a thioredoxin protein fraction having a UV absorbance greater than about 400 nm wavelength.

16. 1. A method for producing a composition comprising a protein or peptide comprising a thioredoxin active site, the method comprising: providing a lysate comprising the protein or peptide comprising the thioredoxin active site; concentrating the protein or peptide in the lysate; and removing a thioredoxin peptide or protein fraction having an absorbance greater than about 400 nm to produce the composition.

17. 17. The method or composition of any of claims 1-16, wherein the thioredoxin active site is a thioredoxin monocysteine ​​active site comprising an amino acid sequence selected from the group consisting of C-X-X-S (SEQ ID NO:24), C-X-X-X (SEQ ID NO:17), X-C-X-X-X-X (SEQ ID NO:19), X-C-G-P-X-X (SEQ ID NO:21), W-C-G-P-X-K (SEQ ID NO:23), X-C-X-X-S-X (SEQ ID NO:25), X-C-G-P-S-X (SEQ ID NO:26) and W-C-G-P-S-K (SEQ ID NO:27), wherein the X residue is any amino acid residue except cysteine.

18. 17. The method or composition of any of claims 1 to 16, wherein the protein or peptide comprises a sequence that is at least about 80% identical to SEQ ID NO:28 or SEQ ID NO:29, and the thioredoxin active site is a thioredoxin monocysteine ​​active site located at a position corresponding to positions 32-35 of SEQ ID NO:28 or SEQ ID NO:

29.

19. 17. The method or composition of any preceding claim, wherein the protein or peptide comprises the sequence of SEQ ID NO:28 or SEQ ID NO:

29.

20. 10. The method of claim 1, wherein the patient has a lung disease in which abnormal or excessive viscosity or stickiness of mucus or sputum is a symptom or cause of the disease.

21. 10. The method of claim 1, wherein the patient has a pulmonary disease in which abnormal or excessive viscosity or stickiness of mucus or sputum is associated with a deficiency in biological reducing agent activity.

22. 10. The method of claim 1, wherein the patient has a disease selected from the group consisting of cystic fibrosis, chronic obstructive pulmonary disease, bronchiectasis, asthma, sinusitis, idiopathic pulmonary fibrosis, pulmonary hypertension, dry eye disease, and gastrointestinal disease.

23. 10. The method of claim 1, wherein the patient has cystic fibrosis.

24. 2. The method of claim 1, wherein the step of contacting the patient's mucus or sputum with the composition is carried out by introducing the composition into the patient by a route selected from the group consisting of nasal, intratracheal, bronchial, direct placement in the lungs, inhalation, oral, and ocular.

25. 10. The method of claim 1, wherein the mucus or sputum to be contacted is in the patient's respiratory tract.

26. The method of claim 1 , wherein the composition further comprises a pharmaceutically acceptable carrier.

27. 10. The method of claim 1, wherein after the step of contacting the patient's mucus or sputum with the composition, the patient has at least about a 2.5% increase in forced expiratory volume (FEV) compared to before the contacting step.

28. 10. The method of claim 1, wherein the thioredoxin monocysteine ​​active site-containing protein or peptide is covalently bound to a cysteine ​​residue in a mucus protein.

29. 29. The method of claim 28, wherein the mucus protein is a mucin.

30. 29. The method of claim 28, wherein the mucus protein is a respiratory mucus protein.

31. The method of claim 1 , wherein the protein comprises human thioredoxin.

32. 10. The method of claim 1, wherein the patient is a human.

33. 3. The pharmaceutical composition of claim 2, wherein the protein or peptide comprises the thioredoxin monocysteine ​​active site sequence of SEQ ID NO:

1.

34. 10. The pharmaceutical composition or method of any of claims 2, 8 or 9, wherein the pharmaceutical composition is formulated for administration to a patient by a route selected from oral, rectal, nasal, inhalation, intratracheal, bronchial, direct instillation, topical and ocular.

35. 6. The composition or method of claim 3, wherein the aqueous solvent is selected from the group consisting of ammonium acetate, ammonium bicarbonate, ammonium formate, triethylammonium acetate, and triethylammonium bicarbonate.

36. 6. The composition or method of claim 3 or 5, wherein the aqueous solvent is ammonium acetate.

37. 6. The composition or method of claim 3, wherein the aqueous solvent is at a concentration of between about 1 mM and about 50 mM.

38. 6. The composition or method of claim 3, wherein the aqueous solvent has a pH between about 4 and about 7.

39. 6. The composition or method of claim 3 or 5, wherein the composition does not include sugars or sugar derivatives.

40. 6. The composition or method of claim 3, wherein the aqueous composition does not contain any compounds other than proteins or peptides that have a vapor pressure of less than about 3 mmHg.

41. 5. The composition of claim 3, wherein the protein or peptide does not contain any cysteine ​​residues except for one or two cysteine ​​residues in the thioredoxin active site.

42. 16. The composition of any of claims 3, 4, 14 or 15, wherein the thioredoxin active site comprises an amino acid sequence selected from the group consisting of C-X-X-C (SEQ ID NO: 16), X-C-X-X-C-X (SEQ ID NO: 20), X-C-G-P-C-X (SEQ ID NO: 22), W-C-G-P-C-K (SEQ ID NO: 3), wherein the X residue is any amino acid residue except cysteine.

43. 5. The composition of claim 3, wherein the thioredoxin active site is a monocysteine ​​thioredoxin active site.

44. 6. The composition or method of claim 3 or 5, wherein the protein or peptide does not contain any cysteine ​​residues except for a single cysteine ​​residue N-terminal to the thioredoxin monocysteine ​​active site.

45. 5. The pharmaceutical composition of claim 4, wherein the sodium chloride is present at about 9 grams of sodium chloride per liter of water.

46. 6. The method of claim 5, wherein the volatilizing step comprises subjecting the composition to conditions selected from the group consisting of reduced pressure, elevated temperature, and combinations thereof.

47. 6. The method of claim 5, wherein the volatilizing step is carried out in a non-oxidizing atmosphere.

48. 6. The method of claim 5, wherein the volatilizing step is carried out under a nitrogen atmosphere.

49. The method of claim 5 , wherein the volatilizing step comprises freeze-drying.

50. 6. The method of claim 5, further comprising solubilizing the dry pharmaceutical composition in a diluent.

51. 51. The method of claim 50, wherein the diluent is a saline solution having a pH between about 4 and about 7.

52. 51. The method of claim 50, wherein the solubilized pharmaceutical composition is at least 80% stable in the reduced form for at least about 1 day at a temperature of about 25°C.

53. 51. The method of claim 50, wherein the solubilized pharmaceutical composition is at least 80% stable in the reduced form for at least about 1 week.

54. 6. The method of claim 5, wherein the thioredoxin comprises a monocysteine ​​thioredoxin active site.

55. 6. The method of claim 5, wherein the composition comprises a protein or peptide comprising a reduced thioredoxin monocysteine ​​active site, and the protein or peptide does not contain any cysteine ​​residues except for a single cysteine ​​residue at the N-terminus of the thioredoxin monocysteine ​​active site.

56. 7. The composition of claim 6, comprising a protein or peptide containing a reduced thioredoxin active site and normal saline.

57. 9. The method of claim 8, wherein administration of the protein or peptide inhibits the release of inflammatory cytokines.

58. 9. The method of claim 8, wherein the proinflammatory cytokine is selected from the group consisting of IL-8, IL-1β, IL-6 and TNFα.

59. 10. The method of claim 9, wherein the composition comprises a crude or purified extract of microbial cells expressing the protein or peptide.

60. The composition of claim 10 , wherein the antimicrobial peptide is a defensin.

61. 12. The method of claim 11, wherein the mucosal surface is a lung surface.

62. 12. The method of claim 11, wherein the mucosal surface is a nasopharyngeal surface.

63. 12. The method of claim 11, wherein the mucosal surface is a gastrointestinal surface.

64. 13. The method of claim 12, wherein the cysteine ​​thiol reduction state is measured using a method selected from the group of a chromogenic assay, a fluorometric assay, and a turbidimetric assay.

65. 13. The method of claim 12, wherein the chromogenic assay is a DTNB assay.

66. 14. The method of claim 13, wherein the viral respiratory disease is selected from the group consisting of acute respiratory distress syndrome (ARDS), severe acute respiratory distress syndrome (SARS), Middle East respiratory syndrome (MERS), SARS-coronavirus-2 (SARS-CoV-19 or COVID-19), influenza, asthma, pneumonia, bronchitis, tuberculosis, reactive airway disease syndrome, and a viral infection associated with interstitial lung disease.

67. 15. The method of any one of claims 13 or 14, wherein the viral respiratory disease is caused by a virus selected from a coronavirus, influenza virus, respiratory syncytial virus (RSV), parainfluenza virus, and respiratory adenovirus.

68. 15. The method of any one of claims 13 or 14, wherein the composition is administered in a form selected from the group consisting of a nebulized form and an aerosolized form.

69. 16. The composition of claim 15, wherein the protein or peptide comprising a thioredoxin active site is in a reduced state, and the composition further comprises an aqueous solvent having a vapor pressure of at least about 3 mmHg.

70. 16. The composition of claim 15, consisting essentially of a protein or peptide containing a reduced thioredoxin active site, water, and sodium chloride.

71. 16. The composition of claim 7 or 15, which is dried to a moisture content of less than about 3.0% by weight.

72. 17. The method of claim 16, wherein the composition comprises a peptide or protein fraction having an absorbance at a light wavelength of less than about 300 nm.

73. 17. The method of claim 16, wherein the removing step comprises hydrophobic interaction chromatography.

74. 17. The method of claim 16, further comprising drying the composition to a moisture content of less than about 3.0% by weight.

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