A 28-day clinical trial of aerosolized hyaluronan in alpha-1 antiprotease deficiency COPD using desmosine as a surrogate marker for drug efficacy.

INTRODUCTION: A previous 2-week clinical trial of aerosolized hyaluronan (HA) in COPD showed a rapid reduction in lung elastic fiber breakdown, as measured by sputum levels of the unique elastin crosslinks, desmosine and isodesmosine (DID). To further assess the therapeutic efficacy of HA and the utility of DID as surrogate markers for the development of pulmonary emphysema, we have conducted a 28-day randomized, double-blind, placebo-controlled, phase 2 trial of HA involving 27 subjects with alpha-1 antiprotease deficiency COPD. METHODS: The study drug consisted of a 3 ml inhalation solution containing 0.03% HA with an average molecular weight of 150 kDa that was self-administered twice daily. DID levels were measured in urine, sputum, and plasma using tandem mass spectrometry. RESULTS: Free urine DID in the HA group showed a significant negative correlation with time between days 14 and 35 (r = -1.0, p = 0.023) and was statistically significantly decreased from baseline at day 35 (15.4 vs 14.2 ng/mg creatinine, p = 0.035). A marked decrease in sputum DID was also seen in the HA group between days 1 and 28 (0.96 vs 0.18 ng/mg protein), but the difference was not significant, possibly due to the small number of adequate specimens. Plasma DID remained unchanged following HA treatment and no significant reductions in urine, sputum, or plasma DID were seen in the placebo group. CONCLUSIONS: The results support additional clinical trials to further evaluate the therapeutic effect of HA and the use of DID as a real-time marker of drug efficacy.

COPD Pathogenesis: Finding the Common in the Complex.

Developing an effective treatment for COPD, and especially pulmonary emphysema, will require an understanding of how fundamental changes at the molecular level affect the macroscopic structure of the lung. Currently, there is no accepted model that encompasses the biochemical and mechanical processes responsible for pulmonary airspace enlargement. We propose that pulmonary emphysematous changes may be more accurately described as an emergent phenomenon, involving alterations at the molecular level that eventually reach a critical structural threshold where uneven mechanical forces produce alveolar wall rupture, accompanied by advanced clinical signs of COPD. The coupling of emergent morphologic changes with biomarkers to detect the process, and counteract it therapeutically, represents a practical approach to the disease.

The Therapeutic Potential of Hyaluronan in COPD.

Insights into the clinical course of COPD indicate the need for new therapies for this condition. The discovery of alpha-1 antitrypsin deficiency (AATD) led to the protease-antiprotease imbalance hypothesis, which was applied to COPD related to AATD as well as COPD not related to AATD. The discovery of AATD brought recognition to the importance of elastin fibers in maintaining lung matrix structure. Two cross-linking amino acids, desmosine and isodesmosine (DI), are unique to mature elastin and can serve as biomarkers of the degradation of elastin. The intravenous augmentation treatment and lung density in severe alpha-1 antitrypsin deficiency (RAPID) study shows a correlation of an anatomic index of COPD (on CT imaging) correlating with a chemical indicator of matrix injury in COPD, DI. The results suggest that preservation of lung elastin structure may slow the progression of COPD. Hyaluronan aerosol decreases the severity of elastase-induced emphysema in animals and has induced reductions in DI levels in preliminary human studies. Hyaluronan deserves further development as a therapy for COPD.

Biomarkers in Alpha-1 Antitrypsin Deficiency Chronic Obstructive Pulmonary Disease.

Biomarkers of pathogenesis in chronic obstructive pulmonary disease (COPD) can significantly accelerate drug development. In COPD related to alpha-1 antitrypsin deficiency, the role of neutrophil elastase and its inhibition by alpha-1 antitrypsin protein focused interest on elastin degradation and the development of pulmonary emphysema. Amino acids desmosine and isodesmosine are unique cross-links in mature elastin fibers and can serve as biomarkers of elastin degradation when measured in body fluids. This review gives a perspective on what has been learned by the earliest measurements of desmosine and isodesmosine followed by later studies using methods of increased sensitivity and specificity and the meaning for developing new therapies. Also included are brief statements on the biomarkers fibrinogen, CC-16, and Aa-Val-360 in COPD.

The Effect of Alpha-1 Proteinase Inhibitor on Biomarkers of Elastin Degradation in Alpha-1 Antitrypsin Deficiency: An Analysis of the RAPID/RAPID Extension Trials.

The RAPID (NCT00261833; N=180) and RAPID Extension (NCT00670007; N=140) trials demonstrated significantly reduced lung density decline in patients with alpha-1 antitrypsin deficiency (AATD) receiving alpha-1 proteinase inhibitor (A1PI) versus placebo. Desmosine and isodesmosine (DES/IDES) are unique crosslinkers of mature elastin fibers and are utilized as measures of elastin degradation. The aim of this post-hoc study was to determine the effect of A1PI therapy on DES/IDES levels in patients from RAPID/RAPID Extension. Plasma levels of DES/IDES were measured using high-performance liquid chromatography and tandem mass spectrometry. Correlation between changes in DES/IDES levels and computed tomography (CT) lung density decline was assessed. Analysis showed that DES/IDES levels were significantly reduced versus baseline in patients receiving A1PI at all time points, from month 3 through month 48. A significant increase from baseline in DES/IDES was observed with placebo at month 24 (n=54; 0.016; p=0.018). DES/IDES change from baseline was significantly different with A1PI versus placebo at months 3 (-0.021; 95% confidence interval [CI] -0.037, 0.004; p=0.026), 12 (-0.040; 95% CI -0.055, 0.025; p<0.001), and 24 (-0.052; 95% CI -0.070, 0.034; p<0.001). Placebo patients started A1PI therapy at month 24 and showed significant reductions in plasma DES/IDES at months 36 (p<0.001) and 48 (p<0.001). Reduced elastin degradation was associated with slower lung density decline (p=0.005), correlating a chemical index of therapy with an anatomical index by CT. In conclusion, A1PI therapy reduced elastin degradation, including pulmonary elastin, in patients with AATD. These data support using DES/IDES levels as biomarkers to monitor emphysema progression and treatment response.

Matrix elastin: a promising biomarker for chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a major health problem worldwide and is now the third leading cause of death in the United States. There is a lack of therapies that can stop progression of the disease and improve survival. New drug discovery can be aided by the development of biomarkers, which can act as indicators of severity in the course of the disease and responses to therapy. This perspective brings together the laboratory and clinical evidence, which suggest that elastin degradation products can fulfill the need for such a biomarker. Elastin is a recognized target for injury in COPD. The amino acids desmosine and isodesmosine exist only in matrix elastin; can be measured specifically and sensitively in plasma, urine, and sputum; and indicate changes in the systemic balance between elastase activity and elastase inhibition brought on by the systemic inflammatory state. The biomarker levels in sputum reflect the state of elastin degradation in the lung specifically. Clinical data accumulated over several decades indicate correlations of desmosine and isodesmosine levels with COPD of varying severity and responses to therapy.

Therapeutic effects of hyaluronan on smoke-induced elastic fiber injury: does delayed treatment affect efficacy?

Aerosolized hyaluronan (HA) has been previously shown to prevent cigarette smoke-induced airspace enlargement and elastic fiber injury in mice when given concurrently with smoke. In the present study, a more stringent test of the therapeutic potential of HA was performed by delaying treatment with this agent for 1 month. After treatment with cigarette smoke for 3 h per day for 5 days per week for 1 month, mice (DBA/2J) began receiving aerosolized HA (0.1%) for 1 h prior to smoke exposure (controls were given aerosolized water). The results indicate that much of the damage to the lung elastic fibers occurred within the first several months of smoke exposure, as measured by levels of desmosine and isodesmosine (DID) in bronchoalveolar lavage fluid (BALF). In contrast to previously published studies, where concurrent administration of aerosolized HA significantly reduced BALF DID levels within 3 months of smoke exposure, the same effect was not seen until 6 months when HA treatment was delayed. However, despite the prolonged breakdown of elastic fibers in the current study, a significant reduction in airspace enlargement was observed after only 2 months of HA treatment. These findings provide further support for testing this agent in patients with pre-existing chronic obstructive pulmonary disease.

Attenuating effect of taurine on lipopolysaccharide-induced acute lung injury in hamsters.

This study has evaluated the ability of the semiessential amino acid taurine to attenuate lipopolysaccharide (LPS)-induced lung inflammation, oxidative stress and apoptosis in a small animal model. For this purpose, bacterial LPS (0.02mg in phosphate buffered saline (PBS) pH 7.4) was instilled intratracheally into female Golden Syrian hamsters, before or after a 3-day intraperitoneal treatment with a single dose (50mg/kg in PBS pH 7.4) of taurine. At 24h after the last treatment, lung tissue and bronchoalveolar lavage fluid (BALF) samples were collected. In comparison to samples from animals receiving only PBS pH 7.4, serving as controls, those of LPS-stimulated animals exhibited a higher count of both total leukocytes and neutrophils in the BALF, and increased incidence of apoptosis, depletion of intracellular glutathione and evidence of inflammation confined to the parenchyma in the lung. In addition, LPS caused cells in the BALF to exhibit a higher expression of tumor necrosis factor-1, a higher activity of caspase-3, marked lipid peroxidation, and altered activities of catalase, glutathione peroxidase and superoxide dismutase relative to control samples. In contrast, a treatment with taurine was found to significantly attenuate all of the cellular and biochemical alterations induced by LPS, more so when given before rather than after the endotoxin. The present results suggest that taurine possesses intrinsic antiinflammatory and antioxidant properties that may be of benefit against the deleterious actions of LPS in the lung.

Endothelin-1 potentiates smoke-induced acute lung inflammation.

The current study examined the role of endothelin-1 (ET-1) in mediating acute lung inflammation induced by short-term cigarette smoke exposure. Hamsters received intraperitoneal injections of ET-1, followed by a 2-hour period of smoke exposure, for 3 consecutive days. The lungs were then evaluated for inflammatory changes, using the following parameters: (1) lung histopathology, (2) neutrophil content of bronchoalveolar lavage fluid (BALF), (3) percent tumor necrosis factor receptor 1 (TNFR1)-labeled BALF macrophages, and (4) alveolar septal cell apoptosis. Results indicate that ET-1 significantly amplified the effect of smoke on each of these inflammatory markers and that these responses could be blocked by pretreatment with a novel endothelin receptor A antagonist, HJP272. In particular, exogenous ET-1 induced a marked increase in BALF neutrophils, consistent with a role for this mediator as an inflammatory cell "gatekeeper."

Preferential recruitment of neutrophils by endothelin-1 in acute lung inflammation induced by lipopolysaccharide or cigarette smoke.

This study examined the role of endothelin-1 (ET-1) in recruiting inflammatory cells to the lung after induction of injury with either lipopolysaccharide (LPS) or cigarette smoke. Hamsters injected with either ET-1 or its precursor peptide (Big ET-1) prior to treatment with LPS or cigarette smoke had markedly increased concentrations of neutrophils in bronchoalveolar lavage fluid (BALF) despite a reduction in total numbers of BALF leukocytes. Furthermore, the effect of ET-1 on smoke-exposed animals was reversed by addition of an endothelin-A receptor antagonist. These results are consistent with preferential recruitment of neutrophils by ET-1, and suggest that inhibition of this proinflammatory mediator may decrease acute pulmonary inflammation associated with cigarette smoke and other pulmonary toxins.

Phosphoramidon, an endothelin-converting enzyme inhibitor, attenuates lipopolysaccharide-induced acute lung injury.

Phosphoramidon blocks the formation of endothelin-1 (ET-1), a proinflammatory mediator implicated in the pathogenesis of a variety of lung diseases. To determine whether phosphoramidon can ameliorate pulmonary inflammation, our laboratory undertook a series of experiments involving treatment of hamsters with either intraperitoneal (i.p.) or aerosolized phosphoramidon prior to induction of acute lung injury by intratracheal administration of lipopolysaccharide (LPS). The results indicate that phosphoramidon significantly reduces LPS-induced pulmonary inflammation as measured by lung histology, neutrophil content of bronchoalveolar lavage (BAL) fluid, percent tumor necrosis factor receptor 1 (TNFR1)-labeled BAL macrophages, and alveolar septal cell apoptosis. In additional experiments, i.p. administration of a novel endothelin A receptor anatgonist (HJP272) similarly decreased BAL neutrophils, whereas i.p. administration of either ET-1, or its precursor peptide, "big" ET-1, had the opposite effect. These findings support further evaluation of phosphoramidon and other ET-1 suppressors as potential treatments for human inflammatory lung disease.

Short-term cigarette smoke exposure predisposes the lung to secondary injury.

Brief exposure to cigarette smoke is not generally associated with pulmonary injury and may adversely affect the lung only if underlying disease is present. To test this hypothesis, our laboratory performed a series of experiments involving exposure of hamsters to second-hand cigarette smoke (2 h/day for 5 days), either immediately before or after induction of acute pulmonary injury by intratracheal administration of amiodarone. Compared to controls receiving amiodarone alone, hamsters pretreated with smoke showed significant increases in the following parameters: (1) lung inflammation graded on a scale of 0-4 (3.4 vs. 1.6; p < 0.001), (2) percentage of neutrophils in bronchoalveolar lavage fluid (BALF) (75.0 vs. 1.3; p < 0.001), (3) percentage of TNFR1-positive BALF macrophages (44.7 vs. 2.7; p < 0.001), and (4) apoptotic lung parenchymal cells per ten high-power microscopic fields (7.3 vs. 0.7; p < 0.001). Animals post-treated with smoke also showed significant increases in these parameters compared to controls but to a lesser degree than pre-exposed animals. With regard to human disease, such synergistic interactions may account for a significant portion of the morbidity associated with second-hand smoke exposure.

Short-term cigarette smoke exposure potentiates endotoxin-induced pulmonary inflammation.

Long-term cigarette smoke exposure is associated with chronic obstructive pulmonary disease. However, the effects of short-term smoke inhalation are less clear, because it may adversely affect the lung only if underlying disease is present. To test this hypothesis, Syrian hamsters were passively exposed to cigarette smoke for 2 hours per day over a period of 3 days either before or after intratracheal instillation of low-dose (20 microg) Escherichia coli endotoxin. The results indicate that short-term smoke exposure can potentiate endotoxin-induced lung inflammation. They also suggest that nonsmokers with underlying lung disease may be particularly vulnerable to the adverse effects of second-hand smoke.

Potential therapeutic applications of hyaluronan in the lung.

Hyaluronan (HA), a long-chain polysaccharide, is currently being evaluated as a potential therapeutic agent for a number of inflammatory disorders. The effect of HA on inflammation appears to be related to its molecular size, with larger polysaccharide chains having anti-inflammatory activity and smaller ones having proinflammatory properties. This dichotomous behavior is particularly relevant to the work of our laboratory on an aerosolized preparation of HA to treat pulmonary emphysema. The breakdown of inhaled HA into smaller fragments could possibly induce an inflammatory reaction in the lung that counteracts any beneficial effect. Consequently, the proposed therapeutic use of HA will require development of treatment strategies aimed at minimizing its proinflammatory activity.

Hyaluronan: the Jekyll and Hyde molecule.

Hyaluronan (HA) is a variable length, long-chain polysaccharide containing repeating disaccharide units of glucuronic acid and n-acetylglucosamine. Long considered a relatively inert component of the extracellular matrix, HA is now coming under scrutiny as a potential therapeutic agent for a number of different diseases, based on its recently discovered role in modulating inflammation. The effect of HA on the inflammatory response appears to be related to its molecular size, with larger polysaccharide chains having anti-inflammatory activity and smaller ones having proinflammatory properties. This dichotomous behavior presents a challenge to investigators seeking to harness the beneficial effects of this molecule. Rapid breakdown of therapeutically administered HA into smaller fragments may conceivably cause further injury to diseased tissues. With this limitation in mind, the authors discuss their own use of HA to treat experimentally induced lung disease, then suggest possible ways of maximizing the therapeutic potential of this molecule.

Synergistic effect of hydrogen peroxide and elastase on elastic fiber injury in vitro.

This laboratory has previously shown that hyperoxia enhances airspace enlargement in a hamster model of elastase-induced emphysema. To further understand the mechanism responsible for this finding, the effect of oxidants on elastase activity was studied in vitro, using a radiolabeled elastic fiber matrix derived from rat pleural mesothelial cells. Matrix samples were treated with either 0.1%, 1%, 3%, or 10% hydrogen peroxide (H2O2) for 1 hr, then incubated with 1.0 microg/ml porcine pancreatic elastase for 2 hrs. Radioactivity released from the matrix was used as a measure of elastolysis. Results indicate that sequential exposure to H2O2 and elastase markedly enhanced elastolysis compared to enzyme treatment alone. A 22% increase in elastolysis was seen with 0.1% H2O2 (325 vs. 396 cpm; P < 0.05), whereas samples pretreated with 1%, 3%, and 10% H2O2 showed increases of 53% (274 vs. 420 cpm; P < 0.05), 71% (381 vs. 653 cpm; P < 0.01), and 38% (322 vs. 443 cpm; P < 0.01), respectively. Exposure to various concentrations of H2O2 alone (0.1% to 10%) produced only minimal elastolysis (<20 cpm). However, 1% H2O2 was capable of degrading peptide-free desmosine and isodesmosine, suggesting that exposure to this oxidant may reduce the stability of the elastic fiber matrix. With regard to lung diseases such as emphysema, H2O2 and other oxidants derived from inflammatory cells or the environment could possibly act as priming agents for elastase-mediated breakdown of elastic fibers, resulting in amplification of lung injury.

Aerosolized hyaluronan limits airspace enlargement in a mouse model of cigarette smoke-induced pulmonary emphysema.

This study was designed to determine if aerosolized hyaluronan (HA) could prevent airspace enlargement and elastic fiber injury in a mouse model of cigarette smoke-induced pulmonary emphysema. Compared to untreated/smoked controls, HA-treated animals showed statistically significant reductions in mean linear intercept (54 versus 65 microm; P < .001) and elastic fiber breakdown products (desmosine and isodesmosine) in bronchoalveolar lavage fluid (0.3 versus 7.0 ng/mL; P < .05). As in previous studies, the aerosolized HA showed preferential binding to elastic fibers, suggesting that it may protect them from injury. These findings support further investigation of the potential use of HA as a treatment for pulmonary emphysema.