Products and methods for mucus consistency standardization

Abstract

Disclosed are compositions and methods for reducing the viscosity and / or stickiness and / or increasing the liquefaction of overly or abnormally viscous or sticky mucus or sputum. The compositions comprise a protein or peptide comprising a reduced thioredoxin single cysteine active site and optionally further comprise a reducing system.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims priority under 35 U.S.C. 119(e) to U.S. provisional application serial number 61 / 792,198, filed March 15, 2013. The entire disclosure of U.S. provisional application serial number 61 / 792,198, filed March 15, 2013, is incorporated herein by reference.

[0003] Reference to Sequence Listing

[0004] This application contains a Sequence Listing submitted electronically via EFS-Web as a text file. The text file is named "7579-1-PCT_sequence_listing_ST25" having a size of 18 KB bytes, and was created on March 17, 2014. The information contained in the text file is incorporated by reference herein in its entirety pursuant to 37 CFR § 1.52(e)(5). TECHNICAL FIELD

[0005] The present invention relates generally to the use of proteins or peptides containing a reduced thioredoxin monocysteine active site for reducing the viscosity of mucus or sputum and inducing, enhancing and / or increasing its liquefaction. BACKGROUND

[0006] There is a large unmet medical need for safe, well-tolerated and effective drugs for treating patients with cystic fibrosis (CF), COPD / emphysema, bronchiectasis, severe asthma and other serious obstructive lung diseases. These diseases are characterized by the overproduction of thickened mucus, leading to impaired lung function (reviewed in Evans, CM. and Koo, J.S., Pharmacology & Therapeutics 121:332-348, 2009). Poor clearance of abnormal, viscous mucus is associated with chronic infection and premature death, especially in CF. Despite advances in antibiotic therapy and other treatments, improved mucus clearance remains an important clinical treatment goal, even though our understanding of the underlying mechanisms of mucus transport capacity remains limited (Verdugo, P., Cold Spring Harb Perspect Med 2012;2:a009597).

[0007] 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 away from the lungs by ciliary action and coughing (Knowles, M. R. and Boucher, R. C. J Clin Invest 109:571-577, 2002; Cone, R. A. Adv Drug Deliv Rev 61 :75-85, 2009). Proper viscoelasticity and mucus layer hydration that enables efficient mucus-ciliary transport is therefore critical for mucus function and prevention of infection and inflammation. Normal mucus is composed primarily of water (97%), with the remaining solids comprising mucin proteins, non-mucin proteins, salts, lipids, and cellular debris (Fahy, J. V. and Dickey, B. F. N Engl J Med 363:2233-47, 2010). The polymer mucin glycoproteins MUC5AC and MUC5B are primarily responsible for the viscoelasticity of respiratory mucus gels (Matsui et al. Cell 95:1005-1015, 1998; review in Kreda et al. Cold Spring Harb Perspect Med 2012;2:a009589). O-linked glycans attached to mucins promote water binding, while the mucins themselves form an entangled network (Verdugo et al. Biorheology 20:223-230, 1983), which can involve both covalent and non-covalent interchain crosslinks, as suggested by detailed studies of the gut mucin MUC2 (Ambort et al. Biochem J 436:61-70, 2011). Mucins are unusually rich in Cys amino acids, with human MUC5AC containing a significant 295 Cys residues out of a total of 5030 amino acids (www.uniprot.org / uniprot / P98088). Mucin Cys residues located near the N- and C-termini are thought to be involved in forming interchain disulfide bonds between mucin subunits, while the role of internal Cys residues is unclear (Thornton et al. Annu Rev Physiol 70:459-486, 2008). Some located in the 'Cys knot' region and potentially susceptible to forming intramolecular disulfide bonds can play a role in non-covalent entanglements important for promoting the mucus gel network (Fahy, J. V. and Dickey, B. F. N Engl J Med 363:2233-47, 2010).

[0008] Recent studies (Button et al., Science 337:937-941, 2012) have generated a new model of the mucosal surface structure based on the finding that certain mucins once thought to be membrane-bound on epithelial cells are actually membranes tethered to the cilia themselves. The significance of this model is that the mobile periciliary fluid layer overlies a more dense periciliary layer, described as "gel-on-brush." The model elegantly explains how fluid moves between the two layers, with the periciliary fluid acting as a reservoir, and sets a new paradigm for understanding the role of periciliary fluid osmotic modulus in determining periciliary transport functionality and periciliary layer hydration. The model also provides a framework for understanding how excess disulfide bonds in the mucin protein backbone can increase the periciliary fluid layer osmotic modulus, which in turn dehydrates the underlying ciliary layer and severely limits normal periciliary transport. Such a scenario can solidify a substantial part of the disease mechanism of CF.

[0009] CF is an autosomal recessive disease. Symptoms of CF result from a defect in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), a critical epithelial membrane transporter of monovalent negatively charged ions, primarily chloride (Riordan et al., Science 245: 1066-1073), but possibly bicarbonate and glutathione. Mutations that cause CF, of which over 1700 are known (www.genet.sickkids.on.ca / cftr / ), include those that cause complete loss of CFTR (the most common CF genotype case) and point mutations that result in partial or complete loss of anion transport activity. In addition, due to the defect in CFTR, epithelial cells in the body are impermeable to chloride transport (Boucher et al., Lung 161: 1-17, 1983; Welsh, Physiol Rev 67: 11443-1184, 1987). While several organs are affected, including the pancreas, intestine, and male reproductive tract, complications in the lung account for 95% of morbidity and mortality (Means, M. Cystic Fibrosis: the first 50 years. Cystic Fibrosis-Current Topics Vol. 1, Edited by Dodge JA, Brock DJH and Widdicombe JH. Chichester: Wiley and Sons, 1992, pp. 217-250). In the diseased lung, chloride transport into the airway lumen causes Na +and excessive absorption of fluid, thereby reducing the volume of airway surface liquid (Jiang et al., Science 262:424-427, 1993). However, attempts to restore chloride channel activity to compensate for the effects of non-functional CFTR (e.g., via agonists of the P2Y2 subtype of purinergic receptors) have failed (Ratjen, F. et al., J Cyst Fibros 11:539-49, 2012). This suggests that the non-chloride effects of CFTR can be more significant than originally thought.

[0010] In an oxidizing environment such as the lung, disulfide bonds readily form between adjacent oxidized Cys residues such as those found in high abundance on the mucin proteins. These bonds are highly stable and breaking (i.e., reducing) them to restore the Cys residues to their free thiol form requires the action of potent chemical or biological reducing agents. In the healthy lung, excess disulfide bond formation is primarily impeded by the reducing form of the biological reducing agent glutathione (GSH), a Cys-containing tripeptide that is abundantly secreted into the mucus layer (Cantin et al., J Appl Physiol 63: 152-157, 1987) and can play a key role in maintaining the normal disulfide bond to free Cys thiol balance in mucins. GSH secretion onto the airway surface is highly dependent on CFTR, which directly and indirectly facilitates GSH export (reviewed in Ballatori et al., Biol Chem 390: 191-214, 2009). Thus, lung GSH levels in CF patients can be 30% or less of the levels found in normal individuals (Roum et al., J Appl Physiol 75: 2419-24, 2003; Wetmore D.R. et al., J Biol Chem 285: 30516-22, 2010). CFTR is also responsible for secreting bicarbonate anions, and the resulting bicarbonate deficiency in the CF lung appears to contribute to the disease. The primary chemical role of bicarbonate is to raise the pH. Because reduction of disulfide bonds by thiol-containing reducing agents requires the formation of the aggressive deprotonated thiolate, which is inhibited at low pH that favors the protonated thiol form (Singh and Whitesides, Sulphur-containing Functional Groups, 5: pp. 633-58, John Wiley & Sons, 1993), a synergy between bicarbonate and the activity of GSH (and other biological reducing agents known to exist in the airway surface environment) is possible. The measured pH in CF tracheobronchial secretions is at most 0.6 units lower than in non-diseased cases (Song et al., Am J Physiol Cell Physiol 290: C741-C749), which is consistent with the environment in the CF lung where reducing agents are present in limited supply and are less active due to impaired ability to form disulfide bond-attacking thiolates. Taking into account the vast number of clustered Cys present in mucins, if the level of secreted reducing agents becomes limited, or if mucin proteins are overproduced and secreted leading to an excess of disulfide bondable Cys, the mucus in an oxidizing respiratory environment would thus be balanced in a higher disulfide bond-linked state.Both scenarios are known to occur in CF and certain other obstructive lung diseases: excess production of mucus proteins in response to lung stress (Rogers, Resp Care 52: 1134-1149), and possible blockage of 70% or more of GSH secretion due to CFTR deficiency (Roum et al., J Appl Physiol 75: 2419-24, 2003; Wetmore D.R. et al., J Biol Chem 285: 30516-22, 2010).

[0011] The potential for this excess mucus disulfide bonding and the general redox imbalance playing a mechanical role in CF has led to clinical evaluation of various thiol-containing agents as mucolytic drugs. These include N-acetylcysteine (NAC) and Nacystelyn (NAL; N-acetylcysteine + L-lysine) (Hurst et al., Am Rev Respir Dis, 96:962-970, 1967; Dasgupta and King, Pediatr Pulmonol, 22:161-166, 1996; Nash, E.F. et al., Cochrane Database of Systematic Reviews, 2010(12):1-49, 2009) and reduced glutathione (Bishop, C. et al., CHEST Journal, 127(1):308-317, 2005; Griese, M. et al., Am J Resp Crit Care Med 169(7):822-828, 2004; Griese, M. et al., Am J Resp Crit Care Med 188(1):83-89, 2013; Roum, J.H. et al., J Appl Physiol, 87:438-443, 1999). While largely safe, these small molecule agents have not exhibited clear clinical benefit in oral or inhaled forms to date (reviewed in Nash, E.F. et al., Cochrane Database of Systematic Reviews, 2010(12):1-49, 2009). This lack of efficacy can be due in large part to loss of potency or activity due to auto-oxidation during delivery, as well as the potential for inactivation by lung enzymes. GSH is subject to rapid auto-oxidation to the inactive GSSG form (Curello, S. et al., Clin Chem, 33:1448-49, 1987) and thus is pharmacologically unstable in the reduced form when aerosolized and inhaled (Carl White M.D., pers comm.), losing much of its activity by the time it reaches the target site in the airways.Additionally, gamma-glutamyltransferase, which is present in high concentrations in the lung space, readily degrades GSH to inactive forms (Corti et al., Am J Resp Crit Care Med 189:233-234, 2014), the abundance of which increases significantly after GSH inhalation (Griese et al., Am J Resp Crit Care Med 188:83-89 Supplemental Information, 2013). Improving thiol agents by combining disulfide targeting with superior pharmacology and biopharmaceutical specificity is thus a critical unmet therapeutic goal.

[0012] While the cause of CF lung disease can be attributed to the altered rheological properties of mucus, lung function impairment at birth is not very pronounced. In contrast, bronchiectasis and airway obstruction progress with age in patients. This chronic lung damage results from the persistent cycles of bacterial infection and inflammatory response. Airway damage produced when neutrophils are recruited into the lung releases matrix-degrading enzymes such as elastase, and harmful reactive oxygen species (reviewed in Konstan and Berger, Pediatr Pulmonol 24: 137-142, 1997). Following persistent infection, interactions of mucin with DNA (Potter et al., Am J Dis Child 100:493-495, 1960; Lethem et al., Am Rev Respir Dis 100:493-495, 1990; Lethem et al., Eur Respir J 3: 19-23, 1990) and f-actin polymers (Sheils et al., Am J Path 148:919-927, 1996; Tomkiewicz et al., DNA and actin filament ultrastructure in cystic fibrosis sputum. Cilia, mucus, and mucociliary interactions, edited by Baum GL, Priel Z, Roth Y, Liron N, and Ostfeld EJ. New York, NY: Marcel Dekker, 1998) released from dead inflammatory cells can also occur and can contribute to some of the tenacious and viscous properties of CF sputum in severe disease. This mucus cannot be cleared by coughing or mucociliary clearance, which promotes further colonization of the lung by opportunistic pathogens, airway remodeling, and ultimately death.

[0013] Interventions designed to directly mitigate the consequences of CFTR deficiency are therefore particularly desirable, as they can prevent or mitigate disease progression. While direct correction of CF through gene therapy has not yet been achieved, recent studies using enhancer and corrector therapies have demonstrated the ability to restore some degree of CFTR function to the defective protein (Sloane, PA, and Rowe, SM, Current Opinion in Pulmonary Medicine 16:591-7, 2010). This therapy is limited to a small percentage of CF patients with specific CFTR deficiencies, such as those with ivacato / Kalydeco. TM Targeting the G551D mutation (Jones, AM and Helm, JM, *Drugs* 69:1903-10, 2009). However, dramatic results have been observed in these few individuals (Accurso, FJ; Rowe, SM; Clancy, JP; Boyle, MP; Dunitz, JM; Durie, PR; Savel, SD; Hornick, DB et al., *The New England Journal of Medicine* 363:1991-2003, 2010), suggesting that mechanical intervention in CF can mitigate late consequences, such as those caused by chronic infection and inflammation. However, currently, symptomatic rather than disease-modifying approaches, including antibiotic regimens combined with drugs that promote the clearance of purulent airway secretions, remain the mainstay of treatment for progressive airway disease. Purification of extracellular DNA present in the airways of CF using rhDNAase (Pulmozyme). TM Inhaled Genentech (USA) is widely used as a decongestant for the airways. Such treatment is clinically effective in reducing sputum viscosity and stabilizing forced expiratory volume (FEV) (Fuchs et al., New England Journal of Medicine 331:637-642, 1994). Other investigational therapies aimed at breaking down mucin or actin polymers, including N-acetylcysteine ​​(NAC), nacystelyn (an N-acetyl-L-cysteine ​​derivative), and gelsole, have also experimentally reduced sputum viscosity, but have not yet been clinically proven or approved in the United States for the treatment of CF (Nash, EF et al., Cochrane Database of Systematic Reviews, 2010(1):CD007168, 2009).

[0014] Other methods that have been utilized to improve mucus clearance include mucus active agents such as inhaled hypertonic saline and inhaled high dose mannitol (Fahy, J. V. and Dickey, B. F., N Engl J Med 363:2233-47, 2010). These agents are thought to act by drawing water into the mucus layer in an osmotic manner to increase hydration, or by inducing the cough reflex to improve clearance. There is some evidence for both mechanisms (Levin, M. H. et al., J Biol Chem 281 :25803-12, 2006; Boucher, R. C., Trends Mol Med 13:231-240, 2007). However, mucus active agents are symptomatic (rather than disease modifying), and efficacy is often only modest, as many patients cannot tolerate the high doses that can have the greatest clinical effect (Aziz, I. and Kastelik, J. A., N Engl J Med 354:1848-1851, 2006).

[0015] The results of studies by White and colleagues (Rancourt et al., Am J Physiol Lung Cell Mol Physiol 286:L931-L938, 2004; Rancourt et al., Free Radical Biol & Med 42:1441-43, 2007) have found that the use of proteins or peptides containing the active site of thioredoxin in the reduced state can be used to increase mucus or sputum liquefaction in patients with excessively viscous or sticky mucus or sputum, including patients with CF, wherein the mucus or sputum is contacted with the protein or peptide (U.S. Patent No. 7,195,766 and U.S. Patent No. 7,534,438, both of which are incorporated herein by reference in their entirety). In this system (see Figure 3), a transient mixed disulfide bond is formed between the N-terminal cysteine of the thioredoxin active site and a cysteine of the target protein (present in mucus or sputum), followed immediately by a nucleophilic attack on the intramolecular mixed disulfide bond and release of oxidized thioredoxin and fully reduced target (Wynn et al., Biochemistry 34(37): 11807-11813, 1995), thus allowing reformation of cysteine disulfide bonds in mucus or sputum but also allowing free access of reduced or oxidized thioredoxin to enter cells and induce unwanted off-target activities followed by re-reduction by the endogenous thioredoxin reductase-NADPH system. In addition, White and colleagues have shown that reduced thioredoxin alleviates the abnormal viscoelasticity of human CF mucus in vitro and in vivo animal implantation studies (Rancourt et al., Free Radical Biol & Med 42: 1441-43, 2007), as well as inhibiting the activity of the pro-inflammatory neutrophil elastase through disruption of the active site disulfide bond (Lee et al., Am J Physiol Lung Cell Mol Physiol 289: L875-L882, 2005). In comparison to GSH and thiol agents such as NAC, thioredoxin is a more potent disulfide reducing molecule and is less susceptible to inactivation by autoxidation. In summary, this creates an opportunity to exploit a pharmacologically stable molecule to restore the normal disulfide reducing state of mucus. Such a therapy can halt or delay the cascade of chronic infection, inflammation, and lung function decline that leads to premature death in CF patients. However, there is also a strong motivation to avoid the potential pro-inflammatory and other intracellular regulatory effects of thioredoxin (Arner, E.S. and A. Holmgren, Eur J Biochem 267: 6102-6109, 2000; Rancourt et al., Free Radical Biol & Med 42: 1441-43, 2007) as well as increasing the efficacy of mucus viscoelasticity modulation by preventing re-oxidation of mucin Cys. These improvements are the subject of the present invention. SUMMARY

[0016] One embodiment of the application relates to a method of reducing the viscosity of mucus or sputum in a patient having mucus or sputum that is excessively viscous or sticky. The method comprises the step of contacting the mucus or sputum of the patient with a composition comprising a protein or peptide comprising a reduced thioredoxin monomercysteine active site, which is effective to reduce the viscosity of the mucus or sputum as compared to prior to the contacting step. In one aspect of this embodiment, 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. In one aspect, the patient has a lung disease selected from the group consisting of cystic fibrosis (CF), chronic obstructive pulmonary disease, bronchiectasis, and asthma. In a preferred aspect, the patient has CF. In another aspect of this embodiment, the patient has a lung disease in which abnormal or excessive viscosity or stickiness of mucus or sputum is associated with a deficiency in bioreductant activity. In another aspect of this embodiment, the patient has a disease of the digestive tract associated with thickened or abnormal mucus, including but not limited to coccidiosis.

[0017] In one aspect, the step of contacting the mucus or sputum of the patient with the composition is performed by introducing the composition into the patient using a route selected from the group consisting of nasal, intratracheal, bronchial, direct installation into the lung, inhalation, and oral. In one aspect, the mucus or sputum to be contacted is located in the respiratory tract, digestive tract (i.e., gastrointestinal tract), or reproductive tract of the patient.

[0018] In another aspect, the composition is administered to the patient in a pharmaceutically acceptable carrier.

[0019] In any of the foregoing aspects, the step of contacting the mucus or sputum of the patient with the composition increases the percentage of free thiols in a sample of mucus or sputum from the patient as compared to prior to contact with the composition.

[0020] In any of the foregoing aspects, following the step of contacting the mucus or sputum of the patient with the composition, the patient has an increase in forced expiratory volume (FEV) of at least about 2.5% as compared to prior to the contacting step.

[0021] In any of the foregoing aspects, the thioredoxin single cysteine active site comprises an amino acid sequence selected from 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), or W-C-G-P-S-K (SEQ ID NO: 27), wherein the C residue is in a reduced state, and wherein the X residues are any amino acid residue other than cysteine. In a preferred aspect, the thioredoxin single cysteine active site comprises the amino acid sequence C-X-X-S (SEQ ID NO: 24) as described above.

[0022] In any of the foregoing aspects, the protein having a thioredoxin single cysteine active site comprises a thioredoxin selected from prokaryotic thioredoxins, fungal thioredoxins, plant thioredoxins, and mammalian thioredoxins. In a preferred aspect, the protein comprises a human thioredoxin.

[0023] In any of the foregoing aspects of the present application, the composition further comprises a reducing agent for reducing the thioredoxin single cysteine active site of the protein. In another aspect, the composition comprises a thioredoxin reductase and NADH or NADPH.

[0024] Another embodiment of the application relates to a composition for reducing the viscosity of mucus or sputum, the composition comprising a protein or peptide comprising a thioredoxin monocysteine active site in a reduced state and at least one additional agent for treating mucus or sputum that is excessively viscous or sticky. In one aspect of this embodiment, the thioredoxin monocysteine active site comprises an amino acid sequence selected from 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), or W-C-G-P-S-K (SEQ ID NO: 27), wherein the C residues are in a reduced state, and wherein the X residues are any amino acid residue other than cysteine. In a preferred aspect, the thioredoxin monocysteine active site comprises the amino acid sequence C-X-X-S (SEQ ID NO: 24) as described above. In any of the foregoing aspects of this embodiment, the protein having a thioredoxin monocysteine active site comprises a thioredoxin selected from prokaryotic thioredoxins, fungal thioredoxins, plant thioredoxins, and mammalian thioredoxins. In one aspect, the protein comprises a human thioredoxin.

[0025] Further, in any of the foregoing aspects of this embodiment, the composition comprises a reducing agent. In another aspect, the composition further comprises a thioredoxin reductase and NADH or NADPH.

[0026] Another embodiment of the application relates to a pharmaceutical composition comprising a protein or peptide comprising a thioredoxin monocysteine active site in a reduced state. In one aspect, the composition is formulated for administration to the lungs in an aerosol form. In another aspect, the composition is formulated for oral administration. In any of the foregoing aspects of this embodiment, the thioredoxin monocysteine active site comprises 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 C residues are in a reduced state, and wherein the X residues are any amino acid residue other than cysteine. In another aspect, the pharmaceutical composition is formulated for administration to the lungs in an aerosol form by a nebulizer device. In one aspect, the nebulizer device is a vibrating mesh nebulizer. In another aspect, the pharmaceutical composition further comprises a reducing agent. In any of the foregoing aspects of the application, the pharmaceutical composition further comprises a thioredoxin reductase and NADH or NADPH.

[0027] Another embodiment of the application relates to a composition comprising a protein or peptide comprising a thioredoxin monocysteine active site, wherein the cysteine in the monocysteine active site is covalently bound to a cysteine residue in a mucus protein. In one aspect, the thioredoxin monocysteine active site comprises 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 C residues are in a reduced state, and wherein the X residues are any amino acid residue other than cysteine. In any of the foregoing aspects, the mucus protein is a respiratory tract mucus protein or a digestive tract mucus protein. In another aspect, the mucus protein is a mucin.

[0028] Another embodiment of the present application relates to a method of reducing the viscosity of mucus or sputum in a patient having mucus or sputum that is overly viscous or sticky. The method includes the step of contacting the mucus or sputum of the patient with a composition comprising a disulfide bond reducing agent and a cysteine blocking agent. In one aspect, the disulfide bond reducing agent and the cysteine blocking agent are the same molecule. In another aspect, the same molecule is a protein or peptide containing a thioredoxin mono-cysteine active site. In another aspect, the disulfide bond reducing agent and the cysteine blocking agent are different molecules. In another aspect, the disulfide bond reducing agent can be dithiothreitol (DTT), ethylenediaminetetraacetic acid (EDTA), glutathione, dithio glycolic acid, 2-mercaptoethanol, N-acetyl cysteine, or tris-(2-carboxyethyl) phospholene. In another aspect, the cysteine blocking agent can be iodoacetamide, iodoacetic acid, or other alkylating agent.

[0029] Another embodiment of the present application relates to a method of treating a patient having mucus that is overly viscous or sticky. The method includes the step of administering to the patient a composition comprising a compound having a thioredoxin active site that cannot be taken up by a cell. In one aspect, the compound can be a protein or peptide comprising a thioredoxin mono-cysteine active site, a fusion protein comprising a thioredoxin moiety and a cell surface receptor ligand moiety, or a combination of a protein or peptide comprising a thioredoxin active site and a blocking compound for the cysteine corresponding to cysteine 35 of SEQ ID NO: 12.

[0030] Another embodiment of the present application relates to a method of preventing systemic exposure of a drug substance in a patient. The method includes the step of administering the drug to the patient by a delivery route including, but not limited to, a pulmonary, oral, or topical delivery route. In another aspect, the drug forms a covalent bond with its target site once administered. In another aspect, the drug substance is a thiol-containing drug and can be, for example, a protein or peptide containing a thioredoxin mono-cysteine active site in a reduced state. In another aspect, the drug substance is an antibiotic or anti-infective agent and is fused or linked to a thioredoxin mono-cysteine active site in a reduced state by a linker. In another aspect, the drug substance is an anti-inflammatory agent and is fused or linked to a thioredoxin mono-cysteine active site in a reduced state by a linker. In another aspect, the drug substance is a nucleic acid hydrolyzing agent and is fused or linked to a thioredoxin mono-cysteine active site in a reduced state by a linker. In another aspect, the drug substance is a chemotherapeutic agent and is fused or linked to a thioredoxin mono-cysteine active site in a reduced state by a linker.

[0031] Another embodiment of the application relates to a pharmaceutical composition comprising a protein or peptide comprising a thioredoxin monocysteine active site in a reduced state and further comprising at least one sugar or sugar derivative capable of stabilizing the reduced oxidoreductively active thiol group. In one aspect, the sugar or sugar derivative can be sucrose, sucralose, lactose, trehalose, maltose, galactose, raffinose, mannose, or mannitol. In one aspect, the thioredoxin monocysteine active site comprises 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 C residue is in a reduced state, and wherein the X residues are any amino acid residue other than cysteine.

[0032] Another embodiment of the application relates to an animal feed composition comprising a protein or peptide comprising a thioredoxin monocysteine active site in a reduced state. In one aspect, the thioredoxin monocysteine active site comprises 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 C residue is in a reduced state, and wherein the X residues are any amino acid residue other than cysteine.

[0033] In one aspect of any embodiment of the application, the patient is a vertebrate, including, but not limited to, mammals and birds. In another aspect, the patient is a human. In another aspect, the patient is a chicken or turkey.

[0034] Another embodiment of the application relates to the use of a composition comprising a protein or peptide comprising a thioredoxin monocysteine active site in a reduced state to reduce the viscosity of mucus or sputum in a patient having mucus or sputum that is excessively viscous or sticky, wherein contacting the mucus or sputum of the patient with the composition reduces the viscosity of the mucus or sputum as compared to prior to the contacting step. In one aspect, the thioredoxin monocysteine active site comprises 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 C residue is in a reduced state, and wherein the X residues are any amino acid residue other than cysteine. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figures la-lb Enzymatic activity of a protein or peptide comprising a thioredoxin monocysteine active site (referred to as r(Cys)hTrx) compared to a protein or peptide comprising a wild-type thioredoxin active site (referred to as WTrhTrx) is shown. Figure la Non-specific 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB or Ellman's reagent) reduction reflects the loss of reducible cysteines in the thioredoxin monocysteine active site relative to wild-type is shown. Figure lb Enzymatic activity of a protein or peptide comprising a thioredoxin monocysteine active site (r(Cys)hTrx) compared to a protein or peptide comprising a wild-type thioredoxin active site (WTrhTrx) is shown.

[0036] Figure 2 Standardized effects of wild-type rhTrx, r(Cys)hTrx, and various controls including dithiothreitol (DTT) at two concentrations (0.58 mM and 1.5 mM), N-acetyl cysteine (NAC), and equimolar concentrations of recombinant human DNase (rhDNase) on patient sputum samples (n = 6 per treatment) in a sputum compression assay are shown.

[0037] Figure 3This illustrates the mechanism by which a protein or peptide with a thioredoxin monocysteine ​​active site at position 35 of SEQ ID NO: 12 forms a disulfide bond. The mechanism of disulfide bond reduction in native Trx involves a two-step reaction. (See diagram below.) Figure 3 As shown, steps I and II illustrate the formation of a transient mixed disulfide bond between the N-terminal Cys of the Trx active site (located at position 32 in the human TRX-1 amino acid sequence) and a Cys of the target protein disulfide bond, followed by step III. Step III illustrates the nucleophilic attack of the intermolecular mixed disulfide bond by the C-terminal Cys of the Trx active site at position 35 in the human TRX-1 amino acid sequence. This secondary reduction dissociates the mixed disulfide bond and releases the oxidized Trx and the target protein for complete reduction. By mutating the Cys residue at position 35 of the Trx C-terminal active site to a non-cysteine ​​amino acid such as a serine residue (monocysteine ​​variant), or by otherwise modifying the protein sequence, for example to interfere with nucleophilic attack by the Cys residue at position 35 of the C-terminal active site, thioredoxin can still act as a reducing agent, but unlike the wild-type enzyme, this monocysteine ​​active site variant remains covalently linked to the target protein to be reduced via the undissociated intermolecular mixed disulfide bond. Detailed Implementation

[0038] This invention generally relates to the use of proteins or peptides containing a reduced-state thioredoxin monocysteine ​​active site to induce, enhance, and / or increase the liquefaction of mucus or sputum. More specifically, the inventors have discovered that proteins or peptides having a reduced-state thioredoxin monocysteine ​​active site reduce the viscosity and / or adhesiveness of sputum or mucus and are therefore effective agents for enhancing or increasing the liquefaction of sputum or mucus. Therefore, proteins or peptides containing a reduced-state thioredoxin monocysteine ​​active site, or nucleic acid molecules encoding these proteins, can be used alone or in combination to treat a variety of conditions or diseases associated with unwanted mucus or viscous and thick sputum. For example, respiratory diseases such as cystic fibrosis, chronic obstructive pulmonary disease, bronchiectasis, and asthma are particularly suitable for treatment using the products and methods of this invention. Furthermore, gastrointestinal diseases associated with thickened or sticky mucus, such as coccidiosis, are also particularly suitable for treatment using the products and methods of this invention. Therefore, this invention relates to the use of a protein containing a reduced-state thioredoxin monocysteine ​​active site for reducing the viscosity of mucus or sputum, particularly abnormally or excessively viscous and / or sticky mucus or sputum. The protein is administered in a manner and amount that effectively reduces the viscosity of mucus or sputum and preferably provides therapeutic benefit to the patient to a patient suffering from or affected by such abnormal or excessive mucus or sputum.

[0039] For use in treating conditions such as cystic fibrosis, thioredoxin and proteins containing the wild-type (or native) thioredoxin active site (also referred to herein as "rhTrx") or containing the thioredoxin single-cysteine active site (also referred to herein as "r(Cys)hTrx") have advantages over other reducing agents. For example, unlike other reducing agents such as N-acetyl cysteine (NAC), Nacystelyn (NAL), dithiothreitol (DTT), or reduced glutathione (GSH), thioredoxin is less susceptible to inactivation by enzymatic or auto-oxidation mechanisms, including reactions that generate superoxide, hydrogen peroxide, hydroxyl radicals, and other toxic oxygen metabolites. In addition, native or wild-type thioredoxin is a naturally occurring compound that is normally secreted extracellularly onto the airway surface, and thus, introduction of thioredoxin into the airway should be non-irritating and unlikely to induce an immune response. Thioredoxin is also not glycosylated, and thus, it is easier to manufacture, and administration of the protein in native or recombinant form should not induce an innate immune response. Perhaps more significantly, reduced thioredoxin more rapidly and effectively restores treated mucus or sputum to normal levels of viscosity, and this standardization persists for a longer period of time, than other reducing agents. NAC, NAL, DTT, and GSH, for example, become "spent" or oxidized over time, and at this stage, standardization of sputum or mucus can revert to a state of abnormal viscosity. In contrast, the reduction in viscosity produced by thioredoxin appears to persist for a longer period of time, likely due to its cyclic re-reduction by its reductase system. In addition, by remaining covalently bound to the mucin Cys residues, r(Cys)hTrx produces a more effective and longer duration reduction in viscosity relative to native rhTrx. Finally, thioredoxin is more effective and specific for disulfide bond reduction than other reducing agents, and thus, it can be used at a significantly lower dose than other agents to achieve a beneficial effect.

[0040] In addition to the above advantages, thioredoxin has other benefits that increase its effectiveness in disease conditions. For example, thioredoxin is known to induce MnSOD (see, e.g., U.S. Patent No. 5,985,261 to White et al., which is incorporated herein by reference in its entirety), which is predicted to reduce the toxicity of certain bacterial toxins (including, but not limited to, endotoxins from the bacterial cell wall of gram-negative bacteria, pyocyanin from Pseudomonas aeruginosa, etc.) in disease sputum (e.g., cystic fibrosis sputum). Additionally, thioredoxin has extracellular anti-inflammatory properties (Lee, R.L. et al., Am J Physiol Lung Cell Mol Physiol, 289(5):L875-82, 2005), which can enhance the overall treatment of respiratory conditions.

[0041] Thioredoxin (Trx) is a protein disulfide reductase that catalyzes numerous thiol-dependent cellular reductive processes. Native thioredoxin contains two redox-active cysteines that are highly conserved among different species. In its oxidized form, these cysteines form a disulfide bridge that protrudes from the three-dimensional structure of the protein (Holmgren, Annu Rev Biochem 54:237-271, 1985). This active center is reduced by NADPH-dependent thioredoxin reductase (TR), allowing Trx to act as an electron carrier with disulfide / thiol exchange capabilities (Oblong et al., Biochemistry 32:7271-7277, 1993). Protein disulfide bonds are the preferred substrates for Trx-mediated reduction. Modification of the C-terminal active site cysteine of Trx results in a single cysteine active site, which, as discussed below, has significant advantages over Trx with a native or wild-type active site. The persistence and viscosity of airway secretions in cystic fibrosis disease results in airway obstruction, opportunistic infection, and deterioration of lung function. Recognizing that respiratory tract mucins contain multiple cysteine domains that are believed to play an important role in polymerization (Bell et al., Biochem J 357:203-209, 2001; Asker et al., Biochem J 333:381-387, 1998) and increased entanglement of mucins via numerous intramolecular disulfide bonds, the present inventors sought to determine whether Trx containing a single cysteine active site could act as an effective mucus viscosity modulator by reducing mucin disulfide bonds.

[0042] There are several advantages and benefits of thioredoxin containing a single cysteine active site over thioredoxin containing a native or wild-type active site. The single cysteine modification was 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). This modification prevents nucleophilic attack on the mixed disulfide formed between thioredoxin and the target protein disulfide bond catalyzed by the N-terminal thioredoxin active site cysteine (e.g., at position 32 of human thioredoxin, SEQ ID NO: 14). Surprisingly, the present inventors have determined that thioredoxin containing a single cysteine active site has greater potency than wild-type thioredoxin in reducing (trending toward liquefaction) and normalizing the viscosity of diseased human mucus. Even though thioredoxin containing a single cysteine active site would logically be expected to have a smaller reduction potential due to the loss of the active site cysteine for total perturbation, and thus after the initial catalytic reaction at the N-terminal thioredoxin active site cysteine, covalently bind to mucus proteins (e.g., heavily disulfide-bonded mucins MUC5AC and MUC5B) and would not be able to be reduced and catalyzed repeatedly, the present inventors have found that thioredoxin containing a single cysteine active site not only does not show impaired activity, but it exhibits greater quantitative ability to reduce human CF mucus viscosity in rheological assays than wild-type thioredoxin. Based on this unexpected result, the present inventors have concluded that the enhanced potency of thioredoxin containing a single cysteine active site should be due to the covalently linked thioredoxin and mucus interaction blocking the reformation of cysteine disulfide bonds in the mucins, thus providing very durable and long-term changes in the mucin oligomer gel structure and pore size. At the same time, the covalent linkage of thioredoxin containing a single cysteine active site to its mucin target sequesters and thus prevents cellular uptake and internalization of inhaled thioredoxin, which has the dual benefit of preventing off-target effects due to intracellular unwanted thioredoxin activity, while at the same time facilitating clearance of the mucus-linked depleted drug from the body.Because the only therapeutic use previously contemplated in the art for Cys-modified mono-cysteine thioredoxins was to facilitate uptake of the non-reduced form via lipid rafts in endothelial cells after injection into the systemic circulation (Hara et al., Antiox Redox Sig 9: 1427-37, 2007; Kondo et al., Antiox Redox Sig 9: 1439-48, 2007; U.S. Patent Applications 20080119398, 20090075871, and 20100184215), and thus diverging from the teachings of the present invention which focus on preventing systemic exposure and intracellular uptake, extracellular mechanisms for enhanced potency and safety of mono-cysteine active site thioredoxins in the reduced form are unexpected and highly novel.

[0043] Mucus plugging of the airways can cause significant morbidity and mortality in CF patients. The present inventors have demonstrated that the viscoelastic properties that favor the persistence of these secretions within the airways are significantly reduced by mono-cysteine active site-containing Trx. This conclusion is supported by two lines of experimental evidence. First, the compression assay results indicate that a substantial amount of liquid is released from the gel matrix of CF sputum during incubation with mono-cysteine Trx. This release is accompanied by a decrease in the volume of solid material, which suggests that the gel that constitutes a component of sputum is being solubilized. This standardization of the viscosity of CF sputum that is generally observable in CF sputum samples over the incubation period and thus, is not an artifact of centrifugation. The liquid release caused by mono-cysteine Trx is expected to have important therapeutic implications because restoration of water quantity at the airway surface can restore the mucociliary transport capacity of CF epithelia (Jiang et al., Science 262: 424-427, 1993), and based on the gel-on-brush model of Button et al. (Science, 2012), reduction of excess viscosity would allow hydration of the underlying periciliary layer and restoration of mucociliary transport, which loss is a major cause of CF pathology. Second, the magnetic micro-rheology measurements provide direct evidence that the decrease in sputum viscoelasticity is due to a reduction in sputum components caused by mono-cysteine Trx.

[0044] CF sputum is a non-Newtonian fluid that exhibits both liquid and solid properties. Polymers that exist as a solution at low concentrations are able to rotate freely. When the polymers become concentrated or cross-linked to the extent that their rotation is hindered, the solution has reached a transition phase, known as the percolation threshold (Forgacs, J Cell Sci 108:2131-2143, 1995). At the percolation threshold, the solution begins to acquire solid properties, and the elastic modulus continues to increase as more cross-linked polymer interactions are added until every filament in the sample is incorporated into the matrix. Biochemical analysis has revealed that the mucins MUC5AC and MUC5B, secreted by the cells lining the respiratory tract, are the primary gel-forming polymer components of airway mucus (Hovenberg et al., Glycoconj J 13:839-847, 1996; Thornton et al., Biochem J 316:967-975, 1996; Thornton et al., J Biol Chem 272:9561-9566, 1997). The cysteine domains present on these mucins promote polymer formation and possibly interact with adjacent mucin chains through intramolecular disulfide bond formation (Bell et al., Biochem J 357:203-209, 2001; Asker et al., Biochem J 333:381-387, 1998), which can be a contributing factor to gel entanglement. Because disulfide bonds on proteins are the preferred substrate for Trx enzyme activity, the mucin polymers are the targets for reduction by Trx during sputum liquefaction. This is supported by PAS staining, which reveals changes in solubility of high molecular weight glycoproteins in Trx-treated sputum. The detection of greater concentrations of glycoproteins in the liquid phase of Trx-exposed sputum is further indicated by a more intense yellow color and greater transparency compared to the liquid phase obtained from diluent-treated samples. The enhanced electrophoretic mobility of PAS-detectable glycoproteins in Trx-exposed sputum also suggests that the size of these macromolecules can decrease during enzymatic reduction. The results from this electrophoretic analysis are consistent with the compression assay measurements by demonstrating that glycoproteins released into the liquid phase are consistent with a decrease in gel matrix mass during exposure to Trx, and the observation of an increase in free thiol labeling in sputum after Trx treatment (Rancourt, R. et al., Free Radic Biol Med, 42(9): 1441-1453, 2007).

[0045] Once the long-term effects of inflammation and infection have been established in CF patients, neutrophil lysis within the airways of diseased CF lungs results in the deposition of extracellular DNA in airway secretions (Lethem et al., Eur Respir J 3: 19-23, 1990). Through non-covalent interactions, this DNA becomes entangled within the mucin glycoproteins, thereby increasing the mucus gel viscoelasticity (Sachdev et al., Chest 81: 41S-43S, 1982). The DNA present in sputum becomes increasingly soluble following Trx treatment. A logical explanation is that Trx activity causes structural changes within the gel matrix sufficient to reduce the entangled interactions between DNA and affected macromolecules. It is not certain how the increased DNA solubility contributes relatively to the observed viscoelasticity changes during CF sputum exposure to Trx. However, from a clinical perspective, the release or removal of DNA from the insoluble gel phase of sputum can make it more susceptible to DNase activity during such CF treatment. In addition, the effects of single cysteine r(Cys)hTrx to reduce mucus viscosity and prevent rapid reformation of disulfide bonds on mucin cysteines would serve to produce a more permeable, accessible mucus layer and de- inflate accumulated mucus plugs. These effects are expected to facilitate the entry of other therapeutic agents into the deep lung and onto the lung epithelial surface. Thus, the mechanical approach of the present invention has strong potential for synergy with existing symptomatic therapies for CF and other obstructive lung diseases such as delivery of inhaled antibiotics, mucus active substances, or mucus dissolving DNA hydrolytic agents.

[0046] Trx containing a single cysteine active site has higher activity and greater oxidative stability than reduced glutathione and acts in the airway mucus outside of cells and does not enter lung cells. Trx containing a single cysteine active site reduces viscosity, increases fluid fraction and reduces the viscoelasticity of sputum (e.g., in CF sputum). Development of a mucus-reducing system that stimulates fluid release and reduces the viscosity of airway secretions is expected to have therapeutic potential for diseases such as CF, as well as for treating excessive or abnormal mucus viscosity and / or stickiness that can be associated with other respiratory conditions (e.g., chronic or acute bronchitis; bronchiectasis; COPD / emphysema; asthma; acute bronchitis; acute or chronic sinusitis; atelectasis resulting from acute or chronic mucus plugging of the airways; bronchiolitis) or various digestive tract disorders (i.e., gastrointestinal tract) such as coccidiosis, or reproductive tract disorders associated with or exacerbated by excessive or abnormal mucus viscosity and / or stickiness (e.g., acute, subacute or chronic intestinal obstruction due to mucus thickening; infertility due to obstruction of vital reproductive structures). Because Trx containing a single cysteine active site in the reduced state becomes covalently linked to mucin, this mechanism of action will also facilitate clearance of the oxidized drug as well as mucus, can prevent or reduce cell renewal and thioredoxin-mediated redox signaling, and can prevent or reduce immune cell presentation, once it reacts with mucin disulfide bonds.

[0047] Accordingly, one embodiment of the present application relates to a method of normalizing and reducing the viscosity of mucus or sputum in a patient having mucus or sputum that is excessively viscous or sticky. The method comprises the step of contacting the mucus or sputum of the patient with a composition comprising a protein or peptide containing a thioredoxin single cysteine active site in the reduced state. The protein can be effective in reducing the viscosity of the mucus or sputum as compared to prior to the contacting step.

[0048] According to the present invention, the term "mucus" generally refers to a normally transparent, viscous fluid secreted by mucous membranes in various tissues of the body, including by the respiratory, gastrointestinal, and reproductive tracts. Mucus moistens, lubricates, and protects the tissue from which it is secreted. It contains mucin macromolecules, including mucin proteins, nucleic acids, and carbohydrates, which are the gel-forming components of mucus. Mucin proteins include, but are not limited to, respiratory tract mucin proteins and digestive tract mucin proteins. The viscoelastic properties of normal mucus depend on the concentration, molecular weight, and degree of entanglement among the mucin polymers. The term "sputum" generally refers to a mixture of saliva and discharges (including mucus) from the respiratory tract. Sputum is typically a coughed mixture of saliva and mucus (and other discharges from respiratory tract tissues). Thus, mucus is a major component of sputum, and as such, the presence of excessively viscous mucus results in sputum that is itself excessively viscous. The present invention relates to reducing the viscosity of mucus or sputum. The term "liquefaction" refers to the act of becoming more fluid. Thus, an increase in liquefaction of mucus or sputum refers to an increase in the liquid phase or liquid state of mucus or sputum as compared to a more solid or viscous phase. In the case of abnormally viscous or excessive mucus associated with disease, the goal is to restore normal levels of mucus viscosity. Thus, liquefaction can also be viewed as a decrease in mucus viscosity.

[0049] It is understood that normal mucus function is achieved by having the proper ratio of bioreductant to oxidizable cysteine. Thus, insufficient bioreductant activity is therefore caused by either excess oxidizable cysteine or lack of bioreductant.

[0050] The general function of mucus and sputum in the body requires that the mucus (and thus the mucus component of sputum) have viscoelastic properties. In individuals with normal mucus and sputum (i.e., healthy individuals, or more specifically, individuals not suffering from a symptom or condition caused or exacerbated by the viscosity or stickiness of mucus or sputum), the viscoelasticity depends on the concentration, molecular weight, and entanglement between mucin polymers (Verdugo et al., Biorheology 20:223-230, 1983). In CF, in particular, when mucin in mucus interacts with DNA released from dead inflammatory cells (Potter et al., Am J Dis Child 100:493-495, 1960; Lethem et al., Am Rev Respir Dis 100:493-495, 1990; Lethem et al., Eur Respir J 3:19-23, 1990) and f-actin polymers (Sheils et al., Am J Path 148:919-927, 1996; Tomkiewicz et al., DNA and actin filament ultrastructure in cystic fibrosis sputum, in Cilia, Mucus, and Mucociliary Interactions, edited by Baum GL, Priel Z, Roth Y, Liron N, and Ostfeld EJ. New York, NY: Marcel Dekker, 1998), the mucus (and thus sputum) can additionally become more dense and viscous. Abnormal, thickened mucus cannot be cleared by coughing or mucociliary clearance, which promotes colonization of the lungs by opportunistic pathogens. Thus, mucus that is abnormal or excessively viscous and / or sticky is characterized by being measurably or detectably more viscous or sticky than mucus from a normal or healthy patient (preferably an age- and gender-matched patient), and / or by causing or contributing to at least one symptom in the patient that causes the patient discomfort or pain, or that causes or exacerbates a condition or disease, due to its level of viscosity and / or stickiness. In other words, sputum that is abnormal or excessively viscous and / or sticky deviates from normal mucus or sputum, where it is desirable to treat the patient to provide some relief of the condition or other therapeutic benefit.

[0051] The methods and compositions of the present application can be used to treat any patient in whom it is desirable to reduce the viscosity of mucus or sputum. In particular, patients having specific lung, sinus, nasal, digestive tract, or gastrointestinal or reproductive tract diseases or conditions can benefit from treatment using the methods of the present application. The present application is most useful in ameliorating or reducing at least one symptom of a condition or disease caused or exacerbated by abnormal or excessive viscosity and / or stickiness of mucus or sputum, which can of course include lung-related diseases such as cystic fibrosis, as well as digestive tract diseases such as coccidiosis. Other diseases can be related to abnormal or excessive viscosity and / or stickiness of mucus or sputum at least some of the time, and when such symptoms arise, the methods of the present application can be used to reduce the viscosity of 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 bronchitis (bacterial, viral, mycoplasmal, or caused by other organisms); acute or chronic sinusitis; atelectasis (collapse of a lung or lobe of a lung) due to acute or chronic mucus plugging of the airways (sometimes seen in a variety of diseases such as asthma); bronchiolitis (viral or other); acute, subacute, or chronic intestinal obstruction due to mucus thickening, including (but not limited to) meconium ileus or meconium ileus equivalent in CF or similar conditions; other diseases of the digestive tract and infertility due to (but not limited to) blockage of the cervix, vas deferens, or other important reproductive structures. In addition, because improved mucus ciliary clearance is associated with clearance of bacteria and other pathogens from the lungs, the compositions and methods of the present application can be used to reduce symptoms associated with excessive viscosity and / or stickiness of mucus or sputum in patients having a variety of respiratory tract infections, including viral and bacterial infections.

[0052] Thus, the therapeutic benefit is not necessarily a cure of a particular disease or condition, but preferably encompasses results that most commonly include: reduction of the disease or condition, elimination of the disease or condition, reduction or elimination of symptoms associated with the disease or condition, prevention or reduction of secondary diseases or conditions arising from the presence of the primary disease or condition (e.g., infectious diseases caused by opportunistic pathogenic microorganisms that take advantage of the excess viscous mucus in the respiratory tract), and / or prevention of the underlying disease or condition or symptoms associated with the disease or condition. As used herein, the phrase "protecting against a disease" means reducing symptoms of a disease; palliative therapy (alleviating or easing symptoms of a disease without affecting a cure); reducing the occurrence of a disease, and / or reducing the severity of a disease or alleviating at least one symptom, sign, or cause of a disease or condition. Prevention refers to the ability of the compositions of the present application to prevent the occurrence of a disease when administered to a patient. Cure (or disease modulation) refers to the ability of the compositions of the present application to cure a disease when administered to a patient. Treating a patient to protect against a disease includes treating a patient who has the disease (therapeutic treatment). Preventing a disease / condition includes preventing the disease from occurring (prophylactic treatment). In particular, protecting a patient against a disease (or preventing a disease) is accomplished by contacting mucus or sputum with a protein or peptide comprising a reduced sulfhydryl protein mono-cysteine active site to increase (standardize) the liquefaction of abnormally viscous mucus or sputum in a patient in order to obtain a beneficial effect. A beneficial effect can be readily assessed by one of ordinary skill in the art and / or a trained clinician treating a patient. The term "disease" refers to any deviation from the normal health of a patient and includes a state when symptoms of a disease are present, as well as conditions in which a deviation has occurred (e.g., infection, genetic mutation, genetic defect, etc.), but symptoms have not yet manifested.

[0053] Contact of a patient's mucus and / or sputum with a protein or peptide comprising a reduced thioredoxin monomercysteine active site (or a composition comprising such a protein) is expected to result in a decrease in the viscosity / increase in liquefaction of the mucus or sputum compared to prior to contact with the composition. According to the present application, an increase in liquefaction of the mucus or sputum can be any measurable or detectable increase in the level of mucus or sputum liquefaction compared to a previous level of liquefaction, and is preferably a statistically significant increase (i.e., the difference in measured level of liquefaction between the patient sample and a baseline control is statistically significant with a confidence level of at least p<0.05). Typically, a "baseline control" is a patient sample prior to administration of the treatment, as normal, healthy individuals typically do not produce sputum in quantities sufficient to serve as a control, but sputum from a normal, healthy individual is not excluded as a baseline control. Additionally, a decrease in viscosity results in an improvement in lung function. Such an improvement can be determined in various ways, including patient reported outcomes, mean time to hospitalization for exacerbation, and / or forced expiratory volume (FEV) increase. In one aspect of the present application, the FEV increase 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 from the patient prior to contact with the composition or protein of the present application. Preferably, contact of the protein or composition of the present application with the mucus or sputum of a patient sample results in an increase of about 2.5% compared to a sample from the patient prior to contact with the composition or protein of the present application. Liquefaction and / or decrease in viscosity of mucus or sputum can be measured using any suitable technique known in the art, including (but not limited to) the compression assay as described in the Examples section. In such an assay, the amount of mucus or sputum in the solid phase (gel) relative to the aqueous phase (liquid) is measured. In other aspects of the present application, other parameters or indicators can be used to measure the relative viscosity or stickiness of mucus or sputum, including (but not limited to) viscoelasticity (e.g., measured by magnetic micro-rheology), glycoprotein content, or DNA content. In another aspect of the present application, changes in mucus protein disulfide bonding can be assessed by using a reagent such as NEM (N-ethylmaleimide), which reacts preferentially with the thiol groups of unbound (free) Cys residues generated by disruption of disulfide bonds (Rancourt, R. et al., Free Radic Biol Med, 42(9): 1441-1453, 2007). In one aspect of the present application, the level of liquefaction is described as the amount of a given mucus or sputum sample 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, contacting the protein or composition of the application with mucus or sputum results in a liquefaction change in the mucus or sputum such that at least about 15% of the total volume is in a liquid phase, and more preferably at least about 20% of the total volume is in a liquid phase, and more preferably at least about 25% of the total volume is in a liquid phase, and more preferably at least about 30% of the total volume is in a liquid phase, and more preferably at least about 35% of the total volume is in a liquid phase, and more preferably at least about 40% of the total volume is in a liquid phase, and more preferably at least about 45% of the total volume is in a liquid phase, and more preferably at least about 50% of the total volume is in a liquid phase, or until the blockage or inhibition of function caused by the mucus has been cleared (e.g., until the patient's airway is sufficiently cleared to begin expectoration of sputum). Generally, it is preferred that the liquefaction of sputum or mucus be increased in small incremental steps until the airway or other obstructed passageway (e.g., in the gastrointestinal tract or reproductive tract) is cleared, but that the sputum not be overly liquefied. Over-liquefaction of mucus or sputum is undesirable because it can be harmful to the patient (e.g., the liquefied sputum can flow back and the thin liquid can flood the small airways, which can also be infected, and then the sputum can be cleared by the patient). Preferably, the protein, peptide, or composition of the application, when contacted with mucus or sputum, produces at least about a 1% increase (by volume) in the liquefaction of the mucus or sputum compared to before treatment, more preferably at least about a 2% increase, and so on (in increments of 1%), until the patient's airway or other obstructed passageway is cleared. Once such clearance is achieved, e.g., by removing the so-called "mucus plug" to improve drug access to the small airways and alveoli, a lower dose maintenance therapy can then be performed to keep newly secreted mucin protein in a normal state of disulfide bonding.

[0054] In one aspect, the method is used in conjunction with therapy to clear the dilute material from the affected tissue (respiratory tract, digestive tract, reproductive tract) of the patient. For example, in the case of the respiratory system, the method of the application can be used in conjunction with postural drainage, forceful coughing, and other respiratory exercises or any other suitable method for coughing up liquefied mucus or sputum.

[0055] According to the present application, mucus or sputum in a patient to be treated is contacted with a protein containing a single cysteine active site in a reduced state (or a composition comprising the protein). The protein can be effective to reduce the viscosity and stickiness of sputum or mucus and / or increase the liquefaction of sputum or mucus as compared to prior to the contacting step. As previously noted, thioredoxin is a protein disulfide reductase found in most organisms that is involved in many thiol-dependent cellular reductive processes. In humans, thioredoxin is also known as adult T-cell leukemia-derived factor (ADF). Within the cell, most of this ubiquitous low molecular weight (11,700) protein remains reduced. Reduced or oxidized thioredoxin can be able to enter intact cells or be absorbed to cell membranes, where small amounts are gradually internalized over time. Native thioredoxin has two vicinal cysteine residues in the active site that form a disulfide bridge in oxidized protein that is located in a protrusion from the three-dimensional structure of the protein. Flavoprotein thioredoxin reductase catalyzes the NADPH-dependent reduction of this disulfide bond. In addition, a designed version of thioredoxin reductase modified for altered co-factor specificity can utilize NADH or in addition to NADPH, as described in U.S. Patent 7,071,307, which is hereby incorporated by reference. Small increases in thioredoxin can cause profound changes in the mercapto-disulfide redox state of proteins.

[0056] In addition to its ability to effect reduction of cellular proteins, thioredoxin is recognized to act directly as an antioxidant (e.g., by preventing oxidation of oxidizable substrates via scavenging of reactive oxygen species), but unlike other thiols, thioredoxin generally does not generate oxidative stress in cells through autoxidation (e.g., production of superoxide radicals through autoxidation). U.S. Patent No. 5,985,261 to White et al. (supra) indicates that thioredoxin directly induces the production of MnSOD and that this induction is effected by thioredoxin in a reduced state.

[0057] The "thioredoxin single cysteine active site" of the present invention comprises the amino acid sequence C-X-X-X (SEQ ID NO: 17) (the native or wild type sequence comprises the amino acid sequence C-X-X-C, having SEQ ID NO: 16). As used herein, the amino acid residue denoted as "C" is a cysteine residue and the amino acid residues denoted as "X" can be any amino acid residue other than a cysteine residue, and in particular any one of the remaining standard 20 amino acid residues. Such thioredoxin single cysteine active site of the present invention preferably comprises the amino acid sequence C-G-P-X (SEQ ID NO: 18), wherein the native or wild type sequence comprises the amino acid sequence C-G-P-C (SEQ ID NO: 1). The thioredoxin single cysteine active site can also comprise the amino acid sequence X-C-X-X-X-X (SEQ ID NO: 19), wherein the native or wild type sequence comprises the amino acid sequence X-C-X-X-C-X (SEQ ID NO: 20). Preferably, the thioredoxin single cysteine active site of the present invention comprises the amino acid sequence X-C-G-P-X-X (SEQ ID NO: 21), wherein such amino acid residue denoted as "G" is a glycine residue, and wherein such amino acid residue denoted as "P" is a proline residue, wherein the native or wild type sequence comprises the amino acid sequence X-C-G-P-C-X (SEQ ID NO: 22). More preferably, the thioredoxin single cysteine active site of the present invention comprises the amino acid sequence W-C-G-P-X-K (SEQ ID NO: 23), wherein such amino acid residue denoted as "W" is a tryptophan residue, and wherein such amino acid residue denoted as "K" is a lysine residue and wherein the native sequence comprises the amino acid sequence W-C-G-P-C-K (SEQ ID NO: 3). Preferably, the thioredoxin single cysteine active site can comprise the amino acid sequence C-X-X-S (SEQ ID NO: 24). Such thioredoxin single cysteine active site of the present invention preferably comprises the amino acid sequence C-G-P-S (SEQ ID NO: 1). The thioredoxin single cysteine active site can also comprise the amino acid sequence X-C-X-X-S-X (SEQ ID NO: 25), X-C-G-P-S-X (SEQ ID NO: 26) or W-C-G-P-S-K (SEQ ID NO: 27), wherein the amino acid residues denoted as "X" can be any amino acid residue other than a cysteine residue. Reference to "thioredoxin active site" includes both the thioredoxin single cysteine active site and the native or wild type thioredoxin active site.

[0058] In one aspect of the application, the protein containing a thioredoxin single cysteine active site is a full-length thioredoxin or any fragment thereof containing a thioredoxin single cysteine active site as described structurally and functionally above. Preferred thioredoxins having a single cysteine active site include prokaryotic thioredoxins, yeast thioredoxins, plant thioredoxins, and mammalian thioredoxins, with human thioredoxin being particularly preferred. Nucleic acid and amino acid sequences for thioredoxins from a variety of organisms are well known in the art and are contemplated as being encompassed by the present application. For example, SEQ ID NOs:4-15 represent the amino acid sequences for 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 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). With reference to each of these sequences, the X-C-G-P-C-X (SEQ ID NO: 22) motif, which includes the CGPC motif of SEQ ID NO: 1, can be seen as follows: SEQ ID NO: 4 (positions 33-38), SEQ ID NO: 5 (positions 28-33), SEQ ID NO: 6 (positions 27-32), SEQ ID NO: 7 (positions 29-34), SEQ ID NO: 8 (positions 31-36), SEQ ID NO: 9 (positions 31-36), SEQ ID NO: 10 (positions 31-36), SEQ ID NO: 11 (positions 31-36), SEQ ID NO: 12 (positions 31-36), SEQ ID NO: 13 (positions 59-64), SEQ ID NO: 14 (positions 88-93), and SEQ ID NO: 15 (positions 94-99).In addition, the three-dimensional structure of several thioredoxins has been resolved, including human and bacterial thioredoxins. Thus, the structure and active site of thioredoxins from a variety of organisms are well known in the art and one of skill in the art would be able to readily identify and produce fragments or homologues of full-length thioredoxins, including thioredoxins having a single cysteine active site, useful in the present application.

[0059] The phrase "in a reduced state" specifically describes the state of the cysteine residues in the active site of the proteins or peptides of the present application. In the reduced state, adjacent cysteine residues form a dithiol (i.e., two free thiol groups, -SH). In contrast, in the oxidized form, these cysteine residues form an intramolecular disulfide bridge; such a molecule can be referred to as a cystine. In the reduced state, a single cysteine thioredoxin active site is capable of participating in redox reactions by virtue of its active site thiol's reversible oxidation to a disulfide bond, and catalyzes thiol-disulfide exchange reactions, which result in covalent linkage to one of the target disulfide Cys. For the proteins or peptides of the present application containing a thioredoxin single cysteine active site, the N-terminal cysteine in the active site is in the reduced state as a monothiol and is thus capable of forming a stable mixed disulfide bond with a cysteine on the target protein.

[0060] As used herein, a protein of the present invention containing a thioredoxin monocysteine active site can be the thioredoxin monocysteine active site itself or a thioredoxin monocysteine active site bound to other amino acids through a glycosidic bond. Thus, the minimum size of a protein or peptide of the present invention is from about 4 to about 6 amino acids in length, with the preferred size depending on whether the full length, fusion, multivalent, or just the functional portion of such a protein is desired. Preferably, the protein or peptide of the present invention is from about 4 to about 100 amino acid residues in length or more, with any intermediate length of peptide in whole numbers (i.e., 4, 5, 6, 7...99, 100, 101) specifically contemplated. It can also be a short thioredoxin mimetic peptide that is capped at the N- and C-termini, as described by Bachnoff et al., Free Radical Biol Med 50: 1355-67, 2011. In another preferred embodiment, the protein of the present invention can be a full length protein or any homolog of such a protein. As used herein, the term "homolog" is used to refer to a protein or peptide that differs from a naturally occurring protein or peptide (i.e., a "prototype" or "wild type" protein) by modifications made to the naturally occurring protein or peptide, but which retains the essential protein and side chain structure of the naturally occurring form, and / or which retains the essential three-dimensional structure of at least the biologically active portion (e.g., the thioredoxin active site) of the native protein. These alterations include, but are not limited to: changes in one or several amino acid side chains; changes in one or several amino acids, including deletions (e.g., truncated forms (fragments) of the protein or peptide), insertions, and / or substitutions; changes in the stereochemistry of one or several atoms; and / or slight derivatization, including but not limited to: methylation, glycosylation, phosphorylation, acetylation, myristoylation, isoprenylation, palmitoylation, amidation, and / or addition of glycosyl phosphatidylinositol. According to the present invention, any protein or peptide useful in the present invention (including homologs of native thioredoxins) has a thioredoxin monocysteine active site such that, in the reduced state, the protein or peptide is capable of participating in redox reactions by oxidizing its active site thiol to a disulfide bond and / or is capable of reducing the viscosity or stickiness or increasing the liquefaction of mucus or sputum. As used herein, a protein or peptide containing a thioredoxin monocysteine active site can have similar properties to thioredoxins and is preferably a thioredoxin selected from prokaryotic thioredoxins, fungal thioredoxins (including yeast), plant thioredoxins, or mammalian thioredoxins. In a particularly preferred embodiment, the protein is a human thioredoxin.

[0061] A homolog can be the result of natural allelic variations or natural mutations. A naturally occurring allelic variant of a nucleic acid encoding a protein is a gene present in the genome at a substantially identical locus (or loci) to the gene encoding such protein, but which has a similar, but not identical sequence due to natural changes caused by, for example, mutation or recombination. Allelic variants generally encode proteins having similar activities to the protein encoded by the gene with which they are compared. One class of allelic variants can encode the same protein, but have different nucleic acid sequences due to the degeneracy of the genetic code. Allelic variants can also include changes in the 5' or 3' untranslated regions (e.g., in regulatory control regions). Allelic variants are well known to those skilled in the art.

[0062] A homolog can be produced using techniques known in the art for making proteins, including, but not limited to, direct modification of an isolated naturally occurring protein, direct protein synthesis, or modification of a nucleic acid sequence encoding a protein using, for example, classical or recombinant DNA techniques to effect random or targeted mutagenesis.

[0063] Modifications in a homolog, as compared to a wild-type protein, agonize, antagonize, or do not substantially alter the essential biological activity of the homolog as compared to the naturally occurring protein. In general, a biological activity or biological effect of a protein refers to any function exhibited or performed by a protein as a result of its naturally occurring form, as measured or observed in vivo (i.e., in the protein's natural physiological environment) or in vitro (i.e., under laboratory conditions). Modifications of a protein, for example, in a homolog or mimetic (discussed below), can result in a protein having the same biological activity as compared to the naturally occurring protein, or a protein having decreased or increased biological activity as compared to the naturally occurring protein. Modifications that result in decreased expression of a protein or decreased activity of a protein can be referred to as inactivation (complete or partial), down-regulation, or reduction of the protein. Similarly, modifications that result in increased expression of a protein or increased activity of a protein can be referred to as amplification, overproduction, activation, enhancement, up-regulation, or increase of the protein.

[0064] In one embodiment, proteins or peptides containing a thioredoxin single cysteine active site can be the product of drug design or selection and can be produced using various methods known in the art. These proteins or peptides can be referred to as mimetics. Mimetics refer to any peptide or non-peptide compound that is capable of mimicking the biological effects of a naturally occurring peptide, usually because the mimetic has a basic structure that mimics the basic structure of the naturally occurring peptide and / or has a significant biological property of the naturally occurring peptide. Mimetics can include, but are not limited to, peptides with substantial modifications compared to the prototype, such as peptides that do not have side chain similarity to the naturally occurring peptide (these modifications can, for example, reduce their susceptibility to degradation); anti-idiotypes and / or catalytic antibodies, or fragments thereof; non-protein portions of isolated proteins (e.g., carbohydrate structures); or synthetic or naturally occurring organic molecules, including, for example, nucleic acids and drugs identified via combinatorial chemistry. These mimetics can be designed, selected, and / or otherwise identified using a variety of methods known in the art. Various drug design methods that can be used to design or select mimetics or other therapeutic compounds useful in the present application 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 effecting efficient and selective redox activity are described by Bachnoff et al., Free Radical Biol Med 50: 1355-67 (2011) and incorporated herein by reference in its entirety.

[0065] Mimetics can be obtained, for example, from molecular diversity strategies (combinations of related strategies that allow rapid construction of large libraries of chemically diverse molecules), libraries of natural or synthetic compounds, particularly from chemical or combinatorial libraries (i.e., libraries of compounds that differ in sequence or size but have similar structural elements) or by rational, directed, or random drug design. See, e.g., Maulik et al., supra.

[0066] In molecular diversity strategies, large libraries of compounds are synthesized using biological, enzymatic, and / or chemical methods, for example, from peptides, oligonucleotides, carbohydrates, and / or synthetic organic molecules. Key parameters in developing molecular diversity strategies include subunit diversity, molecular size, and library diversity. The general goal in screening these libraries is to use sequential applications of combinatorial selection to obtain high affinity ligands for desired targets, followed by optimization of lead molecules through random or directed design strategies. Methods of molecular diversity are described in detail in Maulik et al., supra.

[0067] Maulik et al. also disclose, for example, directed design methods, in which the user guides the generation of novel molecules from a library of fragments of appropriately selected fragments; random design, in which the user uses genetic or other algorithms to randomly mutate fragments and their combinations, while applying selection criteria to evaluate the fitness of candidate ligands; and grid-based methods, in which the user calculates the interaction energies between a three-dimensional receptor structure and small fragment probes, followed by joining favorable probe sites.

[0068] Diversity generation methods such as the foregoing can be combined with other techniques designed to improve function or pharmacology, especially for molecules of reduced size such as active site mimetics. For example, one approach that has shown promise in early studies is hydrocarbon stapled alpha helical peptides, a novel class of synthetic mini-proteins that are locked in their biologically active alpha-helical fold by site-specific introduction of a chemical scaffold (full hydrocarbon stapling). Stapling can greatly improve the pharmacological properties of peptides, increasing their target affinity and proteolytic resistance, while creating a smaller peptide version of a larger protein / enzyme that is amenable to chemical synthesis (Verdine, G. L. and Hilinsky, G. J., Methods Enzymol, 503:3-33, 2012).

[0069] In one embodiment of the application, the proteins suitable for use in the application have an amino acid sequence comprising, consisting essentially of, or consisting of the full-length sequence of a thioredoxin or any fragment thereof having a thioredoxin single-cysteine active site as described herein. For example, the application encompasses any of the natural sequences of SEQ ID NOs 4-15 or fragments or other homologues thereof containing a thioredoxin single-cysteine active site as described herein. These homologues 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, 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, including any percentage between 10% and 100% in whole numbers (10%, 11%, 12%,... 98%, 99%, 100%).

[0070] As used herein, unless otherwise stated, references to the percentage of identity (%) refer to homology assessments performed using the following: (1) BLAST 2.0 Basic BLAST homology search performed using blastp for amino acid search and blastn for nucleic acid search with standard default parameters, where query sequences are filtered for low-complexity regions by default (described in Altschul, SF, Madden, TL, AA, Zhang, J., Zhang, Z., Miller, W. and Lipman, DJ (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; (2) BLAST 2 alignment (using the parameters described below); (3) and / or PSI-BLAST (position-specific iterative BLAST) with standard default parameters. It should be noted that due to some differences in the standard parameters between BLAST 2.0 BasicBLAST and BLAST 2, two specific sequences may be identified as having significant homology using the BLAST 2 program, while a search in BLAST 2.0 Basic BLAST using one of the sequences as the query sequence may not identify the second sequence in the top match. In addition, PSI-BLAST provides an automated, easy-to-use form of "profile" search, which is a sensitive way to find sequence homologs. The procedure begins with a gap BLAST database search. The PSI-BLAST procedure uses information from any significant alignments returned to construct a position-specific score matrix, which replaces the query sequence for the next round of database search. Therefore, it should be understood that any of these procedures can be used to determine the percentage of identity.

[0071] BLAST 2 sequences can be used to align two specific sequences to each other 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, incorporated herein by reference in its entirety. BLAST 2 sequence alignments are performed using the BLAST 2.0 algorithm in blastp or blastn to perform a gapped BLAST search (BLAST 2.0) between two sequences, allowing for the introduction of gaps (deletions and insertions) in the resulting alignment. For clarity herein, BLAST 2 sequence alignments are performed using the following standard default parameters.

[0072] For blastn, use 0 BLOSUM62 matrix:

[0073] Match reward = 1

[0074] Mismatch penalty = -2

[0075] Open gap (5) and extend gap (2) penalties

[0076] Gap x_dropoff (50) It is contemplated (10) word size (11) filter (on)

[0077] For blastp, use 0 BLOSUM62 matrix:

[0078] Open gap (11) and extend gap (1) penalties

[0079] Gap x_dropoff (50) It is contemplated (10) word size (3) filter (on).

[0080] Proteins useful in the present application can also include proteins having an amino acid sequence comprising at least 10 contiguous amino acid residues of any full-length thioredoxin containing a single cysteine active site (10 contiguous amino acid residues of the native sequence represented by SEQ ID NOs: 4-15, i.e., 10 contiguous amino acid residues having 100% identity to 10 contiguous amino acids of the reference sequence). In other embodiments, homologs of thioredoxin include an amino acid sequence comprising at least 15, or at least 20, or at least 25, or at least 30, or at least 35, or at least 40, or at least 45, or at least 50, or at least 55, or at least 60, or at least 65, or at least 70, or at least 75, or at least 80 contiguous amino acid residues, and so on up to the full length of the protein, of the amino acid sequence of a naturally occurring thioredoxin (including any intermediate lengths in whole numbers (10, 11, 12,...)) and which contains a single cysteine active site.

[0081] According to the present application, the term "contiguous" or "adjacent" with respect to sequences described herein means connected in an uninterrupted sequence. For example, for a first sequence comprising 30 contiguous (or adjacent) amino acids of a second sequence, means that the first sequence includes an uninterrupted sequence of 30 amino acid residues having 100% identity to an uninterrupted sequence of 30 amino acid residues in the second sequence. Similarly, for a first sequence having "100% identity" to a second sequence, means that the first sequence matches the second sequence exactly, with no gaps between nucleotides or amino acids.

[0082] In another embodiment, proteins useful in the present application include proteins having an amino acid sequence sufficiently similar to a native thioredoxin amino acid sequence, wherein a nucleic acid sequence encoding the homolog is capable of hybridizing to (i.e., and) a nucleic acid molecule encoding a native thioredoxin (i.e., to the complement of a nucleic acid strand encoding a native thioredoxin amino acid sequence) under conditions of medium, high, or very high stringency (described below). These hybridization conditions are described in detail below.

[0083] A nucleic acid sequence complementary to a nucleic acid sequence encoding a thioredoxin of the present application refers to a nucleic acid sequence of a nucleic acid strand complementary to a strand encoding a thioredoxin. It is understood that a double-stranded DNA encoding a given amino acid sequence includes single-stranded DNA and its complementary strand having a sequence complementary to the single-stranded DNA. Thus, nucleic acid molecules of the present application can be double-stranded or single-stranded, and include those nucleic acid molecules that form stable hybridizes under stringent hybridization conditions to a nucleic acid sequence encoding a thioredoxin amino acid sequence and / or to the complement of a nucleic acid sequence encoding such an amino acid sequence. Methods of inferring complementary sequences are known to those skilled in the art.

[0084] As used herein, reference to hybridization conditions means standard hybridization conditions using nucleic acid molecules to identify similar nucleic acid molecules. These standard conditions are disclosed, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, 1989. Sambrook et al., supra, is incorporated herein by reference in its entirety (see in particular pages 9.31-9.62). In addition, formulas to calculate appropriate hybridization and wash conditions to achieve hybridization allowing for varying degrees of nucleotide mismatch are disclosed, for example, in Meinkoth et al., 1984, Anal. Biochem. 138, 267-284; Meinkoth et al., supra, is incorporated herein by reference in its entirety.

[0085] More specifically, moderately stringent hybridization and wash conditions as referred to herein mean conditions that allow the isolation of nucleic acid molecules having at least about 70% nucleic acid sequence identity to the nucleic acid molecules used to probe the hybridization reaction (i.e., conditions that allow for about 30% or less nucleotide mismatch). Highly stringent hybridization and wash conditions as referred to herein mean conditions that allow the isolation of nucleic acid molecules having at least about 80% nucleic acid sequence identity to the nucleic acid molecules used to probe the hybridization reaction (i.e., conditions that allow for about 20% or less nucleotide mismatch). Very high stringent hybridization and wash conditions as referred to herein mean conditions that allow the isolation of nucleic acid molecules having at least about 90% nucleic acid sequence identity to the nucleic acid molecules used to probe the hybridization reaction (i.e., conditions that allow for about 10% or less nucleotide mismatch). As discussed above, one skilled in the art can use the formulas in Meinkoth et al., supra, to calculate appropriate hybridization and wash conditions to achieve these particular levels of nucleotide mismatch. These conditions will vary depending on whether a DNA:RNA or DNA:DNA hybrid is formed. The calculated melting temperature for a DNA:DNA hybrid is 10°C lower than for a DNA:RNA hybrid. In particular embodiments, stringent hybridization conditions for DNA:DNA hybrids include hybridization at an ionic strength of 6X SSC (0.9 M Na + ) at temperatures of about 20°C to about 35°C (lower stringency), more preferably about 28°C to about 40°C (more stringent), and even more preferably about 35°C to about 45°C (even more stringent), under appropriate wash conditions. In particular embodiments, stringent hybridization conditions for DNA:RNA hybrids include hybridization at temperatures of about 30°C to about 45°C, more preferably about 38°C to about 50°C, and even more preferably about 45°C to about 55°C, under similarly stringent wash conditions. +hybridization. These values are calculated based on the melting temperature of a molecule greater than about 100 nucleotides, 0% formamide, and a G+C content of about 40%. Alternatively, Tm can be empirically calculated as described in Sambrook et al., supra, pages 9.31-9.62. m In general, the wash conditions should be as stringent as possible and should be appropriate for the selected hybridization conditions. For example, the hybridization conditions can include a salt at a temperature about 20-25°C below the calculated Tm of the particular hybrid, and the wash conditions typically include a salt at a temperature about 12-20°C below the calculated Tm of the particular hybrid. m In general, the wash conditions should be as stringent as possible and should be appropriate for the selected hybridization conditions. For example, the hybridization conditions can include a salt at a temperature about 20-25°C below the calculated Tm of the particular hybrid, and the wash conditions typically include a salt at a temperature about 12-20°C below the calculated Tm of the particular hybrid. m One example of hybridization conditions suitable for a DNA:DNA hybrid includes hybridization at about 42°C in 6X SSC (50% formamide) for 2-24 hours, followed by wash steps including one or more washes in about 2X SSC at room temperature, followed by additional washes at higher temperatures and lower ionic strength (e.g., at least one wash in about 0.1X-0.5X SSC at about 37°C, followed by at least one wash in about 0.1X-0.5X SSC at about 68°C).

[0086] The proteins of the present application can also be fusion proteins that include a segment containing a thioredoxin monokanthiol active site and a fusion segment that can have a variety of functions. For example, such a fusion segment can serve as a tool to simplify purification of the proteins of the present application, e.g., to enable purification of the resulting fusion protein using affinity chromatography. Suitable fusion segments can be domains of any size that have a desired function (e.g., to confer increased stability to the protein, to confer increased immunogenicity to the protein, and / or to simplify purification of the protein). The use of one or more fusion segments is within the scope of the present application. The fusion region can be bound to the amino and / or carboxy terminus of the segment containing the thioredoxin monokanthiol active site. The linkage between the fusion region of the fusion protein and the thioredoxin active site-containing domain can be readily cleaved to enable direct recovery of the thioredoxin monokanthiol active site domain of these proteins. Preferably, the fusion proteins are produced by culturing recombinant cells transformed with a fusion nucleic acid molecule that encodes a protein that includes a fusion segment linked to the carboxy and / or amino terminus of a thioredoxin monokanthiol active site domain.

[0087] In one embodiment, the thioredoxin monokanthiol active site-containing proteins or peptides suitable for use in the methods of the present application comprise a thioredoxin monokanthiol active site-containing protein or peptide derived from an animal species substantially similar to the animal species of the subject to which the protein is administered. In another embodiment, any thioredoxin monokanthiol active site-containing protein or peptide from a different source, such as a microorganism, plant, and fungus, can be used in a given patient.

[0088] In one embodiment of the application, any of the amino acid sequences described herein, e.g., a naturally occurring thioredoxin or thioredoxin amino acid sequence containing a single cysteine active site, can be produced in which at least one and up to about 20 additional heterologous amino acids flank each C-terminus and / or N-terminus of the specified amino acid sequence. The resulting protein or polypeptide can be referred to as "consisting essentially of the specified amino acid sequence." According to the application, the heterologous amino acids are amino acid sequences that are not naturally found (i.e., not found in nature, in vivo) flanking the specified amino acid sequence, or that are not functionally relevant to the specified amino acid sequence, or that are not encoded by nucleotides flanking the naturally occurring nucleic acid sequence encoding the specified amino acid sequence when such nucleotides in the naturally occurring sequence are translated using the standard codon usage for the organism from which the given amino acid sequence is obtained, when present in a gene. Similarly, the phrase "consisting essentially of when used with respect to a nucleic acid sequence herein refers to a nucleic acid sequence encoding the specified amino acid sequence that can be flanked at each of the 5' and / or 3' end of the nucleic acid sequence encoding the specified amino acid sequence by at least one and up to as many as about 60 additional heterologous nucleotides. The heterologous nucleotides are not naturally found (i.e., not found in nature, in vivo) flanking the nucleic acid sequence encoding the specified amino acid sequence when present in a naturally occurring gene, or do not encode a protein that confers any additional function to the protein, or alter the function of the protein having the specified amino acid sequence.

[0089] In another embodiment, the thioredoxin single cysteine active site containing protein or peptide suitable for use in the methods of the application comprises an isolated or biologically pure protein. Thus, "isolated" and "biologically pure" do not necessarily reflect the extent to which the protein has been purified. Isolated proteins of the application can be obtained, for example, from their natural source, produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning), or chemically synthesized.

[0090] In another embodiment, chemically synthesized proteins or peptides of the present application containing a thioredoxin monocysteine active site can also refer to stabilized forms, such as proteins or peptides containing the active site constrained structurally by a stapled peptide technology, by cyclization constraints, or by constraints at the N- or C-terminus. Preferably, the proteins containing a thioredoxin monocysteine active site to be used in the methods of the present application have an in vivo half-life sufficient to cause a measurable or detectable increase in mucus or sputum liquefaction (or decrease in viscosity or stickiness) in a patient, and or cause a measurable, detectable or perceived patient treatment benefit associated with mucus and sputum in a patient. Such a half-life can be achieved by the method of delivery of such a protein. The proteins of the present application preferably have a half-life in an animal of greater than about 5 minutes, and more preferably greater than about 4 hours in an animal and even more preferably greater than about 16 hours in an animal. In a preferred embodiment, the proteins of the present application have a half-life in an animal of from about 5 minutes to about 24 hours, and preferably from about 2 hours to about 16 hours in an animal and more preferably from about 4 hours to about 12 hours in an animal.

[0091] Other embodiments of the present application include nucleic acid molecules encoding proteins or peptides containing a thioredoxin monocysteine active site. These nucleic acid molecules can be used to produce proteins that can be used in the methods of the present application, either in vitro or in vivo. Nucleic acid molecules of the present application include nucleic acid molecules comprising, consisting essentially of, or consisting of a nucleic acid sequence encoding any of the proteins described previously herein. In accordance with the present application, an isolated nucleic acid molecule is a nucleic acid molecule (polynucleotide) that has been removed from its natural environment (i.e., has been manipulated by man) and can include DNA, RNA, or a derivative of DNA or RNA, including cDNA. Thus, "isolated" does not reflect the extent to which the nucleic acid molecule has been purified. While the phrase "nucleic acid molecule" refers primarily to a physical nucleic acid molecule and the phrase "nucleic acid sequence" refers primarily to a sequence of nucleotides on a nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule or a nucleic acid sequence capable of encoding a protein. Isolated nucleic acid molecules of the present application can be isolated from their natural source 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, a gene, a natural allelic variant of the gene, a coding region or portion thereof, and coding and / or regulatory regions modified by nucleotide insertions, deletions, substitutions and / or inversions in such a way that the modifications do not substantially interfere with the ability of the nucleic acid molecule to encode a desired protein of the present application or to form stable hybridization with a naturally occurring gene under stringent conditions. Isolated nucleic acid molecules can include degeneracy. As used herein, nucleotide degeneracy refers to the phenomenon that one amino acid can be encoded by different nucleotide codons. Thus, the nucleic acid sequence of a nucleic acid molecule encoding a given protein useful in the present application can vary due to degeneracy.

[0092] According to the present application, reference to a gene includes all nucleic acid sequences associated with a natural (i.e., wild-type) gene and those associated with a thioredoxin monokanthiol active site, such as regulatory regions (e.g., but not limited to, transcriptional, translational, or post-translational control regions) that control the production of a protein encoded by the gene, as well as the coding regions thereof. In another embodiment, the gene can be a naturally occurring allelic variant, which includes sequences similar to, but not identical to, the nucleic acid sequence encoding a given protein. Allelic variants have been previously described above. The phrases "nucleic acid molecule" and "gene" can be used interchangeably when the nucleic acid molecule comprises a gene as described above.

[0093] Preferably, the isolated nucleic acid molecules of the present application are produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis. The isolated nucleic acid molecules include natural nucleic acid molecules and homologues thereof, including, but not limited to, natural allelic variants and modified nucleic acid molecules in which nucleotides have been inserted, deleted, substituted and / or inverted in such a way as to provide a desired effect on the biological activity of the protein. Allelic variants and protein homologues (e.g., proteins encoded by nucleic acid homologues) have been discussed in detail above.

[0094] Nucleic acid molecule homologues can be produced using a variety of methods known to those of skill in the art (e.g., as described in Sambrook et al., supra). For example, nucleic acid molecules can be modified using a variety of techniques, including, but not limited to, by classical mutagenesis and recombinant DNA technology (including, but not limited to, site-directed mutagenesis, chemical treatment, restriction enzyme cleavage, ligation of nucleic acid fragments, and / or PCR amplification), or synthesis and chemical ligation of oligonucleotide mixtures, or in vitro or in vivo recombination of mixtures of molecular building blocks to "build" a reassortant library of nucleic acid molecules comprising various combinations thereof by methods of 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, P.N.A.S. USA 91 :10747-10751, 1994, all of which are incorporated herein by reference in their entireties). These and other similar techniques known to those of skill in the art can be used to effectively introduce a variety of simultaneous changes in a protein. Nucleic acid molecule homologues can then be selected by hybridization to a given moiety, or screened for function and biological activity by direct expression of the proteins encoded by these nucleic acid molecules.

[0095] One embodiment of the present application relates to a recombinant nucleic acid molecule comprising the above-described isolated nucleic acid molecule operably linked to at least one transcription control sequence. More specifically, in accordance with the present application, the recombinant nucleic acid molecule typically comprises a recombinant vector and the isolated nucleic acid molecule as described above. In accordance with the present application, the recombinant vector is an engineered (i.e., artificially produced) nucleic acid molecule that serves as a tool for manipulating a selected nucleic acid sequence and / or for introducing such a nucleic acid sequence into a host cell. The recombinant vector is thus useful for cloning, sequencing, and / or otherwise manipulating a selected nucleic acid sequence, for example, by expressing and / or delivering the selected nucleic acid sequence into a host cell to form a recombinant cell. Such a vector typically contains heterologous nucleic acid sequences, i.e., nucleic acid sequences that are not naturally found adjacent to the nucleic acid sequence to be cloned or delivered, although the vector can also contain regulatory nucleic acid sequences (e.g., promoters, untranslated regions) that are naturally found adjacent to the nucleic acid sequences of the present application or that can be used to express the nucleic acid molecules of the present application (discussed in detail below). The vector can be RNA or DNA, prokaryotic or eukaryotic, and is typically a plasmid. The vector can remain as an extrachromosomal element (e.g., a replicating plasmid), or it can integrate into the chromosome of the recombinant host cell, but preferably the vector remains separate from the genome for most applications of the present application. The entire vector can remain in place within the host cell, or under certain conditions, the plasmid DNA can be lost, leaving the nucleic acid molecule of the present application. The integrated nucleic acid molecule can be under the control of a chromosomal promoter, under the control of a natural or plasmid promoter, or under the control of a combination of several promoters. Single or multiple copies of the nucleic acid molecule can be integrated into the chromosome. The recombinant vector of the present application can contain at least one selectable marker.

[0096] In one embodiment, the recombinant vector used in the recombinant nucleic acid molecule of the present application is an expression vector. As used herein, the phrase "expression vector" is used to refer to a vector that is suitable for producing a coded product (e.g., a protein of interest). In this embodiment, a nucleic acid sequence encoding a product to be produced (e.g., a protein containing a thioredoxin monocysteine active site) is inserted into the recombinant vector to produce the recombinant nucleic acid molecule. The nucleic acid sequence encoding the protein to be produced is inserted into the vector in a manner that operably links the nucleic acid sequence to regulatory sequences in the vector that enable transcription and translation of the nucleic acid sequence within a recombinant host cell.

[0097] In another embodiment of the present application, the recombinant nucleic acid molecule comprises a viral vector. Viral vectors include the isolated nucleic acid molecule of the present application integrated into a viral genome or portion thereof, wherein the nucleic acid molecule is packaged in a viral envelope that allows the DNA to enter a cell. A variety of viral vectors can be used, including, but not limited to, those based on alphaviruses, poxviruses, adenoviruses, herpesviruses, lentiviruses, adeno-associated viruses, and retroviruses.

[0098] Generally, a recombinant nucleic acid molecule comprises at least one nucleic acid molecule of the application operably linked to one or more expression control sequences. As used herein, the phrase "recombinant molecule" or "recombinant nucleic acid molecule" refers primarily to a nucleic acid molecule or nucleic acid sequence that is operably linked to an expression control sequence, but can be used interchangeably with the phrase "nucleic acid molecule" when such nucleic acid molecule is a recombinant molecule as discussed herein. According to the present application, the phrase "operably linked" refers to linking a nucleic acid molecule to an expression control sequence in a manner such that the molecule, when transfected (i.e., transformed, transduced, transfected, conjugated, or conducted) into a host cell, is capable of being expressed. Transcription control sequences are expression control sequences that control the initiation, elongation or termination of transcription. Of particular importance are sequences that control transcriptional initiation, such as promoters, enhancers, operators 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 molecules of the present application can also contain other regulatory sequences, such as translational regulatory sequences, origins of replication, and other regulatory sequences compatible with the recombinant cell. In one embodiment, the recombinant molecules of the present application, including those integrated into the host cell chromosome, also contain a secretion signal (i.e., a signal segment or signal sequence nucleic acid sequence) to effect secretion of the expressed protein from the cell in which the protein is produced. Suitable signal segments include a signal segment naturally associated with the protein to be expressed or any heterologous signal segment capable of directing secretion of a protein according to the present application. In another embodiment, the recombinant molecules of the present application comprise a leader sequence to effect delivery of the expressed protein to and insertion into the host cell membrane. Other signal sequences include those capable of directing periplasmic or extracellular secretion or retention within a desired compartment. Suitable leader sequences include a leader sequence naturally associated with the protein or any heterologous leader sequence capable of directing delivery and insertion of the protein into the cell membrane.

[0099] According to the present application, the term "transfection" is used to refer to any method that can be used to insert an exogenous nucleic acid molecule (i.e., a recombinant nucleic acid molecule) into a cell. The term "transformation" is used interchangeably with the term "transfection" when used in reference 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 genetic change resulting from the uptake of 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 can refer to changes in the growth properties of cells in culture (as described above), for example, after they become cancerous. Thus, 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 both transfection of animal cells and transformation of plant cells and microbial cells to the extent that the term relates 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.

[0100] In one embodiment, a composition comprising a protein or peptide comprising a thioredoxin monocysteine active site in a reduced state is used to reduce the viscosity of excessively viscous mucus or sputum. The composition comprises a protein comprising a thioredoxin monocysteine active site, and can include one or more additional agents or compounds, such as other agents or compounds that can be used to reduce / decrease excessively viscous or sticky mucus or sputum or increase liquefaction of such mucus or sputum. Examples of other agents or compounds are known in the art and include, but are not limited to, purified rhDNAse, N-acetylcysteine, nacystelyn (a N-acetyl-L-cysteine derivative), GSH, and gelofusine. In addition, mucus active agents such as mannitol or hypertonic saline can be used in combination with the monocysteine active site thioredoxin.

[0101] In one embodiment, a composition comprising a pharmaceutical composition can be used to deliver a nucleic acid molecule encoding a protein or peptide comprising a thioredoxin monocysteine active site to cells in a patient to be treated (e.g., epithelial cells in the lungs or airways) so that the cells can become transfected with the protein and express the protein, and thus the protein can contact mucus or sputum in the microenvironment of the cells.

[0102] Compositions, including pharmaceutical compositions, can also include, for example, a pharmaceutically acceptable carrier, which includes a pharmaceutically acceptable excipient and / or a delivery vehicle for delivering a protein or nucleic acid molecule or other modulating compound to a patient. Additionally, compositions, including pharmaceutical compositions of the present application, can be administered to a patient in a pharmaceutically acceptable carrier. As used herein, a pharmaceutically acceptable carrier refers to any material that is suitable for delivery of a therapeutic protein, nucleic acid, or other compound useful in the methods of the present application to a suitable in vivo or ex vivo site. Preferred pharmaceutically acceptable carriers are capable of maintaining a protein, nucleic acid molecule, or compound in a form that, upon reaching the desired site (e.g., a site of secretion or expulsion of mucus or sputum to be treated), is capable of contacting the mucus or sputum (in the case of a protein or compound) or of entering a cell and being expressed and secreted by the cell (in the case of a nucleic acid molecule) such that the expressed protein in a reduced state can contact the mucus or sputum. Suitable excipients of the present application include those that transport or aid in the transport, but do not specifically target a therapeutic agent (protein, nucleic acid, or compound) to a cell, tissue, or fluid (mucus or sputum) (also referred to herein as non-targeted carriers). Examples of pharmaceutically acceptable excipients include, but are not limited to, water, phosphate buffered saline, Ringer's solution, dextrose solution, serum-containing solutions, Hank's solution, other aqueous physiologically balanced solutions, oils, esters, and glycols. An aqueous carrier can contain, for example, suitable auxiliary substances that enhance chemical stability and isotonicity of the solution when it reaches the recipient. Formulations for inhalation of a therapeutic agent can also include a surfactant molecule.

[0103] Suitable auxiliary substances include, for example, sodium acetate, sodium chloride, sodium lactate, potassium chloride, calcium chloride, and other substances for creating phosphate buffered solutions, Tris buffers, and bicarbonate buffers. Auxiliary substances can also include preservatives, such as thimerosal, m-cresol or o-cresol, formalin, and benzyl alcohol. Compositions of the present application can be sterilized and / or lyophilized by conventional methods.

[0104] One type of pharmaceutically acceptable carrier includes controlled release formulations that are capable of slowly releasing a composition of the present application into a patient. As used herein, a controlled release formulation comprises one or more therapeutic agents of the present application in a controlled release vehicle. Suitable controlled release vehicles include, but are not limited to, biocompatible polymers, other polymeric matrices, capsules, microcapsules, bolus formulations, osmotic pumps, diffusion devices, liposomes, niosomes, and transdermal delivery systems. These controlled release vehicles can also incorporate a reducing agent to maintain the thioredoxin single cysteine active site in a reduced state during storage and delivery. Suitable delivery vehicles for nucleic acids include, but are not limited to, liposomes, viral vectors, or other delivery vehicles, including ribozymes.

[0105] A suitable or effective amount of a protein or peptide containing a thioredoxin mono-cysteine active site to be administered to a patient is an amount that is capable of achieving the following: participating in redox reactions by reversibly oxidizing its active site thiol to a disulfide bond, catalyzing thiol-disulfide exchange reactions, and, in particular, reducing the viscosity or stickiness and / or increasing the liquefaction of mucus or sputum in a patient, sufficient to provide a therapeutic benefit to the patient. A reduction in viscosity or stickiness or an increase in mucus or sputum liquefaction can be measured, detected, or assayed as previously described herein or by any suitable method known to one of skill in the art. As discussed above, these measurements include assaying and comparing the percentage of free thiols in a mucus or sputum sample from a patient before and after contact with a suitable or effective amount of a protein or peptide containing a thioredoxin mono-cysteine active site, as well as assaying and comparing the FEV level of a patient before and after contact with a suitable or effective amount of a protein or peptide containing a thioredoxin mono-cysteine active site in a reduced state.

[0106] In one embodiment, a suitable or effective amount of a protein or peptide having a thioredoxin mono-cysteine active site to be administered to a patient comprises about 10 micromole / kilogram, 15 micromole / kilogram, 20 micromole / kilogram, 25 micromole / kilogram, 30 micromole / kilogram, 35 micromole / kilogram, 40 micromole / kilogram, 45 micromole / kilogram, 50 micromole / kilogram, 55 micromole / kilogram, 60 micromole / kilogram, 65 micromole / kilogram, 70 micromole / kilogram, 75 micromole / kilogram, 80 micromole / kilogram, 85 micromole / kilogram, 90 micromole / kilogram, 95 micromole / kilogram, 100 micromole / kilogram, 105 micromole / kilogram, 110 micromole / kilogram, 115 micromole / kilogram, 120 micromole / kilogram, 125 micromole / kilogram, 130 micromole / kilogram, 135 micromole / kilogram, 140 micromole / kilogram, 145 micromole / kilogram, 150 micromole / kilogram, 175 micromole / kilogram, 200 micromole / kilogram, 225 micromole / kilogram, 250 micromole / kilogram, 275 micromole / kilogram, 300 micromole / kilogram, 325 micromole / kilogram, 350 micromole / kilogram, 375 micromole / kilogram, 400 micromole / kilogram, 425 micromole / kilogram, 450 micromole / kilogram, 475 micromole / kilogram, 500 micromole / kilogram, 525 micromole / kilogram, 550 micromole / kilogram, 575 micromole / kilogram, 600 micromole / kilogram, 625 micromole / kilogram, 650 micromole / kilogram, 675 micromole / kilogram, 700 micromole / kilogram, 725 micromole / kilogram, 750 micromole / kilogram, 775 micromole / kilogram, 800 micromole / kilogram, 825 micromole / kilogram, 850 micromole / kilogram, 875 micromole / kilogram, 900 micromole / kilogram, 925 micromole / kilogram, 950 micromole / kilogram, 975 micromole / kilogram, 1000 micromole / kilogram, 1100 micromole / kilogram, 1200 micromole / kilogram, 1300 micromole / kilogram, 1400 micromole / kilogram, 1500 micromole / kilogram, 1600 micromole / kilogram, 1700 micromole / kilogram, 1800 micromole / kilogram, 1900 micromole / kilogram, 2000 micromole / kilogram, 2100 micromole / kilogram, 2200 micromole / kilogram, 2300 micromole / kilogram, 2400 micromole / kilogram, or about 2500 micromole / kilogram based on the weight of the patient.

[0107] In another embodiment, if the route of delivery is aerosol delivery to the lung or similar route, the amount of protein or peptide comprising a thioredoxin mono-cysteine active site to be administered to a patient comprises from about 0.25 mg per dose unit (a dose unit is typically about 2-3 ml for a human) to about 100 mg per dose unit. Preferably, the amount of protein or peptide comprising a thioredoxin mono-cysteine active site 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 dose unit. Depending on the device used for aerosol delivery, some aerosol delivery devices only allow about 10% of the volume of the aerosol to actually be delivered to the lung. However, when the delivery device is a vibrating mesh nebulizer, about 90% of the volume of the aerosol can be delivered. Electronic vibrating mesh nebulizers are able to deliver drugs more rapidly and are a much smaller, more portable device that is highly preferred by CF patients (Geller, D.E., Pediatric Pulmonology, 43(S9):S5-S17, 2008). Vibrating mesh nebulizers also deliver drugs more efficiently with less residual dose than air jet nebulizers. This is particularly significant for reducing the cost of therapy because smaller doses are needed to achieve therapeutic benefit. Devices such as these also do not cause a reduction in the biological activity of the protein (Kesser, K.C. et al., Resp Care, 54(6):754-768, 2009; Scherer, T. et al., J Pharm Sci, 100(1):98-109, 2011). Thus, for other routes of administration, it will be readily apparent that lower doses of the protein or peptide comprising a thioredoxin active site can be used when the volume of composition delivered to the site is larger.

[0108] The optimal amount of a protein of the application to be administered to an animal will vary depending on the route of administration. For example, if the protein is administered by inhalation (aerosol) route, the optimal amount to be administered can be different than the optimal amount to be administered by intratracheal microspray. It is within the ability of one skilled in the art to vary the amount depending on the route of administration. It is important to note that a suitable amount of a protein of the application is an amount that has the desired effect without being toxic to the animal. Other routes of administration include, but are not limited to, oral administration, particularly for treatment of mucus of the digestive tract; or topical administration, for treatment of mucus of the reproductive tract.

[0109] In one embodiment of the application, compositions containing proteins comprising a thioredoxin mono-cysteine reactive site, including the pharmaceutical compositions of the application, are further formulated 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 used in the application include, but are not limited to, dithiothreitol (DTT), lipoic acid, NADH or NADPH dependent thioredoxin reductase, ethylenediaminetetraacetic acid (EDTA), reduced glutathione, dithio glycolic acid, 2-mercaptoethanol, tris-(2-carboxyethyl) phospholene, N-acetyl cysteine, NADPH, NADH, and other biological or chemical reducing agents.

[0110] As discussed above, compositions, including the pharmaceutical compositions of the application, are administered to a patient in a manner effective to deliver the composition and, in particular, the proteins comprising a thioredoxin mono-cysteine active site and / or any other compounds in the composition to the target site (e.g., mucus or sputum to be treated with the proteins and compounds, target host cells in the environment of mucus or sputum to be treated with the recombinant nucleic acid molecules). Suitable administration protocols include any in vivo or ex vivo administration protocol.

[0111] According to the application, an effective administration protocol (i.e., administering the compositions of the application in an effective manner) includes suitable dosage parameters and modes of administration that result in the proteins containing a thioredoxin mono-cysteine active site and / or other compounds in the composition coming into contact with the mucus or sputum to be treated, preferably such that the patient derives some measurable, observable, or perceptible benefit from such administration. In some cases, the effective dosage parameters can be assessed by sampling the mucus or sputum from the patient and using methods as described herein to determine mucus or sputum viscosity or liquefaction. Alternatively, if the patient is a human, the effective dosage parameters can be determined experimentally using in vitro samples, in vivo animal models, and ultimately clinical trials. The effective dosage parameters can be determined using standard methods in the art for the particular disease or condition. These methods include, for example, measuring survival, side effects (i.e., toxicity), and progression or regression of the disease, as well as related physiological parameters such as forced expiratory volume in one second (FEVi).

[0112] According to the present application, suitable methods of administering the compositions of the present application to a patient include any in vivo route of administration suitable for delivering the composition to the desired site in the patient. Preferred routes of administration will be apparent to those skilled in the art, depending on whether the compound is a protein or other compound (e.g., a drug), to what part of the body the composition will be administered, and the disease or condition experienced by the patient. In general, suitable methods of in vivo administration of a single cysteine active site thioredoxin include, but are not limited to, dermal delivery, intratracheal administration, inhalation (e.g., aerosol), nasal, oral, pulmonary administration, and catheter infusion. Otic delivery can include ear drops, intranasal delivery can include nose drops or intranasal injection, and intraocular delivery can include eye drops or use of a device suitable for passing a drug through the sclera. 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, which is incorporated herein by reference in its entirety). Oral delivery can include orally consumable solids and liquids, e.g., in tablet or capsule form, as well as formulated into food and beverage products or animal feed or feed pellets. Other routes of administration available to mucosal tissues include bronchial, intranasal, other inhalation, rectal, topical, transdermal, vaginal, transcervical, pericervical, and urethral routes. In addition, administration protocols can include pretreatment devices, e.g., administration of a protein, peptide, or composition in a septum (e.g., to the cervix) for use in applications such as infertility. In a preferred embodiment of the present application, when a protein or composition of the present application is administered to treat excess or abnormal thick or sticky sputum or mucus in the respiratory tract (airway), the protein or peptide (or composition) or other compound containing a thioredoxin single cysteine active site is administered by a route that includes, but is not limited to, inhalation (i.e., by inhalation of an aerosol, e.g., in or with a surfactant); direct installation to the lung via bronchoscopy, endotracheal intubation, and / or any artificial ventilation device; nasal administration (intranasal or transnasal), bronchial or intratracheal (i.e., by direct injection into the trachea or tracheostomy), directly or via lipid encapsulation or surfactant. The present application encompasses any possible method of introducing the composition or protein into the airway such that it can contact the mucus or sputum therein.

[0113] In the methods of the present application, the compositions (including pharmaceutical compositions) can be administered to any member of the class Vertebrata, including, but not limited to, primates, rodents, livestock, chickens, turkeys, and domestic pets. The preferred protected patient is a human.

[0114] Another embodiment of the application relates to a composition comprising a protein or peptide comprising a single cysteine active site of a thioredoxin covalently bound to a cysteine residue in a mucin protein. Mucin proteins include, but are not limited to, respiratory tract mucin proteins and digestive tract mucin proteins. Mucin proteins include mucins such as the highly disulfide-bonded mucins MUC5AC and MUC5B. For example, after the initial catalytic reaction of the N-terminal thioredoxin active site cysteine with the disulfide bond of a mucin protein in a thiol exchange reaction, the thioredoxin single cysteine-containing protein or peptide can be covalently bound to the mucin protein via the N-terminal cysteine and thus is difficult to reduce and catalyze repeatedly. Covalent attachment of thioredoxin containing a single cysteine active site to its mucin target prevents cellular uptake and internalization of thioredoxin by sequestration on the mucus with multiple valency, which has a dual benefit of preventing epithelial uptake and off-target effects due to unwanted thioredoxin activity in the cell, while facilitating clearance of the mucus-linked, depleted drug from the body.

[0115] Another embodiment of the application relates to a method of reducing the viscosity of mucus or sputum in a patient having overly viscous or sticky mucus or sputum by administering a composition comprising a protein or peptide comprising a single cysteine active site of a thioredoxin covalently bound to a cysteine residue in a mucin protein. In one aspect, the mucin protein is a mucin. In another aspect, the single cysteine active site of the thioredoxin is in a reduced state. In another aspect, the mucin protein can be a respiratory tract mucin protein or a digestive tract mucin protein, or a reproductive tract mucin protein.

[0116] Another embodiment of the application relates to a method of reducing the viscosity of mucus or sputum in a patient having mucus or sputum that is overly viscous or sticky by contacting the mucus or sputum of the patient with a composition comprising a disulfide bond reducing agent and a cysteine blocking agent. The disulfide bond reducing agent and the cysteine blocking agent can be the same molecule, including but not limited to a protein or peptide containing a thioredoxin monocysteine active site, or they can be different molecules. The disulfide bond reducing agent can be dithiothreitol (DTT), ethylenediaminetetraacetic acid (EDTA), GSH, dithio glycolic acid, 2-mercaptoethanol, N-acetyl cysteine, tris-(2-carboxyethyl) phospholene, or other pharmaceutically compatible reducing agents known in the art. The cysteine blocking agent can be iodoacetamide, iodoacetic acid, or other alkylating agents, or cysteine specific antibodies or other affinity encoded protein or peptide compositions or antibody mimics. The cysteine blocking agent binds to the cysteines in the disulfide bonds in the mucus proteins before the bonds are reduced. The cysteine blocking agent prevents the reformation of disulfide bonds from the thiol groups of the cysteines in the mucus.

[0117] Another embodiment of the application relates to a method of treating a patient having mucus that is overly viscous or sticky by administering to the patient a composition comprising at least one compound having a thioredoxin active site that cannot be taken up by cells. The compound can be a protein or peptide comprising a thioredoxin monocysteine active site. The compound can also be a fusion protein comprising a thioredoxin moiety and a cell surface receptor ligand moiety. In this embodiment, the cell surface receptor ligand moiety binds to a cell surface receptor, thereby preventing cellular uptake of the fusion protein. The compound can be a protein or peptide comprising a thioredoxin active site in combination with a blocking compound for the cysteine corresponding to cysteine 35 of SEQ ID NO: 12. In a preferred embodiment, the blocking compound can be an antibody or antibody mimic that binds to the thioredoxin molecule and thereby blocks the cysteine 35 of SEQ ID NO: 12. In this regard, the term "blocks" means to interfere with the ability of the cysteine 35 of SEQ ID NO: 12, for example, to form an intramolecular disulfide bond with the cysteine 32 of SEQ ID NO: 12.

[0118] Another embodiment of the present invention relates to a method of preventing systemic exposure to a drug substance in a patient. The method comprises the step of administering a drug to a patient through a delivery route including, but not limited to, pulmonary, oral or topical delivery routes. The drug, once administered, can form a covalent bond with its target site. This mechanism of action is different from known drug mechanisms of action, as many drugs act through molecular interactions, in which a ligand binds to a receptor in a reversible binding of the molecule to the receptor. In a preferred embodiment, the drug substance is a thiol-containing drug, in which the thiol group forms a covalent bond with another thiol group at its target site. For example, the drug can comprise a protein or peptide containing a thioredoxin single-cysteine active site in a reduced state. In another preferred embodiment, the target site is extracellular and the drug is administered through an extracellular delivery route.

[0119] Another embodiment of the present invention relates to a pharmaceutical composition comprising a protein or peptide containing a thioredoxin single-cysteine active site in a reduced state and further comprising at least one sugar or sugar derivative capable of stabilizing a redox-active thiol group. The sugar or sugar derivative can be sucrose, chloro sucrose, lactose, trehalose, maltose, galactose, raffinose, mannose or mannitol. A redox-active thiol group refers to a thiol group that can exist in a reduced state (-SH) or an oxidized state (-S-S-). The term "stabilizing" includes, for example, reducing the rate of oxidation of a redox-active thiol group in a reduced state relative to a composition in which the sugar or sugar derivative is omitted, when the polypeptide is present in a pharmaceutical composition with the sugar or sugar derivative. "Sugar" refers to any monosaccharide, disaccharide, oligosaccharide or polysaccharide. Examples of sugars are glucose, fructose, sucrose, lactose, maltose, galactose, raffinose, inulin, dextran, trehalose, chloro sucrose, mannose and mannitol. Sugar derivatives refer to compounds that are structurally similar to the sugar from which they are derived. For example, chloro sucrose is a chlorinated sucrose and is considered a sugar derivative of sucrose. Other derivatives include, for example, sugar alcohol derivatives such as mannitol and xylitol. Preferred compositions of the present invention comprise non-reducing sugars such as raffinose, trehalose, stachyose and, in particular, sucrose.

[0120] Another embodiment of the present invention relates to an animal feed composition comprising a protein or peptide containing a thioredoxin single-cysteine active site in a reduced state. Examples of animal feeds include, but are not limited to, glycyrrhiza, silage, compressed and pelleted feed, oil and mixed rations, sprouted grains, legumes, crop stover, grain, cereal crops and corn.

[0121] The following examples are provided for illustrative purposes and are not intended to limit the scope of the present invention.

[0122] Examples

[0123] Example 1

[0124] This example illustrates the expression and purification of wild-type Trx (also referred to herein as "rhTrx") and single-cysteine active site rhTrx (also referred to herein as "r(Cys)hTrx") proteins. The rhTrx and r(Cys)hTrx were codon-optimized to maximize protein expression levels and post-transcriptional expression fidelity by methods described in Harris et al., Biotechnol Bioeng 109: 1987-97 (2012), which is incorporated herein by reference in its entirety. The synthetic constructs were cloned into suitable vectors for production in E. coli, which allowed for production of wild-type and single-cysteine active site r(Cys)hTrx mutant proteins on a pg scale. Several expression tags and purification strategies, as well as affinity cleavage, were evaluated at the laboratory scale to facilitate purification from endotoxins and endogenous host thioredoxins.

[0125] rhTrx and r(Cys)hTrx were produced in E. coli. The rhTrx gene encoding a 105 amino acid mature rhTrx protein (including the initiator methionine) was optimized for E. coli expression and synthesized (DNA2.0, Inc.). The gene was subcloned into a pEV vector containing an inducible T7 promoter, kanamycin resistance marker, 6-histidine tag for Ni affinity purification, and a tobacco etch virus protease cleavage site (TEV). To express rhTrx, the rhTrx pEV plasmid (verified by sequencing) was transformed into C43 E. coli (Lucigen) grown to an optical density of approximately 0.6 (600 nm) and induced with isopropyl β-D-l-thiogalactopyranoside (IPTG). Protein was extracted from E. coli cells using homogenization and detergent lysis followed by centrifugation. rhTrx was purified from E. coli lysate using Ni affinity chromatography. The Ni affinity purified rhTrx was subjected to protease cleavage (TEV) to remove the nickel affinity tag and further purified using ion exchange chromatography and reverse phase high pressure liquid chromatography (HPLC). The purity of the protein was assessed by SDS-PAGE and ion spray mass spectrometry (LC / MS / MS) and the sequence of the protein was verified by trypsin cleavage of rhTrx followed by ion trap mass spectrometry. Protein concentration and endotoxin concentration were determined by the Bicinchoninic Acid Assay (Pierce) and the Limulus Amebocyte Assay (Charles River Lab.), respectively. Similar methods were followed to produce r(Cys)hTrx protein, but wherein the r(Cys)hTrx gene was optimized and synthesized by DNA2.0. These rhTrx and r(Cys)hTrx proteins were reduced in vitro using DTT, followed by removal of DTT by dialysis and desalting column treatment.

[0126] Example 2

[0127] The present example describes a preferred expression, purification, and reduction (activation) strategy for native rhTrx and single-cysteine active site rhTrx (r(Cys)hTrx) proteins using direct expression of mature proteins rather than affinity binding methods. r(Cys)hTrx and native Trx control proteins were produced in shake flask batch and fed-batch fermentations of E. coli BL21 and K12 derived hosts using a variety of common constitutive or inducible promoter systems using standard bacterial fermentation techniques, but sugar inducible or IPTG inducible systems are preferred as these produce the highest expression strains. After cell disruption by microfluidizer, the supernatant containing soluble Trx was collected by centrifugation and treated with ammonium sulfate (AS) to preferentially precipitate host cell proteins. Ultrafiltration / diafiltration (UF / DF) was used to filter the resulting Trx enriched supernatant to buffer exchange into 15 mM Hepes, 10 mM b-mercaptoethanol (b-ME), 250 mM NaCl. In this buffer, Trx does not bind to Q-Sepharose HP anion exchange chromatography (AEC) resin, while DNA and other impurities bind with high affinity. After high salt AEC, the Trx enriched flow through was buffer exchanged into low salt buffer under reducing conditions (10 mM DTT) for a second AEC step on a Hi-Trap Q-HP column (GE Healthcare), followed by a buffer exchange into formulation buffer (described below). Alternatively, after AS precipitation, the soluble fraction was filtered at 30 kD cutoff and loaded onto a hydrophobic interaction chromatography (HIC) column. The eluted material was concentrated and buffer exchanged using a 1 KD UF / DF cassette and loaded onto a Source Q (anion exchange) column. The thioredoxin was eluted from the ion exchange column using a salt gradient and then subjected to another UF / DF step to exchange into storage buffer.

[0128] Endotoxin was quantified using the Limulus Amebocyte Lysate kit (LAL, Pierce) and any residual endotoxin was removed using a 0.2 micron Mustang E filter (Pall). Protein identity and yield were determined at appropriate steps by SDS-PAGE and immunoblotting / ELISA, and total mass and final sequence identity were verified by MALDI and electrospray mass spectrometry. The reduced activity of Trx preparations was quantified using the DTNB (Ellman's reagent) reduction assay. Fifty microliters of 2.5 mM rhTrx or r(Cys)hTrx was added to a 96-well plate, followed by 175 microliters of sample buffer and 25 microliters of 6 mM DTNB (5,5'-dithiobis-(2-nitrobenzoic acid)). After initiating the reaction by adding DTNB, the change in absorbance at 412 nm due to DTNB reduction was measured by spectrophotometry at 30°C after 15 minutes.

[0129] Various storage buffer conditions, such as discussed below, were used for compatibility with lyophilization and their ability to stabilize the reduced state of r(Cys)hTrx. Buffer conditions included low pH with reducing sugar, ammonium acetate pH 5.5 with 10 mM b-ME, and a previously identified buffer formulation (40 mM sodium acetate pH 5.5, 0.05% EDTA, 9.25% sucrose) that enhances the storage stability of reduced native Trx. Trx was lyophilized and stored in the dry form for various times under each buffer condition, and the redox stability of the stored protein was evaluated using the DTNB and protein activity assays as described above. For aerosol stability evaluation, a Penn-Century aerosol or nebulizer can be used. Aerosolized compounds were collected at various incubation time points into 0.1 M Tris buffer pH 8.0 with 1 mM EDTA containing DTNB to determine the aerosol stability of r(Cys)hTrx and native rhTrx. Aggregates are not expected to react with DTNB, so only the reduced form will be detected. The extinction coefficient of DTNB (14150 at 412 nm) was used to calculate the free thiol (SH) groups as a function of the recovered protein concentration. A previous study on the intratracheal (IT) delivery of native rhTrx (Rancourt, R. et al., Free Radic Biol Med, 42(9): 1441-1453, 2007) used physiological saline. The addition of redox stabilizers known to be compatible with inhaled delivery, such as methionine, can also be included.

[0130] Example 3

[0131] This example illustrates the construction and E. coli strains used to express r(Cys)hTrx and native rhTrx. Using different vector systems and laboratory BL21 or K12 strains of E. coli, r(Cys)hTrx and native rhTrx controls are readily expressed as mature soluble proteins. To generate higher yielding (>1 g / L) E. coli strains expressing Trx, codon-optimized r(Cys)hTrx and native rhTrx DNA sequences are synthesized using different intracellular promoter systems including inducible systems based on nutrient depletion, sugar inducible systems (e.g., rhamnose, xylose, melibiose or other inducible promoters known in the art, such as U.S. Patent 7871815), or IPTG inducible systems based on promoters from bacteriophage T3, T5 or T7. After DNA sequence confirmation, the expression constructs are transformed into different commonly used BL21 / K12 E. coli host strains, including strains engineered to lack molecular utilization for induction, or it can also have included overexpressed methionine aminopeptidases to improve fidelity of expression at high titer (Liu, M. et al., Protein Expr Purif 84(1): 130-139, 2012). The resulting production host / vector combinations are grown in high throughput format deep well microtiter plates, shaken at 30-37°C in minimal media and screened for expression using methods commonly used in the art such as gel or capillary electrophoresis. Positive clones are confirmed by SDS-PAGE and immunoblotting and / or ELISA / ELISPOT. The strains are subjected to fed-batch fermentation and the yield of protein (r(Cys)hTrx) is assessed by SDS-PAGE and immunoblotting and / or ELISA / ELISPOT. The protein is purified and reduced using the methods described in Examples 1 and 2 above, and the reduced state of the activated protein is assessed by the DTNB assay and the specific disulfide bond reducing activity is assessed by the insulin reduction assay. Sequence identity is analyzed by mass spectrometry (MALDI or electrospray) to determine fidelity of translation and proper molecular weight.

[0132] Example 4

[0133] This example demonstrates the in vitro activity and function of wild-type and single-cysteine active site rhTrx. The activity of rhTrx was assessed by measuring its reducing activity in a standard colorimetric DTNB assay and by determining its ability to inhibit neutrophil elastase activity. In addition, the ability of r(Cys)hTrx to liquefy human CF mucus compared to rhTrx was determined by using a sputum compression assay, and optionally by examining the decrease in viscoelasticity of CF sputum using a specialized rheometer. rhTrx and r(Cys)hTrx were compared to the standard of care CF mucus-lysing Pulmozyme (rhDNase I; dornase alfa).

[0134] Initial characterization of rhTrx: reducing activity. The general reducing activity of pre-reduced Trx preparations was quantified using the DTNB reduction assay as previously reported (Rancourt, R. et al., Free Radic Biol Med, 42(9): 1441-1453, 2007). For enzymatic reduction of rhTrx and r(Cys)hTrx, 50 microliters of assay buffer (100 mM potassium phosphate pH 7.0, 10 mM EDTA, and 0.05 mg / ml bovine serum albumin) containing 5 or 50 mM rhTrx and 0.5 mM purified TrxR was added to a 96-well plate. This was followed by the addition of 175 microliters of sample buffer containing 720 mM NADPH, followed by 25 microliters of sample buffer containing 6 mM DTNB. To measure the reducing activity of chemically pre-reduced rhTrx, 50 microliters of 2.5 mM rhTrx was added to a 96-well plate, followed by the addition of 175 microliters of sample buffer and 25 microliters of 6 mM DTNB. The reaction was initiated by the addition of DTNB, and the change in dynamic absorbance at 412 nm due to DTNB reduction was monitored by spectrophotometry at 30°C. The same protocol was followed for r(Cys)hTrx and those described below. The total reducing activity of r(Cys)hTrx was less than that of rhTrx, reflecting the presence of one less reduced Cys in r(Cys)hTrx. However, both proteins were reduced to more than 90-95% of their potential reduced state, reflecting full activation.

[0135] Inhibition of neutrophil elastase (NE) activity. The incubation system for determining the effect of rhTrx on NE activity has been previously described (Lee, R. et al., (2005) Am J. Physiol. Lung Cell. Mol. Physiol. 289(5): L875-882). Briefly, Trx was diluted to the desired concentration in phosphate buffered saline (PBS, pH 7.2). The mixture was added to purified human NE (100 mg / ml in PBS, pH 7.2, 0.01% Triton x-100) and incubated at 37°C for 1 hour. The final volume during incubation was 210 microliters, and the concentration of NE was 1.6 microgram / ml. Following incubation, 60 microliter aliquots were tested in triplicate for elastinolytic activity. Elastase activity was determined by adding 120 microliters of PBS (pH 7.2) containing 0.8 mM N-methoxysuccinyl-Ala-Ala-Pro-Val 4-nitroanilide to the experimental samples and monitoring the dynamic absorbance at 405 nm for 4 minutes at 37°C. The concentration of elastase used in these experiments was determined to be within the linear range of the assay. The percent inhibition of NE activity was determined as: percent inhibition = (1 - change in absorbance of experimental group / change in absorbance of PBS control group). The elastase activity of r(Cys)hTrx was found to be at least comparable to rhTrx.

[0136] A compression assay was used to assess liquefaction of human CF sputum. For all manipulations, CF sputum was handled under microbiological hood. Relevant concentrations of rhTrx, r(Cys)hTrx or diluent alone (25 microliters) were added to CF sputum (275 microliters) in 1.5 ml Eppendorf conical tubes and mixed by very brief vortexing. After incubation at 37°C for 30 minutes, the samples were mixed again briefly and loaded into hematocrit tubes and sealed at both ends. After centrifugation in a hematocrit centrifuge for 5 minutes, the percent solid (gel) and percent liquid were determined by direct linear measurement and expressed as percent liquid as follows: 100 x liquid / (liquid + solid). Sputum from at least five different CF patients was measured in triplicate for each condition. The liquefaction ability of r(Cys)hTrx was at least comparable to or greater than rhTrx, reflecting the unexpected potency of the r(Cys)hTrx mechanism of action.

[0137] Sputum viscoelasticity measurements. These measurements were performed using a cone and plate rheometer and qualitatively measured by observing sputum flow directly after treatment with thioredoxin. The same sputum treatment protocol as described above was used for incubation. The viscoelasticity was evaluated using an AR-1000 rheometer (TA Instruments, New Castle, DE) in oscillatory mode at 37°C at an angular velocity of 1 rad / sec. The ability to reduce sputum viscoelasticity was evaluated in triplicate in sputum obtained from at least five different CF patients for each preparation of Trx. The ability to reduce sputum viscoelasticity of r(Cys)hTrx was expected to be at least comparable to or greater than rhTrx, reflecting the unexpected potency of the mechanism of action of r(Cys)hTrx. Sputum viscoelasticity measurements were performed by observing the rate of flow of sputum mixed with rhTrx or r(Cys)hTrx in an inverted Eppendorf tube, and r(Cys)hTrx was found to have a greater magnitude of flow than rhTrx, DTT, and Pulmozyme in this assay, and much greater than the negative (vehicle) control, which exhibited essentially no sputum flow.

[0138] Comparison of efficacy with standard-of-care mucolytic agent (rhDNAse). For these studies, relevant concentrations of rhTrx, r(Cys)hTrx, and rhDNAse were compared by incubation as described above. After 30 minutes, the samples were evaluated for sputum liquefaction changes (compression assay) and viscoelasticity changes. In addition, synergy studies were performed. Therein, sputum samples were exposed to rhTrx for 30 minutes and then to rhDNAse for 30 minutes, and optionally to rhDNAse for 30 minutes followed by rhTrx for 30 minutes. These results were compared to the effects of either agent alone. For each condition, sputum from at least five different CF patients was measured in triplicate. Enough sputum was obtained from separate donors on a given day to test 12 samples each of wild-type and mutant rhTrx variants (rhTrx x 3, rhDNase x 3, rhTrx + rhDNAse x 3, rhDNAse + rhTrx x 3). In practice, this was a minimum of 4.5-5.0 ml of well-mixed sputum from a single donor / day. The liquefaction ability of r(Cys)hTrx was at least comparable to or greater than rhDNAse, rhDNAse, rhTrx, or the DTT positive control.

[0139] Human CF Sputum Collection. Sputum was obtained from adult and pediatric CF patients identified by a healthcare provider. A patient was diagnosed with CF if they exhibited clinical symptoms and had a sweat chloride value exceeding 60 mmol / L in two independent pilocarpine iontophoresis sweat tests and showed two mutations producing the CF allele in subsequent genetic analysis. All samples were provided by spontaneous expectoration or hypertonic saline induction. Sputum samples containing obvious detectable saliva were discarded. Sputum was kept on ice before delivery to the laboratory and then maintained at -80°C until used in O-ring sealed vials to prevent drying.

[0140] Example 5

[0141] This embodiment illustrates the enzymatic activity of proteins or peptides containing a thioredoxin monocysteine ​​active site compared to proteins or peptides containing a wild-type thioredoxin active site. Figure lb ) and non-enzymatic activity ( Figure la ).like Figure la As shown, nonspecific 5,5′-dithiobis-(2-nitrobenzoic acid) reduction (DTNB or Ellman's reagent) reflects the loss of a reducible cysteine ​​residue at the monocysteine ​​active site of thioredoxin compared to the wild type. Figure lb and Figure 2 As shown, proteins or peptides containing the thioredoxin monocysteine ​​active site in human sputum compressibility assays surprisingly exhibit greater efficacy than those containing the wild-type thioredoxin active site, and greater efficacy relative to DNases or NAC on an equimolar basis. Figure 2 This result was unexpected, considering the extensive modifications at the active site due to the Cys35 mutation, and the fact that the total reducing power observed in existing DTNB assays, due to the loss of a single reducible Cys residue, is only 4 / 5 that of native thioredoxin. The surprising increase in the efficacy of the thioredoxin monocysteine ​​active site is due to the covalent linkage with Cys residues in mucinous proteins, which has an unexpected consequence: preventing these Cys residues from reforming into new disulfide bonds.

[0142] Example 6

[0143] This example demonstrates the reduced propensity of r(Cys)hTrx (in the reduced, active state) to stimulate the release of pro-inflammatory cytokines from cultured primary human bronchial epithelial cells (HBE) compared to native rhTrx. Donor tissue and cells were provided under the auspices of an approved protocol for the protection of human subjects' rights. HBE cells from normal (i.e., non-diseased) lung were harvested by enzymatic digestion as previously described (Fulcher, M.L., Methods Mol Med 107: 183-206, 2005). Dispersed HBE cells were seeded onto 12 mm diameter Transwell clear inserts (Corning) at a density of 2.5 x 105 / cm2in well-defined airway cell medium (Fulcher, M.L., 2005, supra). Cultures were maintained at the air-liquid interface until fully differentiated (about 4-6 weeks) prior to use. 5 / cm 2

[0144] Thioredoxin Delivery Protocol: For this study, a device was utilized that is capable of delivering nanoliter volumes of test agent (or control) to the surface of HBE cultures. This system was designed as an in vitro model system to simulate the in vivo delivery of an ultrafine mist of aerosolized drug to the surface of airway epithelial cells, and presents an ideal way to study the effects of adding therapeutic agents such as r(Cys)hTrx. Based on the results of multi-path particle dosimetry modeling (Anjilvel, S. et al., Fundam Appl Toxicol 28(2): 41-50, 1995), which demonstrated that the average deposition rate of a Pari LC Star nebulizer on the first 20 generations of airways, over the course of 15 minutes, the average deposition on the first 6 generations of airways is predicted to be about 50 nl / min / cm 2 , with the majority of particles expected to be delivered. In these studies, a total volume of 750 nl (over 15 minutes) was nebulized to a total of five different experimental groups. These included: 1) vehicle control (isotonic saline), 2) native recombinant Trx (500 μm), and 3) three doses of r(Cys)hTrx (10 μm, 250 μm, and 1000 μm). These concentrations represent the "final" airway surface concentration. Following nebulization of each test agent (or vehicle control), cultures were returned to the tissue culture incubator and incubated for 24 hours prior to cytokine analysis.

[0145] ​Cytokine immunoassays: In these studies, the effects of native and r(Cys)hTrx) on the stimulation of four major cytokines released by HBE cells, including IL-6, IL-8, TNF-α, and IL-1 β, were assessed. Cytokines in cell-free, unconcentrated culture supernatants were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits (R&D systems). Samples of basal side ALI culture medium (Fulcher, M. L., 2005, supra) were obtained 24 hours after all five sprays and frozen prior to analysis. As a background control, clean (unused) ALI medium was subtracted from all values. For each cytokine analysis, a positive control was used in triplicate to generate an appropriate standard curve. In addition, each "unknown" sample was analyzed in duplicate. The cytokines measured were: TNF-α (detection limit of 0.3 pg / ml), IL-6 (detection limit of 0.35 pg / ml), IL-8 (detection limit of 2.4 pg / ml), and IL-1 β (detection limit of 1.5 pg / ml). Cross-reactivity of these assays with each other and with other recombinant human cytokines (IL-la, IL-2, IL-3, IL-4, IL-7, tumor necrosis factor-S, granulocyte colony-stimulating factor, and transforming growth factor-Ill) has previously been shown to be below the detection limit.

[0146] Sample size and statistical analysis: A total of nine separate HBE cultures were evaluated for each of the five conditions. This represents three cultures (n=3) from three different patients (n=3). This approach was statistically sufficient to provide an understanding of both inter- and intra-sample variation for each condition. For data analysis, two-tailed Student's t-tests were used for comparisons between individual samples / groups. For multiple group comparisons, one-way analysis of variance (ANOVA) was used. Significance for all analyses was set at p<0.05.

[0147] Proinflammatory evaluation of r(Cys)hTrx in HBE cultures from CF patient donors. Human CF primary airway epithelial cell culture: The latest technology for human primary airway epithelial cell proliferation and culture at air-liquid interface with mucus ciliary differentiation based on methods reported by Schlegel and colleagues (Liu, M. et al., Protein Expr Purif. 84(1): 130-139, 2012; Suprynowicz, F.A. et al., Proc. Natl. Acad. Sci. U.S.A. 109(49): 20035-20040, 2012). These methods allow for almost unlimited supply of primary airway epithelial cells that are still able to terminally differentiate at the ALI without genetic manipulation. Thirty wells from each of three unique CF donors were cultured to ALI differentiation over 30 days, homozygous for F508del. The apical surface of the cultures was exposed to three concentrations (10 μΜ, 250 μΜ, and 1000 μΜ) of rhTrx (wild type) and r(Cys)hTrx, followed by collection of apical and basolateral side samples at 4 hours and 24 hours post-exposure initiation (see Table 1). The media used was serum free, centrifuged to remove debris and stored at -80°C until use. ELISA assays for human inflammatory cytokines were performed on all samples obtained from apical and basolateral media. The apical was collected by placing 200 μΐ^of sterile PBS on the apical surface and recovering this after a 15 minute incubation. ELISAs for IL-8 and IL-6 were performed in duplicate for each sample (Becker, M.N. et al., Am J Resp Crit Care Med 169(5): 645-653, 2004). The above plan involving 30 ALI cultures was repeated for each of the three CF donors. Comparison of results for native rhTrx and r(Cys)hTrx indicates that the propensity of thioredoxin to induce release of proinflammatory cytokines from differentiated airway epithelial cells of CF patients is attenuated in the single cysteine Trx relative to native at the concentrations tested.

[0148] Table 1

[0149]

[0150]

[0151] Example 7

[0152] This example illustrates the biophysical and biochemical characterization of the effect of r(Cys)hTrx versus native rhTrx on uniform mucus harvested from in vitro HBE cell cultures.

[0153] Preparation of cell culture mucus: Human bronchial epithelial cells were grown and maintained as described (Matsui, H. et al., J Clin Invest 102(6): 1125-1131, 1998). Briefly, mucus harvested from cultures was pooled and stored at 4°C. Samples were loaded into dialysis tubing (MWCO = 3,500) and concentrated with a polymeric absorbent (Spectra / Gel) at 4°C for 1-5 days. The concentrated mucus was then dialyzed against PBS containing 500 μΜ MgCl2and 800 μΜ CaCl2at 4°C to establish proper salt balance (Matsui, H. et al., Proc Natl Acad Sci USA 103(48): 18131-18136, 2006).

[0154] Macro-rheology: The overall, macro-biophysical effects of reduced r(Cys)hTrx and native thioredoxin on the properties of in vitro HBE mucus were assessed using a Bohlin Gemini rheometer with cone-and-plate and parallel-plate geometries in concentration and time course measurements. Creep recovery experiments were performed in which a known stress (0.05 to about 100 Pa) was applied to the treated mucus or control mucus for 10 seconds, and the rheology of the fluid was re-recorded for 50 seconds as it recovered. In a continuous operation, the applied stress was increased logarithmically until the yield stress of the fluid was reached (i.e., the stress at which the viscosity of the fluid suddenly and sharply decreased). 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 two constant stresses and strains and were used to determine the baseline physical properties of the mucus (G' and G", respectively), as well as the viscosity and shear thinning behavior of the fluid. All experiments were performed at 23°C.

[0155] High pressure liquid chromatography: The concentration and molecular mass of mucin in treated mucus and control mucus were assessed by differential refractometry to determine the effect of native thioredoxin and r(Cys)hTrx on the structure of mucin. Samples (500 μΐ) were loaded onto a Sepharose S1000 column (Amersham Pharmacia) and eluted with 200 mM sodium chloride / 10 mM EDTA at a flow rate of 0.5 ml / min. Light scattering and sample concentration were measured using an online Dawn EOS laser photometer coupled to a Wyatt / Optilab DSP interferometric refractometer (Wyatt Technology Corporation). The concentration of mucin was calculated by integrating the refractive index peak associated with the eluted material in the void volume of the column and using a refractive increment (dn / dc) value of 0.165 ml / g, which had previously been measured at 650 nm and found to be reproducible to within 5%. The total protein content (mucin and small proteins) of a given mucus sample was determined by a similar method but utilizing a G-25 column (dn / dc = 0.170). The non-mucin (or small protein) content of a sample was determined by subtracting the mucin content mass (determined by eluting the sample through the S-1000 column) from the total mass of mucin and protein content (detected by eluting the sample through the G-25 column). The advantage of differential refractometry over gel-based methods, such as reducing or non-reducing PAGE, is that accurate molecular weights and molecular weight distributions can be obtained rather than a qualitative comparison. The quantitative advantage of these techniques is particularly important for the study of large glycoproteins such as mucin, where size standards are not available and the molecular weight often exceeds 1 MDa (Gillis, R.B. et al. Carbohydr Polym 93(1): 178-183, 2013).

[0156] Carbohydrate and protein blotting: The potential impact of thioredoxin treatment on the fine structure of mucin was evaluated by Western blot analysis of intact HBE mucus. 50-200 μL aliquots of sample were loaded onto nitrocellulose membranes and vacuum was applied for five minutes to draw the entire loaded sample into the membrane. The samples were washed in distilled water 2 times. For the Periodic Acid Schiff (PAS) assay of carbohydrate content, the loaded and washed samples were incubated in water containing 0.25% periodic acid + 3% acetic acid for 30 minutes. After two washes in distilled water, the samples were incubated in NaMBS solution (0.1% sodium metabisulfite, 1% HC1) twice for five minutes each. Next, the samples were incubated with Schiff reagent for 5-15 minutes and washed with NaMBS twice, distilled water once, and quick vacuum dried. After loading and the first distilled water wash, the protein specific antibody blots were blocked with TBST buffer (1.21% Tris HC1, 8.76% NaCl, and 0.5% Tween, pH 8) containing 1% milk. After two five minute TBST washes, the samples were incubated in primary antibody (MAN-5AC I for MUC5AC, MUC5B III and K5B for MUC5B) for 30 minutes. After two additional five minute washes in TBST, the samples were incubated in secondary antibody for two hours. The membrane blots were developed using a Li-Cor Odyssey infrared detector. Additionally, anti-thioredoxin antibodies can be used to detect native thioredoxin bound to mucin with respect to the single cysteine active site. This direct blotting method allows for a more rapid and accurate determination of mucin content and visualization and quantification of the covalent interaction between the single cysteine active site thioredoxin (r(Cys)hTrx) and mucin disulfide bonds after separation on a PAGE gel as compared to standard Western immunoblotting.

[0157] Example 8

[0158] This example demonstrates the ability of the single cysteine active site Trx (r(Cys)hTrx) to attenuate the propensity of native rhTrx to induce proinflammatory and pathophysiological effects in the lung after intratracheal delivery to rats and mice.

[0159] Proinflammatory cytokine release and cell migration were assessed in normal rats administered r(Cys)hTrx and native rhTrx intratracheally. To determine the ability of r(Cys)hTrx to induce proinflammatory signaling compared to native rhTrx, comparative in vivo studies were performed in rats administered increasing concentrations of thioredoxin delivered intratracheally (IT). Two study components were used: 1) a preliminary study designed to replicate previous study results obtained with purified native rhTrx (Rancourt, R. et al., Free Radic Biol Med, 42(9): 1441-1453, 2007), and 2) a main study comparing the proinflammatory effects of r(Cys)hTrx with native rhTrx. All studies utilized purified endotoxin-free protein that had been treated with DTT or other suitable reducing agent to reduce (activate) the Trx active site Cys residues. After reduction, the DTT or reducing agent was removed by size exclusion chromatography. Complete reduction of the Trx Cys residues was verified by in vitro assay (DTNB reduction) and the catalytic disulfide bond reducing activity of r(Cys)hTrx was determined using insulin reduction or HPLC target binding assays.

[0160] Preliminary Study: Twenty-four rats were randomly assigned to four experimental groups of six animals each, dosed as follows: Group 1, vehicle control; Group 2, oxidized (inactive) rhTrx; Group 3, reduced (active) rhTrx; Group 4, human serum albumin (HSA; negative control). All test articles were delivered by the IT route in a single dose. Endpoints for this study included: 1) cytokine analysis of TNF, cytokine-induced neutrophil chemoattractant-2 (CINC2), and macrophage inflammatory protein-3 (MIP3) by enzyme-linked immunosorbent assay (ELISA); and 2) cell counts from bronchoalveolar lavage (BAL) using Wright's staining to elucidate the percentage of inflammatory cells (neutrophils to macrophages). Group 2 (treated with oxidized rhTrx) had similar levels of cytokine activity and cell counts in the BAL compared to Group 1, vehicle control, and Group 4, HSA. Group 3, treated with reduced rhTrx, had increased levels of cytokine activity and cell counts compared to Groups 1 and 2.

[0161] Primary Study: Comparison of cytokine release in rats when administering native thioredoxin rhTRX versus r(Cys)hTRX. In this comparative study, the relative degree of cytokine release and cell migration in vivo following IT administration of rhTrx versus r(Cys)hTrx was determined. Three dose levels of reduced rhTrx and reduced r(Cys)hTrx were tested (50 μM, 200 μM, and 1000 μM). Endpoints were the same as the pilot study with the addition of immunohistochemical (IHC) analysis of lung tissue. In r(Cys)hTrx, the mutation of the C-terminal active site cysteine motif (CXXC to CXXX) has been shown to eliminate the second thiol-disulfide exchange ability of thioredoxin and results in covalent attachment to disulfide bond targets. The most prominent extracellular disulfide bond targets in the lung (which are the first to encounter IT delivered TRX) are located in the mucus layer as well as epithelial cell-surface proteins. Therefore, r(Cys)hTrx has increased binding to lung epithelial cells and associated mucus compared to native rhTrx, and this binding can be detected by IHC using an anti-human thioredoxin antibody. Lung tissue was harvested from two animals in each group for IHC analysis without BAL and compared to two post-BAL animals from each same group. Therefore, eight animals were involved in each group relationship, and two animals were treated but did not undergo BAL prior to lung harvest and IHC. r(Cys)hTrx exhibited a dose-dependent attenuation of cytokine activity and cell count compared to rhTrx at the same dose levels.

[0162] Toxicity and lung pathology were evaluated in normal rats administered r(Cys)hTrx intratracheally. To evaluate the lung effects of r(Cys)hTrx under exaggerated conditions of administration, multiple single doses of r(Cys)hTrx were administered via the IT route at 0.5 to 20 mg / kg, and evaluated for pathological changes at day 2 and day 14 post-dose. Male and female Sprague-Dawley rats were randomly assigned to three experimental groups with 20 animals per group (10 of each gender). All animals were given a pre-study physical examination. Vehicle (saline formulation) was used as a negative control. Endpoints for these studies included: 1) clinical and histopathological examination for gross adverse changes; and 2) characterization of serum to detect test article and the presence of antibodies to the test article. Single cysteine r(Cys)hTrx produced less severe effects at similar doses compared to native rhTrx.

[0163] Proinflammatory cytokine release and cell migration were assessed in normal and bENaC mice given r(Cys)hTrx and native rhTrx intratracheally. Mice were briefly anesthetized with isoflurane and placed on a tilted rodent table. A rodent laryngoscope fitted with a magnifying lens was used to visualize the larynx directly and a mini-sprayer (Penn Century) was passed into the distal trachea. A solid volume (25-50 μL) of test article solution was administered into the airway and the mice were allowed to recover. Body weights at the time of airway instillation and euthanasia were recorded. Following an Institutional Animal Care and Use Committee (IACUC) approved protocol, five mice per condition were euthanized at 6 hours post-IT instillation and another group of five mice were euthanized at 24 hours post-IT instillation. The airways were lavaged with a total of 1.5 mL of cold sterile saline with protease inhibitors (Pierce). The entire lung was harvested and all specimens were stored on ice until further processing. Bronchoalveolar lavage fluid (BALF) was centrifuged to pellet the leukocytes and other cellular debris. Cell-free BALF was frozen at -80°C until ELISA testing. BAL cell pellets were resuspended in 1 mL of sterile saline and total leukocyte counts were performed using a Coulter counter with differential and 300 cells from stained cytospin preparations were counted manually. When all samples had been collected, BAL fluid was thawed on ice and ELISA was performed for KC (analog of IL-8), TNFα, IL-6, and IL-1 β (ElisaTech, Denver, CO). BALF cellularity was characterized by % and total leukocyte (neutrophils, macrophages, lymphocytes).

[0164] Wild type and mutant TRX, as well as control animals, were tested at two concentrations and two time points to determine the acute airway inflammatory response to mutant and wild type TRX relative to diluent control (Table 2). The responses were characterized by: body weight change, total leukocyte counts in BALF, relative and total neutrophil, macrophage, and lymphocyte counts, and the inflammatory cytokines listed above. r(Cys)hTrx exhibited a dose-dependent attenuation of cytokine activity and cell counts relative to rhTrx at the same dose level, as well as side effects.

[0165] Table 2

[0166]

[0167] Pathophysiological and inflammatory effects were assessed in normal and βENaC mice that were intratracheally administered r(Cys)hTrx and native rhTrx. Previous studies by Rancourt and colleagues (Rancourt, R. et al., Free Radic Biol Med, 42(9): 1441-1453, 2007) showed that the presence of additional (extraneous) viscosity on the airway can significantly reduce the stimulation of cytokine release by native Trx. While this can suggest that CF patients with higher mucus burden can exhibit reduced inflammatory responses due to extracellular reduction of Trx, there is currently no information on the role of thioredoxin in airway models with 1) increased endogenous mucus production and 2) pre-existing inflammatory responses (as in CF). The goal of these studies was to investigate the effects of native Trx and r(Cys)hTrx using a mouse model of chronic mucus obstruction / inflammation. For these studies, the βENaC mouse model that overexpresses the β subunit of the ENaC channel and exhibits high water absorption in the lung (Mall, M. et al., Nat Med. 10(5): 487-493, 2004) was used. The βENaC mice produce excess mucus and develop a CF / COPD-like lung phenotype with mucus airway obstruction and inflammation. These mice have been previously used as a model of CF lung disease in a variety of preclinical studies (e.g., Graeber, S.Y. et al., Am J Respir Cell Mol Biol 49(3): 410-417, 2013). In the present studies, 1) the effects of native Trx versus r(Cys)hTrx on airway / lung tissue pathology and inflammatory status; and 2) the effects of these agents on mucus burden in the βENaC mice were determined (see below) to investigate the relative effects of native and monocysteine Trx (r(Cys)hTrx) at multiple delivery doses and to understand how chronic mucus overproduction and pre-existing inflammation modulate the potential lung toxicity of these compounds.

[0168] WT and βENaC mice are assayed for the effects of native thioredoxin at three concentrations (100 μΜ, 500 μΜ, and 1000 μΜ) in vivo and compared to the toxicity and drug efficacy of the r(Cys)hTrx compound at the same concentrations. A minimum of seven mice per condition is tested. All compounds are administered at a given concentration via intratracheal instillation (10-25 μΐ). A single dose treatment is used and toxicity and drug efficacy are monitored at four time points (4 hours, 24 hours, 72 hours, and 7 days post-dose). Early time points (4 hours - 24 hours) are closely monitored. If drug toxicity persists at 24 hours, later time points (72 hours to 7 days) are monitored. Effects at lower concentrations (100 μΜ, 500 μΜ) are not expected to persist beyond 24 hours or 3 days, respectively. The protocol used allows for reliable instillation of volumes as small as 10 μΐ into the mouse lung. Briefly, animals are anesthetized with isoflurane and placed on a mouse intubation platform where the posterior pharynx is visualized using a small laryngoscope. Drugs are instilled directly into the trachea (intratracheal instillation, IT) or, for more uniform and deeper deposition, 25 μΐ are delivered by micro-spray using a Penn Century device. After treatment, animals are euthanized at the appropriate time points. The whole lung is harvested for histology and bronchoalveolar lavage fluid for various measurements, i.e., cell counts, mucin content and redox state, and cytokines. In more detail, the left lung is tied off by a ligature around the main bronchus and surgically isolated for histology. Bronchoalveolar lavage of the opposite lung (or right lobe) is performed via tracheostomy and intubation using 500 μΐ of sterile PBS. After whole lung fixation, longitudinal sections are analyzed by H&E and AB-PAS staining to assess inflammation and retained mucus. BAL is analyzed for cell counts / differential, mucin content and redox state (agarose gel separation method and immunoblotting), and cytokines.

[0169] Sample size and statistical analysis: A total of 7 mice per condition are evaluated at various concentrations (100 μmol, 500 μmol, 1000 μmol) for each compound. Two groups, wild type (WT) and βENaC mice, are tested (Table 3). As indicated by the "X" in the table below, a total of 36 conditions x 7 mice (a total of 252 mice) are treated and analyzed to provide a sufficient understanding of the inter- and intra-sample variation for each condition. For data analysis, a two-tailed Student's t-test is used for comparisons between individual samples / groups. For multiple group comparisons, a one-way analysis of variance (ANOVA) is used. All analyses are set at p < 0.05 for significance. The single cysteine r(Cys)hTrx produces less severe effects than native rhTrx at similar doses, and this effect is further attenuated due to the presence of endogenous mucus in the βENaC mice.

[0170] Table 3

[0171]

[0172] “X” represents time x concentration tested

[0173] Example 9

[0174] This example demonstrates that r(Cys)hTrx improves mucus standardization activity over wild-type rhTrx. The effect of equimolar concentrations of wild-type rhTrx, r(Cys)hTrx, and various controls including dithiothreitol (DTT), N-acetyl cysteine (NAC), and recombinant human DNase (rhDNase) at two concentrations on the standardization of patient sputum samples (n=6 per treatment) was determined in a sputum compression assay. Sputum studies were performed using 4 to 6 spontaneous coughed-out (non-induced) samples, each from up to three separate CF patients for each experiment. The “favorable” portion of sputum (i.e., mucus) was collected from each sample and combined, and the total sample was gently homogenized by stirring or light vortexing, then aliquoted into tubes and exposed to either diluent (i.e., Tris buffer), DTT-containing diluent (at 0.58 mM and 1.5 mM), NAC-containing diluent (0.58 mM), rhDNase-containing diluent (0.58 mM), rhTrx-containing diluent (0.58 mM), or r(Cys)hTrx-containing diluent (0.58 mM). DTT was freshly prepared for each experiment as a positive control. Figure 2 r(Cys)hTrx was shown to significantly increase liquefaction of patient sputum compared to wild-type thioredoxin (referred to as “rhTrx” in Figure 2 mucus standardization activity relative to native Trx and the care standard recombinant human DNase, Pulmozyme TM("rhDNase") showed improved mucus normalizing activity. Thioredoxin and DNase were both more effective than equimolar amounts of NAC, and are commonly used as mucus-dissolving adjuncts to US. These results using human CF patient sputum indicate that single-cysteine r(Cys)hTrx is still fully competent for CF sputum viscoelasticity normalization (and even more effective than unmodified native rhTrx), but is expected to covalently bind to mucus Cys. However, the likelihood of small 12kDr(Cys)hTrx crossing the lung epithelium should be greatly minimized by binding to mucus. Thus the ability of active r(Cys)hTrx to subsequently enter the nucleus and interact with redox-regulated target proteins such as NFkB is even further attenuated, as any signaling due to interaction with extracellular transmembrane domains on immune cells has been theorized for Trx-mediated CD30 TNF receptor activation (Schwertassek, U. et al., EMBO J 26(13):3086-3097, 2007). The increased potency observed for r(Cys)hTrx Figure la 、 Figure lb and Figure 2 ) is consistent with covalent binding of the molecule to mucus Cys, as the bound Cys will prevent disulfide reformation unlike treatment with native rhTrx (or indeed, any small molecule thiol agent).

[0175] Each publication and other reference discussed or cited herein is incorporated herein by reference in its entirety.

[0176] While various embodiments of the present application have been described in detail, modifications and alterations to those embodiments will be apparent to those skilled in the art. It is understood that the application is not limited to the embodiments set forth herein for purposes of exemplification, but is to be afforded the broadest scope of the claims as set forth below, and that changes can be made in the function and arrangement of elements discussed without departing from the scope of the application.

Claims

1. Use of a composition comprising a protein or peptide comprising a thioredoxin mono-cysteine active site in a reduced state in the preparation of a pharmaceutical composition for reducing the viscoelasticity of mucus or sputum in a patient having mucus or sputum that is excessively viscous or tenacious, wherein the protein or peptide is effective to reduce the viscoelasticity of the mucus or sputum, and wherein the protein or peptide is a thioredoxin variant in which a thioredoxin active site is mutated to a thioredoxin mono-cysteine active site in a wild-type thioredoxin amino acid sequence, wherein the wild-type thioredoxin amino acid sequence is selected from the group consisting of SEQ ID No: 4 and 7-12, and wherein the thioredoxin mono-cysteine active site comprises the amino acid sequence of W-C-G-P-S-K (SEQ ID No: 27), wherein the C residue is in a reduced state.

2. The use of claim 1, wherein the patient has a lung disease in which abnormal or excessive viscosity or tenacity of mucus or sputum is a symptom or cause of the disease.

3. The use of claim 1, wherein the patient has a lung disease in which abnormal or excessive viscosity or tenacity of mucus or sputum is associated with a deficiency in the activity of a biological reducing agent.

4. The use of claim 1, wherein the patient has a disease selected from the group consisting of cystic fibrosis, chronic obstructive pulmonary disease, bronchiectasis, asthma, and a digestive tract disease.

5. The use of claim 1, wherein the patient has cystic fibrosis.

6. The use of claim 4, wherein the digestive tract disease is coccidiosis.

7. The use of claim 1, wherein the pharmaceutical composition is administered by introducing it into the patient by a route selected from the group consisting of nasal, intratracheal, bronchial, direct installation to the lungs, inhalation, and oral.

8. The use of claim 1, wherein the mucus or sputum is located in the respiratory tract, digestive tract, or reproductive tract of the patient.

9. The use of claim 1, wherein the composition is administered to the patient in a pharmaceutically acceptable carrier.

10. The use of any one of claims 1-9, wherein the composition increases the percentage of free thiols in a sample of mucus or sputum from the patient.

11. The use of any one of claims 1-9, wherein the pharmaceutical composition increases the forced expiratory volume (FEV) of the patient by at least about 2.5%.

12. The use of any one of claims 1-9, wherein the protein or peptide comprising a thioredoxin mono-cysteine active site covalently binds to a cysteine residue in a mucus protein.

13. The use of claim 12, wherein the mucus protein is mucin.

14. The use of claim 12, wherein the mucus protein is selected from the group consisting of a respiratory tract mucus protein and a digestive tract mucus protein.

15. The use of any one of claims 1-9, wherein the wild-type thioredoxin amino acid sequence is SEQ ID No:

12.

16. The use of any one of claims 1 to 9, wherein the composition further comprises a reducing agent for reducing the thioredoxin mono-cysteine active site of the protein.

17. The use of claim 16, wherein the composition further comprises a thioredoxin reductase.

18. The use of any one of claims 1-11, wherein the patient is a vertebrate selected from the group consisting of mammals and birds.

19. The use of claim 18, wherein the patient is a human.

20. The use of claim 18, wherein the patient is a chicken or turkey.

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