- Email: [email protected]
Molecularly targeted therapies for asthma: Current development, challenges and potential clinical translation
Accepted Manuscript Molecularly targeted therapies for asthma: Current development, challenges and potential clinical translation Ibrahim Sulaiman, Lim Chee Woei, Soo Hon Liong, Johnson Stanslas PII:
S1094-5539(16)30056-6
DOI:
10.1016/j.pupt.2016.07.005
Reference:
YPUPT 1551
To appear in:
Pulmonary Pharmacology & Therapeutics
Received Date: 9 May 2016 Revised Date:
14 July 2016
Accepted Date: 20 July 2016
Please cite this article as: Sulaiman I, Woei LC, Liong SH, Stanslas J, Molecularly targeted therapies for asthma: Current development, challenges and potential clinical translation, Pulmonary Pharmacology & Therapeutics (2016), doi: 10.1016/j.pupt.2016.07.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT MOLECULARLY TARGETED THERAPIES FOR ASTHMA: CURRENT DEVELOPMENT, CHALLENGES AND POTENTIAL CLINICAL TRANSLATION
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Ibrahim Sulaiman, Lim Chee Woei, Soo Hon Liong and Johnson Stanslas.
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Pharmacotherapeutics Unit, Department of Medicine, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia.
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Corresponding Author: Prof Johnson Stanslas, Pharmacotherapeutics Unit, Department of Medicine, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. Email: [email protected]; [email protected]; Phone: 603-89472310; Fax: 603 89472789
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ACCEPTED MANUSCRIPT Abstract:
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Extensive research into the therapeutics of asthma has yielded numerous effective interventions over the past few decades. However, adverse effects and ineffectiveness of most of these medications especially in the management of steroid resistant severe asthma necessitate the development of better medications. Numerous drug targets with inherent airway smooth muscle tone modulatory role have been identified for asthma therapy. This article reviews the latest understanding of underlying molecular aetiology of asthma towards design and development of better antiasthma drugs. New drug candidates with their putative targets that have shown promising results in the preclinical and/or clinical trials are summarised. Examples of these interventions include restoration of Th1/Th2 balance by the use of newly developed immunomodulators such as toll-like receptor-9 activators (CYT003QbG10 and QAX-935). Clinical trials revealed the safety and effectiveness of chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) antagonists such as OC0000459, BI-671800 and ARRY-502 in the restoration of Th1/Th2 balance. Regulation of cytokine activity by the use of newly developed biologics such as benralizumab, reslizumab, mepolizumab, lebrikizumab, tralokinumab, dupilumab and brodalumab are at the stage of clinical development. Transcription factors are potential targets for asthma therapy, for example SB010, a GATA-3 DNAzyme is at its early stage of clinical trial. Other candidates such as inhibitors of Rho kinases (Fasudil and Y-27632), phosphodiesterase inhibitors (GSK256066, CHF 6001, roflumilast, RPL 554) and proteinase of activated receptor-2 (ENMD-1068) are also discussed. Preclinical results of blockade of calcium sensing receptor by the use of calcilytics such as calcitriol abrogates cardinal signs of asthma. Nevertheless, successful translation of promising preclinical data into clinically viable interventions remains a major challenge to the development of novel anti-asthmatics.
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Keywords:
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Asthma, Inflammation, Airway Remodelling, Anti-Asthmatics, Targets
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Chemical compounds studied in this article:
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Bis-(5-amidino-2-benzimidazolyl)-methane (PubChem CID: 46936860); Calcitriol (PubChem CID: 5280453); JNJ-39758979 (PubChem CID: 24994634); JNJ-7777120 (PubChem CID: 4908365); PF-3893787 (PubChem CID: 24745335); Y-27632 (PubChem CID: 448042); Fasudil (PubChem CID: 3547); Roflumilast (PubChem CID: 449193), YM-341619 (PubChem CID: 10321901); Afzelin (PubChem CID: 5316673).
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Introduction
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Asthma is an intricate inflammatory airway disease that results from activation of numerous
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inflammatory and structural cells, often leading to partially or fully reversible airway
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obstruction, airway inflammation, mucus hypersecretion and acute hyperresponsiveness
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(Prado et al., 2014). The aetiology of asthma may be genetic, environmental or an interplay of
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both factors. It places enormous economic burden on patients, families, and the health care
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system by causing loss of productivity, high number of missed school and/or workdays, high
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medical bills, and premature death (Barnes et al., 2015). About 334 million people globally
78
were estimated to be asthmatic (Global Asthma Report, 2014; Murray et al., 2013) and this
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figure was projected to escalate to 400 million by the year 2025 (Global Asthma Report,
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2014). Statistics have placed asthma as the 14th most important disorder in the world, it
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affects about 14%, 8.6% and 4.5% of world children, young adults and world population
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respectively (Murray et al., 2013; To et al., 2012; Global Asthma Report, 2014). Severe
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uncontrolled asthma account for 5% to 10% of total asthma cases globally, and 50% of total
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asthma cost (Al-Hajjaj, 2011; Dheda et al., 2015). In Europe alone, the direct and indirect
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cost of both controlled and uncontrolled asthma in patients between the age of 15-64 was
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estimated to be €19 billion/annum (Domínguez-Ortega et al., 2015). Asthma, as a global
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disorder is a source of concern to both developed and developing nations (Behera and Sehgal,
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2015). Although improvement in asthma conditions can be achieved by the use of currently
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available conventional therapies such as topical bronchodilators and corticosteroids, these
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drugs have presented a number of unwanted side effects, possible resistance with long term
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use and absolute non-responsiveness in some cases (Abramson et al., 2003; Chung et al.,
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2014). Only a few effective anti-asthma controllers and relievers are currently available
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(example include steroids such as budesonide and fluticasone, bronchodilators such as
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salmeterol and levalbuterol). This may be due to inadequate knowledge of the underlying
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cause of asthma, difficulties in developing inhalable preparations for topical delivery and
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inordinate animal models used in testing new treatments that mostly fail clinical translation
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and relevance. This article discusses targets that could aid in developing new interventions
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with minimal adverse effects as compared to current medications. The role of certain
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receptors, enzymes and cytokines in the aetiology of asthma was further emphasized.
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Pathophysiology of Asthma
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The cardinal pathophysiological manifestations of asthma are airway inflammation,
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intermittent obstruction and acute hyperresponsiveness (AHR) (Holgate, 2008). Depending
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on cellular and molecular characterization of the inflammatory cascade, asthma may either be
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allergic or non-allergic endotype. Allergic asthma is usually eosinophilic, whereas non-
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allergic endotypes, such as aspirin, infection and exercise induced asthma may present with
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neutrophilic or paucigranulocytic phenotype. Most non-allergic asthma is severe, and steroid-
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resistant (Lötvall et al., 2011).
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Fundamental in vitro and in vivo studies have revealed several cellular and molecular events
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involved in development of asthma. These include increased immunoglobulin E (IgE)
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production by B cells, shift in T helper-type 1/T helper-type 2 (Th1/Th2) paradigm,
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upregulation of Th2 cytokines, airway cellular infiltrations, dysfunctional activated
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inflammatory cells, mitochondrial damage, increased production of reactive oxygen species
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(ROS) and reactive nitrite species (RNS) (Aguilera-Aguirre et al., 2009).
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Aeroallergens such as house dust mites, pollens and animal dander exert proteolytic and LPS-
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like activity that permits them to penetrate through the airway epithelium and attach to toll
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like receptors (TLR) to activate some cascade of events (Wilson et al., 2012). Allergen-TLR
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interaction induces release of IL-25, IL-33, Thymic stromal lymphopoietin (TSLP),
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macrophage inflammatory protein-3 (MIP3A) and monocyte chemotactic protein 1 (MCP1)
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by the airway epithelial cells. IL-25, IL-33 and TSLP enhance the development of Th2 cells
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while MIP3A (CCL20) and MCP1 (CCL2) are involved in the recruitment and activation of
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dendritic cells. Activated dendritic cells recognizes and present fragments of allergen peptides
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to lymph nodal naïve T lymphocytes (Th0) through its major histocompatibility complex
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(MHC) class II (Kallinich et al., 2007). The differentiation of Th0 into Th1 or Th2 depends on
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the cytokine milieu. Usually, increased IL-4 (induced by mast cell) and low IL-2 promotes
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differentiation of Th0 to Th2 cells. Th2 cells constitutively expresses GATA3, a crucial
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transcription factor that enhances Th2 production of IL-4, IL-5, IL-9 and IL-13 which further
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improve the suitability of the cytokine milieu for further Th0 - Th2 differentiation (Kaiko et
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al., 2008). Furthermore, IL-4 and IL-13 incites atopy by enhancing the production of IgE
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from B lymphocytes, influences secretion of eosinophil and Th2 recruiting cytokines such as
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IL-4, IL-5, IL-13, granulocyte macrophage colony stimulating factor (GM-CSF) and potent
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chemoattactants such as eotaxin 1 (CCL11), eotaxin 2 (CCL24) and RANTES (CCL5)
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mast cell airway infiltration, maturation and degranulation, while IL-5 induces and augment
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pulmonary eosinophil homing (Fulkerson and Rothenberg, 2013). IL-4 further enhances
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eosinophil migration by activating airway vascular cell adhesion molecule 1 (VCAM-1).
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Pulmonary eosinophils secrete wide array of inflammatory mediators such as cysteinyl
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leukotrienes (LTC4, LTD4, LTE4) and cytotoxic agents such as major basic protein (MBP),
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eosinophil derived neurotoxin, eosinophil cationic protein and eosinophil peroxidases which
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all together further aggravate airway inflammation and oxidative stress damage observed in
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asthma (Hall and Agrawal, 2014; Pelaia et al., 2015). Although eosinophilic asthma is of
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allergic endotype, non-allergic adult onset asthma can present with airway eosinophilia whose
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pattern of development is independent of Th2, rather, it depends on combined transcriptional
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activity of GATA3/RORα transcription factors and type 2 innate lymphoid cells (ILC2) (Woo
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et al., 2014). Nevertheless, both allergic and non-allergic eosinophilic asthma shows elevated
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type 2 cytokine levels, thereby indicating the critical role of IL-4, IL-5, IL-13 in the
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pathogenesis of eosinophilic asthma (Pelaia et al., 2015; Lambrecht and Hammad, 2015;
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Brusselle et al., 2013).
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Apart from airway eosinophilia due to Th2 or ILC2, asthma may present with neutrophilic
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phenotype, which is widely mediated by Th17, a subset of T helper cells that secretes IL-17.
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This category of asthma is often expressed in severe/uncontrolled asthmatics, steroid
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insensitive and it may be triggered by allergens or non-allergenic triggers such as microbes,
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cigarette smoke, diesel exhaust particles and other environmental pollutants (Polosa and
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Thomson, 2013; Newcomb and Peebles, 2013; Vroman et al., 2015). Differentiation of
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thymocytes into Th17 cells depends on RAR-related orphan receptor gammaT (RORɣt), a
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transcription factor that requires IL-1β, IL-6 and transforming growth factor (TGF-β) milieu
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for its upregulation and consequent Th17 production (Dong, 2008; Vroman et al., 2015). The
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heterogeneous nature of asthma pathogenesis has rendered blanket approach to treatment and
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diagnosis of different asthma endotypes ineffective. Therefore, proper understanding of
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asthma pathogenesis is essential for successful drug discovery and repositioning. Figure 1
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describes the cellular pathways involved in the pathogenesis of eosinophilic and neutrophilic
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asthma endotypes.
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Figure 1: Pathophysiology of asthma: Depending on the trigger and cytokine milieu, pathogenesis of asthma may occur through an eosinophilic or neutrophilic pathway. Antigens are presented to dendritic cells (DC) for onward processing and subsequent presentation to the naïve T-helper cells (Th0). Depending on the cytokine milieu, the Th0 may differentiate into T-helper cells type 1 (Th1), Th17 or Th2. A Th independent pathway occurs through the ILC2 to induce eosinophilic asthma. Treg: T regulatory cells, MMP: matrix metalloproteinase, IL: interleukin, TGF-β: Transforming growth factor-beta., LT: leukotriene, MHC: major histocompatibility II, TSLP: Tissue stromal lymphopoietin, GM-CSF: Granulocyte macrophage-colony stimulating factor, PAF: Platelet-activating factor, ILC2: Innate lymphoid cells 2, MHC-II: Major histocompatibility complex II, GR0-α: Growth regulated protein alpha, Cyst-LTs: cysteinyl leukotrienes,15-HETE: 15-Hydroxyicosatetraenoic acid.
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3.
Current Asthma Therapies
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The overall aim of asthma management is to prevent or control acute and chronic symptoms
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(such as exacerbations, airway mucus accumulation, narrowing, squeezing and swelling of
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the airway smooth muscles), stabilize pulmonary function, improve asthma-related quality of
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life, and provide optimum therapeutic interventions with minimal adverse effects (EPR3,
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2007). Current anti-asthmatic drugs function as either bronchodilators or anti-inflammatory,
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or a combination of both. Alternatively, some of the medications are defined according to
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their targets and mode of action, e.g. the leukotriene modifiers, mast cell stabilizer and IgE
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inhibitors. Conventionally, asthma is managed by use of single or multiple agents of
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bronchodilators such as short-acting β2 agonists (e.g pirbuterol, salbutamol and levalbuterol),
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long-acting β2 agonists (e.g salmeterol and formeterol), anti-cholinergics (e.g tiotropium,
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oxitropium and ipratropium bromides), and inhaled corticosteroids (ICS) (Barnes, 2006;
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adverse effects, including sympathomimetic side effects (such as anxiety, tremor and
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tachycardia) ascribed to β2 agonists activity (Abramson et al., 2003; Henderson et al., 2005)
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while anti-cholinergics were associated with induction of xerostomia (Kato et al., 2006;
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Rodrigo and Castro-Rodriguez, 2005).
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Although, steroids are currently the most effective therapy for asthma, they were reported to
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cause a number of unwanted side effects such as glaucoma, cataract, skin bruise,
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telangiectasia, osteoporosis, sore throat, dysphonia (due to laryngeal oedema and muscular
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hypertrophy), hyperglycaemia, adrenal suppression and topical candidiasis (Reddel et al.,
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2015; Saag et al., 2011; Dahl, 2006; Israel et al., 2001). In addition to aforementioned
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adverse effects, a group of asthmatics are resistant to steroid intervention due to variety of
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factors such as alteration in glucocorticoid receptor expression, unstable glucocorticoid
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receptor activity and failure in nuclear translocation of glucocorticoid-receptor complexes.
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Inaccurate binding of complexes to glucocorticoid DNA response elements and faulty RNA
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splicing leading to aberrant cytokine expression or distorted extracellular matrix were also
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suggested to cause steroid resistance (Chung et al., 2014; Beck et al., 2009). The
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unavailability of better alternatives to steroid has made the management of steroid resistant
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asthma to be very challenging.
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Although, omalizumab, a 95% humanized IgE monoclonal antibodies used in moderate to
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severe persistent allergic asthma was proven to be effective in management of uncontrolled
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asthma (Cox, 2009), the antibody has the potential of eliciting anaphylaxis and
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atherothrombotic events such as myocardial infarction and stroke (Ali and Hartzema, 2012).
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Few other classes of drugs used in asthma therapy include the mast cell stabilizers (e.g.
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cromolyn sodium and nedocromil sodium) and leukotriene modifiers (e.g. montelukast,
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pranlukast, zafirlukast and zileuton) all of which are less effective as compared to steroid
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therapy (Fanta, 2009; del Giudice et al., 2009). Identification of safe substitute for current
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asthma medications through targeted therapy is paramount for improved asthma control,
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management and possible cure.
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4.
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Despite the unmet medical needs in prophylaxis and treatment of asthma, very few new
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classes of drugs were introduced and considered safe and effective over the past 40 years. In
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general, development of new drugs in the field of respiratory medicine is slow and scanty due
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Emerging Drug Targets
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preclinical findings into clinically relevant data (Barnes et al., 2015). The probability for a
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newly discovered respiratory drug to enter into the market phase of drug development is only
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3%, as against to 6–14% in other diseases such as HIV/AIDS, cardiovascular disorders,
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cancer, dermatological, haematological and neurological diseases (Mestre-Ferrandiz et al.,
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2012). Meaningful advancements recorded in the understanding of pathophysiology and
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factors associated with asthma development are critical for the successful design and
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development of new and more effective candidate drugs. Thus, targeting of critical cytokines,
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receptors and enzymes implicated in aetiology and progression of specific asthma endotype is
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critical to successful development of novel interventions.
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4.1. Target Receptors
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4.1.1. Toll-like Receptor Activators
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Toll like receptors (TLRs) are pattern recognition receptors (PRRs) capable of detecting and
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responding to microbial and endogenously derived signature molecules. Some TLRs are
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found expressed on surfaces of epithelium, mast cells, fibroblasts, monocytes, macrophages
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and dendritic cells while others are endosomal or phagosomal in nature. Induction of TLR
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signalling promotes Th1 and downregulates Th2 responses, thereby countering allergic
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asthma responses (Hennessy et al., 2010; Meng et al., 2011; Fonseca and Kline, 2009).
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Considering the percentages of asthmatics that are of allergic endotype (50% and 80% of
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asthmatic adults and children respectively), TLR targeting will be beneficial in reducing
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asthma burden (Knudsen et al., 2009).
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Toll like receptor subtypes TLR3, TLR7, TLR8, and TLR9 are strong inducers of Th1
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responses (Hennessy et al., 2010). TLR9 agonist QAX-935 (IMO-2134) was reported to
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decrease airway resistance and inflammation by activating several interferon-dependent genes
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involved in effective Th1 response (Panter, et al., 2009; Kline and Krieg, 2010). Furthermore,
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QbG10, a TLR9 agonist, improved overall asthma symptoms in patients with persistent
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allergic asthma following controlled steroid withdrawal (Casale et al., 2015; Lassen, et al.,
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2014; Beeh, et al., 2013). Similarly, synthetic TLR9 ligands [unmethylated cytosine-guanine
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oligodeoxynucleotides (CpG ODNs)] induced Th1 responses and counterbalanced Th2
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phenotype via Th1 cytokine surge (IFN-γ, IL-10 and IL-12) induced by CpG mediated
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activation of natural killer (NK) cells through dendritic cell stimulation (Gupta and Agrawal,
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2010). In a related development, CpG ODNs analogs (1018 ISS and ASM8) suppressed
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in allergic asthma (Senti et al., 2009; Campbell et al., 2014).
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A potent TLR8 agonist (vTX-1463) reportedly stimulated the production of Th1 polarizing
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cytokines (IFNγ and IL-12), decreased airway eosinophila and pulmonary congestion (figure
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2). Likewise, upregulation and activation of TLR7 markedly suppressed asthma symptoms
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and exacerbations through a Tregs dependent pathway (Hatchwell et al., 2015; Pham Van et
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al., 2011). Immunotherapeutic approach by the use of house dust mite (HDM) sublingual
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allergen immunotherapy (SLIT) tablet was recently reported to reduce the risk of moderate to
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severe asthma exacerbations among adults with HDM allergy–related asthma that respond
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poorly to ICS mono or combination therapy with no report of severe systemic adverse
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reaction (Virchow et al., 2016). This improvement could be credited to allergen
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desensitization and improved allergen tolerance due to shift in Th2 to Treg paradigm.
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Although
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immunotherapy (AIT) is yet to be fully elucidated, the ability of TLR to skew Th2-Th1
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balance towards Th1-treg profile suggests its likely involvement in AIT. Furthermore, unlike
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TLR-4, stimulation of TLR-7 and TLR-9 failed to break allergen-specific T-cell tolerance
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induced by AIT (Akdis and Akdis, 2015). Although, TLR mediated immunotherapy appear
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safe, the potential adverse effects of the intervention include autoimmunity, hypersensitivity,
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exaggerated immune reactions and risk of perpetuating vicious inflammatory cycles as
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obtainable in rheumatoid arthritis (Goh and Midwood, 2012).
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4.1.2. Chemoattractant receptor-homologous molecule expressed on Th2 cells
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(CRTH2)
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Chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) otherwise
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termed D prostanoid receptor 2 (DP2), is a G protein–coupled receptor that has a potential
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role in the pathogenesis of allergic disorders such as asthma. The receptor is highly expressed
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on Th2 cells, while its natural ligand [prostaglandin D2 (PGD2)] was found to be elevated in
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the bronchoalveolar lavage fluid (BALF) of asthmatics and causes a characteristic cough (Fajt
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et al., 2013; Stinson et al., 2015; Maher et al., 2015). Activation of CRTH2 enhances airway
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remodelling, goblet metaplasia, MUC5AC expression and cellular migration as revealed by
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transbronchial biopsy of severe asthma patients (Stinson et al., 2015). The resultant effects of
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CRTH2 activation were blocked by AZD6430 (a potent CRTH2 antagonist), suggesting
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CRTH2 inhibition as remedy to some asthma symptoms. AZD6430 decreased goblet
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allergen
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inhibition of PGD2 driven chemotaxic effects on eosinophils, mast cells and Th2
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lymphocytes (Stinson et al., 2015). Furthermore, downregulation of CRTH2 activity by
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OC0000459 suppressed airway hyperreactivity, improved forced expiratory volume in 1
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second (FEV1), reduced total IgE level, reversed sputum eosinophilia and decreased
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expression of proinflammatory genes in inflamed airways (Lukacs et al., 2008; Barnes et al.,
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2012; Pettipher et al., 2012). According to a randomised, double-blind, placebo-controlled,
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two-way crossover clinical trial study (NCT01056692), OC0000459 inhibited allergic airway
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inflammation by preventing PGD2 mediated airway and serum eosinophilia. In addition,
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improvement of FEV1 among asthma patients was also reported (Singh et al., 2012).
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Moreover, administration of oral CRTH2 antagonist BI 671800 to ICS-naïve patients and
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those on ICS improved asthma symptom by increasing FEV1 in both montelukast and ICS
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treated patients (Hall et al., 2015). Thus, CRTH2 inhibition is potentially suitable for patients
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whose asthma conditions are inadequately controlled by ICS monotherapy.
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4.1.3. Proteinase-activated receptor 2 (PAR-2)
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PAR2 is a G-protein coupled receptor (GPCR) commonly expressed in airway smooth
297
muscle, leukocytes and bronchial epithelial cells. It is usually activated by proteinases that
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arises from aeroallergens, invading pathogens, or by endogenously released proteinases such
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as trypsin, house dust mite (HDM) protein Derp9 and mast cell tryptase (Boitano et al.,
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2011). The attention given to inhibition of protease activity as potential drug target is
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currently on the rise (Alam, 2014; Harvima et al., 2014). Many triggers of asthma (e.g. house
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dust mite, fungal allergens and ovalbumin) exhibit proteinase activity (Corry and
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Kheradmand, 2009; Kauffman and van der Heide, 2003). The activation of PAR2 occurs by
304
cleavage of its extracellular N terminus to reveal its tethered ligand (SLIGRL-NH2/SLIGKV-
305
NH2) that auto-activates the receptor. Upon PAR2 activation, classical GPCR and β-arrestin
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signalling pathways are triggered. The latter is pro-inflammatory and occurs due to immense
307
activation of PAR2 while the former is bronchoprotective and occur due to mild activation of
308
PAR2 (Nichols et al., 2012).
309
Prior reports showed 38% reduction and 52% increase in airway hyper-reactivity following
310
methacholine challenge in asthmatic mice lacking PAR2 and those overexpressing PAR2
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respectively as compared to wild-type (Schmidlin et al., 2002). This implicated the
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involvement of PAR2 in pathogenesis of hyperresponsiveness in asthma. Activated PAR2
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mediators include the granulocyte macrophage- colony stimulating factors (GM-CSF),
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eotaxin and matrix metalloproteinase 9 (Vliagoftis et al., 2000). Bronchial smooth muscles
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(BSM) PAR2 stimulation increased the proliferation of BSM cells from asthmatic subjects
317
only, while BSM cells from non-asthmatic subjects showed no hyper-proliferative property
318
even after lentiviral over-expression of PAR2. Such increased proliferation could be
319
accounted for by increased basal level of PAR2 in asthmatics airway (Allard et al., 2014).
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Furthermore,
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tryptase inhibitor, inhibitors of tryptase (a PAR2 substrate) were found to prevent
322
bronchoconstriction in asthmatics (Sylvin et al., 2002; Hernández-Hernández et al., 2012). It
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could therefore be suggested that mast cells mediated induction of airway hyper
324
responsiveness is not solely dependent on released histamine but also the endpoint effect of
325
mast cell tryptases on PAR2.
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4.1.4. Calcium Sensing Receptor (CaSR) Antagonism
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Calcium-sensing receptor (CaSR) is a calcium-binding G protein-coupled receptor (GPCR),
328
that regulates systemic calcium homeostasis (Breitweiser, 2014). Airway smooth muscle cells
329
contain elevated [Ca2+]i levels while CaSR are upregulated and constitutively activated in
330
airways of asthmatics (Yarova et al., 2015). Increase in [Ca2+]i enhances bronchoconstriction
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that leads to AHR and induces chronic genomic effects that increases ASM cell proliferation
332
and extracellular matrix components depositions that additively results in airway remodelling
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(Koopmans et al., 2014; Mahn et al., 2010). Activation of CaSRs is therefore considered a
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major climactic event leading to asthma pathogenesis. Eosinophilic cationic proteins and
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major basic proteins released by infiltrating eosinophils are considered to primarily induce
336
airway narrowing in asthma (Pégorier et al., 2006), these polycations exert their effect
337
through activation of airway CaSRs which is normally involved in ASM homeostasis of
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divalent cations and polycations (figure 3). Blockade of this receptor by the use of calcilytics
339
(e.g calcityrol), were found to reduce AHR and airway inflammation in mouse asthma models
340
(Yarova et al., 2015). Although the concept of using calcilytics as novel asthma intervention
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it still at its initial stage, the intervention appears promising especially in asthmatics that are
342
non-sensitive to current asthma management strategies (Tanday, 2015).
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APC-366, Bis-(5-amidino-2-benzimidazolyl)-methane
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ACCEPTED MANUSCRIPT 4.1.5. H4 Receptor Antagonism
344
Plasma and airway histamine levels are heightened among asthmatics especially during
345
exacerbations. The biogenic amine mediates through four GPCR subtypes, the histamine H1-,
346
H2-, H3, and H4-receptors (H1-4R). H4R is considered a promising drug target for
347
inflammatory and allergic disorders due to its broad expression in hematopoietic cells (Seifert
348
et al., 2013; Brimblecombe et al., 2010; Zhang et al., 2007). The mediator is usually released
349
by mast cells and it functions by activating DCs, enhance Th2 polarization as well as airway
350
inflammatory cells homing (Reher et al., 2012; Caron et al., 2001). In vivo cellular
351
characterization of H4R in experimental allergic asthma models identified the role of the
352
receptor in the regulation of dendritic cell (DC) activation during sensitization and its role in
353
eosinophil activation during effector phase. This was further proved by the inability of
354
effector T cells to induce eosinophilic infiltration upon adoptive transfer of DCs devoid of
355
H4R at vitro sensitization reaction stage (Hartwig et al., 2015).
356
H4R selective antagonist, JNJ -7777120 H4R ((5-chloro-1H-indol-2-yl)-(4-methyl-piperazin-
357
1-yl)-methanone) ameliorated asthma symptoms in ovalbumin mouse asthma model.
358
Furthermore, H4R gene knock-out caused decreased allergic pulmonary inflammation,
359
prevented Th2 responses and bronchoconstriction, inhibited airway eosinophil and
360
lymphocyte infiltration (Neumann et al., 2013; Dunford et al., 2006; Somma et al., 2013).
361
The interesting results obtained from preclinical assessment of H4R antagonists prompted
362
further exploration of its clinical application in asthma. H4R selective antagonists showed
363
excellent safety profile following preclinical toxicity studies. Likewise, phase 1 clinical trial
364
has shown that selective H4R antagonists such as UR-63325, JNJ-39758979 [(R)-4-(3-amino-
365
pyrrolidin-1-yl)-6-isopropyl-pyrimidin-2-ylamine]
366
(cyclopropylmethyl)-6-(3-(methylamino)pyrrolidin-1-yl)pyrimidine-2,4-diamine] are safe for
367
use in humans. In addition, JNJ-39758979 dose dependently inhibited histamine induced
368
eosinophil morphological changes (Thurmond et al., 2014; Salcedo, 2013; Mowbray et al.,
369
2011). Unfortunate incidence of agranulocytosis observed in two out of eighty-eight Japanese
370
patients that enrolled for safety and efficacy study of JNJ-39758979 in moderate atopic
371
dermatitis led to the withdrawal of a phase 2 clinical trial of JNJ-39758979 in patients with
372
uncontrolled, persistent asthma prior to enrolment (Murata et al., 2015). This adverse effect
373
may be due to off target effect of the intervention or likely due to unascertained
374
pharmacogenomic variation among subjects. Nevertheless, the intervention was found to be
375
effective before the premature discontinuation of the trial.
and
PF-3893787
[(R)-N4-
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Figure 2: Toll like (TLR) and histamine 4 receptor (H4R) targeting in asthma treatment. TLR7, TLR8 and TLR9 engages their Toll–IL-1-resistence (TIR) domains to myeloid differentiation primary-response protein 88 (MYD88). This in turn stimulates downstream IL-1R-associated kinase 4 (IRAK4) and TNF receptor-associated factor 6 (TRAF6) thereby leading the activation of interferon-regulatory factor 7 (IRF-7), which induced type I interferon (IFN-α). IFN-α provide the cytokine milieu that enhances Th1 polarization by inducing IL-12 secretion from monocyte derived dendritic cells (mDC). Histamine 4 receptor (H4R) activation results in mast cell chemotaxis, downstream Ca2+ surge from endoplasmic reticulum (ER) and upregulation of markers such as malonyldialdehyde (MDA), 8-hydroxy-2’-deoxyguanosine (8OHdG) and transforming growth factor β (TGF-β). Alternatively, H4R activation may lead to decreased adenyl cyclase (AC) activity through Gi protein activation. This decreases intracellular cyclic adenosine monophosphate (cAMP) level, which in turnresults in protein kinase A (PKA) activation. Active PKA phosphorylates cAMP-responsive element-binding protein (CREB), which modulates gene transcription.
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ACCEPTED MANUSCRIPT 4.2.
Enzymatic Targets
391
4.2.1. Inhibitors of Rho-Kinases
392
Rho, a member of the Ras superfamily of guanosine triphosphatases (GTPases) and a
393
monomeric G protein associated with regulation of smooth muscle contraction, modulation of
394
myosin light chain phosphorylation and stress fibre formation (primarily actin and non-
395
muscle myosin II fibres). Rho also controls few other physiological events such as
396
cytokinesis, focal adhesion, cell migration, motility, migration and regulation of downstream
397
extracellular signals, such as lysophosphatidic acid (Taki et al., 2007). The contraction of
398
ASM is triggered by phosphorylation of the myosin light chain (MLC) by Ca2+ calmodulin
399
(CaM)-activated MLC kinase in the presence of intracellular calcium, thereby allowing actin-
400
myosin cross-linkage and subsequent shortening of sarcomeres. Whereas, ASM relaxation is
401
caused by MLC dephosphorylation by Rho kinase regulated myosin phosphatase (Chiba et
402
al., 2010; Schaafsma et al., 2006; Burdyga et al., 2003). Phosphorylation of myosin binding
403
subunit of myosin phosphatase by Rho kinases (ROCK) exerts inhibitory effect on MLC
404
dephosphorylation, thereby limiting smooth muscle relaxation (Chiba et al., 2010). Rho-
405
kinases are overexpressed in airway of asthmatics, therefore contributing to intermittence or
406
persistence AHR symptoms in asthma. The additive effect of Rho kinase upregulation results
407
in persistent MLC phosphorylation, contributing to asthma-related cellular remodelling
408
(Holgate, 2008).
409
Aside from ASM tone regulation via MLC phosphatase pathway, Rho kinases also play a
410
vital role in regulating inflammation related cellular infiltration and migrations via their
411
inherent spatiotemporal regulatory potential on the actin cytoskeleton (Biro et al., 2014). In
412
support of this, Rho-kinase inhibition (by use of Y-27632) blocks the cellular expression of
413
NF-κB, an essential transcription factor that contributes to asthma progression by activating
414
genes coding for inflammatory cytokines (Tong and Tergaonkar, 2014).
415
Fasudil (HA-1077), an isoquinoline-based compound is a first generation and the only
416
clinically available ROCK inhibitor that proved to be effective in management of pulmonary
417
arterial hypertension (PAH) and cerebral vasospasm through blockage of ATP dependent
418
domain of Rho-kinase and consequent smooth muscle relaxation (Raja, 2012; Velat et al.,
419
2011; Bain et al., 2007). Fasudil inhibits ROCK induced actomyosin dynamics by
420
competitively binding to the ribose region in the ATP-binding motif of ROCK (Nagumo et
421
al., 2000). It reportedly induced substantial decrease in cellular infiltration, lung
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ACCEPTED MANUSCRIPT inflammatory
index,
423
responsiveness, in addition to downregulation of IL-17, IL-13, and IL-4 in lungs of OVA-
424
challenged mice. Reduction in allergic airway inflammation and mucus hypersecretion by
425
fasudil-mediated inhibition of Rho kinase was reported to occur as a result of decreased
426
expression and phosphorylation of two transcription factors that were critically implicated in
427
asthma, namely; STAT6 and NFκB (Xie et al., 2015; Taki et al., 2007).
428
Another
429
cyclohexane
430
bronchodilation in guinea pigs while maintaining minimal cardiopulmonary side effects
431
(Righetti et al., 2014; Possa et al., 2012). Many studies have shown that administration of Y-
432
27632 by either inhalation or intranasal drops prevented and/or reduced airway
433
hyperresponsiveness and pulmonary eosinophilia in different animal models of asthma (Possa
434
et al., 2012; Schaafsma et al., 2008; Henry et al., 2005; Gosens et al., 2004). Recent reports
435
revealed that co-administration of Y-27632 with corticosteroids preferentially decreased
436
exhaled NO level, bronchial inflammation, airway oxidative stress, extracellular matrix
437
remodelling, airway collagen level as well as TIMP-1 positive cells, alveolar septa
438
eosinophilia, IL-2, 8-iso-PGF2α, IFN-γ and NF-κB activity in distal parenchyma, as
439
compared to corticosteroid or Rho-kinase inhibitor monotherapy (Pigati et al., 2015). Thus,
440
Rho-kinase inhibition (either as monotherapy or combination therapy) is a potential
441
therapeutic tool for asthma management and control.
442
4.2.2. Phosphodiesterase inhibitors
443
Elevation of phosphodiesterase (PDE) activity and alteration in cAMP-PDE pathway in
444
asthmatic airway smooth muscle cells is an indication that PDEs are involved in the
445
regulation of bronchial tone, airway hyperplasia and airway remodeling (Burgess et al., 2006;
446
Trian et al., 2011). PDE4 inhibitor roflumilast, decreased airway muscular contraction,
447
eosinophilia, neutrophilia, bronchial RSV infection and MUC5AC expression (Mata et al.,
448
2013; Gauvreau et al., 2011a). Another PDE4 inhibitor, 3-[4-(3-chlorophenyl)-1-ethyl-7-
449
methyl-2-oxo-1,2-dihydro-1,8-naphthyridin-3-yl] propanoic acid (ASP3258), was reported to
450
have ameliorated airway eosinophilia in a chronic murine asthma model (Kobayashi et al.,
451
2012). Repeated doses of roflumilast (a potent oral PDE4 inhibitor that is well known for its
452
anti-inflammatory property and treatment of chronic obstructive pulmonary disorder)
453
decreased sputum eosinophilia, FeNO, leukotriene (LTE4), improved FEV1 and attenuated
454
exercise-induced asthma (Bardin et al., 2015). Although, roflumilast is considered well
selective
goblet
cell
Rho-kinase
inhibitor,
(Y-27632),
MUC5AC
expression,
and
airway
(+)-(R)-trans–4-(1-aminoethyl)-N-(4-pyridyl)
a
pyridine
derivative,
reportedly
induced
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ACCEPTED MANUSCRIPT tolerated by most asthmatics, few unwanted adverse effects majority of which are related to
456
gastrointestinal disturbances (such as nausea and diarrhoea), insomnia and headache were
457
reported (Bardin et al., 2015; Parikh and Chakraborti, 2016; Chervinsky et al., 2015; Fabbri
458
et al., 2009).
459
In addition to induction of rapid and sustained bronchodilation among asthmatics, reduction
460
in sputum cell titre was recorded in lipopolysaccharide challenged healthy volunteers that
461
were given RPL554, a novel inhalable PDE3/4 inhibitor induced (Franciosi et al., 2013).
462
Unlike most other PDE inhibitors, the safety profile of RPL554 was within the tolerable range
463
and did not produce GI effects often associated with most PDE inhibitors (Franciosi et al.,
464
2013).
465
Xanthines were formally used in asthma treatment, however, considering their non-selectivity
466
in PDE inhibition, narrow therapeutic window, severe side effects (such as cardiac
467
arrhythmias, seizures, nausea, vomiting, and headaches) and following the discovery of better
468
bronchodilators (such as β2 agonists and muscarinic receptor antagonists), the use of
469
xanthines
470
Theophylline (dimethylxanthine) was commonly used as bronchodilator when administered at
471
relatively high dose. However, recent advances have shown that it possesses anti-
472
inflammatory effects in asthma when administered at lower doses (Barnes, 2013).
473
Theophylline molecularly induces bronchodilation by inhibition of PDE3, while it exerts its
474
anti-inflammatory effect by PDE4 inhibition and concomitant upregulation of histone
475
deacetylase-2 (HDAC-2), allowing deactivation of asthma activated inflammatory genes
476
(Barnes, 2013). HDAC-2 is critically lowered in steroid resistant severe asthmatics. The
477
upregulation of HDAC2 by theophylline reverses corticosteroid resistance in steroid
478
insensitive asthma endotype (To et al., 2010). Theophylline mediates HDAC-2 increase is
479
achievable through inhibition of phosphoinositide-3-kinase-δ (PI3Kδ) which is activated by
480
oxidative stress (To et al., 2010; Tobinick, 2009). The co-administration of theophylline with
481
salmeterol/fluticasone propionate combination to asthma patients significantly reduced
482
frequency and number of patients with asthma exacerbations, improved the forced expiratory
483
flow, decreased sputim eosinophil cationic protein concentration and generally improved
484
airway inflammation (Nie et al., 2013). Similarly, an improvement in asthma symptoms was
485
reported in asthmatic smokers that are nonresponsive to ICS following low theophylline dose
486
(Spears et al., 2009). These therefore indicate the antiasthmatic potential of theophylline,
487
especially in control of steroid resistant endotype.
asthma
therapy
gradually
faded
out
(Page,
2014).
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ACCEPTED MANUSCRIPT 4.2.3. iNOS and Arginase Inhibition
489
The enzymatic breakdown of the guanidine group of L-arginine to release of NO and L-
490
citrulline results in generation of nitric oxide, a critical marker of airway inflammation
491
(Verini et al., 2010). This process is catalysed by nitric oxide synthase (NOS). Among the
492
three basic types of NOS, inducible nitric oxide synthase (iNOS) is overexpressed
493
inflammatory conditions. Conversely, NO from iNOS influences localized vasodilatation,
494
plasma extravasation, mucus hypersecretion, promotes eosinophilic homing and consequent
495
activation of Th2 cells, thereby aggravating airway inflammation (Prado et al., 2011; Reis et
496
al., 2012; Souza et al., 2013). Virtually, all cells in the respiratory tract of asthmatics exhibits
497
intensified expression of iNOS, which is correlated with perpetuating and amplified airway
498
inflammation in asthma (Prado et al., 2014). Potent partial and full inhibitors of iNOS
499
effectively decreased the FeNO among asthmatics (Singh et al., 2007). Nevertheless,
500
GW274150, a selective iNOS inhibitor did not effectively ameliorate airway reactivity and
501
inflammation (Singh et al., 2007). The non-desired response observed in upper airway may
502
be due to the paradoxically opposing functions of NO in upper and lower airway. L-NG-
503
Nitroarginine Methyl Ester (L-NAME), a commercially available precursor of NOS inhibitor,
504
was found to increase airway resistance, compliance and elastance, while it decreased both
505
properties (resistance, elastance and compliance) in lung parenchyma following its bio-
506
activation to L-NG-Nitroarginine; NG-nitro-L-Arginine (L-NNA), a non-specific NOS
507
inhibitor. This provide support to the fact that NO is a potent bronchodilator of proximal
508
airways (by acting through the NANC pathways) and as constrictor of distal pulmonary
509
parenchyma. Acute L-NAME therapy was also found to decreased airway eosinophilia (Prado
510
et al., 2005). Alternative treatment with specific iNOS inhibitor (e.g. 1400-W) provided better
511
control of hyperresponsiveness and airway inflammation in the same murine model used
512
(Starling et al., 2009; Marques et al., 2012; Souza et al., 2013).
513
Apart from the NO metabolite of L-arginine produced through the NOS pathway, arginase
514
catabolic cascade appears to play role in development and progression of asthma (Vonk et al.,
515
2010). However unlike NOS, the activity of arginases does not lead to the production of NO,
516
it catabolizes L-arginine to L-ornithine and urea (Ricciardolo et al., 2004). The L-ornithine
517
(product of arginase activity) is further catabolized into polyamines by ornithine
518
decarboxylase. These L-ornithine–derived polyamines are the prime agents through which
519
arginases contribute to AHR. They promote collagen synthesis and cellular proliferation
520
thereby contributing to asthma related airway remodelling (Prado et al., 2014). Moreover,
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ACCEPTED MANUSCRIPT plasma polyamine surge was reported in asthmatics (North et al., 2013). Up-regulation of
522
arginase further complicates AHR by competitively upregulating pro-inflammatory
523
peroxynitrite release due to iNOS activity (Maarsingh et al., 2009). Studies have shown high
524
airway activity and expression of arginases (ARG1 and ARG2) following allergen challenge
525
in mouse asthma models (Vonk et al., 2010). 2(S)-amino-6-boronohexanoic acid, a selective
526
arginase inhibitor, was reported to reduce bronchial hypersensitivity to allergens, prevents
527
airway obstruction and reversed airway inflammation in allergen challenged guinea pigs
528
(Maarsingh et al., 2008). Additionally, S-(2-boronoethyl)-L-cysteine, another potent arginase
529
inhibitor, was reported to have decreased AHR in murine models of allergic asthma (North et
530
al., 2009). Likewise, AHR was reversed in murine model of IL-13-induced AHR by RNA
531
interference intervention against ARG1 (Yang et al., 2006). Thus, the application of specific
532
ARG inhibitors may supposedly be a promising intervention for successful amelioration of
533
asthma symptoms (figure 3).
535 536 537 538 539 540 541 542
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Figure 3: Control of airway remodelling. Ca2+ signalling through calcium sensing receptor (CaSR) and Rho kinase pathways appear critical for asthma control. Inhibition of CaSR by calcilytics reverses bronchoconstriction by deactivating downstream signalling pathways such as the MAPK and the Rho/Rac/Cdc42 pathways. Direct inhibition of Rho kinases permits the dephosphorylation of myosin light chain (MLC) by active myosin phosphatase thereby inducing smooth muscle relaxation. Polyamine and proline induced bronchoconstriction and goblet metaplasia can be averted by inhibiting arginase activity. ODC: ornithine decarboxylase, OAT: Ornithine acetyltransferase, NOS: Nitric oxide synthase.
543 18
ACCEPTED MANUSCRIPT 4.3.
Biologics
545
Mast cells, bronchial epithelium and macrophages are important sources of cytokines,
546
chemokines, and a number of chemical mediators involved in asthma progression (Desai and
547
Brightling, 2009). Targeting individual cytokines that play dominant roles in the
548
pathophysiology of asthma may provide better biologics for prophylaxis and/or treatment of
549
asthma. So far, some few antibodies targeting cytokines are at developmental stages (see table
550
1).
551
Targeting of cytokine effect/activity can be achieved directly by cytokine targeting or
552
indirectly through cytokine receptor modulation. IL-13 is central to development of AHR in
553
asthma and its effect is mediated through its binding to low affinity IL-13 receptor alpha1
554
(IL-13Rα1) and IL-4 receptor alpha (IL-4Rα) complexes (IL-13Rα1/ IL-4Rα complex).
555
While IL-4 also exerts its effect through IL-4Rα. Dupilumab, a human monoclonal antibody
556
against IL-4α/IL-13α receptor complex, inhibited the downstream signalling events induced
557
by IL-4 and IL-13 by binding to the alpha subunit of the IL-4 receptor of the complexes
558
(Hambly and Nair, 2014; Vatrella et al., 2013; Wenzel et al., 2013b; Prado et al., 2014). Add
559
on therapy of dupilumab/ICS/LABA combination was recently reported to effectively
560
improve FEV1, patient’s quality of life and decreased recurrent asthma exacerbations in
561
patients with uncontrolled persistent asthma regardless of baseline blood eosinophil level.
562
Additionally, the antibody has the tendency of ameliorating comorbidities associated with
563
uncontrolled persistent asthma (e.g nasal polyps and acute dermatitis) (Wenzel et al., 2016).
564
Likewise, pitrakinra, a dual IL-4/IL-13 antagonist was evaluated for potential asthma therapy
565
and it was found to dampen the progression of early and late asthmatic response in allergen
566
challenge models (Antoniu, 2010; Wenzel et al., 2007). Pitrakinra competitively inhibited IL-
567
4Rα activity to impede IL-4 and IL-13 activities. It also significantly improved asthma
568
symptoms and decreased exacerbations in patients with airway eosinophilia, nevertheless,
569
phase IIb trial failed to demonstrate clinical efficacy of the intervention in the overall study
570
population (Hambly and Nair, 2014).
571
Effort to target IL-13 alone using lebrikizumab (a humanized anti-IL-13 antibody) suggests
572
asthma endotype specificity in IL-13 dependent asthma treatment. Significant improvement in
573
lung function was reported following treatment with lebrikizumab in patients with high serum
574
periostin (an indicator of eosinophilia in asthmatics) and high fractional exhaled nitric oxide
575
(FeNO) levels (Corren, et al., 2011; Jia et al.,2012; Noonan et al., 2013). Serum periostin
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ACCEPTED MANUSCRIPT level can be used as a critical determinant of lebrikizumab efficacy. Recent randomised,
577
double-blind, placebo-controlled trial revealed reduction in asthma exacerbations rate and
578
improvement in FEV1 among periostin-high patients with moderate-to-severe asthma than in
579
the periostin-low patients following 24 weeks of treatment with different doses of
580
lebrikizumab (37.5, 125, 250 mg every four weeks) (Hanania et al., 2015). Tralokinumab
581
administration dose dependently improved lung function (as measured by FEV1) of patients
582
with moderate-severe asthma. Safety and toxicity studies revealed no serious adverse effect in
583
its phase II trial (Piper et al., 2013; Hanania et al., 2015).
584
An IL-13Rα1 isomer, IL-13 receptor alpha2 (IL-13Rα2) possesses high affinity to IL-13 as
585
compared to IL-13R α1. However, it was proposed to perform a non-signalling decoy role in
586
IL-13 functionality. Upregulation of its soluble isomer (sIL-13Rα2) reportedly inhibited IL-
587
13 induced airway inflammation in murine asthma models (Daines et al., 2006; Chen et al.,
588
2013). Conversely, mouse has both its soluble (sIL-13Rα2) and membrane bound (memIL-
589
13Rα2) forms of IL-13Rα2, humans only have the memIL-13Rα2 form. This may contribute
590
to failure in translation of preclinical trials involving IL-13Rα2 immunomodulation for
591
control of human asthma (Chen et al., 2013).
592
Considering the role of IL-5 in airway eosinophil homing, as well stimulation of mediator
593
release from eosinophil and its effect on tissue survival (Garcia et al., 2013), monoclonal
594
antibodies against interleukin 5 (anti-IL-5) were tried for use in asthma. Anti-IL-5
595
monoclonal antibodies such as mepolizumab and reslizumab revealed beneficial effect in
596
management of persistent airways eosinophilia among corticosteroid resistant subjects. In a
597
randomized, double blind and double dummy trial, reduction in rate of asthma exacerbations
598
and sputum eosinophilia was recorded upon repeated administration of mepolizumab to
599
insensitive asthma patients (Ortega et al., 2014).
600
mepolizumab is an exciting advancement especially for patients with uncontrolled severe
601
eosinophilic asthma because it is safe and effective option that could replace oral
602
corticosteroids (Bel et al 2014; Haldar et al., 2009; Nair et al., 2009; Pavord et al., 2012).
603
According to recent trials on reslizumab, the antibody exhibited tolerable safety profile and
604
improved FEV1, forced vital capacity (FVC), forced expiratory flow (FEF), asthma control,
605
patient’s quality of life and rescued SABA use in uncontrolled asthma with eosinophilic
606
endotype (Corren et al., 2016; Markham, 2016; Castro et al., 2015). Equally, benralizumab
607
(MEDI-563), an afucosylated monoclonal antibody against IL-5Rα, was reported to reduce
608
peripheral eosinophils levels in moderate asthma patients and sustained its effect for about 8–
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The corticosteroid sparing effect of
ACCEPTED MANUSCRIPT 12 weeks through antibody dependent cell-mediated cytotoxicity (Ghazi et al., 2012; Busse et
610
al., 2010; Kolbeck, et al., 2010).
611
Th17 lymphocytes have demonstrated the ability to recruit both eosinophils and neutrophils
612
to the airway through IL-17A and IL-17F release (Tesmer et al., 2008). Although IL-17
613
targeting using secukinumab, and ixekizumab seems promising, there are few clinical trial
614
reports on the use of the antibodies in asthma. However, brodalumab, a monoclonal antibody
615
that targets IL-17RA, produced clinically meaningful responses in moderate to severe
616
asthmatic patients with high bronchodilator reversibility (Busse et al., 2013). Considering the
617
equivocal nature of available data on use of brodalumab in asthma control (Bauer et al.,
618
2015), further studies on its effect on different asthma sub-populations is highly
619
recommended.
620
Tumor necrotic factor-alpha (TNF-α) is mainly secreted by lymphocytes, mast cells, and
621
macrophages. It promotes bronchial hyperresponsiveness and sputum neutrophilia, this make
622
it an attractive target for severe asthma (Wenzel et al., 2009). A number of anti-TNF-α agents
623
exist in the market and they were proven effective in some inflammatory diseases such as
624
rheumatoid arthritis and Crohn disease. It is apparent that the use of inhibitors of TNF-α may
625
be valuable in reversal of asthma symptoms. Etanercept (anti TNF-α agent) showed short-
626
term and modest efficacy for severe and mild asthma respectively (Antoniu, 2009). Slight
627
reduction in number of exacerbations and improvement in wheezing was observed in patients
628
with moderate uncontrolled asthma originally on ICS monotherapy following infliximab or
629
adalimumab administration (Erin et al., 2006; Stoll et al., 2009). Upon withdrawal of TNF-α
630
inhibition, wheezing triumphed (Stoll et al., 2009). Safety concerns may limit the use of anti-
631
TNF-α in asthma therapy. Substantial adverse reactions were observed without achieving
632
fundamental objectives of reducing asthma exacerbations and improving FEV1 among
633
subjects (see table 2). Golimumab lowered risk of asthma exacerbations, nevertheless, its trial
634
on severe persistent asthma ended at phase II due to incidences of malignancies and
635
infections such as pneumonia (Wenzel et al., 2009). Given the role of TNF- α in severe,
636
refractory, or steroid-resistant asthma, future studies on the use of this anti-TNF- are needed
637
to identify whether the long-term risk-benefit profile favours use in asthma.
638
The results of trials involving agents targeting IL-4, IL-13, IL-9, IL-12, IL-10, interferon-γ,
639
granulocyte-macrophage colony-stimulating factor, and even Th17 cells are anticipated
640
(Hansbro et al., 2011; Desai and Brightling, 2009). As understanding of the cytokine
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ACCEPTED MANUSCRIPT 641
networks continues to evolve, so too will the potential targets for antiasthma therapy.
642
4.4.
643
Airway inflammation in asthma is associated with several transcription factors such as the
644
NFkB, nuclear factor of activated T-cells (NF-AT) and glucocorticoid receptor (GR). Cyclic
645
AMP response element binding protein (CREB), activator protein-1 (AP-1), peroxisome
646
proliferator-activated
647
CCAAT/enhancer binding protein (C/EBP), signal transducer and activator of transcription 6
648
(STAT6), GATA-3, doublesex and mab-3 related transcription factor 1 (DMRT1) and nuclear
649
factor erythroid 2-related factor 2 (Nrf2) were also implicated in pathogenesis of asthma
650
(Roth and Black, 2006). Although, most transcription factors play important role in tissue and
651
organ homeostasis, modulation of some of the transcription factors (especially GATA-3,
652
NFkB, Nrf2, NF-AT and STAT6) appeared to be a valid therapeutic approach to control of
653
asthma symptoms. Control of lung inflammatory responses is achievable by application of
654
decoy or antisense oligonucleotides specific for targeted airway transcription factor of
655
interest. Nevertheless, long term suppression or overexpression of these transcription factors
656
could lead to wide array of adverse effects, thus, cell type specific delivery of inhibitors or
657
activators of target factors is critical to obtaining minimal adverse effect that may arise from
658
this approach (Roth and Black, 2006; Haley et al., 2011; Schieck et al., 2016)
659
The characteristic Th2 molecular endotype of allergic asthma is controlled by a zinc finger
660
transcription factor GATA3, which is essential for Th2-cell differentiation and immune
661
pathway activation (Wenzel, 2012). GATA-3 is reportedly overexpressed in broncholaveolar
662
lavage and lung biopsies from severe asthma patients (Bergqvist et al., 2013). Thus,
663
interventions that target GATA3 and its downstream effectors were suggested for treatment
664
of asthma (Holgate, 2012). Afzelin, a flavonol glycoside, was reported to suppress airway
665
inflammation, eosinophil infiltration, airway hyper-responsiveness and BALF Th2 cytokines
666
in murine asthma model by inhibition of GATA-3 activity and increasing T-bet/GATA-3
667
ratio. Considering the role of T-bet transcription factor in regulation of Th1 differentiation
668
and activation, the increase in T-bet/GATA-3 ratio supposedly restored Th1/Th2 balance
669
(Jenner et al., 2009; Zhou and Nie, 2015). In another preclinical study, GATA-3 specific
670
catalytically active, single stranded, synthetic antisense DNA enzyme (DNAzyme) hgd40
671
containing SB010 (active drug component) was tested for efficacy in reduction of Th2
672
cytokine level via inhibition of GATA-3 mRNA and protein level (Turowska et al., 2013; Sel
(PPAR),
runt-related
transcription
factors (RUNX),
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ACCEPTED MANUSCRIPT et al., 2008). The results of the preclinical tests showed remarkable improvement in asthma
674
symptoms with no observable off-target effect therefore allowing for further clinical trial of
675
the DNAzyme (Homburg et al., 2015; Fuhst et al., 2013). In a randomized, double-blind,
676
placebo-controlled, multicenter clinical trial, 10mg of aerosolized SB010 or placebo was
677
administered to asthma patients once daily for 28 days, and a significant improvement in lung
678
function was reported. Furthermore, the intervention attenuated allergen-induced sputum
679
eosinophilia, reduced sputum tryptase and negated plasma IL-5 levels (Krug et al., 2015).
680
GATA-3 activity is linked to few other asthma related transcription factor signalling such as
681
the JAK/STAT6 signalling pathway. Boswellic acid, a specific and non-redox inhibitor of 5-
682
lipoxygenase enzyme, was revealed to suppress allergic airway inflammation by indirectly
683
abrogating GATA3 expression through inhibition of IL-4 dependent pSTAT6 activity (Liu et
684
al, 2015). Furthermore, in an effort to unveil the determinants of asthma development in
685
atopic subjects with or without asthma and identify asthma associated drug targets in HDM-
686
specific T-helper memory responses, a differential gene network analysis revealed that most
687
asthma-associated genes were enriched with targets of STAT6 signaling (Troy et al., 2016).
688
AS1517499 and YM-341619 are small-molecule inhibitors of STAT6 activity, they were
689
found to modulate asthma symptoms by selectively inhibiting Th2 differentiation (Vale,
690
2016; Nagashima et al., 2009). In another development, Ursolic acid reportedly decreased
691
airway eosinophilia, antigen-induced Penh, pulmonary inflammation, pro-inflammatory
692
cytokine production as well as serum antigen-specific IgE by increasing PPARγ expression
693
and decreasing GATA-3/STAT6 expression through NF-kB pathway modulation (Kim et al.,
694
2013). After all, previous studies revealed that ursolic acid suppresses NF-kB activation by
695
inhibition of IκBα kinase and p65 phosphorylation (Shishodia et al., 2003 and Zeng et al.,
696
2012). More recently, integromics studies revealed GRB2-associated binding protein 1
697
(GAB1) as a novel modulator of asthma due to its NF-kB regulatory activity (Sharma et al.,
698
2015).
699
Control of STAT6 activity can be achieved by JAK modulation. Preclinical studies have
700
validated the suitability of JAKs as a therapeutic target for asthma. A pan-JAK inhibitor,
701
Pyridone 6 (P6), suppressed asthmatic symptoms through Th2 inhibition (Matsunaga et al.,
702
2011). Tofacitinib (CP-690,550), a STAT-6 and pan-JAK inhibitor was suggested to reduce
703
eosinophilia, eotaxins and IL-13 by inhibition of IL-4 signaling pathway in murine model of
704
pulmonary eosinophilia (Kudlacz et al., 2008). However, it is established that tofacitinib
705
enhances pulmonary bacterial growth in models of latent tuberculosis infection, thereby
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23
ACCEPTED MANUSCRIPT posing the risk of reactivation of latent form of tuberculosis in asthma patients with
707
comorbidities (Maiga et al., 2012). It is for this reason that development of safer selective
708
JAK inhibitors was proposed. R256, an inhalable selective JAK/1/3 inhibitor proved to be a
709
safer option over inhibitors of pan-JAKs, due to their selective inhibition of Th2
710
differentiation without affecting Th1 and/or Th17 differentiation (Vale, 2016; Ashino et al.,
711
2014). Figure 4 illustrates the targeted JAK-STAT pathway inhibition as it relates to GATA3
712
and CRTh2 signalling pathways in asthma.
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713 714 715 716 717 718 719
Figure 4: Th2 surface receptor directed therapy. Prevention of airway allergic inflammatory cell migration is achievable through modulation of key surface receptors and downstream targets such as intracellular receptors, enzymes and transcription factors. Interaction of Th2 cytokine such as IL-4 with its receptors (IL-4R) results in activation of the JAK/STAT6 pathway that leads to activation and production of GATA3 protein. Prostaglandin D2 (PGD2) released from mast cells interact with CRTh2 to induce inhibition of adenylyl cyclase (AC) and consequent decrease in intracellular cyclic adenosine monophosphate (cAMP) levels through Gi
24
ACCEPTED MANUSCRIPT 720 721 722 723 724 725 726 727
protein activation. This also results in release of βɣ subunit of the G protein, which directly stimulates phospholipase Cβ (PLCβ) isoforms. PLBβ breakdown phosphatidylinositol-4,5-bisphosphate (PIP2) to produce inositol triphosphate (IP3) which mobilizes Ca2+ from endoplasmic-reticulum stores. The Ca2+ activates calcineurin, which then dephosphorylate NFAT to its active state (NFATc). Transcriptional activation of both GATA3 and NFAT exert a net effect of increased Th2 cytokine production, thereby providing the cytokine milieu that is required for aggravated allergic asthma response in a vicious manner. Inhibition of IL-4R, CRTh2, JAK, STAT-6, GATA3, and cytokine inhibition showed remarkable reduction in chemotaxis, goblet metaplasia, Th1/Th2 imbalance, bronchoconstriction and hyperresponsiveness.
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5.
Conclusion
730
Acute asthma exacerbations are arbitrary, unsettling and detrimental to patient’s quality of
731
life. Prevention of exacerbations through adherence to controller drug regimens, avoidance of
732
exposure to known triggers, annual immunization against influenza and pneumococcal
733
polysaccharide (for asthmatics aged 19 years or older) are imperative. This review has
734
highlighted some potential drug targets for the treatment of asthma. However, few more
735
potential candidates for asthma therapy including calcium sensing receptor (CaSR)
736
antagonist, phosphatidylinositol 3-kinase (PI3K) inhibition through direct inhibitors or
737
indirect inhibitors (e.g. theophylline should also be considered. Asthma symptoms are
738
infrequent, short-lived and mild, occasional administration of a quick-acting bronchodilator to
739
reverse bronchoconstriction is an acceptable approach. However, as symptoms become more
740
frequent or more severe, the emphasis changes to prevention of symptoms (and of asthmatic
741
attacks). Inhaled corticosteroid reduces the frequency of episodic bronchoconstriction and
742
lessens the risk of asthmatic attacks through its anti-inflammatory activities. In low-to-
743
moderate doses, corticosteroids administered by inhalation are safe for long-term use, even in
744
young children.
745
Whilst considerable advancement in the understanding pathophysiology of asthma promoted
746
the discovery of novel therapeutic targets, only few are successfully translated into clinical
747
application. The heterogeneous nature of asthma calls for multifaceted approach to its
748
treatment, thus, further investigations into potential therapies are paramount. On a general
749
note, the pleitrophic nature of kinases and their ability to modulate wide array of cellular
750
events increases risk of adverse effects (e.g haematological disorders and cardiovascular
751
complications) as observed in various protein kinase inhibitors currently used for
752
management of cancer. It is therefore pertinent to apply caution while considering kinase
753
inhibitors for asthma therapy. This may include ensuring minimal side effects by
754
administering low doses of inhalable preparations.
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ACCEPTED MANUSCRIPT Most of the candidate drugs discussed in this article have shown potential to fit into stepwise
756
asthma management strategy taking into account the patient’s response to available
757
medication, onset of the disorder (childhood or adult onset asthma), effectiveness and safety
758
of the candidate drug with respect to patient’s asthma endotytpe. Administration of TLR
759
agonist is suitable during ICS step down treatment in patients on medium to high dose ICS
760
whose asthma has been successfully controlled. this is because TLR agonist may contribute to
761
sustaining asthma control during such steroid reduction regime, thereby preventing sudden
762
relapse. The use of CRTh2 antagonist may best be indicated at stages where patients respond
763
poorly to ICS monotherapy (step 2 to 4 of GINA treatment guidelines) and in ICS naïve
764
asthmatics as add on therapy to reliever medications. Additionally, CRTh2 targeted therapies
765
may be more effective in Th2-high asthma subphenotype. The combined bronchodilatory and
766
anti-inflammatory potential of dual PDE3/4 inhibitors may enhance its use as LABA
767
replacement or as add-on therapy to reduced dose of ICS/LABAs controller medication,
768
thereby complementing safety concerns related to long term use of LABAs. While most
769
biologics based interventions will best be administered to patients with uncontrolled
770
persistent asthma that failed to respond to standard therapy. This may include the
771
administration of such biologics as add on therapy to ICS or oral corticosteroids (OCS) to
772
relieve severe recurrent asthma exacerbations
773
Acknowledgment
774
This work was supported by National Key Economic Area Research Grant Scheme
775
(NRGS/NH1014D026), provided by the Ministry of Agriculture, Malaysia.
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ACCEPTED MANUSCRIPT
Table 1: Summary of emerging molecular targets for the treatment of asthma and developmental stage
Drug name
Highest Development phase
Receptor Modulators
CRTH2 antagonist
OC000459
2
CRTH2 antagonist
BI-671800
2
CRTH2 antagonist
ARRY-502
CCR3 receptor blocker
TPI ASM8
TLR-3 antagonists
CNTO 3157
urotensin II receptor antagonist
GSK1440115
Oxagen Ltd
Barnes et al., 2012 NCT01057927 Hall et al., 2015 NCT01103349
2
Array BioPharma Inc.
Wenzel et al., 2014 NCT01561690
2
Pharmaxis,
Gauvreau et al., 2008; Gauvreau et al., 2011b NCT00264966/ NCT01158898
1
Janssen Research and Development, LLC
NCT01704040
1
GlaxoSmithKline
Portnoy et al., 2013 NCT01202214
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Reference/NCT number
Boehringer Ingelheim
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Sponsor
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Mechanism of action
SC
Therapeutic Class
27
ACCEPTED MANUSCRIPT
Mechanism of action
Drug name
Highest Development phase
Enzymatic Targets
PI3K/mTOR inhibitor
GSK2269557
1
PDE4 inhibitor
GSK256066
2
PDE4 inhibitor
CHF 6001 (inhaled)
2
PDE4 inhibitor
roflumilast
PDE4/3 inhibitor
RPL 554
Leukotriene A4 hydrolase inhibitor
JNJ-40929837
IL-5Rα mAb
IL-5 mAb IL-5 mAb
Reference/NCT number
Stark et al., 2015 NCT02294734
GlaxoSmithKline,
Singh et al., 2010 NCT00380354
Chiesi Farmaceutici S.p.A.
Matera et al., 2014 NCT01689571
2
Takeda; Nycomed and Pfizer
NCT01765192/ NCT01365533
2
Verona Pharma
NCT02427165
2
Johnson and Johnson Pharmaceutical Research and Development, L.L.C.
Barchuk et al., 2014 (NCT01241422)
Benralizumab
2/3
AstraZeneca
Nowak et al., 2015 NCT01914757
Reslizumab
3
Teva Pharmaceutical Industries
Castro et al., 2015 NCT01287039/ NCT01285323
Mepolizumab
3
GlaxoSmithKline
Ortega et al., 2014 NCT01691521
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SC
GlaxoSmithKline
AC C
Anti-interleukin agents
Sponsor
RI PT
Therapeutic Class
28
ACCEPTED MANUSCRIPT
Highest Development phase
IL-13 mAb
Lebrikizumab
2
IL-13 mAb
Tralokinumab
3
IL-4Rα mAb
Dupilumab
2
IL-17RA
Brodalumab
1
29
Reference/NCT number
Genentech
Hanania et al., 2015 NCT01545440/ NCT01545453
AstraZeneca
Brightling et al., 2015 NCT02281357
Sanofi and Regeneron Pharmaceuticals Inc.
Wang et al., 2015 NCT01854047
Amgen Kyowa Hakko Kirin Company Limited and AstraZeneca
NCT01902290
Sterna Biologicals GmbH and Co. KG
Homburg et al., 2015 NCT01743768
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SB010
2 (trial terminated during Phase 2)
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GATA-3 Transcription DNAzyme Factor NCT: National Clinical Trial
Sponsor
RI PT
Drug name
SC
Mechanism of action
AC C
Therapeutic Class
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Table 2: Summary of adverse effects of selected anti-asthma candidate drugs as revealed by clinical trials
Mechanism of action
Route of administration
Adverse effects
Reference/Trial number
CYT003
TLR-9 Agonist
Subcutaneous
Injection site reactions, Pyrexia Nasopharyngitis, Head ache.
Casale et al., 2015, NCT01673672
QbG10
TLR-9 Agonist
Subcutaneous
BI 671800
CRTH2 antagonist
Oral
H4R antagonist
SC M AN U
Oral
Oral
Toxic hepatitis in patients with prior elevated hepatic transaminases♯
Hall et al., 2015, NCT01092148 and NCT01103349
Headache♯
Sidharta et al., 2014, EudraCT number 2006-006777-25 Diamant et al., 2014, EudraCT number 2008-001209-41
Drug-induced agranulocytosis*
Murata et al., 2015, NCT01493882, NCT01497119
Headache♯, nausea, diarrhoea, asthenia and dyspepsia
Bateman et al., 2016, NCT01765192. Chervinsky et al., 2015, NCT00073177, NCT00076076, NCT00163527 Gauvreau et al., 2011a, NCT01365533
♯
Roflumilast
PDE-4 inhibitor
AC C
EP
JNJ 39758979
CRTH2 antagonist
Beeh, et al., 2013, NCT00890734
Injection site reaction
TE D
Setipiprant,
RI PT
Candidate drug
Oral
30
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Mechanism of action
Route of administration
Adverse effects
RI PT
Candidate drug
Reference/Trial number Franciosi et al., 2013, EudraCT, numbers 2008005048-17, 2011-001698-22, 2010-023573-18, and 2012000742-34.
Dual PDE-3 and PDE-4 inhibitor
Inhalation
Headache♯, Palpitations, Paraesthesia. Rhinorrhoea, Somnolence
Dupilumab
IL-4Rα antagonist
Subcutaneous
Upper respiratory tract infection, Headache, Injection-site erythema, Sinusitis, Influenza
Wenzel et al., 2016, NCT01854047, EudraCT number 2013-000856-16
Lebrikizumab
IL-13 inhibitor
Subcutaneous
Injection site reaction, Injection-site erythema, Upper respiratory tract infection
Hanania et al., 2015, NCT01545440, NCT01545453
Headache, Injection-site reaction, nasopharyngitis, cough, arthralgia
Pouliquen et al., 2015, NCT01366521 Ortega et al., 2014, NCT01691521
Intravenous/Subcutaneous
Upper respiratory tract infections, headache, nasopharyngitis myalgia, oropharyngeal pain, anaphylaxis and sinusitis
Corren et al., 2016, NCT01508936 Castro et al., 2015 NCT01287039/ NCT01285323 Markham, 2016, NCT02452190, NCT02501629, NCT02559791
Subcutaneous
Injection site erythema, upper respiratory tract infection, sinusitis, nasopharyngitis and oral candidiasis
Busse et al., 2013, NCT01199289
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IL-5 inhibitor
Intravenous/Subcutaneous
IL-5 receptor antagonist
Brodalumab
IL-17 receptorA antagonist
AC C
Reslizumab
EP
Mepolizumab
SC
RPL554
31
ACCEPTED MANUSCRIPT
Adverse effects
Reference/Trial number
Subcutaneous
Injection-site reaction, upper respiratory tract infections, headache, migraine, sinusitis and asthenia
Holgate et al., 2011, NCT00141791
Pneumonia*♯, cellulitis, sepsis, upper respiratory tract infections, chest pain, sinusitis, nausea, and injection-site erythema
Wenzel et al., 2009, NCT00207740
Golimumab
TNF-α inhibitor
Subcutaneous
SB010
GATA-3 DNAzyme
Inhalation
RI PT
TNF-α inhibitor
Route of administration
SC
Etanercept
Mechanism of action
M AN U
Candidate drug
Mild oropharyngeal pain, back pain and bronchospasm♯
Homburg et al., 2015, NCT01743768, NCT01577953
AC C
EP
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*Serious Adverse event, ♯Treatment emergent adverse event, EudraCT: European Clinical Trials Database, NCT: National Clinical Trial
32
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Verini, M., Consilvio, N. P., Di Pillo, S., Cingolani, A., Spagnuolo, C., Rapino, D., ... and Chiarelli, F. (2010). FeNO as a marker of airways inflammation: the possible implications in childhood asthma management. Journal of Allergy, 2010, 1452-1453. http://dx.doi.org/10.1155/2010/691425 Virchow, J. C., Backer, V., Kuna, P., Prieto, L., Nolte, H., Villesen, H. H., ... and de Blay, F. (2016). Efficacy of a house dust mite sublingual allergen immunotherapy tablet in adults with allergic asthma: a randomized clinical trial. Jama, 315(16), 1715-1725. http://dx.doi.org/10.1001/jama.2016.3964 Vliagoftis, H., Schwingshackl, A., Milne, C. D., Duszyk, M., Hollenberg, M. D., Wallace, J. L., ... and Moqbel, R. (2000). Proteinase-activated receptor-2–mediated matrix metalloproteinase-9 release from airway epithelial cells. Journal of Allergy and Clinical Immunology, 106 (3), 537-545. http://dx.doi.org/10.1067/mai.2000.109058 Vonk, J. M., Postma, D. S., Maarsingh, H., Bruinenberg, M., Koppelman, G. H., and Meurs, H. (2010). Arginase 1 and arginase 2 variations associate with asthma, asthma severity and β2 49
ACCEPTED MANUSCRIPT agonist and steroid response. Pharmacogenetics and Genomics, 20 (3), 179-186. http://dx.doi.org/10.1097/fpc.0b013e328336c7fd Vroman, H., van den Blink, B., and Kool, M. (2015). Mode of dendritic cell activation: the decisive hand in Th2/Th17 cell differentiation. Implications in asthma severity? Immunobiology, 220 (2), 254-261. http://dx.doi.org/10.1016/j.imbio.2014.09.016
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S1094-5539(16)30056-6
DOI:
10.1016/j.pupt.2016.07.005
Reference:
YPUPT 1551
To appear in:
Pulmonary Pharmacology & Therapeutics
Received Date: 9 May 2016 Revised Date:
14 July 2016
Accepted Date: 20 July 2016
Please cite this article as: Sulaiman I, Woei LC, Liong SH, Stanslas J, Molecularly targeted therapies for asthma: Current development, challenges and potential clinical translation, Pulmonary Pharmacology & Therapeutics (2016), doi: 10.1016/j.pupt.2016.07.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT MOLECULARLY TARGETED THERAPIES FOR ASTHMA: CURRENT DEVELOPMENT, CHALLENGES AND POTENTIAL CLINICAL TRANSLATION
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Ibrahim Sulaiman, Lim Chee Woei, Soo Hon Liong and Johnson Stanslas.
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Pharmacotherapeutics Unit, Department of Medicine, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia.
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Corresponding Author: Prof Johnson Stanslas, Pharmacotherapeutics Unit, Department of Medicine, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. Email: [email protected]; [email protected]; Phone: 603-89472310; Fax: 603 89472789
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ACCEPTED MANUSCRIPT Abstract:
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Extensive research into the therapeutics of asthma has yielded numerous effective interventions over the past few decades. However, adverse effects and ineffectiveness of most of these medications especially in the management of steroid resistant severe asthma necessitate the development of better medications. Numerous drug targets with inherent airway smooth muscle tone modulatory role have been identified for asthma therapy. This article reviews the latest understanding of underlying molecular aetiology of asthma towards design and development of better antiasthma drugs. New drug candidates with their putative targets that have shown promising results in the preclinical and/or clinical trials are summarised. Examples of these interventions include restoration of Th1/Th2 balance by the use of newly developed immunomodulators such as toll-like receptor-9 activators (CYT003QbG10 and QAX-935). Clinical trials revealed the safety and effectiveness of chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) antagonists such as OC0000459, BI-671800 and ARRY-502 in the restoration of Th1/Th2 balance. Regulation of cytokine activity by the use of newly developed biologics such as benralizumab, reslizumab, mepolizumab, lebrikizumab, tralokinumab, dupilumab and brodalumab are at the stage of clinical development. Transcription factors are potential targets for asthma therapy, for example SB010, a GATA-3 DNAzyme is at its early stage of clinical trial. Other candidates such as inhibitors of Rho kinases (Fasudil and Y-27632), phosphodiesterase inhibitors (GSK256066, CHF 6001, roflumilast, RPL 554) and proteinase of activated receptor-2 (ENMD-1068) are also discussed. Preclinical results of blockade of calcium sensing receptor by the use of calcilytics such as calcitriol abrogates cardinal signs of asthma. Nevertheless, successful translation of promising preclinical data into clinically viable interventions remains a major challenge to the development of novel anti-asthmatics.
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Keywords:
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Asthma, Inflammation, Airway Remodelling, Anti-Asthmatics, Targets
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Chemical compounds studied in this article:
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Bis-(5-amidino-2-benzimidazolyl)-methane (PubChem CID: 46936860); Calcitriol (PubChem CID: 5280453); JNJ-39758979 (PubChem CID: 24994634); JNJ-7777120 (PubChem CID: 4908365); PF-3893787 (PubChem CID: 24745335); Y-27632 (PubChem CID: 448042); Fasudil (PubChem CID: 3547); Roflumilast (PubChem CID: 449193), YM-341619 (PubChem CID: 10321901); Afzelin (PubChem CID: 5316673).
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Introduction
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Asthma is an intricate inflammatory airway disease that results from activation of numerous
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inflammatory and structural cells, often leading to partially or fully reversible airway
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obstruction, airway inflammation, mucus hypersecretion and acute hyperresponsiveness
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(Prado et al., 2014). The aetiology of asthma may be genetic, environmental or an interplay of
75
both factors. It places enormous economic burden on patients, families, and the health care
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system by causing loss of productivity, high number of missed school and/or workdays, high
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medical bills, and premature death (Barnes et al., 2015). About 334 million people globally
78
were estimated to be asthmatic (Global Asthma Report, 2014; Murray et al., 2013) and this
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figure was projected to escalate to 400 million by the year 2025 (Global Asthma Report,
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2014). Statistics have placed asthma as the 14th most important disorder in the world, it
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affects about 14%, 8.6% and 4.5% of world children, young adults and world population
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respectively (Murray et al., 2013; To et al., 2012; Global Asthma Report, 2014). Severe
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uncontrolled asthma account for 5% to 10% of total asthma cases globally, and 50% of total
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asthma cost (Al-Hajjaj, 2011; Dheda et al., 2015). In Europe alone, the direct and indirect
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cost of both controlled and uncontrolled asthma in patients between the age of 15-64 was
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estimated to be €19 billion/annum (Domínguez-Ortega et al., 2015). Asthma, as a global
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disorder is a source of concern to both developed and developing nations (Behera and Sehgal,
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2015). Although improvement in asthma conditions can be achieved by the use of currently
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available conventional therapies such as topical bronchodilators and corticosteroids, these
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drugs have presented a number of unwanted side effects, possible resistance with long term
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use and absolute non-responsiveness in some cases (Abramson et al., 2003; Chung et al.,
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2014). Only a few effective anti-asthma controllers and relievers are currently available
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(example include steroids such as budesonide and fluticasone, bronchodilators such as
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salmeterol and levalbuterol). This may be due to inadequate knowledge of the underlying
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cause of asthma, difficulties in developing inhalable preparations for topical delivery and
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inordinate animal models used in testing new treatments that mostly fail clinical translation
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and relevance. This article discusses targets that could aid in developing new interventions
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with minimal adverse effects as compared to current medications. The role of certain
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receptors, enzymes and cytokines in the aetiology of asthma was further emphasized.
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Pathophysiology of Asthma
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The cardinal pathophysiological manifestations of asthma are airway inflammation,
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intermittent obstruction and acute hyperresponsiveness (AHR) (Holgate, 2008). Depending
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on cellular and molecular characterization of the inflammatory cascade, asthma may either be
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allergic or non-allergic endotype. Allergic asthma is usually eosinophilic, whereas non-
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allergic endotypes, such as aspirin, infection and exercise induced asthma may present with
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neutrophilic or paucigranulocytic phenotype. Most non-allergic asthma is severe, and steroid-
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resistant (Lötvall et al., 2011).
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Fundamental in vitro and in vivo studies have revealed several cellular and molecular events
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involved in development of asthma. These include increased immunoglobulin E (IgE)
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production by B cells, shift in T helper-type 1/T helper-type 2 (Th1/Th2) paradigm,
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upregulation of Th2 cytokines, airway cellular infiltrations, dysfunctional activated
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inflammatory cells, mitochondrial damage, increased production of reactive oxygen species
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(ROS) and reactive nitrite species (RNS) (Aguilera-Aguirre et al., 2009).
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Aeroallergens such as house dust mites, pollens and animal dander exert proteolytic and LPS-
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like activity that permits them to penetrate through the airway epithelium and attach to toll
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like receptors (TLR) to activate some cascade of events (Wilson et al., 2012). Allergen-TLR
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interaction induces release of IL-25, IL-33, Thymic stromal lymphopoietin (TSLP),
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macrophage inflammatory protein-3 (MIP3A) and monocyte chemotactic protein 1 (MCP1)
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by the airway epithelial cells. IL-25, IL-33 and TSLP enhance the development of Th2 cells
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while MIP3A (CCL20) and MCP1 (CCL2) are involved in the recruitment and activation of
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dendritic cells. Activated dendritic cells recognizes and present fragments of allergen peptides
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to lymph nodal naïve T lymphocytes (Th0) through its major histocompatibility complex
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(MHC) class II (Kallinich et al., 2007). The differentiation of Th0 into Th1 or Th2 depends on
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the cytokine milieu. Usually, increased IL-4 (induced by mast cell) and low IL-2 promotes
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differentiation of Th0 to Th2 cells. Th2 cells constitutively expresses GATA3, a crucial
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transcription factor that enhances Th2 production of IL-4, IL-5, IL-9 and IL-13 which further
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improve the suitability of the cytokine milieu for further Th0 - Th2 differentiation (Kaiko et
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al., 2008). Furthermore, IL-4 and IL-13 incites atopy by enhancing the production of IgE
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from B lymphocytes, influences secretion of eosinophil and Th2 recruiting cytokines such as
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IL-4, IL-5, IL-13, granulocyte macrophage colony stimulating factor (GM-CSF) and potent
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chemoattactants such as eotaxin 1 (CCL11), eotaxin 2 (CCL24) and RANTES (CCL5)
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mast cell airway infiltration, maturation and degranulation, while IL-5 induces and augment
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pulmonary eosinophil homing (Fulkerson and Rothenberg, 2013). IL-4 further enhances
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eosinophil migration by activating airway vascular cell adhesion molecule 1 (VCAM-1).
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Pulmonary eosinophils secrete wide array of inflammatory mediators such as cysteinyl
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leukotrienes (LTC4, LTD4, LTE4) and cytotoxic agents such as major basic protein (MBP),
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eosinophil derived neurotoxin, eosinophil cationic protein and eosinophil peroxidases which
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all together further aggravate airway inflammation and oxidative stress damage observed in
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asthma (Hall and Agrawal, 2014; Pelaia et al., 2015). Although eosinophilic asthma is of
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allergic endotype, non-allergic adult onset asthma can present with airway eosinophilia whose
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pattern of development is independent of Th2, rather, it depends on combined transcriptional
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activity of GATA3/RORα transcription factors and type 2 innate lymphoid cells (ILC2) (Woo
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et al., 2014). Nevertheless, both allergic and non-allergic eosinophilic asthma shows elevated
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type 2 cytokine levels, thereby indicating the critical role of IL-4, IL-5, IL-13 in the
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pathogenesis of eosinophilic asthma (Pelaia et al., 2015; Lambrecht and Hammad, 2015;
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Brusselle et al., 2013).
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Apart from airway eosinophilia due to Th2 or ILC2, asthma may present with neutrophilic
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phenotype, which is widely mediated by Th17, a subset of T helper cells that secretes IL-17.
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This category of asthma is often expressed in severe/uncontrolled asthmatics, steroid
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insensitive and it may be triggered by allergens or non-allergenic triggers such as microbes,
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cigarette smoke, diesel exhaust particles and other environmental pollutants (Polosa and
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Thomson, 2013; Newcomb and Peebles, 2013; Vroman et al., 2015). Differentiation of
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thymocytes into Th17 cells depends on RAR-related orphan receptor gammaT (RORɣt), a
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transcription factor that requires IL-1β, IL-6 and transforming growth factor (TGF-β) milieu
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for its upregulation and consequent Th17 production (Dong, 2008; Vroman et al., 2015). The
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heterogeneous nature of asthma pathogenesis has rendered blanket approach to treatment and
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diagnosis of different asthma endotypes ineffective. Therefore, proper understanding of
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asthma pathogenesis is essential for successful drug discovery and repositioning. Figure 1
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describes the cellular pathways involved in the pathogenesis of eosinophilic and neutrophilic
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asthma endotypes.
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Figure 1: Pathophysiology of asthma: Depending on the trigger and cytokine milieu, pathogenesis of asthma may occur through an eosinophilic or neutrophilic pathway. Antigens are presented to dendritic cells (DC) for onward processing and subsequent presentation to the naïve T-helper cells (Th0). Depending on the cytokine milieu, the Th0 may differentiate into T-helper cells type 1 (Th1), Th17 or Th2. A Th independent pathway occurs through the ILC2 to induce eosinophilic asthma. Treg: T regulatory cells, MMP: matrix metalloproteinase, IL: interleukin, TGF-β: Transforming growth factor-beta., LT: leukotriene, MHC: major histocompatibility II, TSLP: Tissue stromal lymphopoietin, GM-CSF: Granulocyte macrophage-colony stimulating factor, PAF: Platelet-activating factor, ILC2: Innate lymphoid cells 2, MHC-II: Major histocompatibility complex II, GR0-α: Growth regulated protein alpha, Cyst-LTs: cysteinyl leukotrienes,15-HETE: 15-Hydroxyicosatetraenoic acid.
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3.
Current Asthma Therapies
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The overall aim of asthma management is to prevent or control acute and chronic symptoms
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(such as exacerbations, airway mucus accumulation, narrowing, squeezing and swelling of
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the airway smooth muscles), stabilize pulmonary function, improve asthma-related quality of
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life, and provide optimum therapeutic interventions with minimal adverse effects (EPR3,
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2007). Current anti-asthmatic drugs function as either bronchodilators or anti-inflammatory,
179
or a combination of both. Alternatively, some of the medications are defined according to
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their targets and mode of action, e.g. the leukotriene modifiers, mast cell stabilizer and IgE
181
inhibitors. Conventionally, asthma is managed by use of single or multiple agents of
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bronchodilators such as short-acting β2 agonists (e.g pirbuterol, salbutamol and levalbuterol),
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long-acting β2 agonists (e.g salmeterol and formeterol), anti-cholinergics (e.g tiotropium,
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oxitropium and ipratropium bromides), and inhaled corticosteroids (ICS) (Barnes, 2006;
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adverse effects, including sympathomimetic side effects (such as anxiety, tremor and
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tachycardia) ascribed to β2 agonists activity (Abramson et al., 2003; Henderson et al., 2005)
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while anti-cholinergics were associated with induction of xerostomia (Kato et al., 2006;
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Rodrigo and Castro-Rodriguez, 2005).
190
Although, steroids are currently the most effective therapy for asthma, they were reported to
191
cause a number of unwanted side effects such as glaucoma, cataract, skin bruise,
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telangiectasia, osteoporosis, sore throat, dysphonia (due to laryngeal oedema and muscular
193
hypertrophy), hyperglycaemia, adrenal suppression and topical candidiasis (Reddel et al.,
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2015; Saag et al., 2011; Dahl, 2006; Israel et al., 2001). In addition to aforementioned
195
adverse effects, a group of asthmatics are resistant to steroid intervention due to variety of
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factors such as alteration in glucocorticoid receptor expression, unstable glucocorticoid
197
receptor activity and failure in nuclear translocation of glucocorticoid-receptor complexes.
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Inaccurate binding of complexes to glucocorticoid DNA response elements and faulty RNA
199
splicing leading to aberrant cytokine expression or distorted extracellular matrix were also
200
suggested to cause steroid resistance (Chung et al., 2014; Beck et al., 2009). The
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unavailability of better alternatives to steroid has made the management of steroid resistant
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asthma to be very challenging.
203
Although, omalizumab, a 95% humanized IgE monoclonal antibodies used in moderate to
204
severe persistent allergic asthma was proven to be effective in management of uncontrolled
205
asthma (Cox, 2009), the antibody has the potential of eliciting anaphylaxis and
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atherothrombotic events such as myocardial infarction and stroke (Ali and Hartzema, 2012).
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Few other classes of drugs used in asthma therapy include the mast cell stabilizers (e.g.
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cromolyn sodium and nedocromil sodium) and leukotriene modifiers (e.g. montelukast,
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pranlukast, zafirlukast and zileuton) all of which are less effective as compared to steroid
210
therapy (Fanta, 2009; del Giudice et al., 2009). Identification of safe substitute for current
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asthma medications through targeted therapy is paramount for improved asthma control,
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management and possible cure.
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4.
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Despite the unmet medical needs in prophylaxis and treatment of asthma, very few new
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classes of drugs were introduced and considered safe and effective over the past 40 years. In
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general, development of new drugs in the field of respiratory medicine is slow and scanty due
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Emerging Drug Targets
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preclinical findings into clinically relevant data (Barnes et al., 2015). The probability for a
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newly discovered respiratory drug to enter into the market phase of drug development is only
220
3%, as against to 6–14% in other diseases such as HIV/AIDS, cardiovascular disorders,
221
cancer, dermatological, haematological and neurological diseases (Mestre-Ferrandiz et al.,
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2012). Meaningful advancements recorded in the understanding of pathophysiology and
223
factors associated with asthma development are critical for the successful design and
224
development of new and more effective candidate drugs. Thus, targeting of critical cytokines,
225
receptors and enzymes implicated in aetiology and progression of specific asthma endotype is
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critical to successful development of novel interventions.
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4.1. Target Receptors
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4.1.1. Toll-like Receptor Activators
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Toll like receptors (TLRs) are pattern recognition receptors (PRRs) capable of detecting and
230
responding to microbial and endogenously derived signature molecules. Some TLRs are
231
found expressed on surfaces of epithelium, mast cells, fibroblasts, monocytes, macrophages
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and dendritic cells while others are endosomal or phagosomal in nature. Induction of TLR
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signalling promotes Th1 and downregulates Th2 responses, thereby countering allergic
234
asthma responses (Hennessy et al., 2010; Meng et al., 2011; Fonseca and Kline, 2009).
235
Considering the percentages of asthmatics that are of allergic endotype (50% and 80% of
236
asthmatic adults and children respectively), TLR targeting will be beneficial in reducing
237
asthma burden (Knudsen et al., 2009).
238
Toll like receptor subtypes TLR3, TLR7, TLR8, and TLR9 are strong inducers of Th1
239
responses (Hennessy et al., 2010). TLR9 agonist QAX-935 (IMO-2134) was reported to
240
decrease airway resistance and inflammation by activating several interferon-dependent genes
241
involved in effective Th1 response (Panter, et al., 2009; Kline and Krieg, 2010). Furthermore,
242
QbG10, a TLR9 agonist, improved overall asthma symptoms in patients with persistent
243
allergic asthma following controlled steroid withdrawal (Casale et al., 2015; Lassen, et al.,
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2014; Beeh, et al., 2013). Similarly, synthetic TLR9 ligands [unmethylated cytosine-guanine
245
oligodeoxynucleotides (CpG ODNs)] induced Th1 responses and counterbalanced Th2
246
phenotype via Th1 cytokine surge (IFN-γ, IL-10 and IL-12) induced by CpG mediated
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activation of natural killer (NK) cells through dendritic cell stimulation (Gupta and Agrawal,
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2010). In a related development, CpG ODNs analogs (1018 ISS and ASM8) suppressed
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in allergic asthma (Senti et al., 2009; Campbell et al., 2014).
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A potent TLR8 agonist (vTX-1463) reportedly stimulated the production of Th1 polarizing
252
cytokines (IFNγ and IL-12), decreased airway eosinophila and pulmonary congestion (figure
253
2). Likewise, upregulation and activation of TLR7 markedly suppressed asthma symptoms
254
and exacerbations through a Tregs dependent pathway (Hatchwell et al., 2015; Pham Van et
255
al., 2011). Immunotherapeutic approach by the use of house dust mite (HDM) sublingual
256
allergen immunotherapy (SLIT) tablet was recently reported to reduce the risk of moderate to
257
severe asthma exacerbations among adults with HDM allergy–related asthma that respond
258
poorly to ICS mono or combination therapy with no report of severe systemic adverse
259
reaction (Virchow et al., 2016). This improvement could be credited to allergen
260
desensitization and improved allergen tolerance due to shift in Th2 to Treg paradigm.
261
Although
262
immunotherapy (AIT) is yet to be fully elucidated, the ability of TLR to skew Th2-Th1
263
balance towards Th1-treg profile suggests its likely involvement in AIT. Furthermore, unlike
264
TLR-4, stimulation of TLR-7 and TLR-9 failed to break allergen-specific T-cell tolerance
265
induced by AIT (Akdis and Akdis, 2015). Although, TLR mediated immunotherapy appear
266
safe, the potential adverse effects of the intervention include autoimmunity, hypersensitivity,
267
exaggerated immune reactions and risk of perpetuating vicious inflammatory cycles as
268
obtainable in rheumatoid arthritis (Goh and Midwood, 2012).
269
4.1.2. Chemoattractant receptor-homologous molecule expressed on Th2 cells
270
(CRTH2)
271
Chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) otherwise
272
termed D prostanoid receptor 2 (DP2), is a G protein–coupled receptor that has a potential
273
role in the pathogenesis of allergic disorders such as asthma. The receptor is highly expressed
274
on Th2 cells, while its natural ligand [prostaglandin D2 (PGD2)] was found to be elevated in
275
the bronchoalveolar lavage fluid (BALF) of asthmatics and causes a characteristic cough (Fajt
276
et al., 2013; Stinson et al., 2015; Maher et al., 2015). Activation of CRTH2 enhances airway
277
remodelling, goblet metaplasia, MUC5AC expression and cellular migration as revealed by
278
transbronchial biopsy of severe asthma patients (Stinson et al., 2015). The resultant effects of
279
CRTH2 activation were blocked by AZD6430 (a potent CRTH2 antagonist), suggesting
280
CRTH2 inhibition as remedy to some asthma symptoms. AZD6430 decreased goblet
detailed
molecular
mechanism
behind
desensitization
in
allergen
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282
inhibition of PGD2 driven chemotaxic effects on eosinophils, mast cells and Th2
283
lymphocytes (Stinson et al., 2015). Furthermore, downregulation of CRTH2 activity by
284
OC0000459 suppressed airway hyperreactivity, improved forced expiratory volume in 1
285
second (FEV1), reduced total IgE level, reversed sputum eosinophilia and decreased
286
expression of proinflammatory genes in inflamed airways (Lukacs et al., 2008; Barnes et al.,
287
2012; Pettipher et al., 2012). According to a randomised, double-blind, placebo-controlled,
288
two-way crossover clinical trial study (NCT01056692), OC0000459 inhibited allergic airway
289
inflammation by preventing PGD2 mediated airway and serum eosinophilia. In addition,
290
improvement of FEV1 among asthma patients was also reported (Singh et al., 2012).
291
Moreover, administration of oral CRTH2 antagonist BI 671800 to ICS-naïve patients and
292
those on ICS improved asthma symptom by increasing FEV1 in both montelukast and ICS
293
treated patients (Hall et al., 2015). Thus, CRTH2 inhibition is potentially suitable for patients
294
whose asthma conditions are inadequately controlled by ICS monotherapy.
295
4.1.3. Proteinase-activated receptor 2 (PAR-2)
296
PAR2 is a G-protein coupled receptor (GPCR) commonly expressed in airway smooth
297
muscle, leukocytes and bronchial epithelial cells. It is usually activated by proteinases that
298
arises from aeroallergens, invading pathogens, or by endogenously released proteinases such
299
as trypsin, house dust mite (HDM) protein Derp9 and mast cell tryptase (Boitano et al.,
300
2011). The attention given to inhibition of protease activity as potential drug target is
301
currently on the rise (Alam, 2014; Harvima et al., 2014). Many triggers of asthma (e.g. house
302
dust mite, fungal allergens and ovalbumin) exhibit proteinase activity (Corry and
303
Kheradmand, 2009; Kauffman and van der Heide, 2003). The activation of PAR2 occurs by
304
cleavage of its extracellular N terminus to reveal its tethered ligand (SLIGRL-NH2/SLIGKV-
305
NH2) that auto-activates the receptor. Upon PAR2 activation, classical GPCR and β-arrestin
306
signalling pathways are triggered. The latter is pro-inflammatory and occurs due to immense
307
activation of PAR2 while the former is bronchoprotective and occur due to mild activation of
308
PAR2 (Nichols et al., 2012).
309
Prior reports showed 38% reduction and 52% increase in airway hyper-reactivity following
310
methacholine challenge in asthmatic mice lacking PAR2 and those overexpressing PAR2
311
respectively as compared to wild-type (Schmidlin et al., 2002). This implicated the
312
involvement of PAR2 in pathogenesis of hyperresponsiveness in asthma. Activated PAR2
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314
mediators include the granulocyte macrophage- colony stimulating factors (GM-CSF),
315
eotaxin and matrix metalloproteinase 9 (Vliagoftis et al., 2000). Bronchial smooth muscles
316
(BSM) PAR2 stimulation increased the proliferation of BSM cells from asthmatic subjects
317
only, while BSM cells from non-asthmatic subjects showed no hyper-proliferative property
318
even after lentiviral over-expression of PAR2. Such increased proliferation could be
319
accounted for by increased basal level of PAR2 in asthmatics airway (Allard et al., 2014).
320
Furthermore,
321
tryptase inhibitor, inhibitors of tryptase (a PAR2 substrate) were found to prevent
322
bronchoconstriction in asthmatics (Sylvin et al., 2002; Hernández-Hernández et al., 2012). It
323
could therefore be suggested that mast cells mediated induction of airway hyper
324
responsiveness is not solely dependent on released histamine but also the endpoint effect of
325
mast cell tryptases on PAR2.
326
4.1.4. Calcium Sensing Receptor (CaSR) Antagonism
327
Calcium-sensing receptor (CaSR) is a calcium-binding G protein-coupled receptor (GPCR),
328
that regulates systemic calcium homeostasis (Breitweiser, 2014). Airway smooth muscle cells
329
contain elevated [Ca2+]i levels while CaSR are upregulated and constitutively activated in
330
airways of asthmatics (Yarova et al., 2015). Increase in [Ca2+]i enhances bronchoconstriction
331
that leads to AHR and induces chronic genomic effects that increases ASM cell proliferation
332
and extracellular matrix components depositions that additively results in airway remodelling
333
(Koopmans et al., 2014; Mahn et al., 2010). Activation of CaSRs is therefore considered a
334
major climactic event leading to asthma pathogenesis. Eosinophilic cationic proteins and
335
major basic proteins released by infiltrating eosinophils are considered to primarily induce
336
airway narrowing in asthma (Pégorier et al., 2006), these polycations exert their effect
337
through activation of airway CaSRs which is normally involved in ASM homeostasis of
338
divalent cations and polycations (figure 3). Blockade of this receptor by the use of calcilytics
339
(e.g calcityrol), were found to reduce AHR and airway inflammation in mouse asthma models
340
(Yarova et al., 2015). Although the concept of using calcilytics as novel asthma intervention
341
it still at its initial stage, the intervention appears promising especially in asthmatics that are
342
non-sensitive to current asthma management strategies (Tanday, 2015).
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344
Plasma and airway histamine levels are heightened among asthmatics especially during
345
exacerbations. The biogenic amine mediates through four GPCR subtypes, the histamine H1-,
346
H2-, H3, and H4-receptors (H1-4R). H4R is considered a promising drug target for
347
inflammatory and allergic disorders due to its broad expression in hematopoietic cells (Seifert
348
et al., 2013; Brimblecombe et al., 2010; Zhang et al., 2007). The mediator is usually released
349
by mast cells and it functions by activating DCs, enhance Th2 polarization as well as airway
350
inflammatory cells homing (Reher et al., 2012; Caron et al., 2001). In vivo cellular
351
characterization of H4R in experimental allergic asthma models identified the role of the
352
receptor in the regulation of dendritic cell (DC) activation during sensitization and its role in
353
eosinophil activation during effector phase. This was further proved by the inability of
354
effector T cells to induce eosinophilic infiltration upon adoptive transfer of DCs devoid of
355
H4R at vitro sensitization reaction stage (Hartwig et al., 2015).
356
H4R selective antagonist, JNJ -7777120 H4R ((5-chloro-1H-indol-2-yl)-(4-methyl-piperazin-
357
1-yl)-methanone) ameliorated asthma symptoms in ovalbumin mouse asthma model.
358
Furthermore, H4R gene knock-out caused decreased allergic pulmonary inflammation,
359
prevented Th2 responses and bronchoconstriction, inhibited airway eosinophil and
360
lymphocyte infiltration (Neumann et al., 2013; Dunford et al., 2006; Somma et al., 2013).
361
The interesting results obtained from preclinical assessment of H4R antagonists prompted
362
further exploration of its clinical application in asthma. H4R selective antagonists showed
363
excellent safety profile following preclinical toxicity studies. Likewise, phase 1 clinical trial
364
has shown that selective H4R antagonists such as UR-63325, JNJ-39758979 [(R)-4-(3-amino-
365
pyrrolidin-1-yl)-6-isopropyl-pyrimidin-2-ylamine]
366
(cyclopropylmethyl)-6-(3-(methylamino)pyrrolidin-1-yl)pyrimidine-2,4-diamine] are safe for
367
use in humans. In addition, JNJ-39758979 dose dependently inhibited histamine induced
368
eosinophil morphological changes (Thurmond et al., 2014; Salcedo, 2013; Mowbray et al.,
369
2011). Unfortunate incidence of agranulocytosis observed in two out of eighty-eight Japanese
370
patients that enrolled for safety and efficacy study of JNJ-39758979 in moderate atopic
371
dermatitis led to the withdrawal of a phase 2 clinical trial of JNJ-39758979 in patients with
372
uncontrolled, persistent asthma prior to enrolment (Murata et al., 2015). This adverse effect
373
may be due to off target effect of the intervention or likely due to unascertained
374
pharmacogenomic variation among subjects. Nevertheless, the intervention was found to be
375
effective before the premature discontinuation of the trial.
and
PF-3893787
[(R)-N4-
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Figure 2: Toll like (TLR) and histamine 4 receptor (H4R) targeting in asthma treatment. TLR7, TLR8 and TLR9 engages their Toll–IL-1-resistence (TIR) domains to myeloid differentiation primary-response protein 88 (MYD88). This in turn stimulates downstream IL-1R-associated kinase 4 (IRAK4) and TNF receptor-associated factor 6 (TRAF6) thereby leading the activation of interferon-regulatory factor 7 (IRF-7), which induced type I interferon (IFN-α). IFN-α provide the cytokine milieu that enhances Th1 polarization by inducing IL-12 secretion from monocyte derived dendritic cells (mDC). Histamine 4 receptor (H4R) activation results in mast cell chemotaxis, downstream Ca2+ surge from endoplasmic reticulum (ER) and upregulation of markers such as malonyldialdehyde (MDA), 8-hydroxy-2’-deoxyguanosine (8OHdG) and transforming growth factor β (TGF-β). Alternatively, H4R activation may lead to decreased adenyl cyclase (AC) activity through Gi protein activation. This decreases intracellular cyclic adenosine monophosphate (cAMP) level, which in turnresults in protein kinase A (PKA) activation. Active PKA phosphorylates cAMP-responsive element-binding protein (CREB), which modulates gene transcription.
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ACCEPTED MANUSCRIPT 4.2.
Enzymatic Targets
391
4.2.1. Inhibitors of Rho-Kinases
392
Rho, a member of the Ras superfamily of guanosine triphosphatases (GTPases) and a
393
monomeric G protein associated with regulation of smooth muscle contraction, modulation of
394
myosin light chain phosphorylation and stress fibre formation (primarily actin and non-
395
muscle myosin II fibres). Rho also controls few other physiological events such as
396
cytokinesis, focal adhesion, cell migration, motility, migration and regulation of downstream
397
extracellular signals, such as lysophosphatidic acid (Taki et al., 2007). The contraction of
398
ASM is triggered by phosphorylation of the myosin light chain (MLC) by Ca2+ calmodulin
399
(CaM)-activated MLC kinase in the presence of intracellular calcium, thereby allowing actin-
400
myosin cross-linkage and subsequent shortening of sarcomeres. Whereas, ASM relaxation is
401
caused by MLC dephosphorylation by Rho kinase regulated myosin phosphatase (Chiba et
402
al., 2010; Schaafsma et al., 2006; Burdyga et al., 2003). Phosphorylation of myosin binding
403
subunit of myosin phosphatase by Rho kinases (ROCK) exerts inhibitory effect on MLC
404
dephosphorylation, thereby limiting smooth muscle relaxation (Chiba et al., 2010). Rho-
405
kinases are overexpressed in airway of asthmatics, therefore contributing to intermittence or
406
persistence AHR symptoms in asthma. The additive effect of Rho kinase upregulation results
407
in persistent MLC phosphorylation, contributing to asthma-related cellular remodelling
408
(Holgate, 2008).
409
Aside from ASM tone regulation via MLC phosphatase pathway, Rho kinases also play a
410
vital role in regulating inflammation related cellular infiltration and migrations via their
411
inherent spatiotemporal regulatory potential on the actin cytoskeleton (Biro et al., 2014). In
412
support of this, Rho-kinase inhibition (by use of Y-27632) blocks the cellular expression of
413
NF-κB, an essential transcription factor that contributes to asthma progression by activating
414
genes coding for inflammatory cytokines (Tong and Tergaonkar, 2014).
415
Fasudil (HA-1077), an isoquinoline-based compound is a first generation and the only
416
clinically available ROCK inhibitor that proved to be effective in management of pulmonary
417
arterial hypertension (PAH) and cerebral vasospasm through blockage of ATP dependent
418
domain of Rho-kinase and consequent smooth muscle relaxation (Raja, 2012; Velat et al.,
419
2011; Bain et al., 2007). Fasudil inhibits ROCK induced actomyosin dynamics by
420
competitively binding to the ribose region in the ATP-binding motif of ROCK (Nagumo et
421
al., 2000). It reportedly induced substantial decrease in cellular infiltration, lung
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ACCEPTED MANUSCRIPT inflammatory
index,
423
responsiveness, in addition to downregulation of IL-17, IL-13, and IL-4 in lungs of OVA-
424
challenged mice. Reduction in allergic airway inflammation and mucus hypersecretion by
425
fasudil-mediated inhibition of Rho kinase was reported to occur as a result of decreased
426
expression and phosphorylation of two transcription factors that were critically implicated in
427
asthma, namely; STAT6 and NFκB (Xie et al., 2015; Taki et al., 2007).
428
Another
429
cyclohexane
430
bronchodilation in guinea pigs while maintaining minimal cardiopulmonary side effects
431
(Righetti et al., 2014; Possa et al., 2012). Many studies have shown that administration of Y-
432
27632 by either inhalation or intranasal drops prevented and/or reduced airway
433
hyperresponsiveness and pulmonary eosinophilia in different animal models of asthma (Possa
434
et al., 2012; Schaafsma et al., 2008; Henry et al., 2005; Gosens et al., 2004). Recent reports
435
revealed that co-administration of Y-27632 with corticosteroids preferentially decreased
436
exhaled NO level, bronchial inflammation, airway oxidative stress, extracellular matrix
437
remodelling, airway collagen level as well as TIMP-1 positive cells, alveolar septa
438
eosinophilia, IL-2, 8-iso-PGF2α, IFN-γ and NF-κB activity in distal parenchyma, as
439
compared to corticosteroid or Rho-kinase inhibitor monotherapy (Pigati et al., 2015). Thus,
440
Rho-kinase inhibition (either as monotherapy or combination therapy) is a potential
441
therapeutic tool for asthma management and control.
442
4.2.2. Phosphodiesterase inhibitors
443
Elevation of phosphodiesterase (PDE) activity and alteration in cAMP-PDE pathway in
444
asthmatic airway smooth muscle cells is an indication that PDEs are involved in the
445
regulation of bronchial tone, airway hyperplasia and airway remodeling (Burgess et al., 2006;
446
Trian et al., 2011). PDE4 inhibitor roflumilast, decreased airway muscular contraction,
447
eosinophilia, neutrophilia, bronchial RSV infection and MUC5AC expression (Mata et al.,
448
2013; Gauvreau et al., 2011a). Another PDE4 inhibitor, 3-[4-(3-chlorophenyl)-1-ethyl-7-
449
methyl-2-oxo-1,2-dihydro-1,8-naphthyridin-3-yl] propanoic acid (ASP3258), was reported to
450
have ameliorated airway eosinophilia in a chronic murine asthma model (Kobayashi et al.,
451
2012). Repeated doses of roflumilast (a potent oral PDE4 inhibitor that is well known for its
452
anti-inflammatory property and treatment of chronic obstructive pulmonary disorder)
453
decreased sputum eosinophilia, FeNO, leukotriene (LTE4), improved FEV1 and attenuated
454
exercise-induced asthma (Bardin et al., 2015). Although, roflumilast is considered well
selective
goblet
cell
Rho-kinase
inhibitor,
(Y-27632),
MUC5AC
expression,
and
airway
(+)-(R)-trans–4-(1-aminoethyl)-N-(4-pyridyl)
a
pyridine
derivative,
reportedly
induced
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ACCEPTED MANUSCRIPT tolerated by most asthmatics, few unwanted adverse effects majority of which are related to
456
gastrointestinal disturbances (such as nausea and diarrhoea), insomnia and headache were
457
reported (Bardin et al., 2015; Parikh and Chakraborti, 2016; Chervinsky et al., 2015; Fabbri
458
et al., 2009).
459
In addition to induction of rapid and sustained bronchodilation among asthmatics, reduction
460
in sputum cell titre was recorded in lipopolysaccharide challenged healthy volunteers that
461
were given RPL554, a novel inhalable PDE3/4 inhibitor induced (Franciosi et al., 2013).
462
Unlike most other PDE inhibitors, the safety profile of RPL554 was within the tolerable range
463
and did not produce GI effects often associated with most PDE inhibitors (Franciosi et al.,
464
2013).
465
Xanthines were formally used in asthma treatment, however, considering their non-selectivity
466
in PDE inhibition, narrow therapeutic window, severe side effects (such as cardiac
467
arrhythmias, seizures, nausea, vomiting, and headaches) and following the discovery of better
468
bronchodilators (such as β2 agonists and muscarinic receptor antagonists), the use of
469
xanthines
470
Theophylline (dimethylxanthine) was commonly used as bronchodilator when administered at
471
relatively high dose. However, recent advances have shown that it possesses anti-
472
inflammatory effects in asthma when administered at lower doses (Barnes, 2013).
473
Theophylline molecularly induces bronchodilation by inhibition of PDE3, while it exerts its
474
anti-inflammatory effect by PDE4 inhibition and concomitant upregulation of histone
475
deacetylase-2 (HDAC-2), allowing deactivation of asthma activated inflammatory genes
476
(Barnes, 2013). HDAC-2 is critically lowered in steroid resistant severe asthmatics. The
477
upregulation of HDAC2 by theophylline reverses corticosteroid resistance in steroid
478
insensitive asthma endotype (To et al., 2010). Theophylline mediates HDAC-2 increase is
479
achievable through inhibition of phosphoinositide-3-kinase-δ (PI3Kδ) which is activated by
480
oxidative stress (To et al., 2010; Tobinick, 2009). The co-administration of theophylline with
481
salmeterol/fluticasone propionate combination to asthma patients significantly reduced
482
frequency and number of patients with asthma exacerbations, improved the forced expiratory
483
flow, decreased sputim eosinophil cationic protein concentration and generally improved
484
airway inflammation (Nie et al., 2013). Similarly, an improvement in asthma symptoms was
485
reported in asthmatic smokers that are nonresponsive to ICS following low theophylline dose
486
(Spears et al., 2009). These therefore indicate the antiasthmatic potential of theophylline,
487
especially in control of steroid resistant endotype.
asthma
therapy
gradually
faded
out
(Page,
2014).
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489
The enzymatic breakdown of the guanidine group of L-arginine to release of NO and L-
490
citrulline results in generation of nitric oxide, a critical marker of airway inflammation
491
(Verini et al., 2010). This process is catalysed by nitric oxide synthase (NOS). Among the
492
three basic types of NOS, inducible nitric oxide synthase (iNOS) is overexpressed
493
inflammatory conditions. Conversely, NO from iNOS influences localized vasodilatation,
494
plasma extravasation, mucus hypersecretion, promotes eosinophilic homing and consequent
495
activation of Th2 cells, thereby aggravating airway inflammation (Prado et al., 2011; Reis et
496
al., 2012; Souza et al., 2013). Virtually, all cells in the respiratory tract of asthmatics exhibits
497
intensified expression of iNOS, which is correlated with perpetuating and amplified airway
498
inflammation in asthma (Prado et al., 2014). Potent partial and full inhibitors of iNOS
499
effectively decreased the FeNO among asthmatics (Singh et al., 2007). Nevertheless,
500
GW274150, a selective iNOS inhibitor did not effectively ameliorate airway reactivity and
501
inflammation (Singh et al., 2007). The non-desired response observed in upper airway may
502
be due to the paradoxically opposing functions of NO in upper and lower airway. L-NG-
503
Nitroarginine Methyl Ester (L-NAME), a commercially available precursor of NOS inhibitor,
504
was found to increase airway resistance, compliance and elastance, while it decreased both
505
properties (resistance, elastance and compliance) in lung parenchyma following its bio-
506
activation to L-NG-Nitroarginine; NG-nitro-L-Arginine (L-NNA), a non-specific NOS
507
inhibitor. This provide support to the fact that NO is a potent bronchodilator of proximal
508
airways (by acting through the NANC pathways) and as constrictor of distal pulmonary
509
parenchyma. Acute L-NAME therapy was also found to decreased airway eosinophilia (Prado
510
et al., 2005). Alternative treatment with specific iNOS inhibitor (e.g. 1400-W) provided better
511
control of hyperresponsiveness and airway inflammation in the same murine model used
512
(Starling et al., 2009; Marques et al., 2012; Souza et al., 2013).
513
Apart from the NO metabolite of L-arginine produced through the NOS pathway, arginase
514
catabolic cascade appears to play role in development and progression of asthma (Vonk et al.,
515
2010). However unlike NOS, the activity of arginases does not lead to the production of NO,
516
it catabolizes L-arginine to L-ornithine and urea (Ricciardolo et al., 2004). The L-ornithine
517
(product of arginase activity) is further catabolized into polyamines by ornithine
518
decarboxylase. These L-ornithine–derived polyamines are the prime agents through which
519
arginases contribute to AHR. They promote collagen synthesis and cellular proliferation
520
thereby contributing to asthma related airway remodelling (Prado et al., 2014). Moreover,
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ACCEPTED MANUSCRIPT plasma polyamine surge was reported in asthmatics (North et al., 2013). Up-regulation of
522
arginase further complicates AHR by competitively upregulating pro-inflammatory
523
peroxynitrite release due to iNOS activity (Maarsingh et al., 2009). Studies have shown high
524
airway activity and expression of arginases (ARG1 and ARG2) following allergen challenge
525
in mouse asthma models (Vonk et al., 2010). 2(S)-amino-6-boronohexanoic acid, a selective
526
arginase inhibitor, was reported to reduce bronchial hypersensitivity to allergens, prevents
527
airway obstruction and reversed airway inflammation in allergen challenged guinea pigs
528
(Maarsingh et al., 2008). Additionally, S-(2-boronoethyl)-L-cysteine, another potent arginase
529
inhibitor, was reported to have decreased AHR in murine models of allergic asthma (North et
530
al., 2009). Likewise, AHR was reversed in murine model of IL-13-induced AHR by RNA
531
interference intervention against ARG1 (Yang et al., 2006). Thus, the application of specific
532
ARG inhibitors may supposedly be a promising intervention for successful amelioration of
533
asthma symptoms (figure 3).
535 536 537 538 539 540 541 542
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Figure 3: Control of airway remodelling. Ca2+ signalling through calcium sensing receptor (CaSR) and Rho kinase pathways appear critical for asthma control. Inhibition of CaSR by calcilytics reverses bronchoconstriction by deactivating downstream signalling pathways such as the MAPK and the Rho/Rac/Cdc42 pathways. Direct inhibition of Rho kinases permits the dephosphorylation of myosin light chain (MLC) by active myosin phosphatase thereby inducing smooth muscle relaxation. Polyamine and proline induced bronchoconstriction and goblet metaplasia can be averted by inhibiting arginase activity. ODC: ornithine decarboxylase, OAT: Ornithine acetyltransferase, NOS: Nitric oxide synthase.
543 18
ACCEPTED MANUSCRIPT 4.3.
Biologics
545
Mast cells, bronchial epithelium and macrophages are important sources of cytokines,
546
chemokines, and a number of chemical mediators involved in asthma progression (Desai and
547
Brightling, 2009). Targeting individual cytokines that play dominant roles in the
548
pathophysiology of asthma may provide better biologics for prophylaxis and/or treatment of
549
asthma. So far, some few antibodies targeting cytokines are at developmental stages (see table
550
1).
551
Targeting of cytokine effect/activity can be achieved directly by cytokine targeting or
552
indirectly through cytokine receptor modulation. IL-13 is central to development of AHR in
553
asthma and its effect is mediated through its binding to low affinity IL-13 receptor alpha1
554
(IL-13Rα1) and IL-4 receptor alpha (IL-4Rα) complexes (IL-13Rα1/ IL-4Rα complex).
555
While IL-4 also exerts its effect through IL-4Rα. Dupilumab, a human monoclonal antibody
556
against IL-4α/IL-13α receptor complex, inhibited the downstream signalling events induced
557
by IL-4 and IL-13 by binding to the alpha subunit of the IL-4 receptor of the complexes
558
(Hambly and Nair, 2014; Vatrella et al., 2013; Wenzel et al., 2013b; Prado et al., 2014). Add
559
on therapy of dupilumab/ICS/LABA combination was recently reported to effectively
560
improve FEV1, patient’s quality of life and decreased recurrent asthma exacerbations in
561
patients with uncontrolled persistent asthma regardless of baseline blood eosinophil level.
562
Additionally, the antibody has the tendency of ameliorating comorbidities associated with
563
uncontrolled persistent asthma (e.g nasal polyps and acute dermatitis) (Wenzel et al., 2016).
564
Likewise, pitrakinra, a dual IL-4/IL-13 antagonist was evaluated for potential asthma therapy
565
and it was found to dampen the progression of early and late asthmatic response in allergen
566
challenge models (Antoniu, 2010; Wenzel et al., 2007). Pitrakinra competitively inhibited IL-
567
4Rα activity to impede IL-4 and IL-13 activities. It also significantly improved asthma
568
symptoms and decreased exacerbations in patients with airway eosinophilia, nevertheless,
569
phase IIb trial failed to demonstrate clinical efficacy of the intervention in the overall study
570
population (Hambly and Nair, 2014).
571
Effort to target IL-13 alone using lebrikizumab (a humanized anti-IL-13 antibody) suggests
572
asthma endotype specificity in IL-13 dependent asthma treatment. Significant improvement in
573
lung function was reported following treatment with lebrikizumab in patients with high serum
574
periostin (an indicator of eosinophilia in asthmatics) and high fractional exhaled nitric oxide
575
(FeNO) levels (Corren, et al., 2011; Jia et al.,2012; Noonan et al., 2013). Serum periostin
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ACCEPTED MANUSCRIPT level can be used as a critical determinant of lebrikizumab efficacy. Recent randomised,
577
double-blind, placebo-controlled trial revealed reduction in asthma exacerbations rate and
578
improvement in FEV1 among periostin-high patients with moderate-to-severe asthma than in
579
the periostin-low patients following 24 weeks of treatment with different doses of
580
lebrikizumab (37.5, 125, 250 mg every four weeks) (Hanania et al., 2015). Tralokinumab
581
administration dose dependently improved lung function (as measured by FEV1) of patients
582
with moderate-severe asthma. Safety and toxicity studies revealed no serious adverse effect in
583
its phase II trial (Piper et al., 2013; Hanania et al., 2015).
584
An IL-13Rα1 isomer, IL-13 receptor alpha2 (IL-13Rα2) possesses high affinity to IL-13 as
585
compared to IL-13R α1. However, it was proposed to perform a non-signalling decoy role in
586
IL-13 functionality. Upregulation of its soluble isomer (sIL-13Rα2) reportedly inhibited IL-
587
13 induced airway inflammation in murine asthma models (Daines et al., 2006; Chen et al.,
588
2013). Conversely, mouse has both its soluble (sIL-13Rα2) and membrane bound (memIL-
589
13Rα2) forms of IL-13Rα2, humans only have the memIL-13Rα2 form. This may contribute
590
to failure in translation of preclinical trials involving IL-13Rα2 immunomodulation for
591
control of human asthma (Chen et al., 2013).
592
Considering the role of IL-5 in airway eosinophil homing, as well stimulation of mediator
593
release from eosinophil and its effect on tissue survival (Garcia et al., 2013), monoclonal
594
antibodies against interleukin 5 (anti-IL-5) were tried for use in asthma. Anti-IL-5
595
monoclonal antibodies such as mepolizumab and reslizumab revealed beneficial effect in
596
management of persistent airways eosinophilia among corticosteroid resistant subjects. In a
597
randomized, double blind and double dummy trial, reduction in rate of asthma exacerbations
598
and sputum eosinophilia was recorded upon repeated administration of mepolizumab to
599
insensitive asthma patients (Ortega et al., 2014).
600
mepolizumab is an exciting advancement especially for patients with uncontrolled severe
601
eosinophilic asthma because it is safe and effective option that could replace oral
602
corticosteroids (Bel et al 2014; Haldar et al., 2009; Nair et al., 2009; Pavord et al., 2012).
603
According to recent trials on reslizumab, the antibody exhibited tolerable safety profile and
604
improved FEV1, forced vital capacity (FVC), forced expiratory flow (FEF), asthma control,
605
patient’s quality of life and rescued SABA use in uncontrolled asthma with eosinophilic
606
endotype (Corren et al., 2016; Markham, 2016; Castro et al., 2015). Equally, benralizumab
607
(MEDI-563), an afucosylated monoclonal antibody against IL-5Rα, was reported to reduce
608
peripheral eosinophils levels in moderate asthma patients and sustained its effect for about 8–
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The corticosteroid sparing effect of
ACCEPTED MANUSCRIPT 12 weeks through antibody dependent cell-mediated cytotoxicity (Ghazi et al., 2012; Busse et
610
al., 2010; Kolbeck, et al., 2010).
611
Th17 lymphocytes have demonstrated the ability to recruit both eosinophils and neutrophils
612
to the airway through IL-17A and IL-17F release (Tesmer et al., 2008). Although IL-17
613
targeting using secukinumab, and ixekizumab seems promising, there are few clinical trial
614
reports on the use of the antibodies in asthma. However, brodalumab, a monoclonal antibody
615
that targets IL-17RA, produced clinically meaningful responses in moderate to severe
616
asthmatic patients with high bronchodilator reversibility (Busse et al., 2013). Considering the
617
equivocal nature of available data on use of brodalumab in asthma control (Bauer et al.,
618
2015), further studies on its effect on different asthma sub-populations is highly
619
recommended.
620
Tumor necrotic factor-alpha (TNF-α) is mainly secreted by lymphocytes, mast cells, and
621
macrophages. It promotes bronchial hyperresponsiveness and sputum neutrophilia, this make
622
it an attractive target for severe asthma (Wenzel et al., 2009). A number of anti-TNF-α agents
623
exist in the market and they were proven effective in some inflammatory diseases such as
624
rheumatoid arthritis and Crohn disease. It is apparent that the use of inhibitors of TNF-α may
625
be valuable in reversal of asthma symptoms. Etanercept (anti TNF-α agent) showed short-
626
term and modest efficacy for severe and mild asthma respectively (Antoniu, 2009). Slight
627
reduction in number of exacerbations and improvement in wheezing was observed in patients
628
with moderate uncontrolled asthma originally on ICS monotherapy following infliximab or
629
adalimumab administration (Erin et al., 2006; Stoll et al., 2009). Upon withdrawal of TNF-α
630
inhibition, wheezing triumphed (Stoll et al., 2009). Safety concerns may limit the use of anti-
631
TNF-α in asthma therapy. Substantial adverse reactions were observed without achieving
632
fundamental objectives of reducing asthma exacerbations and improving FEV1 among
633
subjects (see table 2). Golimumab lowered risk of asthma exacerbations, nevertheless, its trial
634
on severe persistent asthma ended at phase II due to incidences of malignancies and
635
infections such as pneumonia (Wenzel et al., 2009). Given the role of TNF- α in severe,
636
refractory, or steroid-resistant asthma, future studies on the use of this anti-TNF- are needed
637
to identify whether the long-term risk-benefit profile favours use in asthma.
638
The results of trials involving agents targeting IL-4, IL-13, IL-9, IL-12, IL-10, interferon-γ,
639
granulocyte-macrophage colony-stimulating factor, and even Th17 cells are anticipated
640
(Hansbro et al., 2011; Desai and Brightling, 2009). As understanding of the cytokine
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networks continues to evolve, so too will the potential targets for antiasthma therapy.
642
4.4.
643
Airway inflammation in asthma is associated with several transcription factors such as the
644
NFkB, nuclear factor of activated T-cells (NF-AT) and glucocorticoid receptor (GR). Cyclic
645
AMP response element binding protein (CREB), activator protein-1 (AP-1), peroxisome
646
proliferator-activated
647
CCAAT/enhancer binding protein (C/EBP), signal transducer and activator of transcription 6
648
(STAT6), GATA-3, doublesex and mab-3 related transcription factor 1 (DMRT1) and nuclear
649
factor erythroid 2-related factor 2 (Nrf2) were also implicated in pathogenesis of asthma
650
(Roth and Black, 2006). Although, most transcription factors play important role in tissue and
651
organ homeostasis, modulation of some of the transcription factors (especially GATA-3,
652
NFkB, Nrf2, NF-AT and STAT6) appeared to be a valid therapeutic approach to control of
653
asthma symptoms. Control of lung inflammatory responses is achievable by application of
654
decoy or antisense oligonucleotides specific for targeted airway transcription factor of
655
interest. Nevertheless, long term suppression or overexpression of these transcription factors
656
could lead to wide array of adverse effects, thus, cell type specific delivery of inhibitors or
657
activators of target factors is critical to obtaining minimal adverse effect that may arise from
658
this approach (Roth and Black, 2006; Haley et al., 2011; Schieck et al., 2016)
659
The characteristic Th2 molecular endotype of allergic asthma is controlled by a zinc finger
660
transcription factor GATA3, which is essential for Th2-cell differentiation and immune
661
pathway activation (Wenzel, 2012). GATA-3 is reportedly overexpressed in broncholaveolar
662
lavage and lung biopsies from severe asthma patients (Bergqvist et al., 2013). Thus,
663
interventions that target GATA3 and its downstream effectors were suggested for treatment
664
of asthma (Holgate, 2012). Afzelin, a flavonol glycoside, was reported to suppress airway
665
inflammation, eosinophil infiltration, airway hyper-responsiveness and BALF Th2 cytokines
666
in murine asthma model by inhibition of GATA-3 activity and increasing T-bet/GATA-3
667
ratio. Considering the role of T-bet transcription factor in regulation of Th1 differentiation
668
and activation, the increase in T-bet/GATA-3 ratio supposedly restored Th1/Th2 balance
669
(Jenner et al., 2009; Zhou and Nie, 2015). In another preclinical study, GATA-3 specific
670
catalytically active, single stranded, synthetic antisense DNA enzyme (DNAzyme) hgd40
671
containing SB010 (active drug component) was tested for efficacy in reduction of Th2
672
cytokine level via inhibition of GATA-3 mRNA and protein level (Turowska et al., 2013; Sel
(PPAR),
runt-related
transcription
factors (RUNX),
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ACCEPTED MANUSCRIPT et al., 2008). The results of the preclinical tests showed remarkable improvement in asthma
674
symptoms with no observable off-target effect therefore allowing for further clinical trial of
675
the DNAzyme (Homburg et al., 2015; Fuhst et al., 2013). In a randomized, double-blind,
676
placebo-controlled, multicenter clinical trial, 10mg of aerosolized SB010 or placebo was
677
administered to asthma patients once daily for 28 days, and a significant improvement in lung
678
function was reported. Furthermore, the intervention attenuated allergen-induced sputum
679
eosinophilia, reduced sputum tryptase and negated plasma IL-5 levels (Krug et al., 2015).
680
GATA-3 activity is linked to few other asthma related transcription factor signalling such as
681
the JAK/STAT6 signalling pathway. Boswellic acid, a specific and non-redox inhibitor of 5-
682
lipoxygenase enzyme, was revealed to suppress allergic airway inflammation by indirectly
683
abrogating GATA3 expression through inhibition of IL-4 dependent pSTAT6 activity (Liu et
684
al, 2015). Furthermore, in an effort to unveil the determinants of asthma development in
685
atopic subjects with or without asthma and identify asthma associated drug targets in HDM-
686
specific T-helper memory responses, a differential gene network analysis revealed that most
687
asthma-associated genes were enriched with targets of STAT6 signaling (Troy et al., 2016).
688
AS1517499 and YM-341619 are small-molecule inhibitors of STAT6 activity, they were
689
found to modulate asthma symptoms by selectively inhibiting Th2 differentiation (Vale,
690
2016; Nagashima et al., 2009). In another development, Ursolic acid reportedly decreased
691
airway eosinophilia, antigen-induced Penh, pulmonary inflammation, pro-inflammatory
692
cytokine production as well as serum antigen-specific IgE by increasing PPARγ expression
693
and decreasing GATA-3/STAT6 expression through NF-kB pathway modulation (Kim et al.,
694
2013). After all, previous studies revealed that ursolic acid suppresses NF-kB activation by
695
inhibition of IκBα kinase and p65 phosphorylation (Shishodia et al., 2003 and Zeng et al.,
696
2012). More recently, integromics studies revealed GRB2-associated binding protein 1
697
(GAB1) as a novel modulator of asthma due to its NF-kB regulatory activity (Sharma et al.,
698
2015).
699
Control of STAT6 activity can be achieved by JAK modulation. Preclinical studies have
700
validated the suitability of JAKs as a therapeutic target for asthma. A pan-JAK inhibitor,
701
Pyridone 6 (P6), suppressed asthmatic symptoms through Th2 inhibition (Matsunaga et al.,
702
2011). Tofacitinib (CP-690,550), a STAT-6 and pan-JAK inhibitor was suggested to reduce
703
eosinophilia, eotaxins and IL-13 by inhibition of IL-4 signaling pathway in murine model of
704
pulmonary eosinophilia (Kudlacz et al., 2008). However, it is established that tofacitinib
705
enhances pulmonary bacterial growth in models of latent tuberculosis infection, thereby
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23
ACCEPTED MANUSCRIPT posing the risk of reactivation of latent form of tuberculosis in asthma patients with
707
comorbidities (Maiga et al., 2012). It is for this reason that development of safer selective
708
JAK inhibitors was proposed. R256, an inhalable selective JAK/1/3 inhibitor proved to be a
709
safer option over inhibitors of pan-JAKs, due to their selective inhibition of Th2
710
differentiation without affecting Th1 and/or Th17 differentiation (Vale, 2016; Ashino et al.,
711
2014). Figure 4 illustrates the targeted JAK-STAT pathway inhibition as it relates to GATA3
712
and CRTh2 signalling pathways in asthma.
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713 714 715 716 717 718 719
Figure 4: Th2 surface receptor directed therapy. Prevention of airway allergic inflammatory cell migration is achievable through modulation of key surface receptors and downstream targets such as intracellular receptors, enzymes and transcription factors. Interaction of Th2 cytokine such as IL-4 with its receptors (IL-4R) results in activation of the JAK/STAT6 pathway that leads to activation and production of GATA3 protein. Prostaglandin D2 (PGD2) released from mast cells interact with CRTh2 to induce inhibition of adenylyl cyclase (AC) and consequent decrease in intracellular cyclic adenosine monophosphate (cAMP) levels through Gi
24
ACCEPTED MANUSCRIPT 720 721 722 723 724 725 726 727
protein activation. This also results in release of βɣ subunit of the G protein, which directly stimulates phospholipase Cβ (PLCβ) isoforms. PLBβ breakdown phosphatidylinositol-4,5-bisphosphate (PIP2) to produce inositol triphosphate (IP3) which mobilizes Ca2+ from endoplasmic-reticulum stores. The Ca2+ activates calcineurin, which then dephosphorylate NFAT to its active state (NFATc). Transcriptional activation of both GATA3 and NFAT exert a net effect of increased Th2 cytokine production, thereby providing the cytokine milieu that is required for aggravated allergic asthma response in a vicious manner. Inhibition of IL-4R, CRTh2, JAK, STAT-6, GATA3, and cytokine inhibition showed remarkable reduction in chemotaxis, goblet metaplasia, Th1/Th2 imbalance, bronchoconstriction and hyperresponsiveness.
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5.
Conclusion
730
Acute asthma exacerbations are arbitrary, unsettling and detrimental to patient’s quality of
731
life. Prevention of exacerbations through adherence to controller drug regimens, avoidance of
732
exposure to known triggers, annual immunization against influenza and pneumococcal
733
polysaccharide (for asthmatics aged 19 years or older) are imperative. This review has
734
highlighted some potential drug targets for the treatment of asthma. However, few more
735
potential candidates for asthma therapy including calcium sensing receptor (CaSR)
736
antagonist, phosphatidylinositol 3-kinase (PI3K) inhibition through direct inhibitors or
737
indirect inhibitors (e.g. theophylline should also be considered. Asthma symptoms are
738
infrequent, short-lived and mild, occasional administration of a quick-acting bronchodilator to
739
reverse bronchoconstriction is an acceptable approach. However, as symptoms become more
740
frequent or more severe, the emphasis changes to prevention of symptoms (and of asthmatic
741
attacks). Inhaled corticosteroid reduces the frequency of episodic bronchoconstriction and
742
lessens the risk of asthmatic attacks through its anti-inflammatory activities. In low-to-
743
moderate doses, corticosteroids administered by inhalation are safe for long-term use, even in
744
young children.
745
Whilst considerable advancement in the understanding pathophysiology of asthma promoted
746
the discovery of novel therapeutic targets, only few are successfully translated into clinical
747
application. The heterogeneous nature of asthma calls for multifaceted approach to its
748
treatment, thus, further investigations into potential therapies are paramount. On a general
749
note, the pleitrophic nature of kinases and their ability to modulate wide array of cellular
750
events increases risk of adverse effects (e.g haematological disorders and cardiovascular
751
complications) as observed in various protein kinase inhibitors currently used for
752
management of cancer. It is therefore pertinent to apply caution while considering kinase
753
inhibitors for asthma therapy. This may include ensuring minimal side effects by
754
administering low doses of inhalable preparations.
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ACCEPTED MANUSCRIPT Most of the candidate drugs discussed in this article have shown potential to fit into stepwise
756
asthma management strategy taking into account the patient’s response to available
757
medication, onset of the disorder (childhood or adult onset asthma), effectiveness and safety
758
of the candidate drug with respect to patient’s asthma endotytpe. Administration of TLR
759
agonist is suitable during ICS step down treatment in patients on medium to high dose ICS
760
whose asthma has been successfully controlled. this is because TLR agonist may contribute to
761
sustaining asthma control during such steroid reduction regime, thereby preventing sudden
762
relapse. The use of CRTh2 antagonist may best be indicated at stages where patients respond
763
poorly to ICS monotherapy (step 2 to 4 of GINA treatment guidelines) and in ICS naïve
764
asthmatics as add on therapy to reliever medications. Additionally, CRTh2 targeted therapies
765
may be more effective in Th2-high asthma subphenotype. The combined bronchodilatory and
766
anti-inflammatory potential of dual PDE3/4 inhibitors may enhance its use as LABA
767
replacement or as add-on therapy to reduced dose of ICS/LABAs controller medication,
768
thereby complementing safety concerns related to long term use of LABAs. While most
769
biologics based interventions will best be administered to patients with uncontrolled
770
persistent asthma that failed to respond to standard therapy. This may include the
771
administration of such biologics as add on therapy to ICS or oral corticosteroids (OCS) to
772
relieve severe recurrent asthma exacerbations
773
Acknowledgment
774
This work was supported by National Key Economic Area Research Grant Scheme
775
(NRGS/NH1014D026), provided by the Ministry of Agriculture, Malaysia.
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Table 1: Summary of emerging molecular targets for the treatment of asthma and developmental stage
Drug name
Highest Development phase
Receptor Modulators
CRTH2 antagonist
OC000459
2
CRTH2 antagonist
BI-671800
2
CRTH2 antagonist
ARRY-502
CCR3 receptor blocker
TPI ASM8
TLR-3 antagonists
CNTO 3157
urotensin II receptor antagonist
GSK1440115
Oxagen Ltd
Barnes et al., 2012 NCT01057927 Hall et al., 2015 NCT01103349
2
Array BioPharma Inc.
Wenzel et al., 2014 NCT01561690
2
Pharmaxis,
Gauvreau et al., 2008; Gauvreau et al., 2011b NCT00264966/ NCT01158898
1
Janssen Research and Development, LLC
NCT01704040
1
GlaxoSmithKline
Portnoy et al., 2013 NCT01202214
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Reference/NCT number
Boehringer Ingelheim
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Sponsor
RI PT
Mechanism of action
SC
Therapeutic Class
27
ACCEPTED MANUSCRIPT
Mechanism of action
Drug name
Highest Development phase
Enzymatic Targets
PI3K/mTOR inhibitor
GSK2269557
1
PDE4 inhibitor
GSK256066
2
PDE4 inhibitor
CHF 6001 (inhaled)
2
PDE4 inhibitor
roflumilast
PDE4/3 inhibitor
RPL 554
Leukotriene A4 hydrolase inhibitor
JNJ-40929837
IL-5Rα mAb
IL-5 mAb IL-5 mAb
Reference/NCT number
Stark et al., 2015 NCT02294734
GlaxoSmithKline,
Singh et al., 2010 NCT00380354
Chiesi Farmaceutici S.p.A.
Matera et al., 2014 NCT01689571
2
Takeda; Nycomed and Pfizer
NCT01765192/ NCT01365533
2
Verona Pharma
NCT02427165
2
Johnson and Johnson Pharmaceutical Research and Development, L.L.C.
Barchuk et al., 2014 (NCT01241422)
Benralizumab
2/3
AstraZeneca
Nowak et al., 2015 NCT01914757
Reslizumab
3
Teva Pharmaceutical Industries
Castro et al., 2015 NCT01287039/ NCT01285323
Mepolizumab
3
GlaxoSmithKline
Ortega et al., 2014 NCT01691521
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SC
GlaxoSmithKline
AC C
Anti-interleukin agents
Sponsor
RI PT
Therapeutic Class
28
ACCEPTED MANUSCRIPT
Highest Development phase
IL-13 mAb
Lebrikizumab
2
IL-13 mAb
Tralokinumab
3
IL-4Rα mAb
Dupilumab
2
IL-17RA
Brodalumab
1
29
Reference/NCT number
Genentech
Hanania et al., 2015 NCT01545440/ NCT01545453
AstraZeneca
Brightling et al., 2015 NCT02281357
Sanofi and Regeneron Pharmaceuticals Inc.
Wang et al., 2015 NCT01854047
Amgen Kyowa Hakko Kirin Company Limited and AstraZeneca
NCT01902290
Sterna Biologicals GmbH and Co. KG
Homburg et al., 2015 NCT01743768
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SB010
2 (trial terminated during Phase 2)
EP
GATA-3 Transcription DNAzyme Factor NCT: National Clinical Trial
Sponsor
RI PT
Drug name
SC
Mechanism of action
AC C
Therapeutic Class
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Table 2: Summary of adverse effects of selected anti-asthma candidate drugs as revealed by clinical trials
Mechanism of action
Route of administration
Adverse effects
Reference/Trial number
CYT003
TLR-9 Agonist
Subcutaneous
Injection site reactions, Pyrexia Nasopharyngitis, Head ache.
Casale et al., 2015, NCT01673672
QbG10
TLR-9 Agonist
Subcutaneous
BI 671800
CRTH2 antagonist
Oral
H4R antagonist
SC M AN U
Oral
Oral
Toxic hepatitis in patients with prior elevated hepatic transaminases♯
Hall et al., 2015, NCT01092148 and NCT01103349
Headache♯
Sidharta et al., 2014, EudraCT number 2006-006777-25 Diamant et al., 2014, EudraCT number 2008-001209-41
Drug-induced agranulocytosis*
Murata et al., 2015, NCT01493882, NCT01497119
Headache♯, nausea, diarrhoea, asthenia and dyspepsia
Bateman et al., 2016, NCT01765192. Chervinsky et al., 2015, NCT00073177, NCT00076076, NCT00163527 Gauvreau et al., 2011a, NCT01365533
♯
Roflumilast
PDE-4 inhibitor
AC C
EP
JNJ 39758979
CRTH2 antagonist
Beeh, et al., 2013, NCT00890734
Injection site reaction
TE D
Setipiprant,
RI PT
Candidate drug
Oral
30
ACCEPTED MANUSCRIPT
Mechanism of action
Route of administration
Adverse effects
RI PT
Candidate drug
Reference/Trial number Franciosi et al., 2013, EudraCT, numbers 2008005048-17, 2011-001698-22, 2010-023573-18, and 2012000742-34.
Dual PDE-3 and PDE-4 inhibitor
Inhalation
Headache♯, Palpitations, Paraesthesia. Rhinorrhoea, Somnolence
Dupilumab
IL-4Rα antagonist
Subcutaneous
Upper respiratory tract infection, Headache, Injection-site erythema, Sinusitis, Influenza
Wenzel et al., 2016, NCT01854047, EudraCT number 2013-000856-16
Lebrikizumab
IL-13 inhibitor
Subcutaneous
Injection site reaction, Injection-site erythema, Upper respiratory tract infection
Hanania et al., 2015, NCT01545440, NCT01545453
Headache, Injection-site reaction, nasopharyngitis, cough, arthralgia
Pouliquen et al., 2015, NCT01366521 Ortega et al., 2014, NCT01691521
Intravenous/Subcutaneous
Upper respiratory tract infections, headache, nasopharyngitis myalgia, oropharyngeal pain, anaphylaxis and sinusitis
Corren et al., 2016, NCT01508936 Castro et al., 2015 NCT01287039/ NCT01285323 Markham, 2016, NCT02452190, NCT02501629, NCT02559791
Subcutaneous
Injection site erythema, upper respiratory tract infection, sinusitis, nasopharyngitis and oral candidiasis
Busse et al., 2013, NCT01199289
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IL-5 inhibitor
Intravenous/Subcutaneous
IL-5 receptor antagonist
Brodalumab
IL-17 receptorA antagonist
AC C
Reslizumab
EP
Mepolizumab
SC
RPL554
31
ACCEPTED MANUSCRIPT
Adverse effects
Reference/Trial number
Subcutaneous
Injection-site reaction, upper respiratory tract infections, headache, migraine, sinusitis and asthenia
Holgate et al., 2011, NCT00141791
Pneumonia*♯, cellulitis, sepsis, upper respiratory tract infections, chest pain, sinusitis, nausea, and injection-site erythema
Wenzel et al., 2009, NCT00207740
Golimumab
TNF-α inhibitor
Subcutaneous
SB010
GATA-3 DNAzyme
Inhalation
RI PT
TNF-α inhibitor
Route of administration
SC
Etanercept
Mechanism of action
M AN U
Candidate drug
Mild oropharyngeal pain, back pain and bronchospasm♯
Homburg et al., 2015, NCT01743768, NCT01577953
AC C
EP
TE D
*Serious Adverse event, ♯Treatment emergent adverse event, EudraCT: European Clinical Trials Database, NCT: National Clinical Trial
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Al-Hajjaj, M. S. (2011). Difficult-to-treat asthma, is it really difficult? Annals of Thoracic Medicine, 6(1), 1. http://dx.doi.org/10.4103/1817-1737.39633 Ali, A. K., and Hartzema, A. G. (2012). Assessing the association between omalizumab and arteriothrombotic events through spontaneous adverse event reporting. Journal of Asthma and Allergy, 5(1), 1-9. http://dx.doi.org/10.2147/jaa.s29811 Allard, B., Bara, I., Gilbert, G., Carvalho, G., Trian, T., Ozier, A., ... and Berger, P. (2014). Protease activated receptor-2 expression and function in asthmatic bronchial smooth muscle. PloS ONE, 9 (2), e86945. http://dx.doi.org/10.1371/journal.pone.0086945
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Antoniu, S. A. (2009). Cytokine Antagonists for the Treatment of Asthma. BioDrugs, 23 (4), 241-251. http://dx.doi.org/10.2165/11317130-000000000-00000
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Antoniu, S. A. (2010). Pitrakinra, a dual IL-4/IL-13 antagonist for the potential treatment of asthma and eczema. Current Opinion in Investigational Drugs (London, England: 2000), 11 (11), 1286-1294. http://dx.doi.org/10.1517/14712598.2010.524203
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