A patenting perspective on human neutrophil elastase (HNE) inhibitors (2014–2018) and their therapeutic applications

Abstract

Introduction:

Human neutrophil elastase (HNE) is involved in a variety of serious chronic diseases, especially cardiopulmonary pathologies. For this reason, the regulation of HNE activity represents a promising therapeutic approach, which is evident by the development of a number of new and selective HNE inhibitors, both in the academic and pharmaceutical environments.

Areas covered:

The present review analyzes and summarizes the patent literature regarding human neutrophil elastase inhibitors for the treatment of cardiopulmonary diseases over 2014–2018.

Expert opinion:

HNE is an interesting and defined target to treat various inflammatory diseases, including a number of cardiopulmonary pathologies. The research in this field is quite active, and a number of HNE inhibitors are currently in various stages of clinical development. In addition, new opportunities for HNE inhibitor development stem from recent studies demonstrating the involvement of HNE in many other inflammatory pathologies, including rheumatoid arthritis, inflammatory bowel disease, skin diseases, and cancer. Furthermore, the development of dual HNE/proteinase 3 inhibitors is being pursued as an innovative approach for the treatment of neutrophilic inflammatory diseases. Thus, these new developments will likely stimulate new and increased interest in this important therapeutic target and for the development of novel and selective HNE inhibitors.

1. Introduction

Proteases are enzymes that catalyze the hydrolysis of peptide bonds, thus converting proteins into smaller fragments. They are involved in many physiological processes and are considered important signaling molecules [1,2]. Serine proteases represent one-third of all known proteolytic enzymes, and they are grouped into 13 clans (superfamilies) and 40 families [3]. The family name ‘serine’ arises from the amino acid serine located in the active site, which is responsible for the nucleophilic attack on the carbonyl moiety of the substrate peptide bond to form an acyl-enzyme intermediate [4]. The four main clans of serine proteases include the superfamily A or PA clan (e.g. chymotrypsin), the subtilisin clan (SB), clan SC (e.g. carboxypeptidase C), and clan SF (e.g. bacterial leader peptidase I) [2,5]. Significant differences exist in the distribution of each clan across species. For example, proteases of superfamily A are highly represented in eukaryotes and less in prokaryotes and plant genomes, while clans SB and SC are most represented in other organisms. Among serine proteases belonging to the chymotrypsin family are the neutrophil serine proteases (NSP) [6], including neutrophil elastase (NE), cathepsin G (CG), proteinase 3 (PR3), and the recently discovered neutrophil serine protease 4 (NSP4) [7]. In addition, human neutrophils also express azurocidin (AZU), an inactive serine protease homolog with chemotactic and antimicrobial activities [8,9].

Neutrophils are the first line of defense against infection and are efficient phagocytes able to engulf and degrade microorganisms through the combined action of reactive oxygen species (ROS), proteases, and antimicrobial peptides [10,11]. Neutrophils are polymorphonuclear cells containing four types of granules: azurophil granules (also known as primary), specific granules (also known secondary), gelatinase granules (also known as tertiary), and secretory granules [12]. The granules are classified according to their protein content and their different ability to undergo exocytosis after neutrophil activation by inflammatory stimuli or phagocytosis of invading microorganisms. Azurophil granules are mainly involved in the intracellular degradation of microorganisms in the phagolysosome due to their high content of myeloperoxidase (MPO), bactericidal permeability-increasing protein, defensins, and serine proteases, such as NE, CG and PR3 [13]. The extracellular release of serine proteases from azurophil granules plays an important role in modulation of the inflammatory response by proteolytically modifying chemokines and cytokines, activating latent forms of cytokines and growth factors, and cleaving specific cell surface receptors. In addition, neutrophil serine proteases can induce caspase-independent cell apoptosis and initiate adaptive immune responses by activating lymphocytes [14]. Clearly, NSP play an important role in the inflammatory process and immune response and, thus, represent a potential target for therapeutic intervention. In particular, we will discuss human neutrophil elastase (HNE) inhibitors and their therapeutic applications in this review.

2. HNE

HNE (EC 3.4.21.37) is a globular glycoprotein of about 30 kDa belonging to the chymotrypsin family. It consists of a single polypeptide chain of 218 amino acids and two asparagine-linked carbohydrate side chains at Asn95 and Asn144. It is stabilized by four disulfide bridges and exhibits basic properties due to the presence of 19 arginine residues, resulting in an isoelectric point around 10–11. The active HNE structure is composed of an N-terminal activation domain and three flexible loops [15]. The primary structure has homology with PR3 (57%) and CG (37%), since they evolved from a common gene by duplication [16]. ELA2, the gene for HNE, is located in the terminal region of the short arm of chromosome 19 at p13.3 and consists of five exons and four introns. This region also contains the genes for the homologous proteins PR3 and azurocidin [17]. Mutations in ELA2 can be a risk factor for severe congenital and cyclic neutropenia, a rare disease characterized by oscillations in the production of neutrophils and other blood cells [18,19]. HNE is found not only in neutrophils but also in mast cells, monocytes, eosinophils, keratinocytes, and fibroblasts [20,21]. The concentration of HNE in neutrophils exceeds 5 mM, and the total amount in a single cell has been estimated to be up to 3 pg [22], which is sequestered in the cell by compartmentalization in azurophil granules. Active HNE is rapidly released from the granules into the extracellular space, although some released HNE remains associated with the outer plasma membrane [23,24]. HNE performs its proteolytic action through a catalytic triad consisting of Ser195-Asp102-His57, where the powerful nucleophile Ser195 OH group attacks the carbonyl carbon involved in the peptide bond. Close by the catalytic triad, an oxyanion binding site comprised of the backbone amide NH of Ser195 and Gly193 is present (Figure 1) [25]. Hydrolysis of the peptide bond occurs via two tetrahedral intermediates [26]. The OH group of Ser195, activated by His 57 as a general base, attacks the carbonyl group of the substrate leading to the first tetrahedral intermediate. This intermediate is then stabilized by the backbone N atoms of Gly193 and Ser195, which together generate a positively charged pocket within the active site, known as the oxyanion hole. Hydrogen bond interactions in the oxyanion hole contribute 1.5–3.0 kcal/mol to ground and transition state stabilization [27]. His57 transferring a proton to the amine of the tetrahedral intermediate causes this bond to break of this last one, resulting in the formation of the covalent acyl-enzyme complex. A molecule of water attacks the acyl-enzyme complex to generate a new tetrahedral intermediate, stabilized by the oxyanion hole as well. Finally, the collapse of this second intermediate results in the regeneration of active HNE [4,25].

Figure 1.

HNE catalytic triad.

3. Physiological functions of HNE

The term elastase is not quite correct, since HNE does not only degrade elastin, a protein responsible for the elasticity of lung tissue. Rather, HNE degrades a variety of extracellular matrix and plasma proteins, including collagen (types I-IV), fibronectin, laminin, and proteoglycans [28–31]. Other HNE substrates are coagulation factors (fibrinogen and factors V, VII, XII, and XIII), plasminogen, immunoglobulin G (IgG), IgA and IgM, thrombomodulin, platelet, complement factors C3 and C5, complement receptors [32–34], and gp120, the coat protein of the human immunodeficiency virus [35]. HNE can also degrade other proteases present inside neutrophil granules [36], as well as some protease inhibitors, leading to their activation or inactivation [37]. HNE is currently considered a multifunctional enzyme involved in the killing of pathogens, regulation of inflammation, and tissue homeostasis [13] via its proteolytic activity against the proteins listed above. Moreover, HNE appears to play an important role in chemotaxis and migration of neutrophils by the degradation of adhesion molecules at cell junctions [38,39]. Although neutrophils may normally migrate to sites of infection in the absence of elastase, the presence of HNE is required for maximal intracellular killing of Gram-negative bacteria [40,41]. In addition, neutrophils can form neutrophil extracellular traps (NETs), which are web-like chromatin structures extruded from neutrophils and can bind/trap bacteria. These NETs sequester high concentrations of released serine proteases, including elastase, where they can degrade virulence factors and kill bacteria extracellularly [42].

Since a single azurophilic granule activated from neutrophils releases millimolar concentrations of HNE, the potent proteolytic activity of this serine protease must be adequately regulated by endogenous inhibitors. However, why HNE can still degrade extracellular matrix, despite the presence of functioning anti-protease molecules, is still not fully understood. According to the literature, neutrophils could release oxidant molecules that locally turn off anti-proteases, thereby permitting the action of HNE focused on local tissues [43]. Campbell and co-workers [22] measured the function of HNE in regards to quantum proteolysis and found that ~ 67,000 molecules of HNE are stored in each azurophilic granule, reaching a concentration of about 5.33 mM, which exceeds pericellular inhibitor concentrations in vivo by nearly three orders of magnitude. Consequently, other non-oxidative mechanisms must limit the proteolytic activity of HNE together with the endogenous inhibitors [43]. Azurophilic granules contain acidic medium that prevents proteolytic activity. Thus, storage in specialized compartments is important to control inadvertent protease activity inside the cell. Elastase is activated in mild alkaline medium, which results from the fusion of the granules with phagocytic vacuoles to form phagolysosomes where the engulfed bacteria are destroyed [44] or extracellular release.

Upon neutrophil activation, the intracellular granules fuse with the plasma membrane, which dramatically enlarges the neutrophil cell surface area and triggers a neutrophil oxidative burst. At this point, the most of the active enzyme is released in the extracellular space (~10% of the released HNE remains bound to the cell membrane), and it can be neutralized by the endogenous inhibitors in the blood. However, the concentration of elastase at the site of liberation is still higher than that of the extracellular anti-proteases. More distant from the site of degranulation, the elastase concentration decreases due to diffusion/dilution until free elastase is completely neutralized [45]. Consequently, only a small percentage of elastase liberated upon neutrophil activation is involved in a healthy situation [22,46].

The principal class of endogenous proteins which work as HNE inhibitors are serpins (serine protease inhibitors) [47,48], tissue inhibitors of metalloproteases (TIMPS) [49], and cystatins (cysteine protease inhibitors) [50]. The activity of HNE is primarily regulated by serpins, α1-antitrypsin (α1-AT, ATT or α1-proteinase inhibitor, α1-PI) [51], monocyte/NE inhibitor (MNEI, also called serpin B1) [52], and secretory leukocytes proteinase inhibitor (SLPI) [53] and elafin [54], which belongs to the chelonianin family. α1-AT, a blood plasma glycoprotein, regulates the activity of HNE in the low respiratory tract, while SLPI protects the upper airways from proteolysis [55]. Serpin B1, a potent inhibitor of HNE, is expressed at high concentrations mainly in macrophages and neutrophil. Elafin, a small soluble protein of 6 kDa, is a potent HNE inhibitor present in bronchial secretions [52,54]. In addition to regulation of the inflammatory process by direct enzyme inhibition, there is increasing evidence that the serine proteases inhibitors α1-AT, SLPI, and elafin also affect leukocyte chemotaxis and pro-inflammatory mediator release and may contribute to defense against invading pathogens [56].

4. Involvement of HNE in pathologic conditions

As discussed above, the potent proteolytic activity of HNE is strictly controlled by several endogenous inhibitors, which are particularly abundant in the respiratory tract and circulatory system and protect tissues from damage [57,58]. According to the protease/antiprotease imbalance hypothesis, many pathologies of the respiratory system result from damage to the connective tissue that is caused by the massive migration and infiltration of neutrophils and the subsequent release of proteolytic enzymes, including serine proteases (Figure 2). Hence, inadequate control of the protease’s activity due to low levels of their physiological inhibitors ultimately leads to the degradation of elastin, the elastic component of lung connective tissue, and other components of the extracellular matrix [56,59].

Figure 2.

Protease-antiprotease imbalance in pulmonary diseases where HNE plays a key role.

Out-of-balance elastase activity is at the basis of severe diseases, especially those associated with the cardiopulmonary system [60], such as chronic obstructive pulmonary disease (COPD) [61–64], bronchiectasis (BE) [65,66], cystic fibrosis (CF) [43,67], acute lung injury (ALI) [68], acute respiratory distress syndrome (ARDS) [69], pulmonary arterial hypertension (PAH) [70], and idiopathic pulmonary fibrosis (IPF) [71]. HNE significantly contributes to chronic inflammatory airway diseases by inducing mucin production in airway epithelial cells [72]. The exact mechanism at the basis of this effect is unclear, but it was demonstrated to involve protein kinase C and production of ROS. ROS, in turn, activate tumor necrosis factor (TNF)-converting enzyme, leading to TNF-converting enzyme-dependent release of soluble transforming growth factor-α (TGFα) and TGFα stimulation of epidermal growth factor receptor (EGFR), which induces the production of mucin [73].

HNE is present in psoriatic lesions and induces keratinocyte hyperproliferation via the epidermal growth factor receptor signaling pathway [74]. The low concentration of specific elastase inhibitors in psoriatic skin increases the excessive hydrolytic activity of HNE released from migrating cells [75]. Recently, Marto et al. [76] published a promising strategy for the treatment of psoriasis and inflammatory skin disease using a coumarin-based oxo-β-lactam compound to inhibit HNE. This HNE inhibitor, ER-143, was successfully formulated in starch-based nanocapsules with adequate pharmaceutical characteristics (such as particle size distribution, surface charge, and encapsulation efficiency), and it represents a promising approach for topical application to treat inflammatory skin diseases [77,78].

HNE is also involved in rheumatoid arthritis, since it can directly degrade the matrix, destroying cartilage components [79]. Di Cesare Mannelli et al. [80] described the effects of a potent and selective HNE inhibitor, EL-17 (IC₅₀ = 20 nM) [81], in the rat model of rheumatoid arthritis. In this adjuvant-induced arthritis model, a single administration of EL-17 reduced pain evoked by noxious stimuli, and repeated treatment with EL-17 significantly prevented the development of spontaneous pain and hypersensitivity induced by mechanical noxious and non-noxious stimuli. The preventive efficacy seems to be related to a disease-modifying effect, since the articular damage clearly present in the histological examination of the tibiotarsal joint was greatly reduced, at least by the higher dose of EL-17.

HNE also seems to be involved in the development and progression of cancer, which is consistent with the observation that many types of cancer originate in areas of the body where chronic inflammatory states occur [82], and neutrophils and their proteases participate actively, contributing to the progression of the tumor itself [83]. A certain analogy can be found between tumor cells and neutrophils: cancer cells enter the bloodstream, adhere to endothelial cells to pass through them and invade tissues in different organs, and give rise to metastases. Likewise, neutrophils transported by the bloodstream throughout the body adhere to adhesive molecules present on activated endothelial cells to then penetrate through the vessel wall and migrate to the areas of inflammation. The biggest difference between these cells is the lifetime: neutrophils undergo apoptosis after ~72 h from their synthesis in the bone marrow, while the cancer cells do not die [84]. The tumor cells themselves are also able to release proteases, whose proteolytic activity is very important during tissue invasion and metastasis formation, as it allows them to overcome a series of physiological barriers consisting of elastin, collagen, and proteoglycan [85]. In vitro immunoassay studies demonstrate that these proteases, and in particular NE, are directly produced by tumor cells of the breast and lung [86]. In breast cancer, high levels of HNE represent a negative prognosis indicator, as demonstrated by the decrease in survival both in the absence and in the presence of metastasis [87]. In lung cancer, elevated serological levels of HNE are correlated not only with the stage of illness but also with its progression. In fact, the activity of the enzyme is three or five times greater in broncho-alveolar lavage fluid of patients with lung cancer compared to healthy individuals [88]. Furthermore, HNE actively participates in the degradation of elastin fibers, thus promoting the penetration and spread of cancer cells, but it can also generate elastin fragments, called elastochines, with properties similar to cytokines. These fragments act at various levels, promoting the progression of lung cancer [89]. Thus, the inhibition of HNE may be beneficial both in preventing invasion and in metastasis of various types of cancer [84]. In cancer, the production of myeloid cells in the bone marrow is generally accelerated, and the number of circulating neutrophils and myeloid-derived suppressor cells (MDSCs) appears to be correlated with the progression of the disease and patient survival. A large number of MDSCs often correlates with negative clinical outcomes [90,91]. Recently, it has been shown that infiltrating myeloid cells exert pro-tumorigenic action through HNE activity in prostate cancer [92,93]. A recent study demonstrated that the protease-antiprotease imbalance is also related to the pathogenesis of type 1 diabetes [94]. In these patients, α1-AT levels decrease, whereas circulating HNE concentrations and HNE enzymatic activity significantly increase. These changes correlate with diabetes-associated auto-antibody levels [15].

Vicuña et al. [95,96] showed that inhibitors of HNE can reverse ongoing neuropathic pain in mice, demonstrating a new role for this enzyme in the modulation of this type of pain. They also showed that NE contributing to neuropathic pain can be expressed not only by neutrophils but also by T-lymphocytes. In particular, the authors demonstrated that the administration of Sivelestat (see Figure 3) in mice after spinal nerve injury reduced mechanical pain behaviors during the first two weeks after injury as a result of its inhibitory action on serine proteases. Furthermore, treatment with Sivelestat left baseline nociceptive sensitivity unaffected. The evidence that the block of HNE activity by Sivelestat led to positive effects on mechanical allodynia in a model of neuropathic pain suggests a functional involvement of this protease in the complex mechanism of neuropathic pain [97,98]. Lastly, in a recent publication, HNE has been related to chronic kidney disease (CKD) [99], a pathology with an inflammatory basis. Chronic inflammation in patients with CKD under hemodialysis has been associated with an imbalance between oxidant and anti-oxidant mechanisms, including neutrophil activation with release of ROS and granule contents, such as HNE [100,101]. Finally, other important neutrophil-driven inflammatory diseases such as inflammatory bowel disease, Crohn’s disease [102], atherosclerosis [103], severe pneumonia [104], and graft-versus-host disease [105] are related to excessive HNE activity. Based on the discussion above, it is clear that inflammation is a physiological and pathological process that is found in many diseases and that serine proteases, including HNE, are key elements in these processes. Hence, the control of this proteolytic activity represents an important therapeutic aspect for the resolution of these diseases.

Figure 3.

Sivelestat (marketed as Elaspol^® 100), Alvelestat and BAY85-8501 (in Phase II clinical trials).

5. HNE inhibitors

In the last three decades, many pharmaceutical companies and academia have discovered a variety of innovative elastase inhibitors. At present, only two HNE inhibitors are on the market. The first one, Prolastin^® (purified α1-AT), is a peptide drug used in the treatment of α1-AT deficiency [106]. The second is Sivelestat (ONO-5046, Elaspol^® 100), which is a non-peptide inhibitor approved for intravenous use in Japan and South Korea for the treatment of ALI and ARDS associated with systemic inflammatory response syndrome [107]. Sivelestat is a highly specific and effective HNE inhibitor with an IC₅₀ = 44 nM and Ki = 200 nM [108] (Figure 3).

Low-molecular-weight synthetic HNE inhibitors may offer several advantages over peptide inhibitors, including enhanced enzyme selectivity, oral bioavailability, lower susceptibility to proteolytic inactivation, and decreased risk of an immunogenic response. Moreover, a ‘small molecule’ is more easily modifiable in order to allow for the greater ability to modify the inhibitor structure and optimize physical and pharmacokinetic properties for the production of viable clinical candidates [109]. In literature, there are also many HNE inhibitors from natural products reported [110–113], although they generally have low potency, selectivity, and metabolic stability, and none of these compounds have become drug candidates [63]. An important exception to this trend is represented by Lyngbyastatin 7 isolated from Cyanobacteria of marine environment by Salvador et. al in 2013 [114] and later obtained for total synthesis via 31 steps by Luo and co-workers [115]. It shows a 19-membered cyclic hexadepsi-peptide core and is a potent HNE inhibitor (IC₅₀ = 23 nM) provided with a good selectivity for HNE against a panel of 68 proteases.

Patents on HNE inhibitors released in the period 2010–2014 were summarized in an excellent review by Tsai and Hwang (2015). In the current review, we report a perspective on low-molecular-weight HNE inhibitors and their possible therapeutic application by considering the patents registered from 2014 to 2018. Since in this range of time some company have published several licenses on HNE inhibitors, we decided to present the patents and the HNE inhibitors reported therein according to the parent pharmaceutical company.

Before providing a systematic description of the most interesting ‘small molecules’ discovered by the pharmaceutical industry in the selected period, we first report a short presentation of drugs in clinical trials. Table 1 shows the HNE inhibitors currently in clinical trials, and we included the target pathology, the clinical trial phase, the company/institute involved in the clinical trial, and the drug generation. For the latter, we followed the recent classification by Von Nussbaum et al. [45], which categorizes HNE inhibitors into five generations. The peptide inhibitors α1-AT [116–123] and Elafin [124,125] are in clinical trials for different pathologies, including COPD, α1-AT deficiency (ADAT) or PAH. All the others are synthetic non-peptide inhibitors, with the exception of POL6014 [126,127], a new macrocyclic peptide mimetic developed by the University of Zürich and Polyphor Ltd using Protein Epitope Mimetics (PEM) drug discovery [128–130]. The structures of Alvelestat [45,131–133] and BAY-858501 [45,134,135] are reported in Figure 3, while the formula of CHF6333 from Chiesi Farmaceutici is undisclosed [66,136,137]. These latter drugs will be discussed in detail in the corresponding sections.

Table 1.

HNE inhibitors in clinical trials.

HNE Inhibitor	Condition/Disease	Phase Clinical Trials	Generation	Company/Institute	Reference
Alpha 1-Antitrypsin (ATT) (Glassia, Aralast)	COPD Alpha1-antitrypsin Deficiency (ADAT)	III	1	Shire (Baxalta now part of Shire)	[114,115]
Alpha 1-Antitrypsin (ATT) (Prolastin, Aralast)	Acute Myocardial Infarction	II	1	Virginia Commonwealth University	[116,117]
Alpha 1 Anti-Trypsin (AAT) (Zemaria)	Steroid Refractory Acute Graft vs Host Disease	II	1	University of Michigan Cancer Center	[118,119]
Alpha 1 Anti-Trypsin (AAT) (Glassia)	Graft-Versus-Host Disease (GVHD) Acute on Chronic	II	1	Fred Hutchinson Cancer Research Center	[120,121]
ELAFIN (Tiprelestat)	Pulmonary Arterial Hypertension (PAH)	I	1	Roham T. Zamanian, Stanford University	[122,123]
POL6014	Cystic Fibrosis	II	1/2	Santhera Pharmaceuticals	[124–128]
MPH 966 (Alvelestat oral tablet)	ADAT Enphysema COPD	II	3	Mereo BioPharma	[45,129,130]
AZD9668 (Alvelestat)	Bronchiolitis Obliterans Syndrome (BOS) Chronic Graft vs Host Disease	II	3	National Cancer Institute (NCI)	[45,131]
CHF6333	Cystic Fibrosis Bronchiectasis	I	5	Chiesi Farmaceutici S.p.A.	[66,134,135]
BAY 85-8501	Bronchiectasis	II	5	Bayer	[45,132,133]

5.1. Chiesi Farmaceutici S.p.A

Chiesi Farmaceutici S.p.A has been working for years in the development of bronchodilator drugs for the treatment of pulmonary diseases, such as COPD, and is also interested in the identification of new HNE inhibitors to use both alone and in association with other drugs. Before considering the patents, it is important to highlight that the structure of the best compound developed by this company (CHF6333) is not known. CHF6333, currently in phase I clinical trials, is the first inhaled HNE inhibitor under development for the treatment of HNE-driven lung diseases using a dry powder inhaler. In vitro, CHF6333 is a highly potent HNE inhibitor (IC₅₀ = 0.2 nM) with good selectivity against other proteases. In addition, CHF6333 significantly reduces lung neutrophil recruitment induced by cigarette smoke exposure in mice and reduces both lung tissue infection and inflammation when administered intratracheally to P. aeruginosa-infected rats for 7 days [138].

In the literature, there are many 2,3,5,8-tetrahydro-[1,2,4] triazolo[4,3-a]pyrimidine derivatives reported as HNE inhibitors patented by Chiesi [139–143] and active by an inhalatory route. The reported IC₅₀ values indicate compounds endowed with a notable potency, but no clear structure–activity relationships were deduced from the compound library. In Table 2, a general structure of tetrahydrotriazolopirimidine and some significant compounds (1–8) are presented. Compounds 7 and 8 were also tested in at least two separate experiments against activated rat and human neutrophils, with IC₅₀ of <10 nM (rat protease inhibition) and <1 nM (human protease inhibition), respectively [142]. Among the bicyclic compounds patented by Chiesi, the [1,2,4]triazolo[1,5-a]pyridine derivatives 9–11 shown in Figure 4 are active in the nanomolar range [144]. Moreover, there are three patents claiming monocyclic heteroaromatic compounds with imidazolone and pyrazolone scaffolds [145–147]. These compounds exhibit HNE inhibitory activity in the subnanomolar range and are suitable for administration in microparticles using a dry powder inhaler (DPI). Table 3 shows some representative compounds with imidazolone (12–17) and pyrazolone (18,19) scaffolds, all containing a m-trifluoromethylphenyl ring at N-1 of the heterocyclic system and a p-cyanophenyl group [145,146]. Noteworthy, the two pairs of isomers with imidazolone and pyrazolone scaffolds (16 versus 18 and 17 versus 19) retain the same level of inhibitory activity.

Table 2.

Examples of 2,3,5,8-tetrahydro[1,2,4]triazolo[4,3-a]pyrimidine compounds (5th generation) developed by Chiesi Farmaceutici S.p.A.

Compound	R	R1	IC50 (nM)a	Reference
1	CH3		< 20	[137]
2	CH3		< 20	[138]
3	CH3		< 20	[138]
4	CH3		< 20	[139]
5	CH3		< 20	[139]
6	CH3		< 20	[139]
7	CH2CH2OH	H	< 1	[140]
8		H	< 1	[140]

^aThe results are means of two independent experiments, each performed in duplicate (HNE enzyme assay).

Figure 4.

Examples of [1,2,4]triazolo[1,5-a]pyridine derivatives developed by Chiesi Farmaceutici S.p.A.

Table 3.

Examples of representative imidazolone (12–17) and pyrazolone derivatives (18, 19) developed by Chiesi Farmaceutici S.p.A.

Compound	R1	R2	IC50 (nM)a	Reference
12		H	< 1	[143]
13		H	< 1	[143]
14		H	< 1	[143]
15		H	< 1	[143]
16			< 1	[144]
17			< 1	[144]
18			< 1	[144]
19			< 1	[144]

^aThe results are means of two independent experiments, each performed in duplicate (HNE enzyme assay).

5.2. Boehringer Ingelheim GmbH

A number of 4-oxo-1,4-dihydropyridine were patented by Boehringer Ingelheim International GmbH as HNE inhibitors for the treatment and/or prevention of pulmonary, gastrointestinal (GI) and genitourinary diseases, inflammatory diseases of the skin and eye, other autoimmune and allergic disorders, allograft rejection, and oncological diseases [148–150]. In Table 4 the main representative compounds 20–29 are shown. Their HNE inhibitory activity is in the sub/low nanomolar range. The presence of the sulfur atom as chiral center in compounds 20–22 did not influence HNE inhibitory activity, and the reported IC₅₀ values are related to experiments performed on the racemate. In contrast, the chiral center in the substituent at nitrogen in position 1 [the best is the 1-(4-cyanophenyl)ethyl group] had an important effect on activity. For these compounds, it is possible to identify the (R)-enantiomer as the eutomer whose IC₅₀ values are reported in Table 4. The comparison of IC₅₀ values of (R)- and (S)-enantiomers clearly shows that the eutomer (R) is always at least four fold more potent than the (S)-enantiomer [i.e. IC₅₀ < 1 nM for 24(R) and IC₅₀ = 225 nM for 24(S); IC₅₀ = 2.3 nM for 27(R) and IC₅₀ = 1299 nM for 27(S)] [149]. Some compounds reported in this patent exhibited favorable pharmacokinetic properties, metabolic stability, and permeability suitable for oral administration.

Table 4.

Examples of representative 4-oxo-1,4-dihydropyridine derivatives developed by Boehringer Ingelheim GmbH.

Compound	R1	R3	R5	X	IC50 (nM)a	Reference
20	CH2CH3		CF3	C	< 1b	[146]
21	CH(CH3)2		CHF2	C	< 1b	[146]
22			CHF2	C	< 1b	[146]
23			CF3	C	< 1c	[147]
24		CH3	CF3	C	< 1c	[147]
25		CH3	CF3	N	3.7c	[147]
26			CF3	N	2.8c	[147]
27			CF3	N	2.3c	[147]
28			CHF2	C	< 1c	[147]
29	CH(CH3)2		CHF2	C	3	[148]

^aThe results are means of two independent experiments, each performed in duplicate (HNE enzyme assay).

^bIC₅₀ values are related to the racemate.

^cIC₅₀ values are related to eutomer [(R)-enantiomer].

Boehringer Ingelheim also investigated other monocyclic scaffolds, such as 2-pyridone [151], 2-pyrazinone [151], and dihydropyrimidone [152] as HNE inhibitors reaching IC₅₀ values in the nanomolar/picomolar range. In Figure 5 are presented two potent compounds belonging to the 2-oxo-1,2-dihydropyridine (30) and 3-oxo-3,4-dihydropyrazine (31) series, respectively, and a general formula (32) representative of a library of about 215 compounds with 2-oxo-1,2,3,4-tetrahydropyrimidine scaffold, which are very potent HNE inhibitors exhibiting picomolar IC₅₀ values and in some cases, an additional inhibitory effect on neutrophil PR3, which is favorable for pharmacological efficacy. Moreover, the dihydropyrimidone derivatives of structure 32 exhibited favorable metabolic stability, appropriate permeability, and aqueous solubility. Among this library of compounds, the derivative that has R = CH₃ and R₁ = 1-methylpyridin-2(1H)-one exhibited the best properties. In particular, its half-life (t_1/2 in vitro) in human liver microsomes was >130 min, its aqueous stability was 0.073 mg/mL, and its metabolic stability with human hepatocytes calculated as hepatic intrinsic clearance value (Q_h [%]) was 5%. Another important parameter for oral absorption is the measurement of the ability of a compound to pass across the cell membrane. The in vitro model used for determination of drug transport across the membrane is human cancer colon carcinoma cells (Caco-2) grown on permeable filter supports. The selected compound exhibited a permeability coefficient [PE] and drug adsorption from the intestine into the blood [AB] (PEAB) value of 12 × 10⁻⁶ cm/s and a PE and drug secretion from the blood back into intestine [BA] (PEBA) value of 120 × 10⁻⁶. These values make it an optimal candidate for oral administration [152].

Figure 5.

Examples of 2-oxo-1,2-dihydropyridine (30), 3-oxo-3,4-dihydropyrazine (31), and general structure of 2-oxo-1,2,3,4-tetrahydropyrimidine (32) derivatives developed by Boehringer Ingelheim GmbH.

The encouraging results obtained with dihydropyrimidone derivatives (32) encouraged Boehringer researchers to further explore this scaffold and developed new series of tetrahydrocycloalkylpyrimidone analogs [153–155], which are summarized in Tables 5 and 6. These inhibitors were developed for pharmaceutical composition and methods for the treatment of pulmonary disease and other inflammatory pathologies. The representative tetrahydrociclopentane pyrimidone derivatives (33–47) shown in Table 5 are very potent HNE inhibitors, with IC₅₀ values in the low nanomolar/picomolar range. Compounds 33–42 and 47 which contain various groups R₄ belong to the 5th generation [45]. The presence of a group at position 2 on the p-cyanophenyl ‘freezes’ the structure in an ideal bioactive conformation, thus pre-organizing the inhibitor for the forthcoming binding event. This additional substituent increases the potency of inhibitor, although it does not directly interact with the target. Compounds 43–46, although missing the ‘freezing’ group, are equally powerful HNE inhibitors, with IC₅₀ < 1 nM but belong to the 4th generation and, as a result, are less potent and stable than the compounds belonging to the 5th generation.

Table 5.

Examples of 3,4,6,7-tetrahydro-1H-cyclopentane[d]pyrimidine-2,5-dione derivatives developed by Boehringer Ingelheim GmbH.

Compound	R4	R3	X	IC50 (nM)a	Reference
33	SO2CH3	CH3	C	< 1	[151]
34	SO2CH2CH3		N	< 1	[151]
35	SO2CH3	CH2CH2OH	C	<1	[151]
36		CH3	C	2.2	[152]
37		CH3	C	1.8	[152]
38		CH3	C	1.3	[152]
39		CH3	C	7.5	[152]
40		CH3	C	2.1	[152]
41		CH3	C	1.2	[152]
42		CH3	C	< 1	[152]
43	H	H	C	11.5	[153]
44	H	CH3	C	2.4	[153]
45	H		C	<1	[153]
46	H		C	<1	[153]
47	SO2CH3		C	<1	[153]

^aThe results are means of two independent experiments, each performed in duplicate (HNE enzyme assay).

Table 6.

Examples of condensed dihydropyrimidone derivatives developed by Boehringer Ingelheim GmbH.

Compound	R4	R3	X	IC50 (nM)a	Reference
48 b	SO2CH3	H	CH2	4.8	[154]
49 b	H		CH2	< 1	[154]
50 b	H		CH2	< 1	[154]
51 b	SO2CH3		CH2	< 1	[154]
52 b	SO2CH3		CH2	< 1	[154]
53 b	SO2CH3		CH2	1	[155]
54 b	SO2CH3	CH2CH2OH	CH2	< 1	[155]
55 b	CONH2	CH3	CH2	1.6	[152]
56 c	H	H	NH	3.1	[156]
57 c	H		NH	1.3	[156]
58 c	H		NH	< 1	[156]
59 c		CH3	NH	< 1	[156]
60 c	CONH2	CH3	N-CH3	< 1	[156]
61 c	SO2CH3	CH3	NH	< 1	[156]

^aThe results are means of two independent experiments, each performed in duplicate (HNE enzyme assay).

^bThe absolute configuration (S) has been unambiguously assigned by X-ray structure analysis.

^cThe absolute configuration (R) has been unambiguously assigned by X-ray structure analysis.

For the condensed dihydropyrimidones (48–61) presented in Table 6, the absolute configuration responsible for HNE inhibitory activity was unambiguously assigned, leading to IC₅₀ < 1nM [156–158]. They exhibit favorable in vivo potency in models of HNE-induced lung injury in mice, rats, or hamsters; good metabolic stability (t_1/2 > 130 min); good permeability across the cellular membrane; and reduced in vivo intrinsic clearance (CL) [156–158]. Compound 51, with a predicted human hepatic in vivo blood clearance of 0 ml/min/Kg, a PEAB of 9.86 × 10⁻⁶, a PEBA of 53.5 × 10⁻⁶, and aqueous stability of 0.086 mg/mL, represents one of the best pharmacokinetic profiles. However, numerous examples of these compounds containing a carbamoyl substituent at position 3 of the dihydropyrimidine scaffold exhibited improved AB permeability and/or reduced efflux ratio.

5.3. Bayer Healthcare AG

Currently, Bayer Healthcare AG has one HNE inhibitor in phase II clinical trials (BAY 85-8501) (already reported in Figure 3 and Table 1), which belongs to the 5th generation [45]. Analogous to the other pyrimidone derivatives developed by Bayer, BAY 85-8501 contains a methylsulfonyl group at R₄ of the p-cyanophenyl ring, which induces a frozen bioactive conformation leading to a significant increase in potency, selectivity, and stability (Table 7). The high binding efficiency of the pyrimidone class is based on an induced-fit binding mode: upon binding of these molecules, the S2 pocket of HNE widens significantly to accommodate the northern para-cyanoaryl residue. The presence of a substituent R₄ is important to increase potency into picomolar range. Other potent and interesting dihydropyrimidones are shown in Table 7 (compounds 62–67) [159], all exhibiting good in vivo stability, also under oxidative conditions. Due to the high impact of this new class of HNE inhibitors, Bayer started to explore bi-heterocycles containing the pyrimidine nucleus with different substituents (Figure 6) [159,160]. Compounds 68–71 exhibit excellent activities against HNE with IC₅₀ < 0.3 nM. However, pyrimidopyridazine derivative 71 (BAY-8040) [161] is the best in vivo candidate from this series. This compound showed good potency and in vivo pharmacokinetic properties, and it was tested in vivo in a monocrotaline rat model of PAH. Treatment with BAY-8040 (71) (50 mg Kg⁻¹, p.o) from days 14 to 28 significantly decreased right ventricular systolic pressure and right ventricular hypertrophy relative to placebo, with a significant beneficial effect on right ventricular dysfunction and preservation of systemic arterial pressure. Furthermore, compound 71 improved the cardiac output and arterial oxygenation in rats with MCT-induced PAH. Thus, BAY-8040 was able not only to suppress the inflammation at the basis of the pathology but also reverse tissue remodeling in the PAH setting [161].

Table 7.

Examples of 4-(4-cyano-2-thionyl)dihydropyrimidones developed by Bayer HealthCare AG.

Compoundb	R4	R3	IC50 (nM)a	Reference
62	SO2CH3	H	0.5	[157]
63	SO2CH3		< 0.3	[157]
64	SO2CH3	SO2CH3	< 0.3	[157]
BAY 858501	SO2CH3	CH3	< 0.3	[129,130,157]
65	SO2CH3		< 0.3	[157]
66	SO2CH3	CH2CN	< 0.3	[157]
67	SO2Ph	H	< 0.3	[157]

^aThe results are means of two independent experiments, each performed in duplicate (HNE enzyme assay).

^bThe absolute configuration (S) has been unambiguously assigned by X-ray structure analysis.

Figure 6.

Heterocyclic-fused pyrimidine derivatives developed by Bayer Healthcare AG.

5.4. Other companies

AstraZeneca has extensively investigated HNE inhibitors, producing a large number of compounds with a 2-pyridone scaffold. Among these products is Alvelestat (AZD9668) (see Figure 3), which is currently in clinical trials, as previously mentioned in Table 1. AstraZeneca also developed 2-pyrazinone derivatives, and compound 72 (AZD9819), shown in Figure 7, is an interesting and representative example [162]. KRP-109 (73) is claimed to be a selective, water-soluble competitive HNE inhibitor developed by Kyorin Pharmaceutical Co., Ltd. (Tokyo, Japan) [163]. A distinct feature of KRP-109 is its ability to accumulate in high concentrations in lung tissue, thereby inhibiting the activity of HNE released by infiltrating neutrophils under inflammatory conditions. Moreover, KRP-109 reduced lung inflammation in a murine model of severe pneumococcal pneumonia and was also able to decrease the degradation of mucin in CF sputum in vitro [164].

Figure 7.

2-pyrazinone derivative (72) developed by Astrazeneca and KRP-109 (73) developed by Kyorin Pharmaceutical Co.

5.5. Miscellaneous

The California Institute for Biomedical Research has recently developed a series of dimeric compounds with the general formula Y¹-A¹-X-A²-Y² (Figure 8), where A¹ and A² are linear or branched alkyl chains, X is a group such as -C(O)-, -C(O) C(O)-, -NHCO-, and Y¹ and Y² are fragments of drugs, for example, Sivelestat and Alvelestat. These compounds are disclosed in a patent [165] as potentially useful for the treatment of lung diseases, such as COPD and cystic fibrosis, but also for intestinal disease, including inflammatory bowel disease, and a representative product (compound 74) exhibiting IC₅₀ < 150 nM is shown in Figure 8.

Figure 8.

Example of a dimeric compound developed by the California Institute for Biomedical Research.

Figure 9 shows compounds 75–77 with isoxazol-5(2H)-one scaffolds, which were developed by Giovannoni and co-workers [166,167] as potent and stable HNE inhibitors exhibiting IC₅₀ values in the low nanomolar range. These compounds, which were designed as molecular simplifications of other compounds synthesized in the same research laboratory [81,168–172], act as competitive, pseudo-irreversible HNE inhibitors, and docking studies seem to highlight that the attack of Ser195 is directed to the endocyclic CO group at position 5 to form the acyl-enzyme complex.

Figure 9.

Examples of HNE inhibitors reported in the recent literature.

Finally, worthy of mention is a new class of HNE inhibitors with a diazaborine nucleus [173]. Figure 9 shows two examples of these compounds (78, 79). Although endowed with moderate activity in the micromolar range, these compounds exhibit good selectivity toward elastase, and no inhibition was observed against a panel of five closely related serine proteases. Moreover, they are very stable in phosphate buffer at pH 7.4 and in human plasma. Computational studies performed on this system suggest that the mechanism of action involves the formation of a reversible covalent bond between the diazoborine boron center and the catalytic serine oxygen.

6. Conclusion

The selected patents reported for the period 2014–2018 represent a large number of HNE inhibitors obtained through the use of rational drug design and chemical modifications of potent lead compounds and by the study of the structural properties of neutrophil elastase. Mono- and bi-heterocycle nitrogen-containing scaffolds are the most investigated, leading to products with similar chemical characteristics in many cases. Most of the new compounds belong to the 4th and 5th generations, inserting inside the active site in a perpendicular manner. This particular interaction produces a new S2 pocket or allosteric site without interacting with the catalytic triad. Both in the monocyclic and bicycles compounds, a p-cyanophenyl fragment is present at the north position, and a m-trifluorophenyl at nitrogen at the south position, both being important for binding to HNE via an induced fit with a frozen bioactive conformation. These new potent, nonreactive molecules exhibit significant boosts in potency, selectivity, and metabolic stability, especially under pathophysiological conditions.

7. Expert opinion

Human neutrophil elastase (HNE) is a therapeutic target of considerable interest because of its involvement in many inflammatory diseases, as is demonstrated by the number of HNE inhibitors patents developed in the last decade by pharmaceutical companies. Currently, the two HNE inhibitors on the market are used for the treatment of α1-antitrypsin deficiency (Prolastin^®) and ALI and ARDS associated with systemic inflammatory response syndrome (SIRS) (Sivelestat^®). In addition to their use for cardiopulmonary diseases, HNE inhibitors were proposed to be useful for other inflammatory pathologies, such as psoriasis, rheumatoid arthritis, bowel intestinal inflammation, and also cancer, since HNE activity has been directly related to the progression and metastasis of cancer cells.

Several key advances in the last couple of years will likely stimulate further development of newer HNE inhibitors. The first originates from the observation that in addition to HNE, other proteases contribute to tissue damage; thus, dual HNE/PR3 inhibitors have been proposed as an innovative approach for the treatment of neutrophilic inflammatory diseases by Hwang and coworkers [174,175], opening a new window in the discovery of new drugs. Second, the activity of neutrophil serine proteases, including elastase and Pr3, has been implicated in some autoimmune diseases, such as type 1 diabetes (T1D) [94]. Although the role of neutrophils in the development of T1D remains unknown, a recent patent [176] reports that the serum concentration and the enzymatic activities of neutrophil elastase and Pr3 are diagnostic biomarkers for the risk of developing autoimmune diabetes. Another interesting approach being considered to regulate levels of protease activity and restore the protease-antiprotease balance is to indirectly modulate HNE function by inhibiting those peptidases that active this enzyme during development in the bone marrow. For example, DPP1 (dipeptidyl peptidase 1 or Cathepsin C) is a lysosomal cysteine protease that plays a key role in the activation of the proinflammatory neutrophil serine proteases, HNE, PR3, and Cathepsin G by cleaving the N-terminal dipeptide during neutrophil maturation. The inhibition of DPP1 could generate neutrophils lacking these active proteases, thereby reducing the local release and the subsequent damage [177].

Finally, the study published by Marto and coworkers [76] opens a new perspective on the administration routes for HNE inhibitors, till now used intravenously. The recent demonstration of the involvement of HNE in psoriasis and atopic dermatitis stimulated the research in this field, in particular, the use of starch-based nanocapsules as nanobiomaterial for topical drug delivery. The low anti-inflammatory activity of the potent ER143 inhibitor (IC₅₀ = 0.67 nM), when used as a solution, is considerably increased if the same compound is associated with a starch-based nanoparticulate system (StNC). The resulted formulation exhibited interesting characteristics and in vivo studies showed that the topical application of StNC-ER143 strongly inhibited the acute inflammatory response as evident by the decrease of edema (about 92% inhibition), erythema and neutrophil infiltration.

In conclusion, taking into account the interesting results achieved in the field of HNE inhibitors and their medical applications, this topic appears worthy of further in-depth analysis. At the present, the main effort by pharmaceutical companies is pointed to improve quality of life and reduce the risk of death in pulmonary pathologies, overall COPD which in 2020 is expected to be the 4th cause of death in the world. Moreover, the new recent discoveries regarding as the involvement of HNE in other pathologies as the possibility to indirectly modulate HNE function, open new intriguing possibilities.

Article highlights.

• This review focuses on neutrophil elastase (NE) inhibitor patents reported in the literature over 2014–2018.

• The main application of HNE inhibitors concerns the treatment of (cardio)pulmonary inflammatory diseases; other possible uses include skin pathologies, rheumatoid arthritis, and cancer.

• This review follows the recent classification proposed for HNE inhibitors, which considers five generations, starting from biologicals (1st generation) to ‘pre-adaptive pharmacophore derived from 4th generation inhibitors’ (5th generation). Many of these compounds show picomolar activity, high selectivity, and drug-like pharmacokinetic properties.

• In this review, a systematic investigation of pharmaceutical companies working on HNE inhibitors was done. Accordingly, for each company, the most interesting results are reported. A miscellaneous from literature follows.

• Non-peptidic NE inhibitors are low-molecular-mass synthetic compounds containing nitrogen heterocycles such as pyrimidinone, pyrimidinedione, 2-pyrazinone, pyrimidine, pyridine, 4-pyridone, 2-pyridone, and pyridazine.

This box summarizes key points contained in the article.