WO2016205430A1

WO2016205430A1 - Recombinant human cc10 protein facilitates repair and protects against damage to the respiratory epithelium due to exposure to both cigarette and other smoke

Info

Publication number: WO2016205430A1
Application number: PCT/US2016/037714
Authority: WO
Inventors: Aprile L. PILON-CLAYTON
Original assignee: Therabron Therapeutics, Inc.
Priority date: 2015-06-15
Filing date: 2016-06-15
Publication date: 2016-12-22

Abstract

The rhCCI O protein facilitates repair and protects against damage to the respiratory epithelium due to exposure to both cigarette and other smoke. The positive effects of rhCCI O are observed regardless of route of administration.

Description

Title

Recombinant Human CC10 Protein Facilitates Repair and Protects Against Damage to the Respiratory Epithelium Due to Exposure to both Cigarette and Other Smoke

Background

Chronic obstructive pulmonary disease (COPD) is a chronic respiratory disease that affects about 30 million people in the US and hundreds of millions worldwide. The most common cause of COPD is cigarette smoking, and the disease often progresses even after smoking cessation. COPD encompasses two lung pathologies that together make up this disease, including chronic bronchitis and emphysema. Chronic bronchitis involves the large and small airways of the lungs, which undergo thickening of the epithelium, increased mucus production, and increased airway rigidity. Emphysema involves the loss of elasticity in the alveoli, that results in "dead space" in the lungs, preventing exhalation of full breaths of fresh air. Many patients have a some combination of these two pathologies. COPD is medically managed using bronchodilators, beta-agonists, and anti-inflammatory agents, as well as oxygen in advanced cases. These treatments do not significantly impact the long term decline in lung function characteristic of this disease and there is no cure.

It was previously reported that COPD patients had lower levels of circulating CC10, lower levels of CC10 in broncho-alveolar lavage fluid (BALF) and lower numbers of Club cells (formerly known as Clara cells) in their airways. Exposure to cigarette smoke is particularly toxic to Club cells, due to the presence of polychlorinated biphenyls ("PCBs') and other chemicals that are metabolized by cytochrome P450s that are specifically expressed in Club cells. More recently, direct measurement of CC10 expressed in airway epithelial cells demonstrates lower levels in COPD patients versus normal individuals; and that the expression of CC10 in airway epithelium decreases with more advanced disease. The airway remodeling that occurs in chronic bronchitis and other types of lung disease is termed bronchial dysplasia. Patients with bronchial dysplasia were shown to have lower levels of circulating CC10 and CC10 in sputum than normal individuals. More recently, two large observational studies in COPD patients, evaluating nearly 7000 patients combined, evaluated dozens of candidate proteins in a search for circulating biomarkers that were representative of the status of the respiratory epithelium in COPD patients (ECLIPSE cohort, 2083 subjects evaluated over 3 years; LHS cohort, 4724 subjects evaluated over 9 years). They both showed that lower circulating CC10 (aka CC16) correlated with a rapid rate of decline in lung function compared to higher circulating CC10 in patients who did not experience rapid loss of lung function. The "rapid decliners" lost lung function, as measured by spirometry, at a rate of about 40 mis/year or more, while COPD patients that had higher levels of circulating CC10 lost lung function at an average rate of about 20 mis/year. Moreover, the concentration of circulating CC10 corresponded with the rate of decline in COPD patients. Thus, circulating CC10 is representative of the status of the respiratory epithelium, particularly the number of Club cells that express CC10 in the airways, and lower circulating CC10 correlates with rapid decline in lung function and increased mortality, in COPD patients.

Some advances in understanding of homeostasis and repair processes in the lung have been made in the last 10 years which highlight the importance of Club cells in the development, immune regulation, maintenance and repair of the airway epithelium. The epithelium of the lung plays a number of important roles. Under normal circumstances, the lung epithelium protects the lung from bacteria, viruses and environmental toxins. When the lung is diseased or injured, cells from the epithelium are important in the repair process and to the re-establishment of homeostasis. Club cells and the major secretory protein, the Club cell 10 kilodalton protein ("CC10"), may play a critical role in all of these processes. For example, CC10 has been found to be deficient in disease states which are marked by lung injury and disregulated repair. (CC10 is also known as uteroglobin, CC16, CCSP, urine protein-1 , and blastokinin.)

Some epithelial surfaces, like that in the small intestine, are characterized by a rapid rate of cell renewal. The lining of the lung, in contrast, has a slower turnover rate due to its defense mechanisms that mitigate injury. The Club cell, although differentiated, appears to serve as a facultative progenitor cell. Club cells can proliferate in response to injury and restore the ciliated cell population of the conducting airway epithelium as well as self-replace. Because of their broad distribution in the lung, these cells can respond to local perturbations by a loss of differentiation and then proliferation/repair. When Club cells themselves are depleted, they appear to be replaced through the activation of local tissue stem cells residing in NEBs and the bronchioalveolar duct junction (BADJ). Both of these types of stem cells express CC10 protein but they do not produce cytochrome P450 as classical Club cells do. CC10 knockout mice are to demonstrate that the CC10-expressing cells are critical for maintenance and normal repair of the airway epithelium.

Smoke inhalation injury (SI I) is associated with a mortality of approximately 30% and is characterized by airway inflammation mediated by chemical irritants in smoke, which often leads to acute respiratory distress syndrome (ARDS). An estimated 20% to 30% of patients affected by burn injuries also suffer 311 , which is one of the three risk factors associated with high mortality in burn victims.

In the pathogenesis of ARDS, the inflammation in the airway causes the migration of neutrophils and monocytes into the lung followed by the release of cytotoxic enzymes and other oxidative agents. The accumulation of these toxic substances is a major contributor to the damage to the airway epithelium and endothelium of the lung parenchyma. As a consequence, gas exchange by the injured alveoli is impaired, leading to hypoxemia and hypercapnia.

Club cell protein 10-kDa (CC10) is the major protein secreted by Club cells and these CC10-expressing cells have been shown to play a critical role in maintaining the integrity of the airway epithelium and in facilitating epithelial repair. CC10 is a potent anti-inflammatory protein with multiple mechanisms of action, including inhibition of phospholipase A2, inhibition of neutrophil chemotaxis, and suppression of NF-kB signaling.

A decrease in native CC10 has been found in ARDS, as well as in chronic respiratory conditions, such as in asthma, COPD, and cigarette smoking-induced bronchial dysplasia. CC10 is also deficient in respiratory distress of prematurity leading to development of neonatal bronchopulmonary dysplasia (BPD) in severely preterm infants. Decreased circulating CC10 has been proposed as a biomarker for some of these inflammation-associated pathologies and it has also been suggested that CC10 plays a role in the pathophysiology of these conditions.

Efficacy of recombinant human CC10 (rhCCI O) has been evaluated in several animal models of acute lung injury, in which improvements in pulmonary inflammatory mediators, decreases in pulmonary vascular permeability, protection and preservation of pulmonary architecture, mechanical lung function, and gas exchange were observed. Importantly, rhCCI O has been shown to decrease airway inflammation in preterm neonates with respiratory distress.

Reactive oxygen species (ROS) are generated during acute severe inflammatory responses in acute lung injury (ALI) and respiratory distress, including 311. CC10 is oxidized by ROS in tracheal aspirates of preterm infants experiencing respiratory distress and the oxidatively modified CC10 isoforms correlate with greater incidence of death or BPD.

In the present study, we assessed the effects of rhCCI O on survival and lung function in a well-characterized ovine model of ARDS induced by smoke inhalation. Specifically, the potential of rhCCI O protein to control the overwhelming inflammatory response associated with high morbidity and mortality following smoke inhalation lung injury was evaluated. We hypothesized that rhCCI O attenuates pulmonary dysfunction in an ovine ARDS model by reducing an excessive airway inflammatory response.

As discussed above, the lung epithelium can be damaged by smoke inhalation. It was unknown whether CC10 by itself could facilitate repair of this damage or provide any protective effect to damaged lung epithelium. Therefore, what is needed is a reliable effective method of reversing and protecting against damage to the lung epithelium due to cigarette and other smoke using recombinant human CC10.

Summary of the Invention

In aspects of the present invention, secretoglobins modify airway remodeling indirectly by restoring normal numbers of Clara cells and their associated structures, termed neuro-epithelial bodies (aka NEBs) or neuroendocrine cell clusters (aka NECs) that are identified by their immunoreactivity to anti-CGRP1 antibodies, in the airway epithelium. The Clara cells and other CGRP1 + cells, then secrete these secretoglobins and other components of the normal mucosal milieu, contributing to homeostasis and normal functioning of the respiratory mucosa and epithelium that is then more resistant to inhaled challenges without experiencing severe exacerbations."

The difference between the present examples and previous data being that we have directly shown in an animal model that treatment with rhCCI O can maintain circulating CC10 levels in a non-human primate model of cumulative damage to the respiratory epithelium caused by daily exposure to cigarette smoke over an 8 week period of time. Since the primary source of CC10 in the blood is the Club cells in the airway epithelium, loss of circulating CC10 corresponds to loss of Club cells. Therefore, exogenous rhCCI O protected the airway epithelium and Club cells from the cumulative damage caused by daily cigarette smoke exposure over the 8 week period, thereby preventing loss of circulating CC10 levels when the cumulative damage resulted in such loss in the placebo-treated animals.

We provide further direct evidence that rhCCI O protects the airway epithelium in an acute cotton smoke exposure model in sheep.

Further aspects focus on methods of use for recombinant secretoglobin proteins in inhaled smoke and toxin exposures

Brief Description of the Drawings

Fig. 1 is an example of CC10 levels in placebo plasma

Fig. 2 is an example of CC10 levels in plasma of where rhCCI O has been administered. Fig. 3 is an example of bronchial obstruction in experimental groups. Fig. 4 is an example of bronchiolar obstruction in experimental groups.

Fig. 5 is an example of survival portions 48 hours after injury. Fig. 6 is an example of Pa02/Fi02 ratio, oxygenation and shunt fraction.

Fig. 7 is an example of peak inspiratory pressure, bronchial obstruction and bronchiolar obstruction.

Fig. 8 is a fluid balance, plasma protein at 48 hours, alveolar edema and alveolar hemorrhage

Fig. 9 is an example of alveolar neutrophils, myeloperoxidase in lung homogenate, protein carbonyl and tissue samples.

Fig. 10 is an example of CC10 in plasma, CC10 gene expression, CC10 protein level and CC10 in lung tissue by Western blot.

Fig. 1 1 is an example of HPLC monitoring of oxidizing reactions of rhCCI O in vitro. Panel A is rhCCI O reacted with MPO and H202; Panel B is rhCCI O reacted with mCPBA; Panel C: rhCCI O reacted with NaOCI. The arrows show unmodified rhCCI O prior to each reaction. The extent of the reactions was a function of the number of oxidizing equivalents used or the length of time for the reaction (not shown). About 25 micrograms of protein was injected for each run. These represent optimized conditions.

Fig. 12 is an example of isolation of individual peaks for ESI-MS analysis. Panel A are reaction product peaks were numbered and isolated by collecting HPLC fractions, 40- 100 micrograms of each peak were collected from multiple runs. Panel B is isolated peaks were re-injected to verify purity. Panel C is de-convoluted ESI-MS signals for isolated peaks.

Fig. 13 is an example of a Western blot of SDS-PAGE using anti-DNP antibody on DNPH-reacted samples. Lane 1 is NaOCI-reacted rhCCI O; Lane 2 is unreacted rhCCI O (same lot). Approximately 5 micrograms of oxidized rhCCI O per lane were loaded, samples were not reduced.

Fig. 14 is an example of an isoelectric focusing of ROS-reacted rhCCI O. About 25 micrograms of rhCCI O were loaded in each lane. Detailed Description of the Invention

In the following detailed description of embodiments of the invention, numerous specific details are set forth in order to provide a thorough understanding of the embodiment of invention. However, it will be obvious to a person skilled in art that the embodiments of invention may be practiced with or without these specific details. In other instances well known methods, procedures and components have not been described in details so as not to unnecessarily obscure aspects of the embodiments of the invention.

Furthermore, it will be clear that the invention is not limited to these embodiments only. Numerous modifications, changes, variations, substitutions and equivalents will be apparent to those skilled in the art, without parting from the spirit and scope of the invention.

Broadly, the present invention provides that exogenous rhCCI O may be used to both repair and prevent damage to the respiratory epithelium due to chronic inhalation exposure to cigarette smoke or to acute inhalation exposure to other smoke. Smoke inhalation injury induces a severe, often lethal, lung injury characterized by severe pulmonary inflammation, epithelial exfoliation, airway obstruction, pulmonary hemorrhage, and pulmonary edema. The ovine Sll model was established not only to simulate pulmonary injury and dysfunction, but also to simulate clinical practice in critically ill patients experiencing acute lung injury and ARDS.

Treatment with intravenously delivered rhCCI O given 1 hr post-injury reduced pulmonary inflammation, improved gas exchange and lung biomechanics, protected pulmonary architecture, and improved survival at 48 hrs post-injury from 73% to 93%. The improvement in lung function is consistent with previous findings in which intravenously delivered rhCCI O protected pulmonary architecture, reduced pulmonary inflammation, and improved lung function in a rabbit model of ARDS. As a PLA2 inhibitor, rhCCI O also prevents the degradation of lung surfactant, thus preserving lung function. As an inhibitor of NF-kB signaling in airway epithelial cells, rhCCI O can be expected to suppress the downstream inflammatory response in ALI, as observed in several other models of ALI in which rhCCI O was evaluated. CC10 has been reported to be beneficial, in premature infants with respiratory distress.

Native CC10, also known as CCSP, CC16, uteroglobin, and urine protein-1 , is the primary secretory product of non-ciliated respiratory epithelial cells lining the airways, including Club cells. Club cell subpopulations include progenitor stem cells in the airways that re-populate the epithelium following injury. In addition to its antiinflammatory and anti-fibrotic properties; the CC10 protein is thought to play a role in the repair of the respiratory epithelium post-injury. Administration of rhCCI O has been shown to facilitate repair of the pulmonary epithelium following naphthalene injury in a complex mouse model, increasing the number of Club cells.

The histopathology assessment of large and small airway obstruction in the present study suggests that rhCCI O not only suppressed damaging inflammation in the lung but also may have facilitated repair of the airway epithelium post-injury. Although the mechanistic aspects of CC10 are not completely understood, the overall improvement in lung biomechanics mediated by rhCCI O may be a result of the decreased inflammatory response and/or accelerated epithelial repair, resulting in decreased epithelial sloughing, bronchospasm, and mucus obstruction. Whether due to decreased inflammation, accelerated epithelial repair, or a combination of the two, preservation of pulmonary architecture, including airway integrity, and lung compliance mediated by rhCCI O directly correlated with improved gas exchange. Pa02 Fi02 ratio, the main indicator of the gas exchange, improved with rhCCI O treatment in a dose-dependent manner. The improvement of this outcome by itself is particularly relevant, as it is primarily correlated with the mortality of SII-ARDS patients. The deterioration in 01 was also attenuated by rhCCI O, although the difference in 01 was only significant between 24-48 hrs and at the 48 hr endpoint in the high dose group (10 mg/kg/d). Likewise, the shunt fraction was significantly inhibited using high dose rhCCI O during the 24-48 hour timeframe, suggesting that CC10 may have also improved pulmonary oxygenation through inhibiting pulmonary shunt fraction. These parameters integrate both gas exchange and lung biomechanics; contributing to the overall effect of high dose rhCCI O in attenuating the severity of ARDS. These improvements in lung function are consistent with previous experiments using various animal models of acute lung injury, particularly in a rabbit model of ARDS using intratracheal and intravenous routes of administration for rhCCIO, and in a preterm lamb model of infant respiratory distress syndrome showed that intratracheal instillation of rhCCI O, administered shortly after lung surfactant resulted in improvement of lung compliance and PIP, compared to surfactant alone.

Consistent with previous observations, CC10 decreased neutrophil myeloperoxidase activity (MPO) in lung tissue, as well as number of neutrophils in lung tissue. This is consistent with the observation that native rabbit uteroglobin (aka CC10) inhibits fMPL- induced chemotaxis of human neutrophils in vitro. In the absence of CC10, knockout mice exhibit exaggerated neutrophil responses to various respiratory pathogens and inhaled insults. Thus, the property of CC10 and rhCCI O to inhibit neutrophil infiltration of the lungs in ALI is well-documented. CC10 also inhibited the number of neutrophils in tracheal aspirate fluid in a small phase I clinical trial in premature infants that received a single intratracheal dose of rhCCI O.

Carbonyl groups on proteins are generated through interactions with reactive oxygen species (ROS), which are in turn generated by MPO plus H2O2 during ALI. Therefore, the rhCCI O-mediated dose-dependent decrease in protein carbonyl content observed in lung tissue is highly consistent with the decrease in neutrophils and MPO activity in lung tissue. It is also consistent with our observation that rhCCI O can absorb several oxygen radicals, including up to 6 methionines oxidized per dimer after which other amino acids are oxidized, thereby revealing a novel anti-inflammatory and protective mechanism of rhCC10 as a scavenger of ROS.

Another compelling observation was that the rhCCI O decreased systemic vascular leak of both fluid and protein in addition to improved lung function and anti-inflammatory effects. This may also have contributed to improved survival in this model. rhCCI O suppressed pulmonary vascular leak in previously reported models, and in human preterm infants, as measured by total protein in TAF or BAL, but none of those studies evaluated systemic vascular permeability. Since fluid balance was not monitored in these earlier studies, it is unclear whether rhCCI O affected systemic vascular permeability in previous studies. Sheep lung histopathology scores for alveolar edema and pulmonary hemorrhage assessed at 48 hrs confirmed rhCCI O-mediated reduction in pulmonary edema and vascular leak, which was significant in the high dose rhCCIO group.

The pulmonary edema characteristic of ARDS is primarily attributed to the impairment of endothelial barrier integrity and only partially attributable to a defect in the epithelial barrier that maintains homeostasis in the alveoli; therefore, the attenuated systemic vascular leak and the attenuated alveolar edema likely share a common pathway.

As previously mentioned, CC10 is one of the most abundant proteins secreted by Club cells in the healthy airway, and several studies have shown that it decreases in lung tissue, tracheal aspirate fluid, or broncho-alveolar lavage fluid during acute inflammatory airway conditions, while circulating CC10 in plasma or serum increases during acute lung injury. Circulating CC10 is also emerging as a biomarker that correlates with loss of lung function and loss of Club cells with progressive airway remodeling in chronic lung diseases such as COPD and BOS. However, transient increases subsequent to dosing, there were no significant net changes in plasma CC10 between baseline and 48 hrs for any of the groups, including sham, in our study. We measured CC10 gene expression in lung tissue, which was significantly decreased in the control group and the lowest rhCCI O dose group compared to the sham group, while the two highest rhCCI O dose groups were not different than the sham group. Likewise, CC10 protein was significantly decreased in lung tissue (but not in plasma) in all injured groups compared to the sham group, and there was a dose-dependent trend towards increased CC10 in lung tissue, confirming that rhCCI O can be delivered to the lung using intravenous administration, as previously observed in rabbits. The treatment with high doses of rhCCI O attenuated the decrease in lung CC10 mRNA caused by 311 , suggesting that the rhCCI O protected Club cells in the airway epithelium. It is not clear whether the elevated CC10 protein content in lung in the high dose rhCCI O groups is due the presence of ovine CC10 or human CC10, since the two could not be distinguished in our ELISA. However, a previous study in human preterm infants suggested that intratracheal rhCCI O suppressed expression of native human CC10 3-5-fold, with the higher dose mediating greater suppression. The fact that the opposite has been observed in the Sll model suggests that preservation and protection of Club cells in the respiratory epithelium may offset suppression of the native CC10 gene such that a net increase of CC10 mRNA and CC10 protein was observed at higher doses.

With respect to overall effects mediated by rhCCI O, these results demonstrate that the optimal dose tested is 10 mg/kg/d. This dose conferred the greatest potential benefit in greatest number of outcomes, with no apparent adverse effects. The 3 mg/kg/d dose showed a benefit in many of the measured outcomes and even the 1 mg/kg/day dose demonstrated an anti-inflammatory effect in terms of reduced neutrophils in the alveoli. Hence, a suboptimal dose lower than 10 mg/kg/d could provide some benefit as well. A novel mechanism of action for rhCCI O, scavenging of ROS, was also uncovered. In summary, based on the current results and in correlation with previous findings, rhCCI O mediated a therapeutic anti-inflammatory effect in the airway and reduced systemic vascular permeability, which attenuated lung dysfunction and the severity of ARDS, and improved survival in smoke inhalation lung injury.

Example 1

Chronic inhalation exposure to cigarette smoke in non-human primates (NHPs)

Eight female NHPs (Cynomolgus macaques) underwent whole body exposures to cigarette smoke with target exposure concentration of 250 mg/m³ for five days per week (Monday-Friday) over a period of 8 weeks. This treatment is designed to simulated cumulative damage to the respiratory epithelium and lungs experienced in human cigarette smokers and COPD patients. After the first four weeks of cigarette smoke exposure (CSE), animals were treated with either placebo (0.9% NaCI) or rhCCI O (2.25 mg/m I in 0.9% NaCI) for the second four weeks of CSE. The placebo and rhCC10 were administered twice daily, approximately 12 hours apart, for five days per week (Monday - Friday) by intranasal administration. Each dose of either placebo or rhCCI O was given in ~100 microliter volume to each nostril for a total dose of 0 or 900 mg rhCC10/day. The placebo and drug were provided in nasal spray bottles that were labeled only with sequential numbers so that study staff was blinded to the treatment. Plasma samples were collected at 4 timepoints for the purpose of measuring changes in CC10 levels over time as follows: 1 ) three days prior to initiation of CSE, 2) Day 14 after initiation of CSE, 3) Day 42 after initiation of CSE, and 4) Day 53 after initiation of CSE. The CC10 level in the plasma samples was measured using a competitive ELISA to human CC10. This ELISA does not distinguish between human and NHP CC10 proteins, because they are very similar to each other. Fig. 1 shows the change in plasma CC10 levels over time in the placebo group.

Plasma CC10 levels were relatively constant in all four animals in the placebo group until the last two weeks of CSE, when the cumulative exposure resulted in a significant decrease in plasma CC10 between Day 42 and Day 53 of CSE and the mean decrease was 48.7 ng/ml.

However, in the rhCCI O-treated group, this decrease signifying critical damage to the respiratory epithelium and loss of Club cells was not observed. Instead, two of four animals experienced no change and the other two increased plasma CC10, one of them dramatically. The mean plasma CC10 concentration increased between Day 42 and Day 53 was 13.6 ng/ml, as shown in Fig. 2. The two groups were statistically different with respect to the change in CC10 levels between Day 42 and Day 53 (p=0.015; Student T-test), indicating that exogenous rhCCI O facilitated repair, protected, and preserved of the respiratory epithelium despite previous and ongoing CSE.

In order to understand the pharmacokinetic profile of intranasally administered rhCCI O, an additional set of plasma samples were collected on Day 28, the day on which treatment with either placebo or rhCC10 was initiated, after the very first dose. Samples were collected at pre-dosing, 30 minutes, 1 , 2, 4, and 24 hours after dosing and the CC10 in the samples was measured using the competitive CC10 ELISA. However, no significant increases in plasma CC10 were detected, even in the 30 minute post-dose timepoint, indicating that intranasal delivery did not result in measurable increases in plasma CC10. The circulating half-life of CC10 is 2-2.5 hours and that the half-life decreases with increasing dose, suggesting that there is an active mechanism to eliminate excess CC10 from the blood. The lack of any measureable increase in plasma CC10 following the first intranasal dose of rhCCI O, together with the known short half-life of circulating CC10 in mammals, and long term improvements in chronic respiratory morbidity in premature infants following a single large dose of rhCCI O, decreases the likelihood that the stabilization of plasma CC10 in treated animals is due merely to the accumulation of rhCCI O in the blood of treated animals by Day 53. Rather, we consider this as the first proof-of-concept that repeated small doses of rhCCI O can mediate a long term increase in circulating CC10 by protecting and facilitating repair of the respiratory epithelium during chronic lung injury due to CSE. The increase in plasma CC10 between Day 42 and 53 in one of four rhCCI O-treated animals even indicates the reversal of damage due to cumulative CSE and the actual rehabilitation and improvement of respiratory epithelium.

Example 2

Acute exposure to cotton smoke in adult sheep

About 13,000 people die each year in the US due to smoke inhalation in fires. Smoke inhalation lung injury can be simulated in adult sheep. Referring now to Figs. 3 and 4, a total of 36 adult female sheep were exposed to cool cotton smoke insufflation (48 breaths) via tracheostomy, then followed and monitored for 48 hours. Animals were treated after exposure to cotton smoke with either placebo (0.9% NaCI; n=7), 0.5 mg/kg rhCCI O (n=8), 1 .5 mg/kg rhCCI O (n=7), or 5 mg/kg rhCCI O (n=8), and sham-injured animals that were instrumented but not exposed to smoke and not treated with either placebo or rhCCI O. The placebo and rhCCI O treatments were administered by intravenous bolus starting one hour after smoke exposure, then treated every twelve hours for a total of four doses during the 48 hour study period. At the conclusion of the study the animals were euthanized and a histopathology analysis was performed on lung tissue to evaluate cellular and structural changes. The most relevant result with respect to smoke and smoke-toxin induced changes to the airway epithelium were in bronchial and bronchiolar obstruction scores shown in Figs. 3 and 4, respectively. Obstruction of the bronchi and bronchioles represents damage to the respiratory epithelium in the large and small airways of the lungs. The smoke exposure lasts no more than a few minutes, however, it initiates a process of cellular damage and necrotic cell death over the ensuing hours that culminates in sloughing of epithelial cells and debris into the lumen of the airways. This is a dire effect that often results in death within a few hours to a few days; with the mortality rate in this model being about 40%. Treatment with 10 mg/kg/day CC10 showed a significant decrease in bronchial obstruction vs. placebo control (p<0.05); the 1 and 3 mg/kg/d groups were not different than the placebo control. All injured groups had significantly higher bronchial obstruction than the sham group. The control and 1 mg/kg/d groups had significantly increased bronchiolar obstruction vs. sham. The 3 and 10 mg/kg/d groups were not statistically greater than sham but were also not different from control. Pharmacokinetic analyses were consistent with a circulating half-life of 2-3 hours for rhCCI O.

Table 1 shows mortality by 48 hours. The percent mortality in the low dose rhCCI O (1 mg/kg/day) group was essentially the same as in the placebo group. However, the percent mortality decreased by over 4-fold (28.6% vs. 6.7%) in the combined middle and high dose rhCCI O (3 and 10 mg/kg/day) groups. Therefore, rhCCI O lowers the risk of death by at least 50% and appears to impact survival in this model.

Table 1 : Death before 48 hour endpoint

In more detail, thirty-six adult female sheep (30-40 kg) were surgically prepared under deep isoflurane anesthesia and buprenorphine analgesia to locate various access ports for hemodynamic assessment and blood sampling. Catheters were located in the left atrium, pulmonary artery, as previously described. Following surgical preparation, the animals were allowed to recover for 5 to 7 days under buprenorphine analgesia, with free access to food and water and with a basal infusion of lactated Ringer's solution (2 mL/kg/hour).

The day of the study, following the collection of baselines for hemodynamics and blood samples, smoke inhalation injury was induced as previously showed. Injury consists of insufflation of 48 breaths of cool cotton smoke, under deep anesthesia and pain control. Following injury, animals were awakened, placed on a mechanical ventilator and randomized into one of four groups. Control group (injured and treated with 60 mL of saline every 12 hours, n=7), CC10-1 group (injured and treated with rhCCI O at a dose of 1 mg/kg/day, n=8), CC10-3 group (injured and treated with rhCCI O at a dose of 3 mg/kg/day, n=7) and CC10-10 group (injured and treated with rhCCI O at a dose of 1 mg/kg/day, n=8). Sham animals were instrumented but not injured and treated with saline mimicking rhCCI O treatment. rhCCI O and placebo were administrated as an i.v. bolus via the central venous catheter every 12 hours (twice per day) starting one hour after the injury. Hemodynamics, pulmonary function, and blood gasses were recorded every 6 hours. Urine and plasma samples were collected as well. Animals were monitored for 48 hours post-injury, then sacrificed and lungs harvested as previously described. Criteria for euthanasia prior to 48-hour endpoint were Pa0₂ < 50 mmHg or PaC02 > 90 mmHg for one hour regardless of adjustments of F1O2, respiratory rate and pulmonary toilet.

Fluid resuscitation, assessment of fluid balance and plasma protein

After the injury, the animals were resuscitated with Ringer's lactate (4 ml. per percent burned surface area per kilogram of body weight over 24 hours), with half of the daily fluid requirement delivered in the first 8 hours. The majority of patients suffering from smoke inhalation injury are affected by large cutaneous burn injuries; therefore, we provided a fluid plan close to what a burn patient receives. To accurately monitor fluid balance during the study, the sheep had free access to food, but the water was restricted. Urinary output was assessed with a urinary bladder catheter (14Fr). Total protein concentration in fresh plasma was evaluated using a refractometer (National Instrument, Baltimore, MD), as it has shown to correlated well with the colloid osmotic pressure.

Mechanical ventilation and lung biomechanics

All sheep were mechanically ventilated in a volume control mode with a positive end- expiratory pressure (PEEP) of 5 cm H₂0, a tidal volume (Vt) of 12 mL/kg and a respiratory rate (RR) of 20 breaths/minute. During the first 3 hours after injury, the inspired 0₂ (Fi0₂) concentration was maintained at 100% to induce rapid clearance of carbon monoxide after smoke inhalation. After 3 hours, F1O2 was adjusted to keep the arterial Pa0₂ close to 100 mm Hg. The PCO₂ was maintained between 25 and 35 mm Hg by adjusting the RR. The airway pressures were recorded every 6 hours and the mean airway pressure (Paw) and oxygenation index (Ol) calculated with a standard equation. Hemodynamics and blood sample analysis

Through the various catheters surgically placed, mean arterial pressure (MAP), pulmonary artery pressure (PAP), left atrium pressure (LAP), the pulmonary capillary occlusion pressure (PCOP) and the core blood temperature were continuously measure with a hemodynamic monitor and recorded every 6 hours. Simultaneously, blood was sampled from the femoral catheter every 6 hours and the PO2, PCO2, pH, base excess, carboxyhemoglobin (COHb), S0₂, hematocrit, hemoglobin, glucose, lactate and electrolytes measured using a blood gas analyzer.

Lung tissue analysis

Immediately after euthanasia, a section of the right inferior lung was taken and stored in formalin for histopathology analysis using H&E staining as previously described. Lung samples from the same region of the right lung were also taken and frozen immediately in liquid nitrogen and stored at -80°C for biochemical analysis.

Biochemical lung analysis mRNA: Messenger RNA was extracted from lung tissue samples using an RNeasy™ Mini Kit from Qiagen (an RNA extraction kit), then CC10 mRNA was measured using q- PCR. Cyclophilin was used as the control for mRNA quantitation.

CC10 ELISA: Protein was extracted from lung tissue by grinding frozen tissue using a mortar and pestle then homogenized in PBS buffer using a sonicator and 3 cycles of freezing and thawing at -80°C and room temp. The PBS extract was then centrifuged at 12,000 g and the supernatant was analyzed for total protein content by BCA (Pierce- Thermo-Fisher™) and total CC10 content (combination of native ovine and rhCCI O) by competitive CC10 ELISA.

MPO activity: 100 mg of frozen lung tissue was ground using a mortar and pestle, then resuspended in 1 mL cold 50 mM KP0₄, pH 6.0), vortexed, and centrifuged at 10,000g for 10'. The pellet was resuspended in 0.5% hexadecyltrimethylammonium bromide (CTAB), 50 mM KP0₄, pH 6.0, then subjected to 3 freeze-thaw cycles at -80°C and room temperature, sonicated at 20 kHz for 40", then centrifuged at 10,000 g for 5'. MPO activity was measured by incubating 10 μΙ CTAB extract in 100 μΙ substrate buffer (50 mM KP0₄, pH 6.0, 0.2 mg/mL o-dianisidine, 1 mM H₂0₂) in microtiter plates at room temperature for 5' then read at 450 nM. Standard curves were generated with MPO (Calbiochem). Blanks containing CTAB extract plus buffer were also done on each plate and all samples were analyzed in duplicate. MPO activity was normalized to total protein concentration measured by BCA assay (Pierce Thermo-Fisher)) and data were expressed as units MPO activity per milligram of total protein.

Carbonyl ELISA: A Protein Carbonyl ELISA kit (Cell Biolabs) was used to evaluate carbonyl content in CTAB extracts according to the manufacturer's instructions). Data are expressed as nmol of protein carbonyl/mg of total protein.

Example 3

Recombinant ovine CC10 and anti-ovine CC10 antibody

A gene encoding ovine CC10 was synthesized using the reported mRNA sequence (Genebank accession #FJ959385). Recombinant ovine CC10 was over-expressed in bacteria as a fusion with an His-tagged ubiquitin-like protein using a T7 expression system in E. coli strain BL21/DE3, then purified to >95% purity by SDS-PAGE, using IMAC chromatography. Approximately 6 mg of recombinant ovine CC10 (roCCI O) was produced. Two rabbits were immunized with roCCI O and IgG was purified from antisera. Both antibodies recognized reduced and non-reduced roCCI O and native ovine CC10 in lung tissue by Western blot (Fig. 10). Both antibodies also recognized rhCCI O using Western blot (Fig. 10). Rabbit polyclonal anti-rhCC10 antibodies were also found to cross react with roCCI O and native ovine CC10 (not shown).

Pharmacokinetic Analysis

Plasma samples were collected at baseline prior to injury and dosing with rhCCI O, at 3 hours post-injury (2 hrs after the first dose of rhCCI O), 27 hrs post-injury (2 hrs after the second dose), and 48 hrs, post-injury (1 1 hrs after the fourth and last dose). All samples were stored at -80°C until analyzed. Total CC10 (combination of native ovine CC10 and rhCCI O) was measured in plasma by competitive ELISA as previously described (25).

In vitro modification of rhCCIO by ROS

In an effort to simulate in vivo reactions, three types of reactions were used to modify rhCCI O in vitro: 1 ) Neutrophil myeloperoxidase (MPO) + H₂O₂: rhCCI O was pre- incubated for 30' at 37°C in 2 mM CaCI₂ and 10 mM citrate buffer (pH 5.0) prior to addition of 10 mg/mL MPO and 25 equivalents of H₂O₂ then incubated in the dark for 30' at 37°C. After 30', the reaction was boosted with another aliquot of MPO and H₂O₂ and incubated for a further 30' at 37°C. 2) Meta-chloroperbenzoic acid (mCPBA): Reactions were performed at room temperature (24-27°C), incubated for 15' in the dark in a total volume of 0.2 mL. Reactions were initiated by adding the mCPBA. 2-100 oxidizing equivalents were used and the reactions were monitored by RP-HPLC. 3) Sodium hypochlorite (NaOCI): Reactions were performed on ice (~4°C) in the dark in a total volume of 0.2 mL. Reactions were initiated by adding the NaOCI and quenched by adding 0.1 M L-methionine. 1 -100 oxidizing equivalents were used and the reactions were monitored by RP-HPLC. All reactions were stopped by adding 0.1 M L-methionine.

Analysis of ROS-reacted rhCCIO

RP-HPLC was performed on an Agilent™ 1100 system using a VYDAC Polymeric C18 Column 300A, 5 micron, 2.1 mmx250mm, (Cat #218TP52) using a mobile phase as follows: A: water; B: 95% acetonitrile + 5% water (both contain 0.1 % TFA) at a flow rate of 0.3 mL/min. Output was monitored by UV absorption at 214 nm. Individual peaks were isolated by RP-HPLC and analyzed by electrospray mass spectrometry to measure the intact mass of 17 peaks in mCPBA and MPO reactions. The presence of carbonyl groups was demonstrated by reaction with dinitrophenylhydrazine (DNPH) followed by Western blot of SDS-PAGE gels using a rabbit polyclonal anti-DNP antibody. Reaction products were also analyzed by isoelectric focusing using commercially prepared pi 3-7 gels. Statistical analysis

Sampling distribution of the collected data was first assessed by with Shapiro-Wilk normality test, and non-normal distributed variables were log converted for further analysis. Analysis of the treatment effect of one parameter over time was evaluated using a Linear Mixed Model and protected pairwise comparisons were used to determine which of the treatments has a different effect. Comparison between treatment groups at different time points was performed using a two-way analysis of variance (ANOVA) followed by adjusted pairwise comparison. Sets of data of a single time point such as lung tissue samples were analyzed using one-way analysis of variance followed by adjusted pairwise comparison. The mortality among groups was evaluated with a log-rank test adjusted for multiple comparisons. Values reported are expressed as mean ± SEM. The differences were considered significant when the p-value was smaller than 0.05.

Results

The degree of injury was comparable among injured groups as indicated by the COHb levels following smoke inhalation, the percent of bronchial exfoliation and the development of hypoxemia.

COHb levels immediately after smoke inhalation injury were comparable among all injured groups (p=0.28) as the maximal COHb was 70.8 ± 7.6, 79.4 ± 3.8 and 83.2 ± 3.5 and 72.3 ± 5.5% for the Control, CC10-1 , CC10-3 and CC10-10 groups respectively and 5.3 ± 0.6% for the Sham group. Histopathology analysis also indicated a similar exfoliation of the bronchial epithelium among injured groups following the termination of the study (p=0.32, Kruskal-Wallis test). The percentages of bronchial epithelial exfoliation were 85.7 ± 9.2, 93.8 ± 3.2 and 90 ± 7.2 and 79.4 ± 6.6% for Control, CC10- 1 , CC10-3 and CC10-10 groups respectively and 1 .7 ± 1 .7% for the Sham group. PaO₂ FiO₂ ratio decreased below 300 mm Hg without a relevant increase in the left atrium pressure in all injured groups during the study with a PEEP of 5 cm H₂O (Fig. 6A). In this model the hypoxemia with a maintained left heart pressure partially meets the established criteria for ARDS. Dose-dependent increases in CC10 protein concentration in the lung and plasma

Because the anti-human CC10 antibody used in the competitive ELISA is cross-reactive with ovine CC10, the results of the ELISA reflect some combination of native ovine CC10 and rhCCI O. Measurement of total CC10 concentration in plasma at 3 hours post-injury (2 hours after i.v. bolus rhCCI O administration), the concentration of CC10 in the Control group decreased to 3.2 ± 0.7 ng/mL compared to 6.4 ± 2.1 ng/mL in the Sham group (0.5 ± 0.1 fold). There was a dose-dependent increase in total CC10 in the CC10-1 , CC10-3 and CC10-10 treatment groups, with CC10 concentrations of 15.1 ± 2.4, 26.4 ± 6.1 and 66.5 ± 13.6 ng/mL corresponding to 2.4 ± 0.4, 4.2 ± 1 and 10.5 ± 2.1 fold increases, respectively, vs. the Sham group (Fig. 1 1 ).

In lung tissue, the level of CC10 protein was significantly reduced in the Control group vs. the Sham group. The CC10 levels in the CC10-1 , CC10-3, and CC10-10 treatment groups were higher than the Control group and lower than the Sham group, although this difference did not reach significance (Fig. 1 1 ).

CC10 mRNA is decreased in SI I .

Similarly to the CC10 protein levels, the CC10 mRNA was statistically reduced in the Control and CC10-1 groups (74.4 ± 0.1 and 62.3 ± 0.1 % reduction) vs. the Sham group. In the CC10-3 and CC10-10 treatment groups, the CC10 gene expression had a milder reduction with no statistical difference (48.7 ± 0.1 and 52.5 ± 0.1 %) vs. the Sham group and was slightly higher than the Control group (Fig. 1 1 ). The 48-hour survival is improved with rhCCIO (high dose)

All studied animals survived the first 24 hours. The 48 hours survival was 5 out of 7 in the control group, 6 out of 8 in the CC10-1 group, 7 out of 7 in the CC10-3 group, 7 out of 8 in the CC10-10 group and 6 out of 6 in Sham group. Comparisons among groups indicated that the survival of the 15 animals treated in the combined 3-mg/kg/d and 10- mg/kg/d rhCCI O groups was statistically longer than the Control group (93% vs. 73%). The group treated with 1 -mg/kg/d rhCCI O did not improve the survival vs. the Control group (75% vs. 73%) (Fig. 9).

Deterioration of gas exchange is attenuated with the highest doses of rhCCIO

The decrease of Pa0₂/Fi0₂ ratio below the ARDS reference value of 300 mmHg was evident in all Sll-induced animals. Although the decline of this parameter was attenuated in the CC10-3 and CC10-10 treatment groups, as they had a mean ARDS by the end of the study equivalent for mild ARDS (between 200 and 300 mmHg) compared to the CC10-1 and Control groups that exhibited a mean Pa0₂/Fi0₂ ratio equivalent for moderate ARDS (between 100 and 200 mmHg). As indicated by linear analysis, the greatest difference was between CC10-10 group and Control group. Multiple comparisons among groups indicated that the Pa0₂/Fi0₂ ratio decreased in the injured groups vs. the Sham group between 24 and 48 hours. However, comparison among injured groups indicated that the Pa0₂/Fi0₂ ratio was significantly attenuated in the CC10-10 group between 24 and 48 hours post injury (237.4 ± 36.6 mmHg, 48 hours) and in the CC10-3 treatment group between 42 and 48 hours (231.7 ± 53.2 mm Hg, 48 hours) vs. the Control group (128.9 ± 29.8 mm Hg, 48 hours). The Pa0₂/Fi0₂ ratio at 48 hours in the Sham group was 524 ± 26 mm Hg (Fig. 6). The oxygenation index (01) was significantly lower in all injured groups compared to the Sham group, indicated by linear analysis. However, the decrease in 01 was attenuated in the CC10-3 and CC10-10 groups. Multiple comparisons among groups indicated that the CC10-10 treatment group had a significantly lower Ol between 30 and 48 hours (6.6 ± 1 .8, 48 hours) vs. the Control group (1 1.4 ± 2.4, 48 hours). Similarly, the CC10-3 treatment group had a lower Ol between 36 and 42 hours (6.9 ± 3.7, 42 hours) vs. the Control group (13.6 ± 3.7, 42 hours). The Ol at 48 hours in the Sham group was 1 .7 ± 0.1 (Fig. 6).

In the same context, the pulmonary shunt fraction (Qs/Qt) was increased in all injured groups vs. the Sham group (indicated by linear analysis). Multiple comparisons among groups indicated that the Qs/Qt was statistically lower in the CC10-1 group at 42 hours and in the CC10-10 group at 30, 42 and 48 hours vs. the Control group (CC10-10: 0.29 ± 0.03 vs. Control: 0.39 ± 0.05, 48 hours) (Fig. 6). How about CC10-3?

High dose rhCCIO attenuates the increase in airway pressure and airway obstruction.

The peak inspiratory pressure (PIP) was significantly increased in all the injured groups vs. the Sham group (indicated by linear analysis). Treatment with 10 mg/kg/d of rhCCI O showed a significant decrease of the PIP. Furthermore, a decreasing trend (p=0.07) in the PIP was observed in the CC10-3 group. Multiple comparisons among groups showed a significantly decreased PIP in the CC10-10 group vs. the Control group at 30 hours and as well as a slight reduction at 48 hours (CC10-10: 27 ± 3 vs. Control: 30 ± 2 cm H₂O, 48 hours) (Fig. 6). The lung compliance was also significantly decreased in all the injured groups vs. the Sham group, as indicated by linear analysis (Fig. 6). The bronchial obstruction score (determined by histopathology analysis) demonstrated that the large airways were obstructed in all the injured groups vs. the Sham group. However, the increase in bronchial obstruction was significantly reduced in the CC10-10 group vs. the Control group (Fig. 6). Correspondingly, the bronchiolar obstruction score demonstrated that the small airways were significantly obstructed the Control and CC10-1 groups vs. the Sham group. In the CC10-3 and CC10-10 groups, the obstruction was significantly reduced vs. the Control group and had no significant difference vs. the Sham group (Fig. 6). rhCCIO decreased systemic vascular hyperpermeability to proteins and water.

Linear analysis indicated that the injured groups had an increase in fluid retention (determined by fluid balance-fluid in and urinary output) during the 48 hours post-injury time. However, the CC10-1 , CC10-3, and CC10-10 treatment groups had attenuated the fluid retention as compared to the Control group. The fluid balance at 48 hours in surviving animals was 2.2 ± 0.4, 1.4 ± 0.4, 1 .7 ± 0.5 and 1.1 ± 0.4 L in the Control, CC10-1 , CC10-3 and CC10-10 groups respectively. Sham group had a fluid balance of

0.7 ± 0.3 L (Fig. 8). In the same context, linear analysis indicated that the injured groups had a decrease in plasma protein concentration with the exception of the CC10-10 group that had no significant difference vs. the Sham group and was significantly greater with respect to plasma protein concentration compared to the Control group. At 48 hours, the plasma protein was reduced by 13.7 ± 0.4% in the Control group and 17 ±

1 , 5.3 ± 3.6, 2.3 ± 4.3% in the CC10-1 , CC10-3, and CC10-10 groups, respectively. Sham group had only a 2 ± 1.1 % reduction in plasma protein levels at the 48 hour time point (Fig. 8). The hemoglobin at 48 hours was 9.7 ± 0.6, 9.8 ± 0.6, 9.5 ± 0.7, 10 ± 0.6 and 9.5 ± 0.6 g/dL in Sham, Control, CC10-1 , CC10-3 and CC10-10, respectively, demonstrating that the fluid resuscitation was comparable among groups (p=0.98). Similar findings were seen with the hematocrit (data not shown). rhCCIO reduces pulmonary edema

The alveolar edema measured by histopathology analysis indicated that the leakage of plasma content was increased in the Control and CC10-1 group vs. the Sham group. The CC10-3 and CC10-10 groups were statistically similar to the Sham group and statistically lower than the Control group (Fig. 8). The histopathology analysis also showed that pulmonary hemorrhage was increased with the injury since the Control group had a significant increase vs. the Sham group. In contrast, the pulmonary hemorrhage in the treatment groups (CC10-1 , CC10-3, and CC10-10) was statistically similar to the Sham group and significantly reduced vs. the Control group (Fig. 8). rhCCIO decreases pulmonary neutrophil infiltration and myeloperoxidase activity

Histopathology analysis of lung tissue indicated that neutrophil accumulation in the lungs was significantly reduced with the three different doses of rhCCI O. The

histopathology scores for neutrophil accumulation were, 0.8 ±0.3, 0.4 ± 0.3 and 0.6 ± 0.2 in the CC10-1 , CC10-3, and CC10-10 groups, respectively, compared to a score of 1.7 ± 0.5 in the Control group (Fig. 9). In lung tissue homogenate, the levels of MPO activity in the Control group increased vs. the Sham group (3.2 ± 0.7 fold). There was a clear dose-dependent decrease in MPO activity in the rhCCI O treatment groups, which was significant in the CC10-3 and CC10-10 groups relative to the Control group (Fig. 9). However, the CC10-3 and CC10-10 groups were also significantly greater than the Sham group. Representative images of lung tissue for each group is shown in Fig. 9. rhCCIO reduced oxidative stress in lung tissue.

The protein carbonyl levels in the Control group were slightly elevated vs. the Sham group (1 .2 ± 0.1 folds). In comparison, the CC10-1 , CC10-3 and CC10-10 treatment groups (0.9 ± 0.1 , 0.8 ± 0.1 and 0.7 ± 0.1 respectively) showed a dose-dependent decrease in protein carbonyl concentration that was significantly lower compared to the Control group (Fig. 9). rhCCIO is oxidatively modified in vitro by ROS

In order to simulate in vivo reactions between CC10 and ROS during acute lung injury, rhCCI O was reacted with neutrophil myeloperoxidase plus hydrogen peroxide and two chemical oxidants; 1 ) mCPBA, a mild oxidant, and 2) NaOCI, a strong oxidant. In the case of MPO, different amounts of MPO were added to reactions to facilitate the reactions, holding all other reaction parameters the same as shown in Fig. 1 1 . Reactions were monitored by RP-HPLC, and the single starting peak representing the rhCCI O homodimer became several peaks as the reaction progressed, and eventually became a single broad peak. Likewise, the number of new peaks increased with the number of oxidizing equivalents in the mCPBA and NaOCI reactions (Fig. 1 1 ).

The new RP-HPLC peaks from the MPO and mCPBA reactions were isolated from pooled reactions by RP-HPLC as shown in Fig. 12. Then the deconvoluted intact mass for each peak was measured by electrospray mass spectrometry. The data are shown in Table 2; peaks 1 -8 were from the mCPBA reaction and peaks 9-17 were from the MPO reaction.

Table 2

All CC10 isoforms had a greater molecular weight (MW) than the unmodified form, which has a MW of 16, 1 10 daltons (Da). The addition of an oxygen adds 16 Da. mCPBA is a mild oxidant and the mCPBA reaction oxidized methionine residues before modifying other amino acids. This is clear since the average mass of 5 of the 8 peaks was increased by a multiple of 16 (eg. peaks 2, 4, 5, 6, and 8). Peak 3 did not increase by an even multiple of 16; this peak contains dimers in which the average number of oxygens is 5.25 or may represent more complex modification than simple addition of oxygen. Peaks 1 and 7 did not yield usable mass spectra. In contrast to the mCPBA isoforms, none of the MPO-H2O2 isoforms showed molecular weight increases that were multiples of 16 (eg. simple additions of oxygen). Modifications under the conditions tested may include some combination of the addition of oxygen, chlorine, or other adducts, as well as the formation of carbonyl groups. NaOCI is a downstream product of MPO+H₂0₂ reactions and the presence of carbonyl groups in the NaOCI-reacted rhCCI O was verified by Western blot using anti-DNP antibody as shown in Fig. 13. No carbonyl groups were detected in the mCPBA reactions (not shown).

In order to further characterize the oxidized rhCCI O reaction products, isoelectric focusing was done. The predicted isoelectric point (pi) of both native human CC10 protein and unreacted rhCCI O is 4.8. Isoelectric focusing of the MPO+H2O2 reactions, shown in Fig. 14, revealed that only 2 new isoforms with altered pis were generated, including a very faint band at pi 5.5 and one or more isoforms at or below 4.7. Unreacted rhCCI O sometimes appears as a major band at 4.8 plus a minor band at 4.7 (possibly dimer and monomer, respectively, which also appear consistently on SDS- PAGE (not shown)). The multiple peaks observed by RP-HPLC (n=8) do not match the number of new bands on IEF (n>2). This indicates that at least 6 of the isoforms separated by RP-HPLC on the basis of hydrophobic interactions retain the same surface charge as unreacted CC10. Western blot of an identical IEF gel verified that these both new isoforms are recognized by a rabbit polyclonal antibody raised against unreacted rhCCI O (not shown).

IEF analysis of the mCPBA and NaOCI reactions was also done. Multiple new bands were generated and at least one new isoform at pi 5.5 was generated by both oxidants. In addition, reactions with NaOCI generated isoforms at pi 4.2 and 4.5, among others. Arias-Martinez (2012) identified native CC10 isoforms in tracheal aspirates of infants with RDS at 4.2, 4.5, and 5.5, among others. Therefore, some of the rhCCI O isoforms generated by ROS reactions in vitro had the same isoelectric points as native CC10 isoforms found in infant tracheal aspirates.

Administration of exogenous CC10 mitigates the damage to the respiratory epithelium due to severe acute and chronic exposure to smoke, including smoke particles and toxins. Once daily administration of intranasal rhCCI O stabilizes circulating CC10 levels and the respiratory epithelium in prior and ongoing cigarette smoke exposure. Twice daily administration of intravenous rhCCI O improves survival and facilitates rehabilitation and repair of the respiratory epithelium following severe acute smoke exposure. Periodic administration of rhCCI O thus stabilizes the circulating levels of endogenous CC10. Further periodic administration of rhCCI O improves survival and facilitates rehabilitation and repair of the respiratory epithelium following severe acute smoke exposure. Methods of administration may be inhalation, intranasal or intravenous or any further method that passes the mucosal membrane.

Claims

In the Claims:

1. A method of rehabilitating, stabilizing, or preserving the respiratory epithelium in a patient having damaged respiratory epithelium comprising the steps of:

(a) diagnosing the patient with COPD, chronic bronchitis or emphysema;

(b) administering rhCCI O to the patient wherein the respiratory epithelium are rehabilitated, stabilized, or preserved.

2. The method of claim 1 wherein the rehabilitating, stabilizing, or preserving occurs during chronic smoke exposure or chronic cigarette smoke exposure.

3. The method of claim 1 wherein the rehabilitating, stabilizing, or preserving occurs following chronic smoke exposure/chronic cigarette smoke exposure.

4. The method of claim 1 wherein the rehabilitating, stabilizing, or preserving occurs following or during exposure to smoke particles and toxins present in smoke.

5. The method of use of exogenous human CC10 protein or rhCCI O to stabilize the respiratory epithelium in a patient diagnosed with COPD.

6. The method of use of claim 5 wherein the rehabilitating, stabilizing, or preserving occurs during chronic smoke exposure or chronic cigarette smoke exposure.

7. The method of claim 5 wherein the rehabilitating, stabilizing, or preserving occurs following chronic smoke exposure or chronic cigarette smoke exposure.

8. The method of claim 5 wherein the rehabilitating, stabilizing, or preserving occurs following or during exposure to smoke particles and toxins present in smoke.

9. The method of use of exogenous human CC10 protein to rehabilitate, stabilize, or preserve the respiratory epithelium in a patient with chronic lung injury.