Niclosamide—A promising treatment for COVID‐19

Vaccines have reduced the transmission and severity of COVID‐19, but there remains a paucity of efficacious treatment for drug‐resistant strains and more susceptible individuals, particularly those who mount a suboptimal vaccine response, either due to underlying health conditions or concomitant therapies. Repurposing existing drugs is a timely, safe and scientifically robust method for treating pandemics, such as COVID‐19. Here, we review the pharmacology and scientific rationale for repurposing niclosamide, an anti‐helminth already in human use as a treatment for COVID‐19. In addition, its potent antiviral activity, niclosamide has shown pleiotropic anti‐inflammatory, antibacterial, bronchodilatory and anticancer effects in numerous preclinical and early clinical studies. The advantages and rationale for nebulized and intranasal formulations of niclosamide, which target the site of the primary infection in COVID‐19, are reviewed. Finally, we give an overview of ongoing clinical trials investigating niclosamide as a promising candidate against SARS‐CoV‐2.

administered (WHO, 2022). However, vaccines alone are unlikely to be sufficient in vulnerable patient populations (i.e. elderly, cancer, primary immune deficiencies, transplant recipients or dialysis) and patients who receive concurrent medical treatments that supress the immune system may not mount a satisfactory antibody response.
Additionally, global herd immunity is unlikely to be achieved due to the constant evolution of new variants of concern and waning of natural or stimulated antibody responses over time. Geopolitical logistics and vaccine nationalism also make it unlikely that these vaccines will be equitably available across the globe for a few more years. Therefore, parallel strategies such as effective novel therapeutic interventions must be rapidly developed. Intensive and collaborative research efforts have resulted in a number of treatment options for COVID-19.
The antivirals, remdesivir (Beigel et al., 2020) and molnupiravir (Jayk Bernal et al., 2021), neutralizing monoclonal antibodies, casirivimab, imdevimab (Somersan-Karakaya et al., 2021) and sotrovimab (Gupta et al., 2021), IL-6 antibodies, tocilizumab (Gordon et al., 2021; RECOVERY Collaborative Group, 2021) and dexamethasone (Horby et al., 2021)  Coronaviruses (CoVs) are large, enveloped, positive sense and single-stranded RNA viruses belonging to the family Coronaviridae within the order Nidovirales (Y. Chen et al., 2020). They can infect several mammalian hosts and are divided into four genera: -Alpha, Beta, Gamma and Delta, of which Alpha and Beta CoVs are known to infect humans. Full-genome sequencing and phylogenetic analyses have indicated that the CoV that causes COVID-19 was in the same subgenus as the SARS virus (Fehr & Perlman, 2015) and was named on the basis of its appearance under electron microscopy. Human CoV infections usually cause mild, self-limiting respiratory infection. However, the epidemics of SARS-CoV and Middle East respiratory syndrome coronaviruses (MERS-CoV) caused alarming morbidity and mortality in 2002-2003and 2012, respectively (Gao et al., 2016, and COVID-19 has underscored the continued risk of pandemics caused by such viruses. Risk factors for severe COVID-19 across the globe include old age, race, gender, obesity, cardiovascular disease, diabetes, chronic lung disease and immunosuppression. Therefore, drugs that target pleiotropic mechanisms may be important. CoVs have a large genome and a higher mutation rate compared with other RNA viruses, hence eradicating them definitively is difficult (Gralinski & Baric, 2015). Broad-spectrum inhibitors of emerging CoVs are therefore needed and repurposing existing drugs has been validated as a means to tackle the SARS-CoV-2 pandemic, as well as enabling future pandemic preparedness.

| REPURPOSING OLD DRUGS FOR A NEW CAUSE
Drug repurposing has emerged as an attractive alternative to the conventional approach of drug discovery, which is often exhaustive and arduous (Ashburn & Thor, 2004). It is a process of identifying new therapeutic roles for a drug that has already been established for the treatment of another condition. The discovery of a new drug and its journey to the market is a process fraught with risks involving toxicity and lack of efficacy, costing billions of dollars and requiring a long timeline. Repurposing therefore offers several advantages over de novo drug development, such as reduced development timelines, reduced costs and substantially lower risks, as the safety and pharmacokinetic profile of the drug is already established (S. Pushpakom et al., 2019). The risk of failure is lower because the repurposed drug has been shown to be safe in preclinical models and humans, provided early-stage trials have been completed. As a result, the timeframe for drug development is significantly shorter (Breckenridge & Jacob, 2019).
Historically, drug repurposing has been mostly serendipitous, usually after a drug was found to have a newly recognized off-target effect (Nosengo, 2016). Well-known examples are the use of minoxidil for hair loss, sildenafil for erectile dysfunction and thalidomide for multiple myeloma (Pushpakom et al., 2019). However, recent successes have encouraged the development of more systematic approaches resulting in the identification of a number of promising candidate drugs (Hurle et al., 2013). In more recent years, drug repurposing screens have emerged as an attractive strategy to respond swiftly to emerging infectious diseases (Ashburn & Thor, 2004). Food and Drug Administration (FDA; U.S.A.)-approved drugs that can achieve a modest antimicrobial activity are a safe and increasingly popular response mechanism to emerging infections. The drugs concerned can be made immediately available for use in clinical trials as they have known safety profiles at the licensed doses and this has had a huge impact during the COVID-19 pandemic. For example, dexamethasone was repurposed in the RECOVERY trial (Horby et al., 2021) and is estimated to have saved 1 million lives globally by March 2021 (NHS England, 2021) whereas tocilizumab, a repurposed drug for moderate to severe rheumatoid arthritis, is licensed for treating hospitalized patients with moderate to severe COVID-19 (Gordon et al., 2021;RECOVERY Collaborative Group, 2021 and assigns the drugs a 'grade', based on treatment efficacy and whether pre-specified endpoints are met (Venkatesan, 2021).
Here, we review the preclinical models that have demonstrated the pluripotential effects of niclosamide as well as some limited human data in a variety of disease states that suggest it could be an agent of choice in COVID-19.

| HISTORY AND MECHANISM OF ACTION OF NICLOSAMIDE
Structurally, niclosamide (Figure 1) belongs to a large group of lipophilic, weakly acidic molecules called salicylanilides, a derivative of salicylic acid (Pearson & Hewlett, 1985). Niclosamide is listed on the WHO's list of essential medications and chewable tablets have been approved for use as an anti-helminthic agent for cestode (tapeworm) infections for over 40 years with a daily oral dose of 2 g niclosamide (Andrews et al., 1982). Niclosamide inhibits oxidative phosphorylation and stimulates ATP activity in the mitochondria of cestodes, killing both the scolex and the proximal segments of the tapeworm (Al-Hadiya, 2005;Weinbach & Garbus, 1969). It has been shown to affect several signal transduction pathways such as Wnt/β-catenin, mechanistic target of rapamycin (mTOR) complex 1 (mTORC1), signal transducer and activator of transcription 3 (STAT3), nuclear factor kappa-light-chain-enhancer of activated B cells (nuclear factor-κB; NF-κB) and Notch pathways, all indicating its potential to treat conditions such as cancer (summarized in Table 1), chronic medical diseases, and bacterial and viral infections.
Oral niclosamide is only partially absorbed from the intestinal tract with a low bioavailability of 5.5%-10% and is rapidly eliminated by the kidneys with no cumulative toxic effects (Andrews et al., 1982). Distribution studies in rodents with oral doses of 40 mgÁkg À1 niclosamide ethanolamine (i.e. $34 mgÁkg À1 niclosamide) and 50 mgÁkg À1 niclosamide demonstrate that tissue levels are highest in excretory organs (intestines, liver and kidney) and low to negligible levels are achieved in other tissues (such as the brain, heart and lungs) (Duhm et al., 1961;Tao et al., 2014).

| NICLOSAMIDE IN CHRONIC MEDICAL STATES
4.1 | Niclosamide and metabolic syndrome A seminal study from Tao et al. (2014) showed that niclosamide ethanolamine (150 mgÁkg À1 Áday À1 , p.o.) reduced liver fat accumulation (steatosis) in mice fed a high-fat diet. The effects were also studied in human liver cells and demonstrated increased lipid oxidation and upregulation of the AMP-activated protein kinase pathway, suggesting its potential use as an anti-obesity agent. In a murine obesity study, mice were fed either a chow or high-fat diet and niclosamide (150 mgÁkg À1 Áday À1 , p.o., or 75 mgÁkg À1 Áday À1 , p.o.) and niclosamide ethanolamine for 10 weeks (Chowdhury et al., 2017). Both niclosamide and niclosamide ethanolamine significantly improved glucose metabolism by inhibiting the glucagon signalling pathway, without altering total body weight or insulin secretion or sensitivity. In a further study, administration of niclosamide (140 mgÁkg À1 Áday À1 , mixed in food) to high-fat-diet-fed mice, resembling a murine obesity model, for 4 weeks resulted in a significant decline in food intake and body weight compared with control mice (Al-Gareeb et al., 2017).
There was also significant lowering of the fasting blood glucose, fasting plasma insulin and improved insulin resistance.

| Niclosamide and autoimmunity
In a collagen-induced arthritis rat model, niclosamide (100 mgÁkg À1 , p.o.) significantly reduced the arthritis index, footpad thickness and ankle swelling (Al-Gareeb et al., 2019). Histopathological examination revealed reduced infiltration of inflammatory cells along with reduced bone and cartilage destruction. Serum levels of tumour necrosis factor α (TNFα) and IL-1β were also significantly reduced compared with control animals. In a murine lupus study using MRL/lpr mice, daily administration of niclosamide (100 mgÁkg À1 , oral gavage) for 7 weeks improved proteinuria, anti-dsDNA antibody levels, histological features and ameliorated C3 deposition in the kidneys (Jang et al., 2021).
Niclosamide also led to a reduction of splenic follicular helper T cells and plasma cells STAT3 inhibition. Finally, in a hypochlorous acidinduced mouse model of systemic sclerosis, niclosamide (10 mgÁkg À1 5 daysÁweek À1 for 6 weeks, i.p.) reduced clinical markers of skin fibrosis, such as skin thickening and collagen content, as well as skin levels of IL-4 and IL-13. There was a decrease in STAT3, AKT and Wnt/βcatenin pathways in the skin of hypochlorous acid-induced mice (Morin et al., 2016)

| Niclosamide and pulmonary pathology
In a screen of $580,000 compounds, niclosamide was identified as a TMEM16A antagonist, a calcium-activated chloride channel (CaCC) that contributes to mucus hypersecretion and bronchoconstriction in reactive airway disease (Miner et al., 2019). The study tested efficacy, using maximally contracted and cytokine-treated airways,. and confirmed that niclosamide had a potent bronchodilator effect. In transgenic asthmatic mice, niclosamide (13 mgÁkg À1 Áday À1 for 3 days, i.p.) reduced mucus production and bronchoconstriction, and also demonstrated anti-inflammatory and antibacterial effects (Cabrita et al., 2019). Centeio et al. (2021) further investigated these findings, demonstrating that intratracheal instillation of 0.98% niclosamide for 4 days inhibited mucus production and secretion in ovalbumin-sensitized mice.

| Niclosamide and cancer
Several studies have demonstrated that niclosamide has potent in vitro and in vivo effects against a wide range of cancers. We have further summarized animal and preclinical studies that highlight the potential use of niclosamide in cancer in Suppressed proliferation and migration of HCC cells lines Tomizawa et al. (2015) Down-regulation of cyclin D1 via a downregulation of the Wnt-3 pathway Suppressed proliferation of HCC cells by induction of apoptosis Tomizawa et al. (2013) Inhibition of STAT3 and more downstream anti-apoptotic proteins Suppressed cell viability and inhibited clone formation in HCC cells and synergism with cisplatin by promotion of apoptosis Wang, Zhou, et al. (2018) Reduced expression of proteins in the Wnt-βcatenin, STAT3, AKT-mTOR and epidermal growth factor signalling pathways orally bioavailable form in combination with abiraterone and prednisolone in men with castration-resistant prostate cancer (Parikh et al., 2021). Nine patients were recruited and no dose-limiting toxicity was observed at any dose. Five out of eight patients achieved a prostate-specific antigen response, two achieved undetectable prostate-specific antigen levels and a radiographic response. The NIKOLO clinical trial is a Phase II, single-centre, one-arm open-label study to investigate the safety and efficacy of niclosamide in patients with metastatic colorectal cancer progressing under standard therapy Burock, Daum, Tröger, et al., 2018). The study is focused on the feasibility, toxicity and efficacy of niclosamide in order to explore new treatment options. It is worth noting that despite ongoing trials, niclosamide is not approved for the treatment of any oncological condition.

| Niclosamide and other models of disease
In a murine endometriosis model, established by surgically inducing endometriotic-like lesions, niclosamide (100 and 200 mgÁkg À1 , p.o.) decreased the growth rate and progression of endometriosis-like lesions and inhibited STAT3 and NF-κB pathways (Prather et al., 2016). Inhibition of macrophage-induced inflammatory pathways has been observed in primary endometriotic stromal cells (Sekulovski et al., 2020). Niclosamide has also been shown to act as a potential neuroprotective agent such as in Parkinson's disease by activating PINK1 in HeLA and primary cortical neurons (Barini et al., 2018), reduce neuro-inflammation in activated murine primary microglia in vitro (Serrano et al., 2019) and block signalling pathways associated with neuropathic pain in rats receiving 1 mM niclosamide intratracheally .

| NICLOSAMIDE AS AN ANTIBACTERIAL AGENT
Niclosamide has well-identified antimicrobial properties and has been shown to prevent the formation of biofilms of hospital-acquired and device-associated gram-positive bacteria, such as Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) at concentrations as low as 0.01 μgÁml À1 (Gwisai et al., 2017). When applied as a device coating, niclosamide prevented bacterial attachment and demonstrated potent antimicrobial activity (Gwisai et al., 2017). In a screen of 1280 commercially available drugs, niclosamide was one of nine agents that possessed antimicrobial activity against preformed biofilms (Torres et al., 2016). A time-kill study further showed that niclosamide is bacteriostatic against a number of gram-positive bacteria including MRSA, displaying strong in vivo (Caenorhabditis elegans-MRSA liquid infection model) as well as in vitro activity (Rajamuthiah et al., 2015). Niclosamide had activities against clinical isolates of vancomycin-resistant Enterococcus faecium (VRE) and was superior to linezolid as a gastrointestinal decolonizing agent of vancomycinresistant Enterococcus faecium-challenged mice (Mohammad et al., 2018). In this vancomycin-resistant Enterococcus faecium-colonization-reduction mouse model, niclosamide (10 mgÁkg À1 Áday À1 for 8 days, oral gavage) was administered to mice that were sensitized with ampicillin followed by E. faecium infection to establish gastrointestinal colonization. Tam et al. (2018) showed that niclosamide inhibited the pathogenesis of multidrug-resistant Clostridium difficile by targeting entry of its toxins into colonocytes. In a mouse model of primary and recurrent C. difficile infection, post-infection treatment with niclosamide (50 mgÁkg À1 Áday À1 for 4 days, p.o.) ethanolamine (5% in DMSO) reduced both the primary disease and recurrence, without disrupting the gut microbiota (Tam et al., 2018). Niclosamide is stable in acidic pH and synergizes with metronidazole and proton pump inhibitors to eliminate Helicobacter pylori adhesion/invasion via multiple mechanisms such as reducing trans-membrane pH, inhibiting IL-8 (CXCL8) secretion and disrupting H. pylori proton motility (Tharmalingam et al., 2018). A screen of FDA-approved drugs identified niclosamide as an inhibitor of the Pseudomonas aeruginosa quorum sensing signalling molecules (Imperi et al., 2013). Finally, niclosamide has significant activity against multidrug-resistant Mycobacterium tuberculosis strains (Sun & Zhang, 1999) and inhibited its growth in infected human macrophages in a bacteriostatic manner (Fan et al., 2019). The same study also showed that it inhibited HIV replication in human macrophages via transcriptional effects.

| NICLOSAMIDE AS AN ANTIVIRAL AGENT
Niclosamide role in anti-viral host defence was first reported by

| Mechanism of action against SARS-CoV-2
The antiviral activity of niclosamide against SARS-CoV-2 is complex and involves multiple cellular processes as illustrated in Figure 2.
SARS-CoV-2 uses the angiotensin-converting enzyme 2 (ACE2) as a cellular entry receptor in permissive cells of the respiratory tract and the spike proteins initiate the merging of the viral envelope with the host cell cytomembrane (Zhou et al., 2020). Following receptor binding and conformational changes in the spike protein, cathepsin L mediates proteolysis within endosomes leading to viral entry into host cells, as it has been shown for SARS-CoV (Gomes et al., 2020;Huang et al., 2006;Simmons et al., 2005). The protonophoric activity of niclosamide that causes endosomal neutralization is thought to interfere with viral entry and egress preventing SARS-CoV-2 genome release and maturation. This mechanism has been derived from stud-

| CLINICAL TRIALS OF NICLOSAMIDE FOR COVID-19
Based on promising preclinical data, niclosamide is currently being evaluated as a potential COVID-19 treatment in 18 human clinical trials relying on different formulations and/or routes of administration.
Details of these trials can be found on public registries such as ClinicalTrials.gov and the WHO's Trial search website. Twelve of these trials are in Phase 2/3 and investigate the efficacy of niclosamide across the full COVID-19 disease spectrum ( Figure 3 and Table 2).
In the pre-symptomatic and mild COVID-19 disease stage, the potent antiviral activity of niclosamide is thought to limit disease symptomatology and progression. However, as COVID-19 progresses towards moderate and severe disease, the bronchodilatory and anti-F I G U R E 2 Potential mechanisms of antiviral activity have been illustrated in this figure. These include (i) endosomal pH neutralization to prevent viral replication, (ii) promotion of autophagy via inhibition of S-phase kinase-associated protein 2 (SKP2), (iii) decreased mucus plugging via inhibition of TMEM16A (calcium activated chloride channel; CaCC) and (iv) prevention of syncytia formation by ion channel inhibition. A similar trial set-up in the Philippines was terminated (9 December 2020) due to a lack of COVID-19 patients (NCT04541485). Abbreviations: PD, pharmacodynamics; PK, pharmacokinetics; SoC, standard of care.
inflammatory effects of niclosamide might contribute to efficacy. Its antibacterial efficacy could also benefit COVID-19 patients at risk for secondary bacterial infections, which is one of the leading causes of mortality in COVID-19 (Cevik et al., 2020), particularly since the introduction of immunomodulators such as dexamethasone and tocilizumab as standard of care (SoC) medications.

| Intramuscular injections
To address concerns around poor oral bioavailability of niclosamide, a formulation administered via the intramuscular route was developed.
It is currently being investigated in healthy volunteers to assess its safety, tolerability and pharmacokinetics/pharmacodynamics (PK/PD) in 40 COVID-19 patients (

| Inhalational and intranasal administration
In another approach, the poor oral availability of niclosamide has been and 424 ngÁml À1 (single dose). This is lower compared with plasma levels of the (much higher) 2 g, p.o., dose used in anti-helminthic treatment (Andrews et al., 1982;Schweizer et al., 2018aSchweizer et al., , 2018b. The lower C max would potentially limit the systemic side effects seen with oral dosing, a route that has been in use since the 1960s.  (Fisk et al., 2021;Lu et al., 2020;TACTIC-E Trial, 2020). For the latter, combined intranasal and intra-pulmonary (via the nebulized route) administration of niclosamide has the potential to be an efficacious approach as aerosol application of niclosamide via the inhaled and intranasal routes enables local delivery at the site of disease. Using this local delivery approach, niclosamide levels in target tissues are expected to be higher than plasma levels (Backer et al., 2021;UNION therapeutics, 2020) and achieve therapeutic concentrations at the primary site of infection. The targeted nasal administration is crucial because the nasopharynx and nasal cavity are both an entry point and a reservoir for SARS-CoV-2 (Gallo et al., 2021;Sungnak et al., 2020).
Overall, the breadth and spectrum of clinical trials utilizing niclosamide across the different disease stages of COVID-19 will provide valuable human clinical and pharmacological data and have the potential to enable the development of niclosamide as an effective anti-COVID-19 agent, either in its own right or an as adjunct.

| LIMITATIONS AND CHALLENGES TO THE CLINICAL USE OF NICLOSAMIDE
The favourable safety profile of niclosamide in treating humans with tapeworm infection could be due to the fact that the organ of interest was the gut and the drug did not need to be absorbed systemically and therefore did not get the chance to negatively modulate systemic signalling cascades. Systemic delivery is required for infections, cancers and metabolic diseases that have shown to be responsive to niclosamide. The safety profile of niclosamide in these conditions is largely unexplored and future studies are required for a clearer picture of its toxicity. The oral dose of niclosamide as a cestocidal agent is 2 g as a single dose, and this leads to a wide range of serum concentrations as described above (Andrews et al., 1982), mainly due to variable absorption rates. The combination of a low oral bioavailability and a wide range of serum concentrations results in unpredictable efficacy in clinical studies. Additional studies with a formulation that gives high bioavailability are required before niclosamide can be used more widely. One of the obstacles in this direction is that a direct target of niclosamide remains undiscovered.
There is great interest in conducting studies to elucidate the structure-activity relationship of niclosamide and thereby to identify novel derivatives of niclosamide that might have better bioavailability (H. Chen et al., 2013). A recent study that combined structureassisted drug design identified a mechanism-based inhibitor (N3) and then determined the crystal structure of M pro (main protease of SARS-CoV-2) in complex with this inhibitor (Z. Jin et al., 2020).
Through a combination of structure-based virtual and highthroughput screening, the investigators assayed more than 10,000 compounds as inhibitors of M pro and demonstrated that a robust screening strategy can rapidly discover drugs for new infections.
Although these crystal structures provide useful new insights into drug discovery, extensive efforts are still needed to identify effective binding pockets for small molecules such as niclosamide and thereby validate the drug targets.
The challenges involved in repurposing niclosamide begin with its stable crystalline structure and its lipophilicity that restrict its solubil-  (Schweizer et al., 2018a(Schweizer et al., , 2018b. The adverse events relating to the two patients who discontinued treatment (in the 1000 mg three times per day group) were most likely driven by the high local niclosamide concentrations in the gastrointestinal tract rather than the high systemic exposure. Peak plasma levels for the 1000 mg three times per day dose (C max = 149-182 ngÁml À1 , AUC 0 À t = 629-676 ngÁh À1 Áml À1 [min-max]) were below or similar to the plasma levels of studies using a 1.5 or 2 g daily dose, which is well tolerated (Burock, Daum, Tröger, et al., 2018). Of note, the COVID-19 trials with niclosamide are using the approved 2 g p.o. daily dose (or lower inhalation/intranasal and intramuscular doses), and therefore, one would expect even smaller proportions of the drug to reach the gastrointestinal tract.

| CONCLUSIONS AND FUTURE DIRECTIONS
Evidence has accumulated that niclosamide is a multi-functional drug that can modulate several signalling pathways and biological processes. It has shown preclinical activity in many disease models, from