key: cord-295156-trzkb9ne
authors: Cheong, Dorothy H.J.; Daniel Tan, W.S.; Fred Wong, W.S.; Tran, Thai
title: Anti-malarial drug, artemisinin and its derivatives for the treatment of respiratory diseases
date: 2020-05-13
journal: Pharmacol Res
DOI: 10.1016/j.phrs.2020.104901
sha: 
doc_id: 295156
cord_uid: trzkb9ne

Artemisinins are sesquiterpene lactones with a peroxide moiety that are isolated from the herb Artemisia annua. It has been used for centuries for the treatment of fever and chills, and has been recently approved for the treatment of malaria due to its endoperoxidase properties. Progressively, research has found that artemisinins displayed multiple pharmacological actions against inflammation, viral infections, and cell and tumour proliferation, making them effective against diseases. Moreover, it has displayed a relatively safe toxicity profile. The use of artemisinins against different respiratory diseases has been investigated in lung cancer models and inflammatory-driven respiratory disorders. These studies revealed the ability of artemisinins in attenuating proliferation, inflammation, invasion, and metastasis, and in inducing apoptosis. Artemisinins can regulate the expression of pro-inflammatory cytokines, nuclear factor-kappa B (NF-κB), matrix metalloproteinases (MMPs), vascular endothelial growth factor (VEGF), promote cell cycle arrest, drive reactive oxygen species (ROS) production and induce Bak or Bax-dependent or independent apoptosis. In this review, we aim to provide a comprehensive update of the current knowledge of the effects of artemisinins in relation to respiratory diseases to identify gaps that need to be filled in the course of repurposing artemisinins for the treatment of respiratory diseases. In addition, we postulate whether artemisinins can also be repurposed for the treatment of COVID-19 given its anti-viral and anti-inflammatory properties.

Artemisinins are sesquiterpene lactones with a peroxide moiety that are isolated from the herb Artemisia annua. It has been used for centuries for the treatment of fever and chills, and has been recently approved for the treatment of malaria due to its endoperoxidase properties. Progressively, research has found that artemisinins displayed multiple pharmacological actions against inflammation, viral infections, and cell and tumour proliferation, making them effective against diseases. Moreover, it has displayed a relatively safe toxicity profile. The use of artemisinins against different respiratory diseases has been investigated in lung cancer models and inflammatory-driven respiratory disorders. These studies revealed the ability of artemisinins in attenuating proliferation, inflammation, invasion, and metastasis, and in inducing apoptosis. Artemisinins can regulate the expression of pro-inflammatory cytokines, nuclear factor-kappa B (NF-κB), matrix metalloproteinases (MMPs), vascular endothelial growth factor (VEGF), promote cell cycle arrest, drive reactive oxygen species (ROS) production and induce Bak or Bax-dependent or independent apoptosis. In this review, we aim to provide a comprehensive update of the current knowledge of the effects of artemisinins in relation to respiratory diseases to identify gaps that need to be filled in the course of repurposing artemisinins for the treatment of respiratory diseases. In addition, we postulate whether artemisinins can also be repurposed for the treatment of COVID-19 given its anti-viral and anti-inflammatory properties.

ATP-binding cassette subfamily member 2 ACE2

angiotensin-converting enzyme 2 AEC alveolar epithelial cells AHR airway hyperresponsiveness AIF apoptosis-inducing factor ALI acute lung injury AP- 

Respiratory diseases refer to any disease or disorder of the airways and the lungs that interfere with respiration [1] . The respiratory system, comprising of the nose, nasal cavities and the lung, is the sole internal system that is exposed to the external environment. Hence, it is easily susceptible to environmental agents (such as bacterial or viral infections, smoking, air pollution or cold weather [2] ), that can cause respiratory diseases. Importantly, chronic respiratory diseases are a great cause of J o u r n a l P r e -p r o o f concern as approximately one billion people suffer from them while four million succumb to these illnesses prematurely every year [2] . The Forum of International Respiratory societies identified chronic obstructive pulmonary disorder (COPD), asthma, acute respiratory infections, and lung cancer as the top few respiratory diseases that heavily burden society [2] . In brief, COPD is an obstructive lung disease characterized by long term breathing issues and poor airflow. It affects 200 million people and is the fourth leading cause of death worldwide [3] . It is currently being treated using inhaled bronchodilators and glucocorticoids [4] . Asthma is a condition in which the airways narrow and swell, and is accompanied by increased mucus production. It affects 300 million people worldwide [5] . The cause of asthma is still unknown, and it is furthermore uncurable. Treatment is limited to symptom relief using inhaled corticosteroids and bronchodilators [5] . Acute respiratory infections include pneumonia and viral respiratory infections. Annually, respiratory tract infections like influenza kill 250,000 to 500,000 people and cost billions of dollars. In addition, it occasionally causes epidemics that threaten the health of the global population [6] . Lung cancer is a malignant lung tumour that is characterized by uncontrolled cell growth in the lung tissues, and this growth can spread to other parts of the body, causing death. It has the highest fatality rate amongst the major cancers, killing more than 1.4 million people a year [7] . Patients are diagnosed and their disease is classified into different stages where earlier-stage patients are treated with surgery to remove the lung tumour, while late-stage patients are treated with chemotherapy or radiotherapy but often succumb to the disease [2] . Currently, the global approach to managing respiratory diseases is to provide better healthcare, reduce environmental pollution, and to create public awareness of the prevalence and risks of such diseases. Research in this field explores the causes of these respiratory diseases, prognostic markers to better diagnose patients and new therapies that can maintain and contain the disease [2] . Nonetheless, a lot more work will need to be done to find safer and more effective treatment methods.

Natural products have been used to treat respiratory diseases as far back as 2600BC with the first records indicating that oils of Cedrus (cedar), Commiphora (myrrh), Cupressus sempervirens (cypress)

and Glycyrrhiza glabra (licorice) were being used to treat inflammation, coughs, and colds [8] . Male newborns of the Indian tribes of southern California were bathed in hot Salvia ashes as it was believed to provide lifetime immunity from all respiratory diseases [9] . In 1952, erythromycin, derived from Saccharopolyspora erythraea, was launched commercially for bacterial infections affecting the upper J o u r n a l P r e -p r o o f respiratory tract [10] . Umckaloabo contains root extract of Pelargonium sidoides and was marketed in 1897 against tuberculosis but was later superseded by antibiotics. In the 2000s, it regained popularity for the treatment of acute bronchitis and is now one of the most commonly prescribed childhood medications [11] . Today, many natural-based products are still being investigated for its beneficial properties against respiratory diseases. In this review, we will provide a comprehensive update of the current knowledge of artemisinin, and its derivatives, for the treatment of various respiratory diseases.

Artemisinin is a sesquiterpene lactone with a peroxide constituent [12] . It is isolated from the leafy parts of Artemisia annua, a herb and medicinal plant that has been used for the treatment of chills and fever for centuries [13] . In the 1960s, the search for new anti-malarial drugs began in lieu of the increasing resistance of Plasmodium falciparum to chloroquine. Artemisinin, also known as Qinghaosu, was first isolated. Dihydroartemisinin (DHA) was subsequently the first generation of derivatives, made by modifying the carbonyl groups into hydroxyl groups [12] . Others like the more water-soluble artesunate and more oil-soluble artemether and arteether followed [14] . These derivatives were ten times more potent than artemisinin [12] , with artesunate having a more favourable pharmacokineticpharmacodynamic profile [15] . They are also more easily produced [12] (Table 1) . Artemisinins and its derivates are selectively taken up by parasites-infected erythrocytes and later localized in the parasite membranes, including that of the mitochondria, digestive vacuole and the parasite limiting membrane [12, 14] . All forms of the drug contain an endoperoxide bridge (C-O-O-C) that is crucial for its antimalarial activity, where the compound itself is catalyzed by heme or iron to form free radicals. These free radicals then alkylate malaria membrane-associated proteins, killing the parasite [14] . Artemisinin and its derivatives are found to be effective against different severities of malaria, especially those resistant to traditional gold standard drugs. They are highly efficacious, requiring only nanomolar concentrations in vitro [14] . They are also fast-acting, showing therapeutic potential as early as 20 hours after administration. Moreover, artemisinins display a relatively safe toxicity profile, with the LD50 being 4223 mg/kg. In addition, whilst there was some evidence for neurotoxicity in neuronal cells and animals at high dosages, this was never reported in humans despite the wide usage of the drug [12, 14] .

Apart from its anti-malarial effects, artemisinin and its derivatives also exhibited additional properties in other diseases. For example, artesunate had anti-cancer effects as shown by its cytotoxic activity against 55 cancer cell lines through its regulation of various processes, including DNA damage and repair, apoptosis, and proliferation [16, 17] . Artesunate displayed anti-inflammatory properties, as seen by its attenuation of the production of interleukin (IL)-1β, IL-6 and IL-8 in tumour necrosis factor (TNF)α-stimulated rheumatoid arthritis fibroblast-like synoviocytes (RA FLS) via the regulation of NF-κB and phosphoinositide 3 kinase (PI3K) pathways [18] . It also displayed anti-viral properties where artemisinin inhibited the replication of human cytomegalovirus (HCMV) through a reduction in the DNA binding activity of NF-κB and Sp1, and subsequently downstream activities of Akt1 and p70S6K. [19] . Many of these pathophysiological processes are also present in respiratory diseases. Thus, artemisinin and its derivatives could potentially be repurposed for the treatment of respiratory diseases as well.

The effects of artemisinin and its derivatives have been examined in various in vitro models (Tables 2-5 ) and these include: inhibition of cell proliferation; inductions of cell cycle arrest and apoptosis;

inhibition of inflammation and oxidative stress; inhibition of angiogenesis, invasion and metastasis, and chemosensitization of cancer cells to chemotherapeutic agents.

The anti-proliferative effect of artemisinin and its derivatives are observed in a variety of lung cancer cell lines, including the non-small cell lung cancer (NSCLC) cell lineslung adenocarcinoma A549 [20] [21] [22] [23] [24] , PC-9 [21] , PC-14 [25] , H1299 [22] , ASTC-a-1 [26] [27] [28] and Spc-A-1 [29] cells, squamous carcinoma SK-MES-1 cells and large cell lung cancer NCI-H661 cells [29] . Interestingly, one study noted that low concentrations (1.25-5 μg/L) of artesunate were unable to prevent the proliferation of A549 cells [30] , suggesting that there may be a therapeutic window by which the drug would have anti-proliferative effects.

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In addition, artesunate was found to have anti-proliferative effects in non-cancer cell types. Pretreatment with artesunate reduced mitogen-stimulated increases in cyclin D1 protein expression and cell number in both asthmatic and non-asthmatic human cultured airway smooth muscle (ASM) cells.

This effect was mediated by reductions in p-Akt and p-p70S6K protein expressions, which were not observed with dexamethasone treatment [31] . In HCMV, artesunate, but not ganciclovir, reduced the proliferation rates of infected human embryonic lung fibroblasts (HELF) [32] . It is worthy to note that they did not have cyotoxic effects on healthy, non-diseased cells, such as in normal human lung fibroblast WI-38 cells [21, 33] , non-cancerous human dermal fibroblasts CCD-1108Sk cells [34] and normal hepatic L-02 cells [35] . This finding lends support for the desired clinical property of arteminismins in regimes where inhibiton of cell growth is needed (such as in the cancer setting) without affecting the healthy, non-disease state condition.

Various studies have shown that artemisinins induce cell cycle arrest at different phases in lung and nasopharyngeal cancer. Artemisinin, artesunate, and DHA inhibited cell proliferation in A549 and H1299 cells via cell cycle arrest in the G1 phase [22] , with corresponding downregulation of p-Akt, p-glycogen and H1299 cells [34] . Collectively, these studies show that whilst artemisinins bring about cell cycle arrest, caution should be taken into the study of artemisinins on cell cycle arrest as they affect different stages of the cell cycle and the effects may be cell-type dependent.

The apoptotic effects of artemisinins and its derivatives are largely observed in lung cancer cell lines and have been found to induce both the intrinsic and extrinsic pathways of apoptosis (Table 3) .

DHA induces apoptosis in A549 and PC-9 cells. The glycolytic metabolism was attenuated, together with the inhibition of glucose uptake, and lactate and ATP production. DHA treatment also reduced the levels of p-S6 ribosomal protein, p-mammalian target of rapamycin (mTOR), and glucose transporter (GLUT)1. These effects were enhanced together with the glycolysis inhibitor 2-Deoxy-D-glucose (2DG), inducing apoptosis through the activation of caspases 3, 8, and 9, cytochrome c and apoptosis-inducing factor (AIF), without raising ROS levels [21] . Other studies found that artemisinins and its derivatives 

Interestingly, Xiao and colleagues observed that blockade of caspases 8 or 9, but not caspase 3, largely inhibited the pro-apoptotic effects of artemisinin [26, 27] . In contrast, Gao and colleagues found that silencing either caspase led to almost no activation of all three caspases, suggesting the role of an amplification loop among these caspases [45] . In the latter, there was no loss of mitochondrial membrane potential and cytochrome c release, but an activation of Smac and AIF release [45] .

Artesunate similarly induced ROS-mediated apoptosis through the release of Smac and AIF, but this was accompanied by the loss of mitochondrial membrane potential. Here, inhibiting caspases 8 or 9 did not have any effect whilst silencing AIF did prevent artesunate-induced apoptosis [44].

Certain differences regarding the apoptotic effects involving Bak and Bax were uncovered. Xiao and colleagues found that silencing Bax and Bak by RNAi did not have any effect on artemisinin-induced apoptosis, suggesting a Bax/Bak-independent apoptotic process [26, 27] . However, others observed that silencing Bak, but not Bax inhibited artesunate-induced apoptosis and AIF release. In fact, artesunate was found to only activate Bak, not Bax [44, 45] . On the other hand, silencing pro-apoptotic

Bax, but not Bak hampered DHA-induced apoptosis [39] .

Interestingly, whilst artesunate treatment in ASTC-a-1 and A549 cells did not induce a significant levels of ferrous ion and endogenous oxidation stress in A549 cells [35] . Together, these studies show that artemisinins induce apoptosis but utilize very different pathways to induce apoptosis even within the same cell lines itself.

Several studies have indicated the anti-inflammatory effects of artemisinins in vitro with most studies mainly in vivo (Table 4 ). BEAS-2B cells were found to be insensitive to dexamethasone after being exposed to cigarette smoke extract (CSE) and TNF-α stimulation. Treatment with artesunate was able to reverse this effect and restore HDAC2 deactivation that was induced by CSE [50] . BEAS-2B cells Artemisinin-daumone hybrid 15 (ARTD) was able to inhibit the invasion and metastasis of A549 cells, coupled with downregulation of E2F transcription factor 1 (E2F1) and hepatocyte nuclear factor 4 alpha (HNF4A), and upregulation of tumour-suppressive activating transcription factor 3 (ATF3) [58] . These show that artemisinins have the potential to impair angiogenesis and metastasis, but its effects were largely explored in the lung cancer setting. Thus, it would be interesting to see if similar effects are observed in other respiratory diseases that are implicated with angiogenic and metastatic events.

Multiple studies have shown that artemisinin and its derivatives could chemosensitize other drugs. 10 µg/ml of DHA and 10 µg/ml of doxorubicin was found to be the most optimal concentrations that could reduce A549 cell viability [59] . DHA promoted the cytotoxic and apoptotic levels of carboplatin in LLC cells via the phosphorylation of p38 [36] . DHA together with ABT-263 could activate Bax-dependent apoptosis in NSCLC cells. This was because DHA induced downregulation of survivin and an upregulation of Bim, contributing to cotreatment-induced cytotoxicity. Also, DHA downregulated Mcl-1 expression which is responsible for drug resistance to ABT-263. This anti-tumour effect was also observed in vivo on H1975 xenograft growth in nude mice [48] . Similarly, DHA also upregulated Bax expression in the presence of gefitinib in H1975 cells, alongside an attenuation of p-Akt, p-mTOR, psignal transducers and activators of transcription (STAT)3 and Bcl-2 to prevent migration and invasion [38] . Surprisingly, the JNK inhibitor SP600125 synergistically promoted DHA-induced cell apoptosis in A549 and ASTC-a-1 cells by activating Bax translocation, mitochondrial membrane depolarisation, cytochrome c release and caspase 3 and 9, unlike its usual anti-apoptotic function that suppresses c-Jun N-terminal kinase (JNK) and Bax [60] . DHA also interestingly promoted dictamine-induced apoptosis via a caspase 3-mediated pathway in A549 cells, even though dictamine alone induces S phase cell cycle arrest at low concentrations and cell apoptosis at higher concentrations without the J o u r n a l P r e -p r o o f involvement of caspases or mitochondria [61] . DHA and cisplatin ablated cell proliferation and induced apoptosis in both A549 and cisplatin-insensitive A549/DDP cells [56] . DHA could also reverse the high resistance of A549 cells to arsenic trioxide to reduce cell viability and promote cell death via higher levels of ROS and DNA damage, with no adverse effects on normal human bronchial epithelial cells [62] . On the contrary, apoptosis triggered by a combination of DHA and gemcitabine in A549 cells was not associated with additional generation of ROS as compared to either treatments alone. Instead, the combination strongly activated both the Bak-mediated intrinsic apoptosis pathway as well as the Fascaspase 8-mediated extrinsic apoptosis pathway [63] . Moreover, DHA can enhance radiosensitization in GLC-82 lung cancer cells, inducing apoptosis with heightened expressions of p53 and p21, and lowered expression of Bcl-2 [37] . DHA coupled with a low dose of ionizing radiation led to irreparable G2/M phase cell cycle arrest as well as apoptosis due to ROS generation and the activation of caspases 3 and 8 [39] . In SCLC, pre-treatment with transferrin sensitized the multi-resistant H69VP phenotype to artemisinin as they had double the number of transferrin receptors. This combination induced DNA fragmentation and apoptosis [64] . Treating A549 cells with chloroquine prior to artesunate treatment synergistically promoted cell death, where an increase in the sub-G1 population of cells was observed, and the build-up of acidic vacuoles and ROS resulted in cytochrome c release followed by caspase 3mediated apoptosis [33] . Conversely, artesunate did not induce A549 cell apoptosis when administered alone or in the presence of local radiotherapy. Instead, it induced G2/M phase cell cycle arrest, with heightened NO protein, and lessened cyclin B1 and cdc2 mRNA expression [40] . All these studies show that various therapies can be used in conjunction with artemisinins to promote its therapeutic effectiveness.

The effects of artemisinins in in vivo models are summarized in Tables 2-5 and these include disease models of pulmonary fibrosis, acute lung injury (ALI), asthma, COPD, lung cancer, and NPC. In general, the underlying mechanisms implicated in these models include inhibition of oxidative stress, inflammation, airway remodelling features, and tumour formation. Artemisitene reduced bleomycin-induced acute inflammatory responses through the activation of the Nrf2 pathway, as seen by a reduction in the total number of inflammatory cells, neutrophils, macrophages and lymphocytes, together with lower IL-4, IL-6, tumomur growth factor (TGF)β and monocyte chemoattractant protein-1 (MCP-1) mRNA expressions [66] . Artesunate treatment attenuated lung injury in paraquat-intoxicated rats via reductions in TGFβ1, IL-10 and TNF-α [70] . 30 mg/kg of artesunate suppressed total, eosinophil and neutrophil inflammatory cell counts as well in an OVAinduced model of allergic asthma [68] . It also reduced IL-8 levels and total inflammatory and neutrophil cell counts that were increased in a 40 days cigarette smoke-induced lung oxidative damage mouse model. IL-8 levels were similarly lowered by artesunate in 16HBE cells exposed to cigarette smoke extract [69] . Interestingly, the activation of NLRP3 inflammasome was dependent on pulmonary ROS generation accompanied by higher ASC and caspase 1 levels [53]. In another study, Liu and colleagues also found that artesunate suppressed many RIR-stimulated factors involved in lung inflammation, including the production of serum and pulmonary NO, MDA, macrophage inflammatory protein 2 (MIP-2) and

prostaglandin E2 (PGE2), and attenuated NF-κB translocation [72] . Artesunate also protected against sepsis-induced lung injury by reducing IL-6 and TNF-α levels in both the serum and BALF. In the lung tissues, artesunate suppressed cyclooxygenase-2 (COX-2), iNOS and NF-κB levels and activated Nrf2 through and increase in HO-1 expression and enzymatic activity [73] . The effect of artesunate on COPD was similar to that of ALI where artesunate dose-dependently suppressed total and differential 

Whether or not changes in inflammation brought about by artesunate was associated with changes in lung function parameters have been reported by one group. Here, artesunate treatment in mice exposed to cigarette smoke and OVA saw a reduction in methacholine-induced AHR, with efficacies similar to the extent produced by dexamethasone [50] . Both artesunate and DHA treatment in an OVA-induced model of allergic asthma brought about a reduction in AHR [74, 75] . Another study found that 120 µg of artesunate relieved OVA-induced airway resistance with comparable efficacy to 3 µg of salbutamol through an increase in [Ca 2+ ]i and reduced traction force in airway smooth muscle cells, mediated by bitter taste receptor signaling [78] . However, the concentration and dose used in this study are high;

suggesting the need to explore whether the same effects could be observed at the lower therapeutic range. Artemisinin was also able to improve the behavior scores (sneezing, nasal rubbing) in a mouse model of allergic rhinitis, where mice were given nasal drip of 500 µg of OVA [77] .

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Artemisitene inhibited bleomycin-induced collagen and hydroxyproline expression in mice. The expression of key players of fibrosis, smooth muscle (sm)-α actin and TGFβ were also reduced in bleomycin-treated mice [66] . Similar observations were made with DHA, which reduced the Szapiel fibrotic score and hydroxyproline content with comparable efficacy to dexamethasone in bleomycininduced pulmonary fibrosis in rats [79] . Another study also showed that DHA treatment reduced interstitial fibrosis, leukocyte infiltration, collagen deposition and sm-α actin expression in lung tissues with heightened E-cadherin expression. The reduction in sm-α actin, normally heightened in the event of oxidative stress, was also seen in DHA-treated rat alveolar epithelial cells (AECs) cultured in hypoxia, which shows that DHA could inhibit the hypoxia-induced increase in myofibroblastic-like process [65] .

Artesunate attenuated bleomycin-induced pulmonary fibrosis in Sprague Dawley rats through a reduction in pro-fibrotic proteins such as TGFβ1, Smad3, heat shock protein 47 (hsp47), sm-α actin, and collagen I [80] . The same group also observed that artesunate upregulated MMP2 and MMP9 expressions while reducing tissue inhibitor of metalloproteinases (TIMP) and TIMP2 levels, which then contributed to a decrease in collagen IV protein expression, which is otherwise heightened in bleomycininduced pulmonary fibrosis [81] .

Artemisinin, artesunate, and DHA inhibited processes that contribute to tumour malignancy, including migration, invasion, cancer stem cells and epithelial-mesenchymal transition (EMT) transition. This was through attenuation of the Wnt/β-catenin pathway that contributes to tumour cell proliferation and malignancy, as seen by a reduction in Wnt5-a/b protein level and a simultaneous increase in naked cuticle homolog 2 (NKD2) and axis inhibition protein 2 (Axin2) that eventually led to a drop in β-catenin levels [22] . Artesunate post-treatment also reportedly prevented TGFβ1-induced EMT in RLE-6TN alveolar epithelial cells by reducing p-Smad3 and Smad3 and upregulating Smad7 protein expressions [82] . Artesunate impaired tumour growth and metastasis in a chicken embryo metastasis model, secreted levels of MMP9 and cathepsin K that contribute to the bone-resorbing activity [58] . In addition, oral administration of artemisinin inhibited lymph node and lung metastasis, with no effect on tumour growth in a LLC mouse model, promoting longer survival. Tumour lymphangiogenesis was also inhibited, with corresponding reduction in VEGF-C levels [57] . Interestingly, studies done by two different groups found that a combination of DHA with either cisplatin or onconase could more effectively ablate the density of the microvasculature and microvessels in an A549 mouse xenograft model [29, 56] .

Studies by Wong and colleagues found that artesunate mitigated mucus hypersecretion via a reduction in muc5ac mRNA expression in the lung tissues of OVA-challenged asthmatic mice [74, 75] . Whether or not similar effects on mucus production and muc5ac expression can be observed using a more clinically relevant allergen such as house dust mite remains to be observed. Unfortunately, not much research has looked at the effect of artemisinins on mucus production and alleviating it would be beneficial since excessive mucus production occurs in many lung diseases and impede on patients' comfort levels.

Tong and colleagues observed that artemisinin, DHA and artesunate were all able to reduce tumour growth in an A549-induced mouse xenograft model via inhibition of the Wnt-5a/b/β-catenin signaling pathway [22] . Artesunate dose-dependently attenuated A549 xenograft growth in mice with a reduction in EGFR, Akt and ATP-binding cassette subfamily member 2 (ABCG2) mRNA and protein expressions [83] . In addition, artesunate radiosensitized tumour cells to the effects of local radiotherapy [40] .

Conversely, another group found that 10 mg/kg of artesunate was not sufficient to inhibit A549-induced xenograft growth in mice, although it could potentially block invasion as observed by a reduction in J o u r n a l P r e -p r o o f ICAM-1 and MMP9 protein abundance [30] . Unexpectedly, oral administration of either DLAe or artesunate was able to inhibit A549 xenograft growth but only DLAe was able to inhibit PC-9 induced tumour growth [34] . The tumour-inhibiting rate of DHA as studied in nude mice bearing A549 cells was 54.3% [24] . A combination of DHA and ABT-263 reduced xenograft growth in nude mice [48] . Using an acute model of allergic asthma in mice, artesunate pre-treatment was found to reduce the area of smα actin positive cells in the airways and cyclin D1 protein expression [31] .

Currently, only one study has investigated the use of artesunate for lung cancer in humans. Adding on 120 mg/day of artesunate treatment to vinorelbine and cisplatin chemotherapy was found to promote better disease control and slow the time to disease progression as compared to advanced stage NSCLC patients treated with vinorelbine and cisplatin chemotherapy alone. However, no significant differences to short term survival rate, mean survival time and one-year survival rates were observed. Importantly, this treatment combination did not produce significant toxic effects [84] .

The recent Coronavirus Disease 2019 (COVID-19) pandemic has affected and taken many lives [85] .

Since vaccines against the novel SARS-CoV-2 virus, which causes COVID-19, may take a long time to be developed, many are repurposing drugs for its treatment. Chloroquine (CQ) and hydroxychloroquine (HCQ) are anti-malarial drugs being tested for COVID-19 [86, 87] that have also been used against autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus (SLE). Whilst HCQ has displayed a safer toxicity profile than CQ [86] , there are still side effects that are of concern.

One example is cardiac toxicity, which would be especially dangerous for patients with pre-existing health conditions, like that of cardiovascular diseases, as they would have poorer prognosis for COVID-19 [88] . Whilst the anti-malarial mode of action of artemisinins are different from CQ or HCQ, their immunomodulant effects against inflammatory disorders and viral replications are overlapping.

Traditionally, artemisinins have been used for the treatment of fevers, and could be useful given that 83.3% of patients with COVID-19 have fever [89] . Given its ability to reduce TNF-α and IL-6, key J o u r n a l P r e -p r o o f mediators of acute respiratory distress syndrome (ARDS) that leads to the worsening of COVID-19 patient conditions [90] , artemisinins may be a promising therapy. Other molecular targets of artemisinin and its derivatives, as shown in Fig. 1 , may also be involved in the pathogenesis of COVID-19 and thus may have other benefits that may not yet be known. Moreover, artemisinins are known to display a safe toxicity profile so higher doses can be prescribed with less worry about potential side effects. With the understanding that CQ and HCQ are affective against viruses due to the pH altering activities that affect viral replication, artemisinins could alternatively be used as adjunct therapy to lower the dose required of CQ or HCQ, and reduce side effects, while also suppressing the cytokine storm. Unfortunately, no study to date has investigated the effects or interactions of artemisinins on the angiotensin-converting enzyme 2 (ACE2) receptor, that is known to be the critical binding cellular receptor of SARS-CoV-2 [91] .

This can greatly influence the favourability of trying out the effectiveness of artemisinins for COVID-19.

Artemisinins have been used for a long time with high efficacies and relatively safe toxicity profiles.

Some groups have looked into modifications to artemisinins in order to improve its efficacy and lower the risk of toxic side effects. DHA was observed to display poor water solubility and short plasma halflife. Dai and colleagues connected DHA with a multiarm polyethylene glycol (PEG) to produce PEG-DHA and found that it was 82 to 163 times more water-soluble and its blood circulation half-time was 5.75 to 16.75 times that of DHA, all while retaining or improving its anti-cancer efficacy [92] . Sun and colleagues encapsulated DHA with gelatin or hyaluronan nanoparticles using an electrostatic field system and observed that it inhibited proliferation and promoted apoptosis of A549 cells better than DHA [93] . DHA loaded with nanostructured lipid carriers (DHA-NLC) resided more greatly in organs such as the lung, liver, spleen, brain, and muscle, and less in the heart and kidneys, promoting sustained-release and better drug-targeted effects, therefore allowing for lower dosages and systemic toxic side effects [94] . A C-10 acetal artemisinin synthesized using the Sonogashira cross-coupling reaction displayed higher growth inhibition of A549 cells compared to artemisinin. However, it only had moderate effects on other cancer cell lines such as breast, prostate, and neuroblastoma [95] . Lastly,

Yang and colleagues noted that transferrin receptors were overexpressed in cancer cells. They, therefore, developed adducts of transferrin with artemisinin, DHA or artesunate and found that their J o u r n a l P r e -p r o o f anti-cancer effects were stronger in A549 cells with improved cellular uptake, whilst having minimal effects on normal human liver HL-7702 cells [96] .

We present here an up-to-date overview of the current knowledge of artemisinins and its derivatives as potential therapeutic targets for the treatment of respiratory diseases. Fig. 1 

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 

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The antimalarial drug artesunate inhibits primary human cultured airway smooth muscle cell proliferation

Human embryonic lung fibroblasts treated with artesunate exhibit reduced rates of proliferation and human cytomegalovirus infection in vitro

Inhibition of autophagy by chloroquine potentiates synergistically anti-cancer property of artemisinin by promoting ROS dependent apoptosis

Dried leaf Artemisia annua efficacy against nonsmall cell lung cancer

Synthesis and in vitro antitumor evaluation of dihydroartemisinincinnamic acid ester derivatives

Dihydroartemisinin sensitizes Lewis lung carcinoma cells to carboplatin therapy via p38 mitogen-activated protein kinase activation

Dihydroartemisinin and gefitinib synergistically inhibit NSCLC cell growth and promote apoptosis via the Akt/mTOR/STAT3 pathway

Ionizing radiation potentiates dihydroartemisinininduced apoptosis of A549 cells via a caspase-8-dependent pathway

Artesunate enhances radiosensitivity of human non-small cell lung cancer A549 cells via increasing NO production to induce cell cycle arrest at G2/M phase

Artemisinin-Daumone Hybrid Inhibits Cancer Cell-Mediated Osteolysis by Targeting Cancer Cells and Osteoclasts

In vitro and in vivo inhibition of tumor cell viability by combined dihydroartemisinin and doxorubicin treatment, and the underlying mechanism

The JNK inhibitor SP600125 enhances dihydroartemisinin-induced apoptosis by accelerating Bax translocation into mitochondria in human lung adenocarcinoma cells

Dihydroartemisinine enhances dictamnine-induced apoptosis via a caspase dependent pathway in human lung adenocarcinoma A549 cells

Dihydroartemisinin Sensitizes Human Lung Adenocarcinoma A549 Cells to Arsenic Trioxide via Apoptosis

Synergistic induction of apoptosis in A549 cells by dihydroartemisinin and gemcitabine

Transferrin overcomes drug resistance to artemisinin in human small-cell lung carcinoma cells

Dihydroartemisinin alleviates oxidative stress in bleomycininduced pulmonary fibrosis

Artemisitene activates the Nrf2-dependent antioxidant response and protects against bleomycin-induced lung injury

Untargeted Proteomics and Systems-Based Mechanistic Investigation of Artesunate in Human Bronchial Epithelial Cells

Anti-malarial drug artesunate ameliorates oxidative lung damage in experimental allergic asthma

Effects of artesunate on cigarette smoke-induced lung oxidative damage in mice and the expression of Nrf2 and the possible mechanism

Artesunate Attenuates Lung Injury in Paraquat-Intoxicated Rats via Downregulation of Inflammatory Cytokines

Artesunate Inhibits Renal Ischemia Reperfusion-Stimulated Lung Inflammation in Rats by Activating HO-1 Pathway. Inflammation

Artesunate Protects Against Sepsis-Induced Lung Injury Via Heme Oxygenase-1 Modulation. Inflammation

Anti-malarial drug artesunate attenuates experimental allergic asthma via inhibition of the phosphoinositide 3-kinase/Akt pathway

Dihydroartemisinin suppresses ovalbumin-induced airway inflammation in a mouse allergic asthma model

Dihydroartemisinin Ameliorated Ovalbumin-Induced Asthma in Mice via Regulation of MiR-183C. Medical science monitor : international medical journal of experimental and clinical research

Effect of artemisinin and neurectomy of pterygoid canal in ovalbumininduced allergic rhinitis mouse model

Artesunate attenuates airway resistance in vivo and relaxes airway smooth muscle cells in vitro via bitter taste receptor-dependent calcium signalling

Dihydroartemisinin supresses inflammation and fibrosis in bleomycine-induced pulmonary fibrosis in rats

Anti-profibrotic effects of artesunate on bleomycin-induced pulmonary fibrosis in Sprague Dawley rats

Artesunate modulates expression of matrix metalloproteinases and their inhibitors as well as collagen-IV to attenuate pulmonary fibrosis in rats

Relationship between artesunate influence on the process of TGF-beta1 induced alveolar epithelial cells transform into mesenchymal cells and on idiopathic pulmonary fibrosis

The effects of artesunate on the expression of EGFR and ABCG2 in A549 human lung cancer cells and a xenograft model

Artesunate combined with vinorelbine plus cisplatin in treatment of advanced non-small cell lung cancer: a randomized controlled trial

World Health Organisation. Coronavirus Disease (COVID-19) Pandemic

Hydroxychloroquine and azithromycin as a treatment of COVID-19: results of an open-label non-randomized clinical trial

Candidate drugs against SARS-CoV-2 and COVID-19

Chloroquine and hydroxychloroquine to treat COVID-19: between hope and caution

Clinical characteristics of coronavirus disease 2019 (COVID-19) in China: a systematic review and meta-analysis

Could chloroquine /hydroxychloroquine be harmful in Coronavirus Disease 2019 (COVID-19) treatment?

A pneumonia outbreak associated with a new coronavirus of probable bat origin

Novel multiarm polyethylene glycol-dihydroartemisinin conjugates enhancing therapeutic efficacy in non-small-cell lung cancer

Enhanced apoptotic effects of dihydroartemisinin-aggregated gelatin and hyaluronan nanoparticles on human lung cancer cells

Dihydroartemisinin loaded nanostructured lipid carriers (DHA-NLC): evaluation of pharmacokinetics and tissue distribution after intravenous administration to rats

Synthesis of a novel series of artemisinin dimers with potent anticancer activity involving Sonogashira cross-coupling reaction

Enhanced delivery of artemisinin and its analogues to cancer cells by their adducts with human serum transferrin

Orally formulated artemisinin in healthy fasting Vietnamese male subjects: a randomized, four-sequence, open-label, pharmacokinetic crossover study

Review of the clinical pharmacokinetics of artesunate and its active metabolite dihydroartemisinin following intravenous, intramuscular, oral or rectal administration

Determination of artemisitene in rat plasma by ultra-performance liquid chromatography/tandem mass spectrometry and its application in pharmacokinetics