Neutrophil elastase inhibitors: a patent review and potential applications for inflammatory lung diseases (2010 - 2014).

Review

1.

Introduction

2.

Physiological functions of NE

3.

Pathogenesis caused by a protease--antiprotease

Expert Opin. Ther. Patents Downloaded from informahealthcare.com by University of Otago on 07/07/15 For personal use only.

imbalance 4.

NE inhibitors

5.

Expert opinion

Neutrophil elastase inhibitors: a patent review and potential applications for inflammatory lung diseases (2010 -- 2014) Yung-Fong Tsai & Tsong-Long Hwang† †

Chang Gung University, Graduate Institute of Natural Products, School of Traditional Chinese Medicine, College of Medicine, Taoyuan, Taiwan

Introduction: The proteolytic activity of neutrophil elastase (NE) not only destroys pathogens but also degrades host matrix tissues by generating a localized protease--antiprotease imbalance. In humans, NE is well known to be involved in various acute and chronic inflammatory diseases, such as chronic obstructive pulmonary disease, emphysema, asthma, acute lung injury, acute respiratory distress syndrome and cystic fibrosis. The regulation of NE activity is thought to represent a promising therapeutic approach, and NE is considered as an important target for the development of novel selective inhibitors to treat these diseases. Areas covered: This article summarizes and analyzes patents on NE inhibitors and their therapeutic potential based on a review of patent applications disclosed between 2010 and 2014. Expert opinion: According to this review of recent NE inhibitor patents, all of the disclosed inhibitors can be classified into peptide- and non-peptide-based groups. The non-peptide NE inhibitors include heterocyclics, uracil derivatives and deuterium oxide. Among the heterocyclic analogs, derivatives of pyrimidinones, tetrahydropyrrolopyrimidinediones, pyrazinones, benzoxazinones and hypersulfated disaccharides were introduced. The literature has increasingly implicated NE in the pathogenesis of various diseases, of which inflammatory destructive lung diseases remain a major concern. However, only a few agents have been validated for therapeutic use in clinical settings to date. Keywords: elastase inhibitor, inflammatory diseases, neutrophil elastase, protease--antiprotease imbalance Expert Opin. Ther. Patents [Early Online]

1.

Introduction

Polymorphonuclear neutrophils play an essential role in innate immunity and are a part of the first line of defense against pathogens [1]. Activated neutrophils destroy invading pathogens by releasing oxidative free radicals and non-oxidative proteases to neutralize phagocytized pathogens. In addition to their intracellular activities, proteases are also degranulated into the extracellular space to help kill bacteria. Neutrophil elastase (NE) is especially known to perform this function. Infiltrated neutrophils secrete a high level of proteolytic NE, which can degrade proteins from invading pathogens or damage the elastin-rich connective tissue of targeted organs, especially the lungs [2,3]. NE also cleaves collagens, fibronectin, proteoglycans [4,5] and many plasma proteins [6,7]. Overactive NE function may disrupt the integrity of the permeability barrier between alveolar epithelial and endothelial cells and may induce edematous changes in inflamed lungs. In addition to infectious or 10.1517/13543776.2015.1061998 © 2015 Informa UK, Ltd. ISSN 1354-3776, e-ISSN 1744-7674 All rights reserved: reproduction in whole or in part not permitted

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Y.-F. Tsai & T.-L. Hwang

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Article highlights. .

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In this review, we report all 20 neutrophil elastase (NE) inhibitor patents that were published from 2010 to 2014. In total, 42 general schemes or significant derivatives were demonstrated or structurally illustrated. All of the disclosed compounds are divided into two major categories: peptide- and non-peptide-based inhibitors. The peptide-based NE inhibitors include 3 linear peptides, 13 cyclic template-fixed b-hairpin peptidomimetics, and a mutant plasminogen activator inhibitor-1 protein. All of the non-peptide-based NE inhibitors are low-molecular-mass synthetic inhibitors, including heterocyclic inhibitors, a uracil derivative and deuterium oxide. The non-peptide-based heterocyclic NE inhibitors include pyrimidinone, pyrimidinedione, pyrimidine, 2-pyrazinone, pyridine, benzoxazinone and hypersulfated disaccharide derivatives.

This box summarizes key points contained in the article.

inflammatory lung diseases [8-11], increasing evidence supports the notion that NE participates in the pathogenesis of several extrapulmonary diseases, such as ischemia-reperfusion injury [9], rheumatoid arthritis, autoimmune diabetes [12], delayed wound healing and glomerulonephritis [13]. Notably, specific NE inhibitors have been developed as drugs over the past two decades. Sivelestat sodium hydrate (sivelestat, Elaspol) is a synthetic NE inhibitor that was suggested to treat acute lung injury associated with pathogen infections; this compound is presently marketed as such [14]. However, in clinical trials, intravenous sivelestat treatment of acute lung injury and acute respiratory distress syndrome (ARDS) patients failed to improve clinical outcomes, including the number of ventilator-free days and the 28-day mortality rate [15,16]. In addition to direct NE inactivation by inhibitors, other therapeutic interventions that limit the activation and recruitment of neutrophils in the lungs can reduce excessive proteolytic activity and the levels of other reactive noxious molecules, such as superoxide anions and proinflammatory mediators, in inflammatory pulmonary diseases. For instance, PDE4 inhibitors suppress neutrophil activation, reactive oxygen species (ROS) generation and cell migration, and these compounds have been used in treating chronic obstructive pulmonary disease (COPD) patients [17,18]. Although increasingly selective NE inhibitors have demonstrated significant protective effects against acute or chronic lung injury in animal models, few of these inhibitors have been approved for clinical treatment. The invention of novel NE inhibitors with high potency and low toxicity that are suitable for patient therapy is encouraged. 2

Physiological functions of NE

Elastase is found not only in neutrophils but also in monocytes, mast cells, eosinophils [19,20], keratinocytes and fibroblasts [21]. The concentration of NE in neutrophils is estimated to be ~ 5 mM, and NE in its active form is tightly compartmentalized in granules [22]. The largest type of granule is the azurophilic granule (peroxidase-positive granule), also referred to as the primary granule; it is the first granule produced during neutrophil maturation. It is named for its ability to be stained by the dye azure A, and it contains myeloperoxidase (MPO), which is an essential enzyme for oxidative respiratory burst. As neutrophils are activated to defend against invading pathogens or to react with stimuli, azurophilic granules translocate to and fuse with the phagosome or the plasma membrane to release their contents into their respective environments. Azurophilic granules contain MPO and four chymotrypsin family serine proteases: NE, cathepsin G (CG), proteinase 3 (PR3), and neutrophil serine protease 4 (NSP4) [23,24]. They participate in the degradation and digestion of engulfed microorganisms and in the remodeling of connective tissue in inflamed areas. The enzyme commission number of human NE is EC 3.4.21.37. There are 218 residues in NE, and its molecular mass is ~ 29.5 kDa. The catalytic triad, which consists of aspartate (Asp102), histidine (His57) and serine (Ser195) residues, is responsible for the potent catalytic activity of NE. It is a cationic protein with a basic isoelectric point, and the optimal pH for its enzymatic activity is ~ 8.0 -- 8.5. The active NE structure is composed of an N-terminal activation domain and three flexible loops. ELA2, which is the gene that encodes human NE, is located on chromosome 19 at p13.3 [25]. The mutation of this gene is a risk factor for congenital neutropenia [26]. NE is implicated in immune responses and signal transduction [27,28]. Human NE activates Toll-like receptor-4 to induce IL-8 expression [29]. In addition to its ability to degrade pathogen and matrix proteins, NE can also modulate active cytokines and their receptors at inflammation sites [30]. NE stimulates the apoptosis of lung endothelial cells and regulates their barrier integrity via proteinase-activated receptor (PAR)1 signaling [31,32]. Moreover, NE induces mucus hypersecretion from airway endothelial cells by binding to PAR2 [33]. NE cleaves lung surfactant protein [34] and receptors or surface antigens on immune cells [35,36]. It not only induces cytokine and chemokine activities but also induces submucosal gland secretion [37] and keratinocyte proliferation in psoriasis [38]. NE also contributes to the neutrophilmediated activation of platelets [39] and to wrinkle formation via decreased skin elasticity [21]. NE was shown to disrupt matrix barrier integrity, which likely facilitates cancer development, and NE inhibitors were shown to suppress skin tumorigenesis in mice [40,41].

Expert Opin. Ther. Patents (2015) 25(10)

Neutrophil elastase inhibitors

Pathogenesis caused by a protease--antiprotease imbalance


3.

4.

Several endogenous secretory NE inhibitors neutralize the proteolytic activity of NE, and these inhibitors are especially abundant in the respiratory tract and circulation system naturally. They function by closely maintaining the protease--antiprotease balance to protect tissues from proteolysis [42]. The 1-antitrypsin deficiency results in emphysema, which can be successfully treated via exogenous supplementation of 1-antitrypsin [43]. The intrinsic physiological inhibitors of NE can be divided into three main families: the serpin, chelonianin and macroglobulin families. They exert potent control over the proteolytic activity of NE, PR3, CG and NSP4. The serpins are the most abundant NE inhibitors, and they play a major role in fundamental biological reactions. They are implicated in inflammation, tumor suppression, coagulation and complement activation [44]. In the serpin family, a1-protease inhibitor (a1-PI), serpin B1 and proteinase inhibitor 9 are able to inhibit NE. Additionally, secretory leukocyte protease inhibitor (SLPI) and elafin, which are members of the chelonianin family, are able to inhibit NE. SLPI and elafin are primarily produced by respiratory tract epithelial cells. a2-Macroglobulin also inhibits NE activity. However, it does not easily diffuse into inflammatory tissues because of its high molecular weight; therefore, it predominantly regulates NE activity in the blood circulation [45,46]. Several in vitro studies have shown that NE induces proinflammatory mediator release from immune cells and airway epithelial cells. NE has the potential to degrade elastin, basement membrane proteins and interstitial collagens in vitro [47]; thus, NE activity enlarges the lung airspace [48]. NE also induces mucin production, which leads to airway obstruction [33,49]. Overactive NE damages epithelial cells and decreases mucociliary function in vitro [50]. NE is also thought to induce subepithelial fibrosis in airways. The protease--antiprotease balance may be affected by a decrease in a1-antitrypsin availability, insufficient production of a1-antitrypsin due to genetic defects or the inactivation of a1-antitrypsin by smoking-induced oxidants. The endogenous inhibitor a1-PI is administered to a1-PIdeficient patients who suffer from lung damage or COPD. This strategy effectively reduces bronchial inflammation, alleviates lung function deterioration and increases quality of life [51]. In animal studies, synthetic NE inhibitor administration via the oral, inhaled or systemic route was confirmed to suppress the progression of lung damage and inflammation [52-54]. A recent study demonstrated that the protease--antiprotease imbalance is also related to the pathogenesis of type 1 diabetes [12]. In these patients, the a1-antitrypsin levels are decreased; however, circulating NE concentrations and NE enzymatic activities are significantly increased. These changes correlate with diabetes-associated auto-antibody levels.

NE inhibitors

In inflammatory lung diseases such as COPD, cystic fibrosis, emphysema, acute lung injury and ARDS, excess neutrophil accumulation and activation disrupt the protease--antiprotease balance and trigger lung destruction. Therefore, effective treatments can be targeted to inhibit excessive NE activity or to indirectly limit the NE burden by restraining neutrophil influx into the lungs. Several therapeutic strategies have been proposed, such as the use of specific NE inhibitors to directly reduce unwanted proteolysis and to perturb the intracellular concentrations or trafficking of active NE. These approaches may reduce extracellular or intracellular NE activity levels while not affecting neutrophil recruitment and infiltration, which are required for innate defense in infected tissues. Another approach to decrease neutrophil accumulation is to inhibit dipeptidyl peptidase I (cathepsin C) activity [55]. Dipeptidyl peptidase I functions by post-translationally processing inactive NE into an active form before it is stored in granules. This alternative strategy to indirectly decrease total NE activity is thought to be advantageous in non-infectious or inflammatory diseases. Based on the pathogenic mechanisms mentioned above, NE has been targeted for the development of low-molecularmass organic and peptide-based inhibitor drugs. We comprehensively review the patents registered from 2010 to 2014 and their possible applications in the following sections. Peptide-based inhibitors Although low-molecular-mass NE inhibitors represent mainstream potential therapeutics, several novel peptide-based NE inhibitors with a larger molecular mass have been developed. Peptides with the general formula R1-W p-X n-AA1-AA2-AA3-AA4-Y m-R2 (where AA1 to AA4 are selected amino acids) (1) tend to inhibit human NE activity and/or stimulate collagen synthesis in the skin; thus, they are useful in cosmetic applications [56]. The minimum inhibitory concentrations (IC50 values) of these peptides against human NE are 0.094 mM for H-L-Arg-Phg-L-Val-Phg-OH (2), 0.407 mM for H-L-Arg-Phg-L-Val-Gly-OH (3) and 0.103 mM for H-L-Arg-Phg-L-Trp-Phg-OH (4) (Table 1). Compared with linear peptides, template-fixed b-hairpin peptidomimetics [57], which are cyclic peptides, provide more stability and structural rigidity in physiological settings because of their macrolactam-cystine knot scaffold [58]. For stabilizing the structure, a selected undecapeptide sequence helps to form a heterocyclic structure by anchoring the C and N termini to a heterochiral template that is composed of proline together with proline, glutamine or aspartic acid. This modification results in potent activity against NE, CG or tryptase, which are significant therapeutic targets. There are 13 template-fixed peptidomimetic structures (5 -- 17) that exert inhibitory effects on NE, and 4 of them are disclosed as selective inhibitors in this patent application 4.1


3


Table 1. Inhibitory effects of selected peptides claimed by Lipotec, Inc. [56].


General scheme: R1-W p-X n-AA1-AA2-AA3-AA4-Y m-R2 (1) AA1: -Arg-, -Phg- and -Nle- or is a bond; AA2: -Ala-, -Phg-, -Cit- and -Nle-; AA3: -Trp-, -Val- and -Tyr-; AA4: -Phg- and -GlyPeptide sequence

NE (IC50, mM)

(2) H-L-Arg-Phg-L-Val-Phg-OH (3) H-L-Arg-Phg-L-Val-Gly-OH (4) H-L-Arg-Phg-L-Trp-Phg-OH

0.094 0.407 0.103

(Table 2). These effective synthetics inhibit NE activity in the nanomolar range with high stability in the circulation. The aamino acid residues in positions 1 -- 11 are involved in serine protease inhibitory activity. The relative selectivity between these proteases relies on the choice of a-amino acid residues and their position in the side chain. The template induces the H-bond network to fix a b-hairpin mimetic conformation in the Z-chain of the peptide. The template-fixed b-hairpin is also important for selective inhibitory activity. b-Hairpin peptidomimetics can be applied to pulmonary diseases or infections and immunological, cardiovascular or neurodegenerative diseases that result from NE activity. These formulations can be administered via systemic injection or via oral or pulmonary administration. Plasminogen activators (PAs), consisting of tissue-type and urokinase-type PAs, belong to the serine protease superfamily and activate the proenzyme of plasminogen such that plasmin is formed [59]. PAs are expressed in mammals and are involved in many biological reactions, including inflammation, coagulation, fibrinolysis, angiogenesis, tissue remodeling and tumor metastasis. PA inhibitors (PAIs) belong to the serpin superfamily. A mutant PAI-1 protein in which positions 331 and 346 in the reactive center loop of the mature PAI-1 were substituted (18) was shown to inhibit NE activity and vitronectindependent cell migration [60]. In this mutation, the residue at position 346 is substituted with Ala, Val, Asp, Phe and Gly, and the residue at position 331 is substituted with Gly. This mutant protein, which notably inhibited NE, could be used to treat NE-associated diseases such as emphysema, cystic fibrosis and inflammatory lung injury. Because of its additional suppression of vitronectin, it is also promising as a treatment for diseases that are associated with unwanted cell migration and proliferation, including atherosclerosis, post-angioplasty restenosis, tumor invasion and chronic inflammatory fibrosis [60]. Non-peptide-based inhibitors Compared with higher molecular mass physiological NE inhibitors, low-molecular-mass synthetic inhibitors more easily reach protected interstitial microenvironmental spaces and 4.2

4

reach and inhibit membrane-associated NE [61]. Low-molecular-mass protease inhibitors are also more structurally resistant to ROS [62]. Moreover, low-molecular-mass protease inhibitors reduce the activity of not only free NE but also substrate-bound NE, which endogenous high-molecularmass NE inhibitors are typically unable to inhibit [63]. Sivelestat, a low-molecular-mass (528.51 g/mol) NE inhibitor, is the only synthetic inhibitor on the clinical market that is approved (in Japan and South Korea) to treat acute lung injury associated with systemic inflammatory response syndrome. Additionally, sivelestat has been suggested to protect against pulmonary microcirculation disturbances and interstitial edema [64]. The active compound is only suitable for intravenous administration. Heterocyclic inhibitors Among the low-molecular-mass NE inhibitors, many structurally diverse classes of heterocyclic NE inhibitors, particularly reversible competitive NE inhibitors, have been disclosed in recent patent applications. Many of the claimed pharmaceutical compounds have been adapted for pulmonary or oral administration. The pyrimidine moiety is a significant characteristic of heterocyclic nitrogen, which is extensively applied as a foundation for pharmaceutical designs. Pyrimidine derivatives display therapeutic properties such as antifungal, calcium-blocking and anti-inflammatory activities [65]. The dihydropyrimidinones function as calcium channel modulators and selective a1a-adrenoceptor antagonists. Recently, derivatives of these compounds have been found to be potent human NE inhibitors and have thus been proposed as potential COPD therapeutics [66]. Twenty-nine 4-(4-cyano-2-thioaryl)dihydropyrimidinone (Table 3) derivatives (19) and 273 other substituted bicyclic dihydropyrimidinone derivatives (20) claimed in reviewed patents potently inhibit NE, and the IC50 values for most of these compounds are in the picomolar to low nanomolar range [67]. 4-(4-Cyano-2-thioaryl)-dihydropyrimidin-2-ones were claimed for the treatment or prevention of lung and cardiovascular system diseases [67]. Substituted bicyclic dihydropyrimidinones are applicable for treating pulmonary, gastrointestinal and inflammatory diseases [68]. Argenta Discovery Ltd disclosed a patent for multimeric heterocyclic pyrimidinone derivatives in which the compound formula is M-L-M, where L is a linker and each M is an independent group (21). These derivatives were revealed to have IC50 values for human NE in the range of 1 -- 1,000 nM [69]. NE selectivity assays showed selectivity ranging from 1- to > 300-fold for various proteases and demonstrated desirable human NE inhibitory activity in an NE-induced lung hemorrhage model in rats. The IC50 values for the inhibition of human NE by N,N¢-[ethane-1,2-diylbis(oxyethane-2,1-diyl)] bis {5-[4-cyanophenyl)sulfinyl]-6-methyl-2-oxo-1-[3-(trifluoromethyl)phenyl]-1,2 dihydropyridine-3-carboxamide} (22) and N,N¢-(2-hydroxypropane-1,3-diyl)bis(5-(1-(4-cyanophenyl)-1H-pyrazol-5-yl)-6-methyl-2-oxo-1-(3-(trifluoromethyl)phenyl)-1,2-dihydropyridine-3-carboxamide) (23) are 4.2.1



Table 2. Peptide sequences and anti-proteolytic activity of derivatives from the claimed template-fixed b-hairpin scheme [57].


Sequence of P1 -- P11 Selective (5) OctG Cys Thr Ala Ser Cha Pro Pro Gln Cys Phe (6) OctG Cys Thr Ala Ser Cha Pro Pro Gln Cys Gln (7) hPhe Cys Thr Ala Ser OctG Pro Pro Gln Cys Tyr (8) OctG Cys Thr Ala Ser OctG Pro Pro Gln Cys Gln Non-selective (9) Nle Cys Thr Ala Ser Ile Pro Pro Gln Cys Gln (10) Nle Cys Thr Ala Ser Ile Pro Pro Gln Cys Tyr (11) hPhe Cys Thr Ala Ser OctG Pro Pro Gln Cys Gln (12) OctG Gln Thr Ala Ser Ile Pro Pro Gln Thr Tyr (13) hPhe Cys Thr Ala Ser Cha Pro Pro Gln Cys Phe (14) OctG Glu Thr Ala Ser Ile Pro Pro Gln Lys Tyr (15) hPhe Cys Thr Ala Ser Cha Pro Pro Gln Cys Gln (16) OctG Cys Thr Ala Ser Cha Pro Pro(4NHCOPhe) Gln Cys Gln (17) OctG Cys Thr Ala Ser Ile Pro Pro Gln Cys Tyr

Template

NE (IC50, nM)

CG (IC50, nM)

D

ProLPro ProLPro D ProLPro D ProLGln

7 7 6 6

ND ND ND ND

D

28 18 8.5 8.3 8.2 7.5 7.1 6 8

45* 11155 56* 27* 52* 46* 55* 55* 54*

D

ProLPro ProLAsp D ProLPro D ProLPro D ProLPro D ProLPro D ProLPro D ProLPro D ProLGln D

*Represents the enzymatic activity of the derivatives, which was calculated as the percentage inhibition by the inhibitor at 100 µM. CG: Cathepsin G; ND: Not determined; NE: Neutrophil elastase.

0.61 and 0.26 nM, respectively. The protective effects of these two compounds were also confirmed in human NE- and lipopolysaccharide (LPS)-induced rat lung injury models [70]. Most of the tetrahydropyrrolopyrimidinediones (24) display human NE inhibitory activity in the 1 -- 50 nM range, and they were shown to protect rat lungs in a human NE-induced acute lung injury model [71]. The IC50 for the inhibition of human NE is < 20 nM [72] for most tetrahydrotriazolopyrimidines (25) and 5 nM -- 5 µM for 1,4-diaryldihydropyrimidin-2-ones (26) [73]. Various derivatives of these compounds have been claimed as potent human NE inhibitors for the treatment of COPD, acute coronary syndrome, myocardial infarction and heart failure. Other pyrimidine derivatives (27) that were suggested as COPD treatments exhibited human NE inhibitory activity in the range of 1 -- 200 nM [74]. Two patent applications disclosed several structurally similar derivatives of 2-pyrazinones, which are illustrated in Table 4 as compounds (28) and (29 -- 34); all of these derivatives display IC50 values for human NE activity in the nanomolar range. The patented compounds may be administered orally or as a dry powder for inhalation [75,76]. 2-Pyridone compounds (35,36) were demonstrated to inhibit human NE with IC50 values in the nanomolar to picomolar range [77,78]. Hundreds of related structural variants, which are known as substituted 4-pyridone derivatives, were claimed in patents to display IC50 values for human NE inhibition predominantly in the nanomolar range [79]. The main compounds and their formulas are illustrated as compounds (37,38) in (Table 5). A group of 204 representative compounds derived from benzoxazinone groups exhibited inhibitory activity on human NE in the < 15 -- 150 nM range [80]; the general formula of

these patented compounds is shown as compound (39) (Table 6). Their beneficial effects were tested in a rat LPSinduced acute lung injury model. The benzoxazinones are known as heterocyclic acylating compounds that inhibit human NE via a mechanism involving the formation of an acyl enzyme intermediate [81,82]. In a previous study, a new series of benzoxazinone analogs were identified, and these analogs exerted potent dual inhibitory effects on NE activity and superoxide anion generation in formyl-L-methionyl-Lleucyl-L-phenylalanine -activated human neutrophils. The inhibitory effects of these compounds on NE were related to the position of the chloro-substituent in the A ring. These compounds also demonstrated antiplatelet aggregation effects and protected rats from hemorrhagic shock-induced lung injury [81,83]. Heterocyclic sulfide derivatives were disclosed to potently inhibit the activities of serine proteases [84]. A synthetic group of hypersulfated disaccharides (Table 7) (40) has been claimed to potently inhibit human NE [85]. These synthetic disaccharide compounds were derived from heparin, which is a polysaccharide. A previous study proposed that heparin fragments possess NE inhibitory activity when the chain length is at least 12 -- 14 saccharides. In an in vivo study, the disclosed compounds effectively protected NE-treated sheep, restored tracheal mucus velocity and suppressed lung mucociliary dysfunction. However, the evidence of NE inhibition was not disclosed in this patent. The hypersulfated disaccharides disclosed in this patent were recommended for the treatment of COPD and cystic fibrosis. The claimed compositions are deliverable to the lungs via an aerosol route or via nebulization, and oral intake is also suitable.


5



Table 3. Pyrimidinone, pyrimidinedione and pyrimidine derivatives. Human NE inhibitors

IC50 (nM)

Ref.

Dihydropyrimidinones (19)

< 0.3 -- 10

[67]

(20)

< 1 -- 7015

[68]

Pyrimidinones (21)

1 -- 1000

[69]

(22)

0.61

[70]

NE: Neutrophil elastase.

6




Table 3. Pyrimidinone, pyrimidinedione and pyrimidine derivatives (continued). Human NE inhibitors

IC50 (nM)

Ref.

(23)

0.26

[70]

Tetrahydropyrrolopyrimidinediones (24)

1 -- 50

[71]

Tetrahydrotriazolopyrimidine (25)

< 20

[72]

1,4-diaryl-dihydropyrimidin-2-ones (26)

5 -- 5000

[73]



7


Table 3. Pyrimidinone, pyrimidinedione and pyrimidine derivatives (continued). Human NE inhibitors

IC50 (nM)

Ref.

Other pyrimidine derivatives (27)

1 -- 200

[74]



4.2.2

Uracil derivative

Uracil is one of the nitrogen bases that form RNA. Uracil derivatives were reported to exhibit human NE and CG inhibitory activity in in vitro studies [86]. An issued patent illustrated the representative formula, which is shown as compound (41) (Table 8); 96 of the example compounds were disclosed to be useful as therapeutic agents for treating various inflammatory diseases that are associated with NE, and their inhibitory IC50 values were in the nanomolar range (specifically, 3.5 -- 353.1 nM) [87]. In addition to their utility for the treatment of lung inflammation, topically administered 5-substituted uracil derivatives exerted anti-inflammatory effects on picryl chloride-induced contact hypersensitivity reactions in animals [88] and showed promising results for the treatment of atopic dermatitis and contact dermatitis. In addition, 5-substituted uracil derivatives are suggested as radiation-sensitizing, anticancer and anti-herpes virus agents [89]. Deuterium oxide Deuterium oxide (D2O) is known to exert antitumor effects, and a patent demonstrated that D2O (42) acts as a potent human NE inhibitor (Table 8) [90]. D2O has similar properties to water (H2O). The difference between these compounds is that the hydrogen atoms in H2O are substituted with isotopic deuterium atoms, thus altering the binding strength of the hydrogen bonds, which stabilize the structure of the NE catalytic triad [91]. Because of the different binding energy and binding pitch of D2O, the catalytic triad complex becomes fractured, which causes morphological changes to the enzyme, thereby blocking the catalytic triad and inhibiting the catalytic activity of the enzyme. D2O showed a higher specificity for NE inhibition than trypsin, metalloprotease and chymotrypsin. In an animal study, D2O administration protected animals from human NE-induced lung bleeding. 4.2.3

5.

Expert opinion

Recently, selective NE inhibitors have attracted significant attention as treatments for inflammatory lung diseases including COPD, cystic fibrosis, emphysema and acute lung injury. New applications were also developed to treat conditions in 8

other fields, such as skin aging and for chronically unhealed wounds. Many experts agree that an imbalance of local protease--antiprotease activity may be at least partly responsible for destructive lung inflammation progression or pathogenesis. However, there remains no dependable tool or biomarker that precisely balances protease and antiprotease activity. Treatment of the protease--antiprotease imbalance with natural or synthetic NE inhibitors via the intravenous or the aerosol route has produced incomplete and unconvincing results. These discrepancies may arise from the physicochemical constraints in tissue structures or matrix components. Additionally, several serine proteases aside from NE are degranulated from activated neutrophils. Specific NE inhibitors may not interact with all of these proteases to restore the protease--antiprotease imbalance. The mechanisms of inflammatory lung diseases are complicated and involve multiple pathways. In addition to NE, other proteases such as CG, PR3 and MMPs contribute to tissue damage. Moreover, proteases are not the only molecules that destroy connective tissues; oxidative radicals also exert such effects. Treatment solely with a selective NE inhibitor may not be sufficient; thus, treatments that are combined with other agents, such as MMP-12 or PDE4 inhibitors, are encouraged, especially in COPD patients. In this review, we only present recent patent applications that were disclosed from 2010 to 2014, which may not be the most promising patents compared with patents that were issued previously. Additionally, most of these compounds were newly synthesized; thus, limited results for their clinical application, toxicity and bioavailability in the human body are available. Many potent NE inhibitors with IC50 values for human NE in the picomolar to nanomolar range have been disclosed, including template-fixed b-hairpin peptidomimetics, pyrimidinone, tetrahydropyrrolopyrimidinedione, pyrazinone, benzoxazinone and uracil derivatives. The compounds disclosed in patent applications showed potent inhibitory effects in in vitro or in vivo studies. Based on the templates of these novel designs, we believe that efficient therapeutic NE inhibitors will become available for an increasing number of clinical applications in the near future. Phagocytosis and degranulation were long considered as antimicrobial strategies for neutrophils until neutrophil




Table 4. 2-Pyrazinone derivatives.

Table 4. 2-Pyrazinone derivatives (continued).

Human NE inhibitors

IC50 (nM)

Ref.

Human NE inhibitors

IC50 (nM)

Ref.

(28)

0.33 -- 1.0

[75]

(32)

3.5

[76]

(29)

2.2

[76]

(33)

4.2

[76]

(34)

1.4

[76]

(30)

11

[76]

(31)

2.8

[76]



extracellular trap (NET) formation was first described by Brinkmann et al. in 2004 [92]. NETs are considered to be involved in a cell death program termed NETosis. This process is different from apoptosis and necrosis [92,93]. NE also plays an essential role in NETosis, which is implicated in the pathogenesis of many autoimmune diseases because of its poor regulation, and NE inhibitors may thus serve as a therapeutic target for NETosis, although little is known about this process at present. NETs form an extracellular web-like complex, and they are composed of decondensed chromatin, histones, NE, MPO, CG and other antimicrobial factors [92,94]. On the initiation of NETosis, NE translocates to Expert Opin. Ther. Patents (2015) 25(10)

9



Table 5. Pyridone derivatives.

Table 7. Hypersulfated disaccharide compound.

Human NE inhibitors

IC50 (nM)

Ref.

2-pyridone derivatives (35)

0.57 -- 3.48

[77]

(36)

3 -- 48

[78]

Human NE inhibitors

IC50 (nM)

Ref.

(40)

Not available

[85]


Table 8. Miscellaneous inhibitors.

Substituted 4-pyridone derivatives (37)

< 1 -- 1885

[79]

(38)

3 -- 98

[79]

Human NE inhibitors

IC50 (nM)

Ref.

Uracil derivatives (41)

3.5 -- 353.1

[87]

Deuterium oxide (42)

40%

[90]

NE: Neutrophil elastase. NE: Neutrophil elastase.

Table 6. Benzoxazinone derivatives. Human NE inhibitors

IC50 (nM)

Ref.

(39)

< 15 -- 150

[80]


the nucleus from azurophilic granules and then degrades scaffolded histones to decondense chromatin [95]. Extracellular chromatin fibers bind Gram (+) and (-) microorganisms, limit their spreading, and equip various antimicrobial molecules 10

to neutralize virulence factors and eliminate bacteria. This mechanism is important when an individual is infected with large microbes and parasites that are difficult to phagocytose or clear [96]. When a patient suffers from severe neutropenia, opportunistic fungal pathogens can cause lethal infections [97,98]. Although NETs help to sequester pathogens and inhibit their spread, excessive expression of NETs results in tissue damage and further inflammation [99-101]. The treatment of lung injury caused by aberrant NET release using an NE inhibitor increased the survival rate of dectin-1-deficient mice that were infected with a fungus [96]. NETs and NETotic neutrophils notably appear in the sputa of COPD patients with acute exacerbation [102]. NETs in the respiratory tract are also thought to be associated with decreased lung function during cystic fibrosis [103]. The aberrant generation or failed clearance of NETs may be involved in the pathogenesis of various inflammatory and autoimmune diseases [104,105].



Thetreatment of NET-related diseases with NE inhibitors may represent a promising approach in the future.

Acknowledgments The authors would like to thank Taylor & Francis Editing Services for their language check of the paper.


Bibliography Papers of special note have been highlighted as either of interest () or of considerable interest () to readers. 1.

2.

3.

Nauseef WM. How human neutrophils kill and degrade microbes: an integrated view. Immunol Rev 2007;219:88-102

12.

Henriksen PA. The potential of neutrophil elastase inhibitors as antiinflammatory therapies. Cur Opin Hematol 2014;21(1):23-8 Hoenderdos K, Condliffe A. The neutrophil in chronic obstructive pulmonary disease. Am J Respir Cell Molecul Biol 2013;48(5):531-9

4.

Pipoly DJ, Crouch EC. Degradation of native type IV procollagen by human neutrophil elastase. Implications for leukocyte-mediated degradation of basement membranes. Biochemistry 1987;26(18):5748-54

5.

Stein RL, Strimpler AM. Catalysis by human leukocyte elastase. Aminolysis of acyl-enzymes by amino acid amides and peptides. Biochemistry 1987;26(8):2238-42

6.

11.

Anderssen T, Halvorsen H, Bajaj SP, Osterud B. Human leukocyte elastase and cathepsin G inactivate factor VII by limited proteolysis. Thromb Haemost 1993;70(3):414-17

7.

Turkington PT. Degradation of human factor X by human polymorphonuclear leucocyte cathepsin G and elastase. Haemostasis 1991;21(2):111-16

8.

Doring G. The role of neutrophil elastase in chronic inflammation. Am J Respir Crit Care Med 1994;150(6 Pt 2):S114-17

9.

Moraes TJ, Chow CW, Downey GP. Proteases and lung injury. Crit Care Med 2003;31(4 Suppl):S189-94

10.

Stockley RA. The role of proteinases in the pathogenesis of chronic bronchitis. Am J Respir Crit Care Med 1994;150(6 Pt 2):S109-13

..

Declaration of interest The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents, received or pending, or royalties.

19.

Wang Y, Xiao Y, Zhong L, et al. Increased neutrophil elastase and proteinase 3 and augmented NETosis are closely associated with beta-cell autoimmunity in patients with type 1 diabetes. Diabetes 2014;63(12):4239-48 An important work demonstrating the relationship between neutrophil elastase (NE) and type 1 diabetes.

Campbell EJ, Silverman EK, Campbell MA. Elastase and cathepsin G of human monocytes. Quantification of cellular content, release in response to stimuli, and heterogeneity in elastasemediated proteolytic activity. J Immunol 1989;143(9):2961-8

20.

Lungarella G, Menegazzi R, Gardi C, et al. Identification of elastase in human eosinophils: immunolocalization, isolation, and partial characterization. Arch Biochem Biophys 1992;292(1):128-35

21.

Tsuji N, Moriwaki S, Suzuki Y, et al. The role of elastases secreted by fibroblasts in wrinkle formation: implication through selective inhibition of elastase activity. Photochem Photobiol 2001;74(2):283-90

22.

Liou TG, Campbell EJ. Nonisotropic enzyme–inhibitor interactions: a novel nonoxidative mechanism for quantum proteolysis by human neutrophils. Biochemistry 1995;34(49):16171-7

23.

Faurschou M, Borregaard N. Neutrophil granules and secretory vesicles in inflammation. Microbes Infect 2003;5(14):1317-27

24.

Perera NC, Schilling O, Kittel H, et al. NSP4, an elastase-related protease in human neutrophils with arginine specificity. Proc Nat Acad Sci USA 2012;109(16):6229-34 A work revealing a new branch of the chymotrypsinogen superfamily of serine proteases in neutrophils.

13.

Schrijver G, Schalkwijk J, Robben JC, et al. Antiglomerular basement membrane nephritis in beige mice. Deficiency of leukocytic neutral proteinases prevents the induction of albuminuria in the heterologous phase. J Experim Med 1989;169(4):1435-48

14.

Yokoyama T, Tsushima K, Ushiki A, et al. Acute lung injury with alveolar hemorrhage due to a novel swine-origin influenza A (H1N1) virus. Int Med 2010;49(5):427-30

15.

Tagami T, Tosa R, Omura M, et al. Effect of a selective neutrophil elastase inhibitor on mortality and ventilator-free days in patients with increased extravascular lung water: a post hoc analysis of the PiCCO Pulmonary Edema Study. J Inten Care 2014;2(1):67

16.

disease: what does the future hold? Drugs 2014;74(17):1983-92

Wiener-Kronish JP, Gropper MA, Matthay MA. The adult respiratory distress syndrome: definition and prognosis, pathogenesis and treatment. Br J Anaesth 1990;65(1):107-29

Zeiher BG, Artigas A, Vincent JL, et al. Neutrophil elastase inhibition in acute lung injury: results of the STRIVE study. Crit Care Med 2004;32(8):1695-702

17.

Kroegel C, Foerster M. Phosphodiesterase-4 inhibitors as a novel approach for the treatment of respiratory disease: cilomilast. Expert Opin Investig Drugs 2007;16(1):109-24

18.

Matera MG, Rogliani P, Calzetta L, Cazzola M. Phosphodiesterase inhibitors for chronic obstructive pulmonary


.

25.

Zimmer M, Medcalf RL, Fink TM, et al. Three human elastase-like genes coordinately expressed in the myelomonocyte lineage are organized as a single genetic locus on 19pter. Proc Nat Acad Sci USA 1992;89(17):8215-19

26.

Horwitz MS, Duan Z, Korkmaz B, et al. Neutrophil elastase in cyclic and severe

11


Taggart CC, Greene CM, Carroll TP, et al. Elastolytic proteases: inflammation resolution and dysregulation in chronic infective lung disease. Am J Respir Crit Care Med 2005;171(10):1070-6

38.

Wiedow O, Meyer-Hoffert U. Neutrophil serine proteases: potential key regulators of cell signalling during inflammation. J Int Med 2005;257(4):319-28

39.

29.

Devaney JM, Greene CM, Taggart CC, et al. Neutrophil elastase up-regulates interleukin-8 via toll-like receptor 4. FEBS Lett 2003;544(1-3):129-32

40.

30.

Bank U, Ansorge S. More than destructive: neutrophil-derived serine proteases in cytokine bioactivity control. J Leukoc Biol 2001;69(2):197-206

Starcher B, O’Neal P, Granstein RD, Beissert S. Inhibition of neutrophil elastase suppresses the development of skin tumors in hairless mice. J Invest Dermatol 1996;107(2):159-63

41.

Sun Z, Yang P. Role of imbalance between neutrophil elastase and alpha 1-antitrypsin in cancer development and progression. Lancet Oncol 2004;5(3):182-90

42.

Gadek JE, Pacht ER. The proteaseantiprotease balance within the human lung: implications for the pathogenesis of emphysema. Lung 1990;168 Suppl:552-64

27.


28.

31.

32.

Mihara K, Ramachandran R, Renaux B, et al. Neutrophil elastase and proteinase3 trigger G protein-biased signaling through proteinase-activated receptor-1 (PAR1). J Biol Chem 2013;288(46):32979-90 Suzuki T, Yamashita C, Zemans RL, et al. Leukocyte elastase induces lung epithelial apoptosis via a PAR-1-, NFkappaB-, and p53-dependent pathway. Am J Respir Cell Molecul Biol 2009;41(6):742-55

33.

Zhou J, Perelman JM, Kolosov VP, Zhou X. Neutrophil elastase induces MUC5AC secretion via proteaseactivated receptor 2. Mol Cell Biochem 2013;377(1-2):75-85

34.

Hirche TO, Crouch EC, Espinola M, et al. Neutrophil serine proteinases inactivate surfactant protein D by cleaving within a conserved subregion of the carbohydrate recognition domain. J Biol Chem 2004;279(26):27688-98

35.

36.

37.

12

Beaufort N, Leduc D, Rousselle JC, et al. Proteolytic regulation of the urokinase receptor/CD87 on monocytic cells by neutrophil elastase and cathepsin G. J Immunol 2004;172(1):540-9 Doring G, Frank F, Boudier C, et al. Cleavage of lymphocyte surface antigens CD2, CD4, and CD8 by polymorphonuclear leukocyte elastase and cathepsin G in patients with cystic fibrosis. J Immunol 1995;154(9):4842-50 Nadel JA. Role of mast cell and neutrophil proteases in airway secretion.

induced by neutrophil elastase in human airway epithelial cells. Cell Physiol Biochem 2015;35(1):326-38

Am Rev Respir Dis 1991;144(3 Pt 2):S48-51

congenital neutropenia. Blood 2007;109(5):1817-24

43.

44.

Rogalski C, Meyer-Hoffert U, Proksch E, Wiedow O. Human leukocyte elastase induces keratinocyte proliferation in vitro and in vivo. J Invest Dermatol 2002;118(1):49-54 Renesto P, Chignard M. Enhancement of cathepsin G-induced platelet activation by leukocyte elastase: consequence for the neutrophil-mediated platelet activation. Blood 1993;82(1):139-44

Perlmutter DH. Alpha-1-antitrypsin deficiency: biochemistry and clinical manifestations. Ann Med 1996;28(5):385-94 Silverman GA, Bird PI, Carrell RW, et al. The serpins are an expanding superfamily of structurally similar but functionally diverse proteins. Evolution, mechanism of inhibition, novel functions, and a revised nomenclature. J Bio Chem 2001;276(36):33293-6

45.

Sottrup-Jensen L. Alpha-macroglobulins: structure, shape, and mechanism of proteinase complex formation. J Biol Chem 1989;264(20):11539-42

46.

Travis J. Structure, function, and control of neutrophil proteinases. Am J Med 1988;84(6A):37-42

47.

Owen CA, Campbell EJ. The cell biology of leukocyte-mediated proteolysis. J Leuk Biol 1999;65(2):137-50

48.

49.

Sarker RS, John-Schuster G, Bohla A, et al. CARM1 regulates alveolar epithelial senescence and elastase-induced emphysema susceptibility. Am J Respir Cell Molecul Biol 2015. [Epub ahead of print] Li Q, Li N, Liu CY, et al. Ezrin/Exocyst complex regulates mucin 5AC secretion Expert Opin. Ther. Patents (2015) 25(10)

50.

Sabater JR, Lee TA, Abraham WM. Comparative effects of salmeterol, albuterol, and ipratropium on normal and impaired mucociliary function in sheep. Chest 2005;128(5):3743-9

51.

Juvelekian GS, Stoller JK. Augmentation therapy for alpha(1)-antitrypsin deficiency. Drugs 2004;64(16):1743-56

52.

Hwang TL, Wang WH, Wang TY, et al. Synthesis and pharmacological characterization of 2-aminobenzaldehyde oxime analogs as dual inhibitors of neutrophil elastase and proteinase 3. Bioorg Med Chem 2015;23(5):1123-34

53.

Tsai YF, Yu HP, Chang WY, et al. Sirtinol inhibits neutrophil elastase activity and attenuates lipopolysaccharide-mediated acute lung injury in mice. Scientific Reports 2015;5:8347

54.

Wright JL, Farmer SG, Churg A. Synthetic serine elastase inhibitor reduces cigarette smoke-induced emphysema in guinea pigs. Am J Respir Crit Care Med 2002;166(7):954-60

55.

Korkmaz B, Horwitz MS, Jenne DE, Gauthier F. Neutrophil elastase, proteinase 3, and cathepsin G as therapeutic targets in human diseases. Pharmacol Rev 2010;62(4):726-59 A review that analyzes the roles of neutrophilic proteases as therapeutic targets for curing human diseases.

..

56.

Garcia SN, Van DNW, Carreno SC, et al. Peptides used in the treatment and/ or care of the skin, mucous membranes and/or scalp and their use in cosmetic or pharmaceutical compositions. CA2751436A1; 2010

57.

DeMarco SJ, Moehle K, Henze H, et al. Template-fixed beta-hairpin peptidomimetics with protease inhibitory activity. US8658604B2; 2014

58.

Thongyoo P, Bonomelli C, Leatherbarrow RJ, Tate EW. Potent inhibitors of beta-tryptase and human leukocyte elastase based on the MCoTIII scaffold. J Med Chem 2009;52(20):6197-200

59.

Saksela O. Plasminogen activation and regulation of pericellular proteolysis. Biochimica et Biophysica Acta 1985;823(1):35-65


Ray NC, Finch H, Edwards C, O’connor E. Tetrahydropyrrolopyrimidinediones and their use as human neutrophil elastase inhibitors. EP2024367B1; 2010

72.

Blench TJ, Edwards C, Heald RA, et al. Tetrahydrotriazolopyrimidine derivatives as human neutrophil elastase inhibitors. WO037809A1; 2013

Kawabata K, Suzuki M, Sugitani M, et al. ONO-5046, a novel inhibitor of human neutrophil elastase. Biochem Biophys Res Commun 1991;177(2):814-20

73.

Gielen-Haertwig H, Albrecht B, Keldenich J, et al. 1,4-diaryldihydropyrimidin-2-ones and their use as human neutrophil elastase inhibitors. EP1723121B1; 2012

Kawabata K, Moore AR, Willoughby DA. Impaired activity of protease inhibitors towards neutrophil elastase bound to human articular cartilage. Ann Rheum Dis 1996;55(4):248-52

74.

Edwards C, Kulagowski J, Finch H. Pyrimidine derivatives and their use in the treatment of respiratory diseases such as copd. WO110858A1; 2011

75.

Hansen P, Ivarsson M, Lawitz K, et al. 2-pyrazinone derivatives for the treatment of disease or condition in which inhibition of neutrophil elastase activity is beneficial. CA2650553C; 2014

Lawrence DA, Stefansson SP. Mutant plasminogen activator-inhibitor type 1 (pai-1) and uses thereof. CA2233670C; 2012

61.

Shinguh Y, Imai K, Yamazaki A, et al. Biochemical and pharmacological characterization of FK706, a novel elastase inhibitor. Eur J Pharmacol 1997;337(1):63-71

62.


71.

60.

63.

64.

65.

66.

67.

68.

69.

70.

Iba T, Kidokoro A, Fukunaga M, et al. Pretreatment of sivelestat sodium hydrate improves the lung microcirculation and alveolar damage in lipopolysaccharideinduced acute lung inflammation in hamsters. Shock 2006;26(1):95-8 Jagwani D, Joshi P. A greener chemistry approach for synthesis of 4-(4hydroxyphenyl)-6-methyl-2-oxo1,2,3,4 tetrahydropyrimidine-5-carboxylic acid ethyl ester. Int J Pharmaceut Sci Res 2015;6(2):783-90 Hansen G, Gielen-Haertwig H, Reinemer P, et al. Unexpected active-site flexibility in the structure of human neutrophil elastase in complex with a new dihydropyrimidone inhibitor. J Mol Biol 2011;409(5):681-91 Von Nussbaum F, Karthaus D, Anlauf S, et al. 4-(4-cyano-2-thioaryl) dihydropyrimidinones and their use. US8288402B2; 2012 GNAMM C, Oost T, Peters S, Rudolf K. Substituted Bicyclic Dihydropyrimidinones And Their Use As Inhibitors Of Neutrophil Elastase Activity. US0249129A1; 2014 FINCH HADLTD. Edwards C, Fitzgerald MF, et al. Multimers of pyrimidinone derivatives and their use as human neutrophil elastase inhibitors. EP1856059B1; 2010 Bergstr€om L, L€onn H, Lundkvist M, Sj€o P. Multimeric heterocyclic compounds useful as neutrophil elastase inhibitors. US0003858A1; 2011

benzoxazinone derivatives as antiinflammatory and anti-platelet aggregation agents. Bioor Med Chem Let 2005;15(11):2786-9 84.

Wong T, Groutas CS, Mohan S, et al. 1,2,5-Thiadiazolidin-3-one 1,1-dioxidebased heterocyclic sulfides are potent inhibitors of human tryptase. Arch Biochem Biophys 2005;436(1):1-7

85.

Ahmed T, Abraham W. Hypersulfated disaccharides to treat elastase related disorders. CN103269585A; 2013

86.

Groutas WC, Huang H, Epp JB, et al. Mechanism-based inhibition of human leukocyte elastase and cathepsin G by substituted dihydrouracils. Biochim Biophys Acta 1994;1227(3):130-6

87.

Shiro T, Tobe M, Kubota K, et al. Uracil derivative and use thereof for medical purposes. US0179670A1; 2014

88.

Isobe Y, Tobe M, Inoue Y, et al. Structure and activity relationships of novel uracil derivatives as topical antiinflammatory agents. Bioor Med Chem 2003;11(23):4933-40

76.

Ainge D, Chapman D, Lindsjo M, et al. Some 2-pyrazinone derivatives and their use as inhibitors of neutrophile elastase. US0280048A1; 2010

89.

77.

Murugan A, Ashton M, Bergstrom LK, et al. 2-pyridone compounds used as inhibitors of neutrophil elastase. EP2483244A1; 2012

Walker RT, Jones AS, De Clercq E, et al. The synthesis and properties of some 5-substituted uracil derivatives. Nucleic Acids Symp Ser 1980(8):s95-102

€ Andersson M, Hansen P, LONN H, et al. 2-pyridone derivatives as neutrophil elastase inhibitors and their use. EP1663973B1; 2013

90.

78.

Bayerl T. Use of deuterium oxide as an elastase inhibitor. WO2009130615A3; 2010

91.

Oost T, FIEGEN D, GNAMM C, et al. Substituted 4-pyridones and their use as inhibitors of neutrophil elastase activity. US0057916A1; 2014

Perona JJ, Craik CS. Structural basis of substrate specificity in the serine proteases. Protein Sci 1995;4(3):337-60

92.

Shreder K, Hu Y, Fraser A, et al. Serine hydrolase inhibitors. EP2066662B1; 2012

..

Brinkmann V, Reichard U, Goosmann C, et al. Neutrophil extracellular traps kill bacteria. Science 2004;303(5663):1532-5 An important paper revealing how NE is implicated in the formation of neutrophil extracellular traps (NETs) and in the pathogenesis of diseases.

79.

80.

81.

82.

83.

Hsieh PW, Yu HP, Chang YJ, Hwang TL. Synthesis and evaluation of benzoxazinone derivatives on activity of human neutrophil elastase and on hemorrhagic shock-induced lung injury in rats. Eur J Med Chem 2010;45(7):3111-15 Shreder KR, Cajica J, Du L, et al. Synthesis and optimization of 2-pyridin3-yl-benzo[d][1,3]oxazin-4-one based inhibitors of human neutrophil elastase. Bioor Med Chem Let 2009;19(16):4743-6 Hsieh PW, Hwang TL, Wu CC, et al. The evaluation of 2,8-disubstituted Expert Opin. Ther. Patents (2015) 25(10)

93.

Amulic B, Hayes G. Neutrophil extracellular traps. Cur Biol 2011;21(9):R297-8

94.

Zawrotniak M, Rapala-Kozik M. Neutrophil extracellular traps (NETs) formation and implications. Acta Biochim Pol 2013;60(3):277-84

95.

Papayannopoulos V, Metzler KD, Hakkim A, Zychlinsky A. Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps. J Cell Biol 2010;191(3):677-91

13


96.


97.

Branzk N, Lubojemska A, Hardison SE, et al. Neutrophils sense microbe size and selectively release neutrophil extracellular traps in response to large pathogens. Nat Immunol 2014;15(11):1017-25 Arendrup MC. Epidemiology of invasive candidiasis. Curr Opin Crit Care 2010;16(5):445-52

98.

Low CY, Rotstein C. Emerging fungal infections in immunocompromised patients. F1000 Med Repor 2011;3:14

99.

Almyroudis NG, Grimm MJ, Davidson BA, et al. NETosis and NADPH oxidase: at the intersection of host defense, inflammation, and injury. Front Immunol 2013;4:45

100. Hahn S, Giaglis S, Chowdhury CS, et al. Modulation of neutrophil NETosis: interplay between infectious agents and underlying host physiology. Semin Immunopathol 2013;35(4):439-53 101. Mesa MA, Vasquez G. NETosis. Autoimmune Dis 2013;2013:651497

..

the structure of neutrophil extracellular traps from chronic obstructive pulmonary disease and in vitro generation. PLoS One 2014;9(5):e97784 A study analyzing the antimicrobial and cytotoxic activities of NETs in chronic obstructive pulmonary disease.

103. Yoo DG, Floyd M, Winn M, et al. NET formation induced by Pseudomonas aeruginosa cystic fibrosis isolates measured as release of myeloperoxidaseDNA and neutrophil elastase-DNA complexes. Immunol Lett 2014;160(2):186-94 104. Branzk N, Papayannopoulos V. Molecular mechanisms regulating NETosis in infection and disease. Semin Immunopathol 2013;35(4):513-30 105. Caza T, Oaks Z, Perl A. Interplay of infections, autoimmunity, and immunosuppression in systemic lupus erythematosus. Int Rev Immunol 2014;33(4):330-63

102. Obermayer A, Stoiber W, Krautgartner WD, et al. New aspects on

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Affiliation Yung-Fong Tsai1,2 MD & Tsong-Long Hwang†1,3,4,5 † Author for correspondence 1 Chang Gung University, Graduate Institute of Natural Products, School of Traditional Chinese Medicine, College of Medicine, Taoyuan 33302, Taiwan 2 Chang Gung Memorial Hospital, Department of Anesthesiology, Kweishan, Taoyuan, Taiwan 3 Chang Gung University, Healthy Aging Research Center, Chinese Herbal Medicine Research Team, Taoyuan 33302, Taiwan 4 Chang Gung University of Science and Technology, Department of Cosmetic Science and Research Center for Industry of Human Ecology, Taoyuan 33302, Taiwan 5 Professor, Chang Gung University, Graduate Institute of Natural Products, School of Traditional Chinese Medicine, College of Medicine, Taoyuan 33302, Taiwan Tel: +88 6 3211 8506; Fax: +88 6 3211 8506; E-mail: [email protected]

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