Determining exacerbation rates in patients receiving therapy is an additional parameter that is employed to determine the efficacy of AAT replacement therapy. In a web based question‐ naire prepared by Lieberman and colleagues, key questions addressed issues such as frequen‐ cy of respiratory infections pre- and post-AAT augmentation therapy [139]. Out of eighty nine individuals who had received augmentation therapy for more than one year, seventy four felt that the treatment was of benefit, with almost half accrediting the benefits to a reduction in the number of infectious exacerbations from approximately five down to one per year (P<0.001). CT lung density measurements are currently considered to be a more sensitive outcome to measure the impact of AAT replacement therapy. The first of these studies was a double blind trial with 26 Danish and 30 Dutch ex-smokers, randomized to either AAT (250 mg/kg) or albumin (625 mg/kg) infusions at 4 week intervals for at least 3 years [140]. Using selfadministered spirometry, no difference was found in FEV1 decline between treatment and placebo. Conversely, upon analysis of CT scans (slices 5 cm below carina), a trend towards a slower rate in loss of lung density was observed in augmentation receiving patients with measurements of 2.6 ± 0.41 g/L/yr for placebo as compared with 1.5 ± 0.41 g/L/yr for AAT

in the placebo group.

80 COPD Clinical Perspectives

Despite the accumulation of encouraging data supporting the use of AAT augmentation therapy, significant questions remain regarding the cost-effectiveness of this intravenous strategy for treatment of AAT deficient related COPD, which is estimated to cost the US \$100,000 per patient per year worldwide [143]. Such deliberation has prompted the develop‐ ment of new strategies, including the use of recombinant and transgenic protein to boost the natural antiprotease screen. A variety of systems have been employed to produce humanized recombinant AAT including plants, yeast fungi, animals, insect cells, bacteria and mammalian cells. One of the approaches involved expression of recombinant AAT in *E. coli,* but AAT produced via this system lacked glycosylation and consequently exhibited a short half-life within the circulation. To address this issue the presence of a single thiol residue at the surface of AAT was exploited by researchers, who conjugated maleimido-polyethylene glycol to the thiol group. Results revealed that site-specific conjugation with polyethylene glycol at Cys232 of nonglycosylated recombinant human AAT gave rise to active inhibitor with prolonged *in vivo* stability [164]. An alternative approach included production of a secreted fully functional AAT by the yeast *Saccharomyces cerevisiae* [165]. This secreted protein had high mannose-type glycosylation [166], which was thought to give rise to an immune response in humans because of non-human glycan residues, an adverse effect shared by active recombinant AAT protein expressed in the insect cell baculovirus expression vector system [167]. In an attempt to overcome the issue of glycosylation, a method of hyper glycosylation of the protein was introduced. This was achieved by adding terminal sialic acids to the existing glycans on the human AAT molecule. Such modification conferred increased protein half-life while maintaining the anti-NE capacity of the protein when injected into a mouse model [168]. In a further attempt to prepare fully glycosylated human AAT, Blanchard *et al* employed a human neuronal cell line (AGE1.HN®) to produce a recombinant AAT protein which retained biological activity and displayed a similar glycosylation patterns to native AAT [169]. Moreover, the recombinant AAT protein was biologically active and maintained anti-elastase activity and anti-inflamma‐ tory capacity, including modulation of TNFα production in neutrophils and monocytes [169]. *In vitro* testing of this protein revealed that the inhibitory effect of the recombinant AAT was comparable to that of Prolastin, however this has yet to be confirmed *in vivo*. More recently, AAT has been successfully produced and purified from the human PER.C6 cell line. The resultant protein shares the same primary, secondary and tertiary structures and *N*-linked glycosylation sites as AAT derived from human plasma and exhibits equivalent anti-NE capacity [170, 171]. While these studies strengthen the argument for the therapeutic use of recombinantly produced AAT, issues still remain with regards to the safety and efficacy of protein produced in this manner.

administration, remained elevated from 48-72 hours, and then declined slowly by 144 hours after administration [180]. Comparable results were reported by Hubbard *et al.* who evaluated aerosol administration of recombinant AAT to COPD individuals with AAT deficiency. Post aerosolization of single doses of 10-200mg of AAT, AAT anti- NE defences in ELF were augmented and aerosolized AAT was detectable in serum indicating that recombinant AAT

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Further support for the use of aerosolized AAT in treatment of COPD was provided by Griese and colleagues who examined the effect of four weeks of plasma purified AAT inhalation on lung function, protease–antiprotease balance and airway inflammation in patients with cystic fibrosis [181]. The authors reported an increased concentration of aerosolized AAT accumu‐ lation in the airways. Post treatment, levels of NE activity, numbers of infiltrating neutrophils, pro-inflammatory cytokines levels and the numbers of bacteria (*P. aeruginosa*) were reduced culminating in a marked reduction in airway inflammation. In a second study by this research group, the authors documented that the inability of neutrophils to eradicate *Pseudomonas* infection despite the abundance of neutrophils in the cystic fibrosis lung was due to cleavage of CXCR1 from the neutrophil. *In vivo* administration of aerosolized AAT was shown to restore CXCR1 expression and improve the killing capacity of neutrophils as assessed by sputum levels of *P. aeruginosa*, thus supporting the beneficial effects of aerosolized AAT therapy [179]. In a further study employing a mouse model of smoke induced emphysema, inhaled AAT resulted in reductions in airspace enlargement of up to 73%. The results of this investigation indicated that delivery of AAT directly to the lung can prevent NE mediated tissue damage. Moreover, further evidence of the ability of aerosolised AAT to directly protect the lung tissue from proteolytic breakdown was demonstrated as aerosolised AAT abrogated NE-induced expression of cathepsin B and MMP-2 in BAL fluid, thus indirectly protecting key antiinflammatory and antimicrobial peptides including SLPI and lactoferrin from cathepsin mediated proteolysis within the milieu of the COPD lung [36]. In terms of clinical efficacy of aerosolised AAT, a phase III trial of inhaled Kamada AAT is currently on-going with results on outcome measures including safety and efficacy, exacerbation frequency and progression of emphysema to be released presently. The outcome of this study will be pivotal to shaping

the future development of aerosolised AAT augmentation therapy (NCT01217671).

A number of studies have generated data to support the use of naturally occurring antipro‐ teases, other than AAT, as potential anti-inflammatory therapies to modulate the high level of inflammatory within the lungs of COPD patients. Within the literature reports suggest that serine protease inhibitors may potentially be used as a treatment option for COPD. One such antiprotease is elafin, a small cationic protein (6kDa) which is a member of the chelonianin family. Throughout the literature it is often referred to as skin derived anti-leukoprotease (SKALP) due to its first identification in psoriatic epidermis [182]. More recently however, it has been shown to be produced and released by epidermal keratinocytes [183], lung derived

**4.4. Naturally occurring antiproteases elafin and SLPI as therapies for COPD**

epithelia [184], T-cells [185], macrophages [186] and also neutrophils [187].

was capable of gaining access to lung interstitium [176].

Subsequently, it was envisaged that sources of recombinant AAT from transgenic animals might overcome this posttranslational glycosylation challenge and fulfil the demand for mass production of AAT. To this end rabbits [172], mice [173] and sheep transgenic for a fusion of the ovine beta-lactoglobulin gene promoter to the human AAT genomic sequences were generated [174]. Human AAT purified from the milk of these animals appeared to be fully *N*glycosylated and biologically active. While the use of transgenic sheep certainly tackled the issue of cost effectiveness, 5,000kg of AAT could be produced in one year, unfortunately however, a trial of inhaled AAT derived from sheep milk proved disappointing and observa‐ tions of a systemic antibody response in recipients to residual native sheep AAT and alpha-1 antichymotrypsin were observed [175]. Thus it is clear that transgenic preparations of AAT will only be of therapeutic value if they are of high purity in order to avoid immunogenicity.

While enhancing production of AAT seems promising from a cost and mass production point of view, significant challenges remain for the development of recombinant and transgenic AAT and they cannot compete with the current source of AAT from pooled human plasma for safety and efficacy in the treatment of AAT deficiency.

#### **4.3. Aerosolized AAT augmentation therapy**

Interest in the administration of AAT in aerosolized form has increased in recent years due to the advantageous properties provided such as a direct route to the lungs, avoidance of systemic deliver and a reduction in costs. Aerosolization of AAT has been shown to increase AAT levels and to restore anti-NE capacity in lung epithelium lining fluid of both COPD and cystic fibrosis individuals [176]. AAT administered in aerosolized form was found to positively impact upon neutrophil mediated killing of *Pseudomonas* [177], possibly by preventing cleavage of neutro‐ phil complement receptors by serine proteases, a previously reported adverse effect of NE [178] or by preventing cleavage of CXC chemokine receptor 1 (CXCR1) [179]. In addition, the presence of increased AAT in serum post aerosolization supports the hypothesis that aug‐ mented AAT is capable of diffusing across the pulmonary interstitium after administration [180] and affords anti-NE protection to the interstitial compartment [176]. Measurements of the rate of transfer of AAT from the lung and of the rate of appearance of AAT in plasma resulted in a calculated permeability of the alveolar-capillary membrane to AAT of 3.49-6.39 X 10-10 cm/s. In a canine model, levels of AAT rose to a maximum value at 48 hours after administration, remained elevated from 48-72 hours, and then declined slowly by 144 hours after administration [180]. Comparable results were reported by Hubbard *et al.* who evaluated aerosol administration of recombinant AAT to COPD individuals with AAT deficiency. Post aerosolization of single doses of 10-200mg of AAT, AAT anti- NE defences in ELF were augmented and aerosolized AAT was detectable in serum indicating that recombinant AAT was capable of gaining access to lung interstitium [176].

displayed a similar glycosylation patterns to native AAT [169]. Moreover, the recombinant AAT protein was biologically active and maintained anti-elastase activity and anti-inflamma‐ tory capacity, including modulation of TNFα production in neutrophils and monocytes [169]. *In vitro* testing of this protein revealed that the inhibitory effect of the recombinant AAT was comparable to that of Prolastin, however this has yet to be confirmed *in vivo*. More recently, AAT has been successfully produced and purified from the human PER.C6 cell line. The resultant protein shares the same primary, secondary and tertiary structures and *N*-linked glycosylation sites as AAT derived from human plasma and exhibits equivalent anti-NE capacity [170, 171]. While these studies strengthen the argument for the therapeutic use of recombinantly produced AAT, issues still remain with regards to the safety and efficacy of

Subsequently, it was envisaged that sources of recombinant AAT from transgenic animals might overcome this posttranslational glycosylation challenge and fulfil the demand for mass production of AAT. To this end rabbits [172], mice [173] and sheep transgenic for a fusion of the ovine beta-lactoglobulin gene promoter to the human AAT genomic sequences were generated [174]. Human AAT purified from the milk of these animals appeared to be fully *N*glycosylated and biologically active. While the use of transgenic sheep certainly tackled the issue of cost effectiveness, 5,000kg of AAT could be produced in one year, unfortunately however, a trial of inhaled AAT derived from sheep milk proved disappointing and observa‐ tions of a systemic antibody response in recipients to residual native sheep AAT and alpha-1 antichymotrypsin were observed [175]. Thus it is clear that transgenic preparations of AAT will only be of therapeutic value if they are of high purity in order to avoid immunogenicity.

While enhancing production of AAT seems promising from a cost and mass production point of view, significant challenges remain for the development of recombinant and transgenic AAT and they cannot compete with the current source of AAT from pooled human plasma for safety

Interest in the administration of AAT in aerosolized form has increased in recent years due to the advantageous properties provided such as a direct route to the lungs, avoidance of systemic deliver and a reduction in costs. Aerosolization of AAT has been shown to increase AAT levels and to restore anti-NE capacity in lung epithelium lining fluid of both COPD and cystic fibrosis individuals [176]. AAT administered in aerosolized form was found to positively impact upon neutrophil mediated killing of *Pseudomonas* [177], possibly by preventing cleavage of neutro‐ phil complement receptors by serine proteases, a previously reported adverse effect of NE [178] or by preventing cleavage of CXC chemokine receptor 1 (CXCR1) [179]. In addition, the presence of increased AAT in serum post aerosolization supports the hypothesis that aug‐ mented AAT is capable of diffusing across the pulmonary interstitium after administration [180] and affords anti-NE protection to the interstitial compartment [176]. Measurements of the rate of transfer of AAT from the lung and of the rate of appearance of AAT in plasma resulted in a calculated permeability of the alveolar-capillary membrane to AAT of 3.49-6.39 X 10-10 cm/s. In a canine model, levels of AAT rose to a maximum value at 48 hours after

protein produced in this manner.

82 COPD Clinical Perspectives

and efficacy in the treatment of AAT deficiency.

**4.3. Aerosolized AAT augmentation therapy**

Further support for the use of aerosolized AAT in treatment of COPD was provided by Griese and colleagues who examined the effect of four weeks of plasma purified AAT inhalation on lung function, protease–antiprotease balance and airway inflammation in patients with cystic fibrosis [181]. The authors reported an increased concentration of aerosolized AAT accumu‐ lation in the airways. Post treatment, levels of NE activity, numbers of infiltrating neutrophils, pro-inflammatory cytokines levels and the numbers of bacteria (*P. aeruginosa*) were reduced culminating in a marked reduction in airway inflammation. In a second study by this research group, the authors documented that the inability of neutrophils to eradicate *Pseudomonas* infection despite the abundance of neutrophils in the cystic fibrosis lung was due to cleavage of CXCR1 from the neutrophil. *In vivo* administration of aerosolized AAT was shown to restore CXCR1 expression and improve the killing capacity of neutrophils as assessed by sputum levels of *P. aeruginosa*, thus supporting the beneficial effects of aerosolized AAT therapy [179]. In a further study employing a mouse model of smoke induced emphysema, inhaled AAT resulted in reductions in airspace enlargement of up to 73%. The results of this investigation indicated that delivery of AAT directly to the lung can prevent NE mediated tissue damage. Moreover, further evidence of the ability of aerosolised AAT to directly protect the lung tissue from proteolytic breakdown was demonstrated as aerosolised AAT abrogated NE-induced expression of cathepsin B and MMP-2 in BAL fluid, thus indirectly protecting key antiinflammatory and antimicrobial peptides including SLPI and lactoferrin from cathepsin mediated proteolysis within the milieu of the COPD lung [36]. In terms of clinical efficacy of aerosolised AAT, a phase III trial of inhaled Kamada AAT is currently on-going with results on outcome measures including safety and efficacy, exacerbation frequency and progression of emphysema to be released presently. The outcome of this study will be pivotal to shaping the future development of aerosolised AAT augmentation therapy (NCT01217671).

#### **4.4. Naturally occurring antiproteases elafin and SLPI as therapies for COPD**

A number of studies have generated data to support the use of naturally occurring antipro‐ teases, other than AAT, as potential anti-inflammatory therapies to modulate the high level of inflammatory within the lungs of COPD patients. Within the literature reports suggest that serine protease inhibitors may potentially be used as a treatment option for COPD. One such antiprotease is elafin, a small cationic protein (6kDa) which is a member of the chelonianin family. Throughout the literature it is often referred to as skin derived anti-leukoprotease (SKALP) due to its first identification in psoriatic epidermis [182]. More recently however, it has been shown to be produced and released by epidermal keratinocytes [183], lung derived epithelia [184], T-cells [185], macrophages [186] and also neutrophils [187].

Elafin is a subunit derived from the precursor molecule trappin-2 which has a molecular weight of 12.3 kDa [188]. The conversion of trappin-2 into elafin can occur by cleavage by several proteases including cathepsin L, cathepsin K, plasmin, trypsin but the most efficient protease at releasing elafin is tryptase [189]. The mature form of trappin-2 consists of two domains; the first domain is unique to only trappin-2, the *N*-terminal cementoin domain. This domain aids as a transglutaminase substrate which allows trappin-2 to interact and bind to extracellular matrix proteins such as fibronectin, collagen IV, fibrinogen, laminin V, vitronec‐ tin, thus prolonging its presence within in the lung [188]. The second domain, which is present in both trappin-2 and elafin, is the whey acidic protein (WAP) domain. The WAP domain provides antiprotease activity with specificity against NE [18] and PR3 [17], two key proteases that play a role in the lung pathophysiology associated with COPD [56, 190].

studies it was demonstrated that the impact of elafin on chemokine production was cell specific [199]. Results demonstrated that elafin reduced TNFα secretion by human macrophages and IL-8 release by human umbilical cord endothelial cells (HUVECs) in response to TNFα, LPS and oxidized LDL, through modulation of NFκB signalling. In contrast, in the same study transfecting an alveolar epithelial cell line with elafin had no significant effect on LPS induced

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In addition to possessing antiprotease and anti-inflammatory properties, elafin also demon‐ strates anti-microbial properties which may prove beneficial in the treatment of infective exacerbations in COPD patients. In a study carried out by Simpson *et al*., the anti-bacterial capability of elafin was evaluated and the results demonstrated that elafin maintained bactericidal effect against both *P. aeruginosa* and *S. aureus* [200]. This effect could in part be due to the cationic charge of elafin which may destabilize bacterial membranes similar to classical antimicrobial proteins [201]. Moreover, a study by Baranger *et al*., demonstrated that trappin-2 had antibacterial properties against other clinically important bacteria including *Klebsiella pneumoniae, Haemophilus influenzae, Streptococcus pneumoniae* and *Branhamella catarrhalis* [202]. Furthermore, this study highlighted the novel anti-fungal effect of trappin-2 against *Aspergillus fumigatus*, with the outcome of this study demonstrating the anti-microbial properties of trappin-2 to be independent of its antiprotease property. Aside from direct activity against microbes, trappin-2 in turn has been shown to enhance the clearance of *P. aeruginosa* from the lungs of infected mice [203], an effect mediated via opsonisation of bacteria with trappin-2 for more efficient CD14-dependent clearance by macrophages [197]. In additional studies it has been shown that elafin can modulate inflammation associated with LPS, through inhibition of AP-1 and NFκB activation [198], possibly by direct interaction with this microbial inflamma‐

These innate attributes of elafin make it an ideal candidate for a replacement therapy in treatment of inflammatory lung diseases such as COPD which is associated with excess protease burden and/or infection. However caution must be exercised, as like many other endogenous protease inhibitors, elafin is susceptible to inactivation by neutrophil-derived oxidants resulting in loss of its antiprotease activity [205]. Moreover, elafin can be cleaved by NE [206], which is present in high concentrations in the COPD lung [207] and cleavage can result in diminished ability of elafin to bind LPS and its capacity to be immobilized by transglutamination [206]. A similar observation was made in regard to the *P. aeruginosa*derived proteases pseudolysin and aeruginolysin which can cleave elafin thereby negatively impacting on its biological functions [208]. Collectively, these findings suggest that within an environ‐ ment of high protease burden, as found in the COPD lung, this could have a negative impact

An additional native antiprotease that has been well documented for its potential as a therapy for COPD is human SLPI, which was originally identified in parotid secretions [20]. Being a member of the antileukoprotease family, SLPI shares 40% homology with elafin. A number of cell types have been documented to produce SLPI including macrophages [186], monocytes, neutrophils [187] and lung epithelial cells [209]. It is well accepted that SLPI is a potent inhibitor of an array of serine proteases including NE, cathepsin G, trypsin and chymotrypsin. Similar

IL-8 production [199].

tory mediatory [204].

upon the clinical efficacy of elafin.

Several studies have demonstrated that elafin is produced under inflammatory conditions suggesting it to be an acute phase reactant [191]. Interestingly, NE can impact on elafin expression and it has been demonstrated that exposing human alveolar epithelial cells to NE can result in increased protein expression and secretion of elafin [192]. Additional studies have demonstrated that pro-inflammatory mediators such as TNFα and IL-1β increase elafin expression in lung derived cell lines, indicating that in response to cytokine induced inflam‐ mation, epithelial cells can increase their antiprotease shield [191]. This induced increase in elafin expression is not limited to only host derived inflammatory mediators as it has been shown that bacterial LPS can increase elafin expression in murine airways [193] and in macrophages from transgenic mice expressing human elafin [194].

The immuno-modulatory impact of elafin on inflammation has been established in a number of studies and animal models. For example, Vachon and colleagues utilised a murine model and following intranasal administration of LPS, pre-treatment with recombinant human elafin was shown to diminish neutrophil infiltration into the airways [195]. In the same study elafin was shown to reduce gelatinase activity, macrophage inflammatory protein-2 (MIP-2), keratinocyte chemoattractant and significantly reduced mRNA levels of three members of the IL-1 ligand family [195]. Conversely, in an earlier study involving a transgenic murine model expressing human trappin-2, an increase in neutrophil recruitment to the lungs of LPS challenged mice was observed [193]. The discrepancy between these two latter studies could in part be due to differences in the mode of elafin administration and the murine models employed. Of interest, the positive impact of elafin on COPD related complications such as pulmonary hypertension has been reported [196]. Moreover, in a hypoxia associated pulmo‐ nary hypertension murine model the protease activity, muscularization of pulmonary arteries and right ventricular pressure were reduced in transgenic mice that over expressed elafin [197]. Indeed, both elafin and its precursor molecule trappin-2 have anti-inflammatory properties, as evident by the inactivation of key inflammatory signalling pathways and immune cell activity associated with COPD pathogenesis. In a study using a human myelo‐ monocytic cell line, elafin was shown to exert its anti-inflammatory effect intracellularly [198]. The mode of action involved direct impact on the ubiquitin-proteasome pathway, delaying turnover of polyubiquitinated proteins thus affecting NFκB activation and the AP-1 pathway leading to reduced MCP-1 expression in response to LPS [198]. Moreover, in transfection studies it was demonstrated that the impact of elafin on chemokine production was cell specific [199]. Results demonstrated that elafin reduced TNFα secretion by human macrophages and IL-8 release by human umbilical cord endothelial cells (HUVECs) in response to TNFα, LPS and oxidized LDL, through modulation of NFκB signalling. In contrast, in the same study transfecting an alveolar epithelial cell line with elafin had no significant effect on LPS induced IL-8 production [199].

Elafin is a subunit derived from the precursor molecule trappin-2 which has a molecular weight of 12.3 kDa [188]. The conversion of trappin-2 into elafin can occur by cleavage by several proteases including cathepsin L, cathepsin K, plasmin, trypsin but the most efficient protease at releasing elafin is tryptase [189]. The mature form of trappin-2 consists of two domains; the first domain is unique to only trappin-2, the *N*-terminal cementoin domain. This domain aids as a transglutaminase substrate which allows trappin-2 to interact and bind to extracellular matrix proteins such as fibronectin, collagen IV, fibrinogen, laminin V, vitronec‐ tin, thus prolonging its presence within in the lung [188]. The second domain, which is present in both trappin-2 and elafin, is the whey acidic protein (WAP) domain. The WAP domain provides antiprotease activity with specificity against NE [18] and PR3 [17], two key proteases

Several studies have demonstrated that elafin is produced under inflammatory conditions suggesting it to be an acute phase reactant [191]. Interestingly, NE can impact on elafin expression and it has been demonstrated that exposing human alveolar epithelial cells to NE can result in increased protein expression and secretion of elafin [192]. Additional studies have demonstrated that pro-inflammatory mediators such as TNFα and IL-1β increase elafin expression in lung derived cell lines, indicating that in response to cytokine induced inflam‐ mation, epithelial cells can increase their antiprotease shield [191]. This induced increase in elafin expression is not limited to only host derived inflammatory mediators as it has been shown that bacterial LPS can increase elafin expression in murine airways [193] and in

The immuno-modulatory impact of elafin on inflammation has been established in a number of studies and animal models. For example, Vachon and colleagues utilised a murine model and following intranasal administration of LPS, pre-treatment with recombinant human elafin was shown to diminish neutrophil infiltration into the airways [195]. In the same study elafin was shown to reduce gelatinase activity, macrophage inflammatory protein-2 (MIP-2), keratinocyte chemoattractant and significantly reduced mRNA levels of three members of the IL-1 ligand family [195]. Conversely, in an earlier study involving a transgenic murine model expressing human trappin-2, an increase in neutrophil recruitment to the lungs of LPS challenged mice was observed [193]. The discrepancy between these two latter studies could in part be due to differences in the mode of elafin administration and the murine models employed. Of interest, the positive impact of elafin on COPD related complications such as pulmonary hypertension has been reported [196]. Moreover, in a hypoxia associated pulmo‐ nary hypertension murine model the protease activity, muscularization of pulmonary arteries and right ventricular pressure were reduced in transgenic mice that over expressed elafin [197]. Indeed, both elafin and its precursor molecule trappin-2 have anti-inflammatory properties, as evident by the inactivation of key inflammatory signalling pathways and immune cell activity associated with COPD pathogenesis. In a study using a human myelo‐ monocytic cell line, elafin was shown to exert its anti-inflammatory effect intracellularly [198]. The mode of action involved direct impact on the ubiquitin-proteasome pathway, delaying turnover of polyubiquitinated proteins thus affecting NFκB activation and the AP-1 pathway leading to reduced MCP-1 expression in response to LPS [198]. Moreover, in transfection

that play a role in the lung pathophysiology associated with COPD [56, 190].

84 COPD Clinical Perspectives

macrophages from transgenic mice expressing human elafin [194].

In addition to possessing antiprotease and anti-inflammatory properties, elafin also demon‐ strates anti-microbial properties which may prove beneficial in the treatment of infective exacerbations in COPD patients. In a study carried out by Simpson *et al*., the anti-bacterial capability of elafin was evaluated and the results demonstrated that elafin maintained bactericidal effect against both *P. aeruginosa* and *S. aureus* [200]. This effect could in part be due to the cationic charge of elafin which may destabilize bacterial membranes similar to classical antimicrobial proteins [201]. Moreover, a study by Baranger *et al*., demonstrated that trappin-2 had antibacterial properties against other clinically important bacteria including *Klebsiella pneumoniae, Haemophilus influenzae, Streptococcus pneumoniae* and *Branhamella catarrhalis* [202]. Furthermore, this study highlighted the novel anti-fungal effect of trappin-2 against *Aspergillus fumigatus*, with the outcome of this study demonstrating the anti-microbial properties of trappin-2 to be independent of its antiprotease property. Aside from direct activity against microbes, trappin-2 in turn has been shown to enhance the clearance of *P. aeruginosa* from the lungs of infected mice [203], an effect mediated via opsonisation of bacteria with trappin-2 for more efficient CD14-dependent clearance by macrophages [197]. In additional studies it has been shown that elafin can modulate inflammation associated with LPS, through inhibition of AP-1 and NFκB activation [198], possibly by direct interaction with this microbial inflamma‐ tory mediatory [204].

These innate attributes of elafin make it an ideal candidate for a replacement therapy in treatment of inflammatory lung diseases such as COPD which is associated with excess protease burden and/or infection. However caution must be exercised, as like many other endogenous protease inhibitors, elafin is susceptible to inactivation by neutrophil-derived oxidants resulting in loss of its antiprotease activity [205]. Moreover, elafin can be cleaved by NE [206], which is present in high concentrations in the COPD lung [207] and cleavage can result in diminished ability of elafin to bind LPS and its capacity to be immobilized by transglutamination [206]. A similar observation was made in regard to the *P. aeruginosa*derived proteases pseudolysin and aeruginolysin which can cleave elafin thereby negatively impacting on its biological functions [208]. Collectively, these findings suggest that within an environ‐ ment of high protease burden, as found in the COPD lung, this could have a negative impact upon the clinical efficacy of elafin.

An additional native antiprotease that has been well documented for its potential as a therapy for COPD is human SLPI, which was originally identified in parotid secretions [20]. Being a member of the antileukoprotease family, SLPI shares 40% homology with elafin. A number of cell types have been documented to produce SLPI including macrophages [186], monocytes, neutrophils [187] and lung epithelial cells [209]. It is well accepted that SLPI is a potent inhibitor of an array of serine proteases including NE, cathepsin G, trypsin and chymotrypsin. Similar to elafin, SLPI expression levels are increased post inflammatory mediator exposure; for example TNFα and IL-1β treatment of lung epithelial cells [191]. Other host derived inflam‐ matory proteins including proteases from neutrophils (e.g. NE and defensins [184] ) can increase and induce SLPI expression in bronchial epithelial cells. In more recent studies, aside from host derived inflammatory mediators, bacteria have also been shown to induced SLPI expression in macrophages by a TLR2-dependent but MyD88-independent signalling pathway [210]. Additionally, bacterial products such as LPS can increase SLPI expression in macro‐ phages and neutrophils [211], suggesting that SLPI functions as an acute phase reactant.

*vitro* [226]. Alternatively, co-treatment of SLPI with surfactant protein A has been proposed, which aids in the preservation of SLPI and protects it from cleavage by matrix metalloproteases

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**4.5. Synthetic and semi-synthetic engineered protease inhibitors as alternative therapies for**

In a COPD animal model, ADAM-17 (a membrane bound MMP) has been shown to contribute to progression of COPD lung disease through activation of TNFα [228]. Recent studies have demonstrated that AAT, a natural serine protease inhibitor, also acts as an ADAM-17 inhibitor thereby regulating soluble immune complex induced neutrophil chemotaxis [148] and TNFα production in lung endothelial cells [229]. However, other therapeutic options for the modulation of inflammation associated with excessive pro‐ tease activity is the use of synthetic or semi synthetic protease inhibitors. These mole‐ cules, for example the non-peptide inhibitor ONO-5046 or sivelestat, offer better accessibility into the lung milieu due to their reduced size. Sivelestat is a specific inhibitor of NE and in LPS animal studies was shown to significantly reduce the number of infiltrating neutrophils and elastase activity levels, thereby decreasing lung tissue damage [230-232]. Moreover, sivelestat has been reported to reduce neutrophil-mediated endothelial cell injury by inactivating extracellular elastase and by suppressing release of this serine protease by neutrophils [231]. A recent study demonstrated the ability of sivelestat to inhibit bleomy‐ cin induced pulmonary fibrosis and apoptosis in human epithelial cells [233]. The au‐ thors of this study also verified that sivelestat reduced pulmonary neutrophil counts by decreasing BAL fluid levels of cytokine-induced neutrophil chemoattractant (CINC)-1. So far there have been no *in vitro* or *in vivo* investigations on the potential use of sivelestat in COPD, but studies involving other pulmonary conditions including post-cardiopulmona‐ ry bypass lung injury [234], acute lung injury with sepsis [235] and adult respiratory distress syndrome [236] have shown some therapeutic success with this synthetic inhibitor. To date, Japan has been the only country to approve sivelestat for the above conditions [237].

An alternative synthetic inhibitor that has been utilized in several studies is the cyclic thiol compound MR889. This synthetic inhibitor demonstrated inhibition against several serine proteases *in vitro* and in sputum from patients with chronic bronchitis [238]. When used in clinical studies however, although MR889 was considered safe for COPD patients, the results demonstrated that there was no significant reduction in the level of biomarkers of lung disease except for a small subset of individuals who had a short disease duration [239]. Other synthetic elastase inhibitors such as ZD0892 and FR134043 have been shown to reduce lung inflamma‐ tion associated with cigarette smoke [240, 241] or NE [242] in animal models and in cultured cells [243]. ZD0892 was shown not to exert an adverse effect on neutrophil function including uptake and killing of *S. aureus*, but use of this compound in clinical studies in humans has not

More recently, the synthetic selective inhibitor of elastase, AZD9668, has attracted a lot of attention and was shown to have significant impact on NE activity *in vitro* [244]. In rodent models, AZD9668 was shown to prevent NE-induced lung injury, cigarette smoke induced

[227].

**COPD**

been documented.

SLPI exhibits an array of anti-inflammatory properties including inhibition of monocyte production of MMPs [212, 213] which play an important pathophysiological role in tissue remodelling and are observed to be in high abundance in COPD [95, 107, 214]. The range of anti-inflammatory properties of SLPI includes inhibition of nitric oxide and TNFα production in macrophages in response to LPS [215] and inhibition of IFN-γ induced cathepsin S expres‐ sion in macrophages [216]. In monocytes it has been demonstrated that SLPI can cross the plasma membrane and enter the cell cytoplasm and nucleus [217]. Localization to these cell compartments facilitates the ability of SLPI to inhibit degradation of key proteins that activate NFκB and to competitively compete with p65 binding to NFκB consensus sequences thereby preventing NFκB binding to promoter regions of inflammatory genes [217]. In a more recent study involving neutrophils from COPD patients, the intracellular inhibitory activity of SLPI was further expanded as it was shown that SLPI modulated calcium flux and inositol 1,4,5 triphosphate generation thereby reducing cell migration [218] Additionally, SLPI possesses anti-microbial capabilities against *P. aeruginosa, S. aureus, S. epidermidis, E. coli and S. aureus* [219, 220]. SLPI's antifungal properties have been observed against *A. fumigatus* and *Candida albicans* [221] and in this latter study SLPIs fungicidal and fungistatic activity were shown comparable to that of human defensins and lysozyme.

Despite the potential advantages, delivery of SLPI to the lungs has proven unimpressive [222]. Aerosolisation of recombinant SLPI (100mg) twice daily to individuals with cystic fibrosis was associated with reduced NE activity on the respiratory epithelial surface, as well a reduction in the level of IL-8 and neutrophil numbers [223]. However, a greater concentration of recombinant SLPI compared to AAT is required to suppress NE activity in individuals with lung disease. Furthermore accumulation of recombinant SLPI on respiratory epithelial surfaces does not occur and SLPI does not penetrate significantly into the interstitium following aerosolization. Further drawbacks include the fact that most of the anti-NE effects are gone within 12 hours of administration. Reasons for this latter phenomenon have been proposed and may be due to uptake of SLPI by epithelial cells and macrophages, and/or binding of recombinant SLPI to molecules in the interstitium after passing through the epithelium [222, 224].

More recently, it has also been shown that SLPI is vulnerable to degradation and inactivation by cysteinyl cathepins [225] and NE [21], and thus the utilization of SLPI as effective therapy for COPD presents some challenges. One approach to this challenge is delivery of recombinant SLPI via a liposomal carrier which protects the activity of SLPI against cathepsin L mediated degradation, whilst having no adverse effect on SLPI access to intracellular sites of action *in* *vitro* [226]. Alternatively, co-treatment of SLPI with surfactant protein A has been proposed, which aids in the preservation of SLPI and protects it from cleavage by matrix metalloproteases [227].

to elafin, SLPI expression levels are increased post inflammatory mediator exposure; for example TNFα and IL-1β treatment of lung epithelial cells [191]. Other host derived inflam‐ matory proteins including proteases from neutrophils (e.g. NE and defensins [184] ) can increase and induce SLPI expression in bronchial epithelial cells. In more recent studies, aside from host derived inflammatory mediators, bacteria have also been shown to induced SLPI expression in macrophages by a TLR2-dependent but MyD88-independent signalling pathway [210]. Additionally, bacterial products such as LPS can increase SLPI expression in macro‐ phages and neutrophils [211], suggesting that SLPI functions as an acute phase reactant.

SLPI exhibits an array of anti-inflammatory properties including inhibition of monocyte production of MMPs [212, 213] which play an important pathophysiological role in tissue remodelling and are observed to be in high abundance in COPD [95, 107, 214]. The range of anti-inflammatory properties of SLPI includes inhibition of nitric oxide and TNFα production in macrophages in response to LPS [215] and inhibition of IFN-γ induced cathepsin S expres‐ sion in macrophages [216]. In monocytes it has been demonstrated that SLPI can cross the plasma membrane and enter the cell cytoplasm and nucleus [217]. Localization to these cell compartments facilitates the ability of SLPI to inhibit degradation of key proteins that activate NFκB and to competitively compete with p65 binding to NFκB consensus sequences thereby preventing NFκB binding to promoter regions of inflammatory genes [217]. In a more recent study involving neutrophils from COPD patients, the intracellular inhibitory activity of SLPI was further expanded as it was shown that SLPI modulated calcium flux and inositol 1,4,5 triphosphate generation thereby reducing cell migration [218] Additionally, SLPI possesses anti-microbial capabilities against *P. aeruginosa, S. aureus, S. epidermidis, E. coli and S. aureus* [219, 220]. SLPI's antifungal properties have been observed against *A. fumigatus* and *Candida albicans* [221] and in this latter study SLPIs fungicidal and fungistatic activity were shown

Despite the potential advantages, delivery of SLPI to the lungs has proven unimpressive [222]. Aerosolisation of recombinant SLPI (100mg) twice daily to individuals with cystic fibrosis was associated with reduced NE activity on the respiratory epithelial surface, as well a reduction in the level of IL-8 and neutrophil numbers [223]. However, a greater concentration of recombinant SLPI compared to AAT is required to suppress NE activity in individuals with lung disease. Furthermore accumulation of recombinant SLPI on respiratory epithelial surfaces does not occur and SLPI does not penetrate significantly into the interstitium following aerosolization. Further drawbacks include the fact that most of the anti-NE effects are gone within 12 hours of administration. Reasons for this latter phenomenon have been proposed and may be due to uptake of SLPI by epithelial cells and macrophages, and/or binding of recombinant SLPI to molecules in the interstitium after passing through the

More recently, it has also been shown that SLPI is vulnerable to degradation and inactivation by cysteinyl cathepins [225] and NE [21], and thus the utilization of SLPI as effective therapy for COPD presents some challenges. One approach to this challenge is delivery of recombinant SLPI via a liposomal carrier which protects the activity of SLPI against cathepsin L mediated degradation, whilst having no adverse effect on SLPI access to intracellular sites of action *in*

comparable to that of human defensins and lysozyme.

epithelium [222, 224].

86 COPD Clinical Perspectives

#### **4.5. Synthetic and semi-synthetic engineered protease inhibitors as alternative therapies for COPD**

In a COPD animal model, ADAM-17 (a membrane bound MMP) has been shown to contribute to progression of COPD lung disease through activation of TNFα [228]. Recent studies have demonstrated that AAT, a natural serine protease inhibitor, also acts as an ADAM-17 inhibitor thereby regulating soluble immune complex induced neutrophil chemotaxis [148] and TNFα production in lung endothelial cells [229]. However, other therapeutic options for the modulation of inflammation associated with excessive pro‐ tease activity is the use of synthetic or semi synthetic protease inhibitors. These mole‐ cules, for example the non-peptide inhibitor ONO-5046 or sivelestat, offer better accessibility into the lung milieu due to their reduced size. Sivelestat is a specific inhibitor of NE and in LPS animal studies was shown to significantly reduce the number of infiltrating neutrophils and elastase activity levels, thereby decreasing lung tissue damage [230-232]. Moreover, sivelestat has been reported to reduce neutrophil-mediated endothelial cell injury by inactivating extracellular elastase and by suppressing release of this serine protease by neutrophils [231]. A recent study demonstrated the ability of sivelestat to inhibit bleomy‐ cin induced pulmonary fibrosis and apoptosis in human epithelial cells [233]. The au‐ thors of this study also verified that sivelestat reduced pulmonary neutrophil counts by decreasing BAL fluid levels of cytokine-induced neutrophil chemoattractant (CINC)-1. So far there have been no *in vitro* or *in vivo* investigations on the potential use of sivelestat in COPD, but studies involving other pulmonary conditions including post-cardiopulmona‐ ry bypass lung injury [234], acute lung injury with sepsis [235] and adult respiratory distress syndrome [236] have shown some therapeutic success with this synthetic inhibitor. To date, Japan has been the only country to approve sivelestat for the above conditions [237].

An alternative synthetic inhibitor that has been utilized in several studies is the cyclic thiol compound MR889. This synthetic inhibitor demonstrated inhibition against several serine proteases *in vitro* and in sputum from patients with chronic bronchitis [238]. When used in clinical studies however, although MR889 was considered safe for COPD patients, the results demonstrated that there was no significant reduction in the level of biomarkers of lung disease except for a small subset of individuals who had a short disease duration [239]. Other synthetic elastase inhibitors such as ZD0892 and FR134043 have been shown to reduce lung inflamma‐ tion associated with cigarette smoke [240, 241] or NE [242] in animal models and in cultured cells [243]. ZD0892 was shown not to exert an adverse effect on neutrophil function including uptake and killing of *S. aureus*, but use of this compound in clinical studies in humans has not been documented.

More recently, the synthetic selective inhibitor of elastase, AZD9668, has attracted a lot of attention and was shown to have significant impact on NE activity *in vitro* [244]. In rodent models, AZD9668 was shown to prevent NE-induced lung injury, cigarette smoke induced inflammatory responses, airspace enlargement and small airways remodelling [244]. Despite these positive findings however, results of a recent clinical trial involving AZD9668, queried the efficiency of this inhibitor in COPD patients as no significant change in lung function and clinical measurements was observed [245]. Although these later results suggest that targeting NE activity alone may not be an effective treatment option in COPD, a subsequent smaller study indicated the potential clinical efficacy of AZD9668 in the treatment of bronchiectasis [246].

Within this expanding field of potential antiprotease therapies lies the development of novel chimeras that are active against NE, PR3 and CathG. A study by Zani *et al*., reported on the development of a chimera consisting of domains from both elafin and SLPI and a trappin-2 variant (A62L) [259]. The authors demonstrated that both A62L and the elafin-SLPI chimera retained their polypotent inhibition of protease activity while covalently cross-linked to fibronectin or elastin by a tissue transglutaminase, in a similar fashion to wild-type elafin and trappin-2 [188]. A recent extensive study on A62L in an epithelial cell line demonstrated that this engineered trappin-2 molecule prevented neutrophil and protease induced epithelial cell injury by inhibiting cell detachment, tight junction disruption and ultimately reducing apoptosis [260]. Although the preliminary data has been generated for these engineered antiproteases, overall their effect on the inflammatory status and clinical parameters in COPD

Antiproteases as Therapeutics to Target Inflammation in Chronic Obstructive Pulmonary Disease

Extensive studies have been performed to extend our knowledge of the involvement of proteases in respiratory manifestations of COPD, the outcome of which has served to illustrate the multifactorial complexity of the disease (Figure 2). Nevertheless, this clinical and scientific knowledge has advanced the design of both fundamental and novel therapeutic strategies, directed at relieving the protease burden associated with this disorder (Table 2). There have been major advances in the evaluation of AAT augmentation therapy in the treatment of the lung disease associated with AAT deficiency with newer focus on aerosolization approaches. These studies will inevitably show within the next number of years whether this approach works clinically. No single treatment that effectively prevents activity of the broad spectrum of proteases present in the airways is available as yet. Such pharmacological approaches including the development of antiprotease chimeras would essentially correct lung disease processes and conceivably exert anti-inflammatory effects. However, this would require a more complete understanding of the mechanism of disease and of the action of presently

**inactivators**

Alpha-1 antitrypsin (AAT) Monocyte neutrophil elastase inhibitor (MNEI/

Serpin B1) Elafin

Secretory leucocyte protease inhibitor (SLPI) **Clinical trials and evidence for antiproteases as therapeutics**

http://dx.doi.org/10.5772/57455

89

Intravenous augmentation therapy of AAT.(Multiple Clinical Trials in Humans, see Table 1) Clinical effects of IV AAT in

Slows rate of FEV1 decline Reduces elastin degradation Reduces exacerbation frequency

AATD:

patients remains to be investigated.

accessible and newly discovered drugs.

Neutrophil Elastase Elastolysis/destruction of

laminin

receptors

**Protease Role in COPD pathogenesis Inhibitors and**

extracellular matrix proteins including elastin, collagens, fibronectin, proteoglycans &

Cleaves epithelial cell surface

Cleaves signalling cytokines

**5. Conclusion**

Aside from serine proteases, and as already mentioned, MMPs also play a key role in the development of COPD. TIMPs are endogenous MMP inhibitors and in COPD, MMP activity levels associated with lung disease are not counteracted by TIMPs [247]. To date no native TIMPs have been used as potential therapeutics, but synthetic and selective MMP inhibits have been well documented. The orally administered MMP-9 and MMP-12 inhibitor AZD1236 was well tolerated by patients in two separate clinical trials but overall it was deemed to have no effect on clinical parameters [248] or disease related biomarkers in COPD patients [249]. In a recent study by Wu *et al*., several potential MMP-12 inhibitors were analysed *in vivo*, with one specific inhibitor, Compound 26, modulating cell infiltration into the lungs of mice with MMP-12 induced inflammation [250]. Similar findings were observed for a second MMP-12 inhibitor, Compound 14 [251]. An additional *in vivo* study involved the use of the broad spectrum MMP inhibitor RS113456, which possessed comparable anti-inflammatory proper‐ ties to AAT [252]. In the latter study the authors noted multi-facetted attributes of RS113456, whereby this MMP inhibitor reduced neutrophil influx, modulated NFκB activity and reduced MCP-1 and MIP-2 expression in the short term [252].

Other synthetic MMP inhibitors evaluated in cigarette smoke induced models of COPD have shown significant reductions in lung macrophage numbers [126], neutrophilia [253, 254], elastase/ MMP levels [254], air space enlargement [126, 254] and inflammatory markers [90, 126]. To date the benefits of MMP inhibitors has only been assessed in animal models and their successful use as therapeutics for treatment of COPD in humans has not been reported. Thus caution needs to be exercised due to the potential for tumour growth promotion as a result of inhibition of angiogenesis factors [255].

An alternative to synthetic inhibitors is the production of semi-synthetic protease inhibitors. These agents are created through the chemical modification of naturally occurring protease inhibitors. One such semi-synthetic inhibitor is the engineered protein inhibitor of human NE (EPI-HNE-4) which is derived from the Kunitz type domain from the naturally occurring inter α inhibitor. EPI-HNE-4 has been shown resistant to degradation by both human and bacterial MMPs and its antiprotease activity was reported impervious to oxidative inhibition [256]. Further studies involving the use of a rat model and sputum from cystic fibrosis patients demonstrated the anti-NE capacity of EPI-HNE-4 along with its ability to decrease neutrophil migration towards the bacterial peptide *N*-formyl-L-methionyl-L-leucyl-phenylalanine [257]. Subsequently however, contrary data on the immuno-regulatory effects of this inhibitor have been documented as Honoré *et al*., demonstrated that EPI-HNE-4 had little effect on neutrophil migration or impact on bacterial clearance in a *P. aeruginosa* model of pneumonia [258].

Within this expanding field of potential antiprotease therapies lies the development of novel chimeras that are active against NE, PR3 and CathG. A study by Zani *et al*., reported on the development of a chimera consisting of domains from both elafin and SLPI and a trappin-2 variant (A62L) [259]. The authors demonstrated that both A62L and the elafin-SLPI chimera retained their polypotent inhibition of protease activity while covalently cross-linked to fibronectin or elastin by a tissue transglutaminase, in a similar fashion to wild-type elafin and trappin-2 [188]. A recent extensive study on A62L in an epithelial cell line demonstrated that this engineered trappin-2 molecule prevented neutrophil and protease induced epithelial cell injury by inhibiting cell detachment, tight junction disruption and ultimately reducing apoptosis [260]. Although the preliminary data has been generated for these engineered antiproteases, overall their effect on the inflammatory status and clinical parameters in COPD patients remains to be investigated.

## **5. Conclusion**

inflammatory responses, airspace enlargement and small airways remodelling [244]. Despite these positive findings however, results of a recent clinical trial involving AZD9668, queried the efficiency of this inhibitor in COPD patients as no significant change in lung function and clinical measurements was observed [245]. Although these later results suggest that targeting NE activity alone may not be an effective treatment option in COPD, a subsequent smaller study indicated the potential clinical efficacy of AZD9668 in the treatment of bronchiectasis

Aside from serine proteases, and as already mentioned, MMPs also play a key role in the development of COPD. TIMPs are endogenous MMP inhibitors and in COPD, MMP activity levels associated with lung disease are not counteracted by TIMPs [247]. To date no native TIMPs have been used as potential therapeutics, but synthetic and selective MMP inhibits have been well documented. The orally administered MMP-9 and MMP-12 inhibitor AZD1236 was well tolerated by patients in two separate clinical trials but overall it was deemed to have no effect on clinical parameters [248] or disease related biomarkers in COPD patients [249]. In a recent study by Wu *et al*., several potential MMP-12 inhibitors were analysed *in vivo*, with one specific inhibitor, Compound 26, modulating cell infiltration into the lungs of mice with MMP-12 induced inflammation [250]. Similar findings were observed for a second MMP-12 inhibitor, Compound 14 [251]. An additional *in vivo* study involved the use of the broad spectrum MMP inhibitor RS113456, which possessed comparable anti-inflammatory proper‐ ties to AAT [252]. In the latter study the authors noted multi-facetted attributes of RS113456, whereby this MMP inhibitor reduced neutrophil influx, modulated NFκB activity and reduced

Other synthetic MMP inhibitors evaluated in cigarette smoke induced models of COPD have shown significant reductions in lung macrophage numbers [126], neutrophilia [253, 254], elastase/ MMP levels [254], air space enlargement [126, 254] and inflammatory markers [90, 126]. To date the benefits of MMP inhibitors has only been assessed in animal models and their successful use as therapeutics for treatment of COPD in humans has not been reported. Thus caution needs to be exercised due to the potential for tumour growth promotion as a result of

An alternative to synthetic inhibitors is the production of semi-synthetic protease inhibitors. These agents are created through the chemical modification of naturally occurring protease inhibitors. One such semi-synthetic inhibitor is the engineered protein inhibitor of human NE (EPI-HNE-4) which is derived from the Kunitz type domain from the naturally occurring inter α inhibitor. EPI-HNE-4 has been shown resistant to degradation by both human and bacterial MMPs and its antiprotease activity was reported impervious to oxidative inhibition [256]. Further studies involving the use of a rat model and sputum from cystic fibrosis patients demonstrated the anti-NE capacity of EPI-HNE-4 along with its ability to decrease neutrophil migration towards the bacterial peptide *N*-formyl-L-methionyl-L-leucyl-phenylalanine [257]. Subsequently however, contrary data on the immuno-regulatory effects of this inhibitor have been documented as Honoré *et al*., demonstrated that EPI-HNE-4 had little effect on neutrophil migration or impact on bacterial clearance in a *P. aeruginosa* model of pneumonia [258].

MCP-1 and MIP-2 expression in the short term [252].

inhibition of angiogenesis factors [255].

[246].

88 COPD Clinical Perspectives

Extensive studies have been performed to extend our knowledge of the involvement of proteases in respiratory manifestations of COPD, the outcome of which has served to illustrate the multifactorial complexity of the disease (Figure 2). Nevertheless, this clinical and scientific knowledge has advanced the design of both fundamental and novel therapeutic strategies, directed at relieving the protease burden associated with this disorder (Table 2). There have been major advances in the evaluation of AAT augmentation therapy in the treatment of the lung disease associated with AAT deficiency with newer focus on aerosolization approaches. These studies will inevitably show within the next number of years whether this approach works clinically. No single treatment that effectively prevents activity of the broad spectrum of proteases present in the airways is available as yet. Such pharmacological approaches including the development of antiprotease chimeras would essentially correct lung disease processes and conceivably exert anti-inflammatory effects. However, this would require a more complete understanding of the mechanism of disease and of the action of presently accessible and newly discovered drugs.

**Protease Role in COPD pathogenesis Inhibitors and**

extracellular matrix of the lung Induces mucus secretion Impairs T-cell function

Parenchymal destruction and emphysema development Proteolytic degradation of beta-defensins thus impairing

Induce airway inflammation

Degradation of collagen and

Monocyte recruitment Activates TNFα Induces IL-8 production Can degrade AAT

Degradation of collagens, elastin, gelatin, pro-MMP9,

TACE (TNFα converting enzyme) like activity Induces TGFβ-1 – leading to

Induces mucus hypersecretion

Degradation of collagens, gelatin, pro-MMP9, pro-

TACE (TNFα converting enzyme) like activity

pro-MMP13

fibrotic changes

MMP13

bacterial killing

elastin

Cathepsin G Proteolysis and destruction of

Other Cathepsins (Cathepsin B, D, H, K, L and S)

Matrix

Matrix

(MMP-1)

Other Matrix Metalloproteases (MMP-2, MMP-8, MMP 10, MMP-13)

Metalloprotease-1

(MMP-12)

Metalloprotease-12

**inactivators**

Antiproteases as Therapeutics to Target Inflammation in Chronic Obstructive Pulmonary Disease

Alpha-1 antitrypsin (AAT) Secretory leucocyte protease inhibitor (SLPI)

Cystatins are a group of endogenous reversible, tight-binding competitive cysteine protease inhibitors

cathepsins B, H, and L. SLPI inhibits cathepsin S expression in macrophages

Tissue inhibitors of metalloproteases (TIMP) Secretory leucocyte protease inhibitor (SLPI) AZD1236 (synthetic inhibitor of MMP-12 and

Compound 26 (synthetic

Compound 14 (synthetic

MMP-9)

compound)

compound) RS113456 (synthetic MMP-12 inhibitor)

MMP-9)

Tissue inhibitors of metalloproteases (TIMP) Secretory leucocyte protease inhibitor (SLPI) AZD1236 (synthetic inhibitor of MMP-12 and

Tissue inhibitors of metalloproteases (TIMP)

for

**Clinical trials and evidence for antiproteases as therapeutics**

http://dx.doi.org/10.5772/57455

91

See above for clinical effects of AAT therapy in AATD and COPD

SLPI – Anti-inflammatory properties and specifically inhibits monocyte production of

AZD1236 – Well tolerated in COPD but not effect on clinical parameters (248,249) Compound 26, Compound 14,

MMPs (212, 213)

RS113456 – Reduced neutrophilic inflammation but no human trial data (250-252)

SLPI – Anti-inflammatory properties and specifically inhibits monocyte production of

AZD1236 – Well tolerated in COPD but no effect on clinical parameters (248,249)

MMPs (212, 213)

**Protease Role in COPD pathogenesis Inhibitors and**

Induces IL-8 expression in bronchial epithelial cells Upregulates TGF-β in airway

production of cathepsin B and

Induces expression of MUC5AC leading to hypersecretion of

Decreases ciliary beat frequency of bronchial epithelial cells

smooth muscle Impairs T-cell function Induces macrophage

MMP-2

90 COPD Clinical Perspectives

mucus.

Proteinase-3 (PR3) Elastolysis/destruction of

NE

extracellular matrix proteins including elastin, collagens, fibronectin, proteoglycans & laminin. Less destructive than

Pro-inflammatory, activates

Hypersecretion of mucus Cellular apoptosis

TNFα and IL-1β

**inactivators**

Sivelestat (synthetic nonpeptide inhibitor) MR889 (synthetic cyclic thiol compound)

ZD0892 (synthetic elastase

FR134043 (synthetic elastase inhibitor) AZD9668 (synthetic selective elastase inhibitor)

inhibitor)

**Clinical trials and evidence for antiproteases as therapeutics**

Attenuates loss of lung density Clinical effects of aerosolized

Restores anti-NE capacity (176,

Improved neutrophil killing of

Outcome of phase III clinical trial pending (NCT 0217671) SLPI – Reduced NE activity and reduced IL-8 levels in CF patients

Sivelestat – Reduced number of

Has shown therapeutic benefit in acute lung injury and ARDS, but no trials in COPD to date (230-232, 235, 236)

MR889 – Safe but no significant reduction in the level of biomarkers of lung disease in COPD patients (239)

ZD0892 & FR134043 – Reduce cigarette induced lung inflammation, but no human trials to date (240,2410) AZD9668 – Decreased NE activity

No change in clinical parameters

AAT therapy in AATD and COPD

*in vitro* (244)

in COPD (245)

Alpha-1 antitrypsin (AAT) See above for clinical effects of

*P. aeruginosa* (179)

neutrophils in BAL.

AAT:

177)

(223)

**Acknowledgements**

**Author details**

Emer P. Reeves\*

**References**

Education Authority and Science Foundation Ireland.

\*Address all correspondence to<EMAIL_ADDRESS>
2002;102(12):4501-24. Epub 2002/12/12.

2225-36. Epub 2005/06/28.

475-88. Epub 2009/08/04.

This work was supported by the Medical Research Charities Group/Health Research Board, Science Foundation Ireland (grant number 11/RFP/BMT/3094), the US Alpha One Foundation and the Program for Research in Third Level Institutes (PRTLI) administered by the Higher

Antiproteases as Therapeutics to Target Inflammation in Chronic Obstructive Pulmonary Disease

http://dx.doi.org/10.5772/57455

93

Respiratory Research Division, Department of Medicine, Royal College of Surgeons in Ire‐

[1] Hedstrom L. Serine protease mechanism and specificity. Chemical reviews.

[2] Rooney CP, Taggart C, Coakley R, McElvaney NG, O'Neill SJ. Anti-proteinase 3 anti‐ body activation of neutrophils can be inhibited by alpha1-antitrypsin. American jour‐ nal of respiratory cell and molecular biology. 2001;24(6):747-54. Epub 2001/06/21.

[3] Stoller JK, Aboussouan LS. Alpha1-antitrypsin deficiency. Lancet. 2005;365(9478):

[4] Gooptu B, Ekeowa UI, Lomas DA. Mechanisms of emphysema in alpha1-antitrypsin deficiency: molecular and cellular insights. The European respiratory journal : official journal of the European Society for Clinical Respiratory Physiology. 2009;34(2):

[5] Hiemstra PS, van Wetering S, Stolk J. Neutrophil serine proteinases and defensins in chronic obstructive pulmonary disease: effects on pulmonary epithelium. The Euro‐ pean respiratory journal : official journal of the European Society for Clinical Respi‐

[6] Kelly E, Greene CM, McElvaney NG. Targeting neutrophil elastase in cystic fibrosis.

Expert opinion on therapeutic targets. 2008;12(2):145-57. Epub 2008/01/23.

ratory Physiology. 1998;12(5):1200-8. Epub 1998/12/24.

Cormac McCarthy, Ciara A. O'Dwyer, David A. Bergin, Noel G. McElvaney and

land, Education and Research Centre, Beaumont Hospital, Dublin, Ireland

**Table 2.** Summary table listing the various proteases, their targets, their inhibitor/inactivators, and clinical trials.

**Figure 2. The effect of protease activity on the pathophysiology of COPD.** A diagramatic illustration of the pri‐ mary effects of excessive protease activity on inflammation in COPD. The effects include activation of signalling path‐ ways and proteases, to cleavage and degradation of matrix proteins and antiproteases. The secondary effect of the excessive protease burden in the COPD lung can result in tissue destruction/ remodelling and excessive cytokine and mucin production. Consequenrly proteases play a key role in emphysema, neutrophil influx, inflammation, mucus pro‐ duction and infection. As shown by the red lines, antiprotease therapy (AAT, SLPI and elafin) can impact on several aspects of this complex and intertwined pathway and potentially modulate disease progression.

## **Acknowledgements**

**Protease Role in COPD pathogenesis Inhibitors and**

Degradation of collagens, elastin, gelatin and fibronectin

92 COPD Clinical Perspectives

**inactivators**

Tissue inhibitors of metalloproteases (TIMP)

**Table 2.** Summary table listing the various proteases, their targets, their inhibitor/inactivators, and clinical trials.

**Figure 2. The effect of protease activity on the pathophysiology of COPD.** A diagramatic illustration of the pri‐ mary effects of excessive protease activity on inflammation in COPD. The effects include activation of signalling path‐ ways and proteases, to cleavage and degradation of matrix proteins and antiproteases. The secondary effect of the excessive protease burden in the COPD lung can result in tissue destruction/ remodelling and excessive cytokine and mucin production. Consequenrly proteases play a key role in emphysema, neutrophil influx, inflammation, mucus pro‐ duction and infection. As shown by the red lines, antiprotease therapy (AAT, SLPI and elafin) can impact on several

aspects of this complex and intertwined pathway and potentially modulate disease progression.

**Clinical trials and evidence for antiproteases as therapeutics**

> This work was supported by the Medical Research Charities Group/Health Research Board, Science Foundation Ireland (grant number 11/RFP/BMT/3094), the US Alpha One Foundation and the Program for Research in Third Level Institutes (PRTLI) administered by the Higher Education Authority and Science Foundation Ireland.

## **Author details**

Cormac McCarthy, Ciara A. O'Dwyer, David A. Bergin, Noel G. McElvaney and Emer P. Reeves\*

\*Address all correspondence to<EMAIL_ADDRESS>
Respiratory Research Division, Department of Medicine, Royal College of Surgeons in Ire‐ land, Education and Research Centre, Beaumont Hospital, Dublin, Ireland

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