Niclosamide for Covid-19: bridging the gap

Abstract

Aim/purpose

Niclosamide (NCL) is an anthelminthic drug, which is widely used to treat various diseases due to its pleiotropic anti-inflammatory and antiviral activities. NCL modulates of uncoupling oxidative phosphorylation and different signaling pathways in human biological processes. The wide-spectrum antiviral effect of NCL makes it a possible candidate for recent pandemic SARS-CoV-2 infection and may reduce Covid-19 severity. Therefore, the aim of the present study was to review and clarify the potential role of NCL in Covid-19.

Methods

This study reviewed and highlighted the protective role of NCL therapy in Covid-19. A related literature search in PubMed, Scopus, Web of Science, Google Scholar, and Science Direct was done.

Results

NCL has noteworthy anti-inflammatory and antiviral effects. The primary antiviral mechanism of NCL is through neutralization of endosomal PH and inhibition of viral protein maturation. NCL acts as a proton carrier, inhibits homeostasis of endosomal PH, which limiting of viral proliferation and release. The anti-inflammatory effects of NCL are mediated by suppression of inflammatory signaling pathways and release of pro-inflammatory cytokines. However, the major limitation in using NCL is low aqueous solubility, which reduces oral bioavailability and therapeutic serum concentration that reducing the in vivo effect of NCL against SARS-CoV-2.

Conclusions

NCL has anti-inflammatory and immune regulatory effects by modulating the release of pro-inflammatory cytokines, inhibition of NF-κB /NLRP3 inflammasome and mTOR signaling pathway. NCL has an anti-SARS-CoV-2 effect via interruption of viral life-cycle and/or induction of cytopathic effect. Prospective clinical studies and clinical trials are mandatory to confirm the potential role of NCL in patients with Covid-19 concerning the severity and clinical outcomes.

Introduction

Coronavirus disease 2019 (Covid-19) is a worldwide pandemic disease caused by severe acute respiratory coronavirus 2 (SARS-CoV-2). Covid-19 affects diverse organs, chiefly the respiratory system, and presented with acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) [1]. The systemic effect of Covid-19 is due to the broad allocation and distribution of angiotensin-converting enzyme 2 (AEC2), a primary receptor for SARS-CoV-2 [2, 3]. ACE2 receptor is primarily expressed in lung alveolar cells type II, proximal renal tubules, immune cells, and intestines [4]. Binding of SARS-CoV-2 with ACE2 is linked with down-regulation of these protective receptors with significant intensification in the level of vasoconstrictors angiotensin II (Ang II) and decreasing of vasodilator angiotensin (Ang 1–7), (Ang 1–9) with induction release of pro-inflammatory cytokines. These changes elicit hyper-inflammation and the development of cytokine storms that linked with ARDS development and multi-organ failure (MOF) [5].

Since the World Health Organization (WHO) declaration of this disease as a pandemic and until late July 2021, the total confirmed cases are 194,250,977, with 4,258,789 deaths. In this international predicament, extraordinary efforts, advancing investigations, and research are meticulously performed to find an effective drug against SARS-CoV-2 from recent or old standard and/or approved drugs as repurposing drug strategy [6].

Niclosamide (NCL) is an anthelminthic drug, inhibits glucose uptake by the worms, used to treat tapeworm infestations, including taeniasis, hymenolepiasis, and diphyllobothriasis. However, it was not effective against other types of worms [7]. NLS was discovered in 1953 in Bayer Research Laboratories as a molluscicidal agent against schistosomiasis snail and was marketed as Bayluscide in 1959 [8]. Later on, it was to be effective for tapeworms in 1962 and marketed as Yomesan for human use; in 1982, it was approved by Food and Drug Administration (FDA) for treatment of tapeworms and regarded as one of essential medicine by World Health Organization (WHO) [9]. NCL is widely used to treat various metabolic disorders, cancers, viral infections, and immunological disturbance due to its pleiotropic effects antiviral and anti-inflammatory properties with modulation of uncoupling oxidative phosphorylation and various signaling pathways in human biological processes [10]. Several experimental studies confirmed that NCL is a safe and well-tolerated drug with a toxicity profile of more than 5 g/kg in rats [11]. NCL is well tolerated drug in humans, can be used orally at 2 g/day for worm treatment leading to serum concentrations of 0.25–6.0 µg/mL that corresponding to 0.76–18.35 µM respectively. This concentration is not toxic and within range of active antiviral concentration in humans [8]. Since, NCL has potential antiviral and anti-inflammatory properties, therefore, the aim of the present study was to review and clarify the potential role of NCL in Covid-19.

Methods and search strategy

An interrelated literature search in PubMed, Scopus, Web of Science, Google Scholar, and Science Direct was prepared. All of published articles related to the role of NCL and Covid-19 were included in this mini-review. We seek out the international database by using the medical subject heading (MeSH) to recognize the applicable articles published up to 2021. The listed keywords used in this search included; [Covid-19 OR SARS-CoV-2] AND [NCL], [Covid-19 OR SARS-CoV-2] AND [NCL], [antiviral, anti-inflammatory effects], AND [Covid-19 severity]. The entire types of published articles with dissimilar languages were integrated, and the absolute findings were sum up in a mini-review study.

Antiviral activity of Niclosamide

The antiviral activity of NCL against influenza virus and human rhinovirus was explained to be PH-dependent and related to the protonophore/proton carrier activity. NCL has neutralizing effects on the endo-lysosomal PH with significant acidification of extracellular medium [12]. NCL did not affect cell H-ATPase but neutralize coated vesicles indicating a proton carrier inhibition independent of any target proteins [12].Wang et al. [13] illustrated that NCL inhibits entry and transmission of the Chikungunya virus, which enters the host cells through receptor-mediated endocytosis, suggesting that NCL might be effective in treatment of Chikungunya virus and other alphaviruses. Similarly, NCL inhibits replication of Zika virus and attenuates Zika virus-induced cell death through attenuation of PH-dependent membrane fusion [14]. Indeed, NCL suppresses viral-lytic replication of Epstein-Barr virus (EBV) through inhibition of the mammalian cellular target of rapamycin (mTOR) activation, which is necessary for pathogenesis of EBV infection [15]. Recently, Kao et al. [16] observed that NCL has a potential effect against Dengue infection by inhibiting various viral life-cycle and virulence steps, including binding, receptor-mediated endocytosis entry, fusion, uncoating, replication, assembly, and RNA release. In addition, NCL inhibits Dengue and Zika infections through targeting of NS2B-NS3 protease [66]. Furthermore, more extensive experimental studies illustrated that NCL has more extended antiviral spectrums. NLS inhibits replication of hepatitis C virus (HCV) through modulation of host immune response [17] and regarded as a potent inhibitor of Ebola virus [18]. As well, NLS reduces replication of human adenovirus by blocking of viral protein-endosomal transport [19]. Of note, most of in vitro studies revealed short-term antiviral effects of NLS; however long-term effect was confirmed against HIV infection through inhibition replication of human HIV in the macrophage [67].

This wide-spectrum antiviral effect of NCL makes it possible for recent pandemic SARS-CoV-2 infection and may reduce Covid-19 severity.

Coronaviruses and niclosamide

Coronaviruses (CoVs) are a group of non-segmental enveloped, positive-sense RNA viruses with a sizeable genomic size of about 27–34 kb [20]. Human strain of CoVs like HCoV-229E, HCoV-HKU1, HCoV-OC43, and HCoV-NL63 may cause self-limiting and mild upper respiratory tract infection [21]. In July 2003, the SARS-CoV outbreak was emerged and led to pandemics in China and associated with a 9% mortality rate [22]. In 2012, Middle East Respiratory Syndrome (MERS) emerged in Saudi Arabia, which was caused by MERS-CoV led to more fatality with a 34% mortality rate [23]. At the time of the SARS-CoV outbreak, there was no specific effective against replication of Coronaviruses (CoVs). NCL has been reported to be effective against SARS by inhibiting SARS-CoV replication with protection from viral cytopathic effects at 1.56 µM concentration. The anti-cytopathic effect of NCL was demonstrated at a lower concentration of 1.0 µM in Vero E6 cells [24]. The effective concentration of NCL that reduces 50% of cell viability of infected cells was 1-3 µM. As well, NCL was able to inhibit replication of SARS-CoV when the cells infected, suggesting that NCL does not interferes with SARS-CoV attachment and entry to the affected cells [24]. Besides, 5 µM leads to more potent inhibition of SARS-CoV as compared to 1-3 µM, indicating a dose-dependent anti-SARS-CoV activity of NCL [24, 68].

Various molecular studies regarding SARS-CoV genomic analysis confirmed that SARS-CoV 3CL-protease is an important enzyme for processing viral polyprotein and is regarded as a potential drug target [69]. However, NCL showed no noticeable effect on SARS-CoV 3CL-protease even at a higher concentration [25]. Furthermore, NCL inhibits MERS-CoV replication by inhibiting E3-ligase S-phase kinase associated protein 2 (SKP2), which is involved in the degradation of proteasomes [26, 70].

Covid-19 and niclosamide

It has been shown that SARS-CoV-2 has a genomic similarity with SARS-CoV in about 79% and with that of bat CoV in about 96% [27]. Therefore, experimental studies that confirmed the potential drug effects on SARS-CoV could be applied for SARS-CoV-2 due to relative genomic similarity. Molecular docking and in vitro studies illustrated that NCL is an effective agent against SARS-CoV-2 replication by inhibiting protease enzyme [28, 29]. Different recent studies illustrated that NCL inhibits SARS-CoV-2 replication through inhibition of intracellular acidification and viral protein assembly [30] with autophagy activation [63] thereby; it can be used in the management of Covid-19.

The major limitation in using of NCL is low aqueous solubility, which reduces oral bioavailability and therapeutic serum concentration that reduces in vivo effect of NCL against SARS-CoV-2[31]. Therefore, different pharmaceutical formulations and/or routes are applied for NCL to be more effective in the management of SARS-CoV-2 infections and to overcome the low bioavailability of NCL. Brunaugh et al. [32] revealed that inhalation of NCL in combination with human lysosome increases in vitro and vivo activity of NCL against SARS-CoV-2 that ensures a rapid clinical improvement. Besides, Wang et al. [33] confirmed that lipid nanoparticles of NCL are cost-effective and highly scalable against SARS-CoV-2 in Vero E6 and AEC2 expressing cells. Jara et al. [34] experimental study involved multi-dose pharmacokinetics illustrated that inhalational route of NCL is more effective against pulmonary SARS-CoV-2 infection in the Syrian hamster model. Therefore, mitigation of NCL through these pharmaceutical formulations may improve its solubility and pharmacokinetic profile.

The main antiviral mechanisms of NCL are neutralization of endosomal PH and inhibition of viral protein maturation in the Golgi apparatus, chiefly in CoVs and hepatitis C virus (HCV). [12] NCL acts as a proton carrier and inhibits homeostasis of endosomal PH, limiting viral proliferation, and release [12]. Therefore, the antiviral mechanisms of NCL are either of PH-dependent, which inhibit endosomal acidification, or PH-independent pathway. NCL inhibits mitochondrial oxidative phosphorylation with subsequent depletion of intracellular ATP, which trigger adenosine monophosphate protein kinase (AMPK) which directly blocks mTOR signaling pathway [76, 77] (Fig. 1).

Fig. 1.

Inhibitory effects of niclosamide on SARS-CoV-2 infection and associated inflammatory pathways: SARS-CoV-2 through activation of macrophage, mammalian target of rapamycin (mTOR) and signal transducer and activator transcription 3 (STAT-3) leads to macrophage activation syndrome (MAS), cell apoptosis, and activation release of plasminogen activator inhibitor-1(PAI-1) respectively. As well, SARS-CoV-2 directly activates nuclear factor kappa B (NF-κB) and nod-like receptor pyrin 3 (NLRP3) inflammasome leading to release of pro-inflammatory cytokines with development of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Niclosamide has inhibitory effects () on the SARS-CoV-2-mediated inflammatory signaling pathways and release of pro-inflammatory cytokines

On the other hand, NCL has anti-inflammatory and immune regulatory effects that may affect the propagation of different inflammatory and immune-mediated disorders through inhibition of pro-inflammatory cytokine, mainly tumor necrosis factor-alpha (TNF-α) [35]. Moreover, experimental studies demonstrated that NCL is a potent inhibitor of Ca⁺²- activated Cl⁻ channel TMEM16A expressed in the airway smooth muscles and mucus-producing goblet cells that mediates bronchoconstriction and mucus hypersecretion in chronic inflammatory airway diseases[36, 64]. Miner et al., revealed that NCL had beneficial effect on constricted airways and mucus hyper-secretion during airway inflammation by antagonizing of airway TMEM16A channels [65].

In addition, NCL inhibits progression of inflammatory airway diseases like asthma, chronic obstructive pulmonary disease, and cystic fibrosis by suppression release of IL-8 and intracellular ca⁺² signaling [36].

What’s more, NCL is effective in treating pulmonary secondary bacterial infections caused by pseudomonas species [37]. Of note, NCL might be an effective agent against Covid-19 pneumonia through anti-SARS-CoV-2 activity and modulation of associated inflammatory disorders [38]. NCL improves anti-inflammatory and inhibits pro-inflammatory cytokines, limiting the development of cytokine storm-induced ALI in Covid-19 [39]. Therefore, NCL may reduce the severity of Covid-19 by attenuating the development of ALI and ARDS induced by SARS-CoV-2 infection [40]. Braga et al. [41] showed that NCL reduces the development of pulmonary hypertension (PH) in rats through inhibition of signal transducer and activator transcription 3 (STAT-3). It has been reported that PH is developed during SARS-CoV-2 infection and associated with Covid-19 severity and high mortality [42]. Recent studies showed that SARS-CoV-2 proteins, mainly ORF6 induce dysfunction of STAT-3 with compensatory over-activation of STAT-3-induced activation of plasminogen activator inhibitor-1(PAI-1) and subsequent coagulopathy and thrombosis [43]. In this way, overactivated PAI-1 stimulates macrophages to produce pro-inflammatory cytokines, leading to more inhibition of STAT-3 in a vicious cycle [44].

High circulating IL-6 level in patients with Covid-19 is implicated in the development of cytokine storm and production of PAI-1, so inhibition of IL-6 by tocilizumab may alleviate clinical outcomes through reduction synthesis of PAI-1 in severely affected Covid-19 patients [45]. For that reason, inhibition of STAT-3, PAI-1, and IL-6 is related with better clinical outcomes in Covid-19 patients, and the use of NCL might be beneficial in this context. Different studies reported that NCL attenuates the production of STAT-3, PAI-1, and IL-6 through the inhibition of associated inflammatory pathways during cancer metastasis [46, 47]. At present, there are no clinical studies accounted for the potential effects of NCL on the inflammatory cytokines and pathway in Covid-19 patients. In SARS-CoV-2, the mammalian target of rapamycin (mTOR) is activated with inhibition of p53. Activation of mTOR inhibits host cells for interferon (INF) production, while inhibition of p53 is associated with high viral survival, so mTOR inhibitors and p53 activators might attenuate SARS-CoV-2 [48]. Gassen et al. [49] found that NCL activates autophagy and inhibits the mTOR pathway in SARS-CoV-2, which might explain the prolonged antiviral effect of NCL. NCL plays a crucial role in the activation of p53 in small cell lung cancer [50] and viral infections [51]; therefore, it may attenuate the survival of SARS-CoV-2. Of interest, most of enveloped RNA viruses including SARS-CoV-2 and HIV-1 activate mTOR pathway during their replications, thus disruption of this pathway may interfere with viral replication and pathogenesis [72].

Also, stimulation of nod-like receptor pyrin 3 (NLRP3) inflammasome and nuclear factor kappa B (NF-κB) signaling pathway are associated with SARS-CoV-2 infection, leading to the release of pro-inflammatory cytokines, which initiate cytokine storm with development of ALI and ARDS [52, 53]. Recently, Hu et al. [54] observed that chemical mitochondrial uncouplers like NCL have a potential inhibitory effect on the NLRP3 inflammasome via suppressing NF-κB signaling pathway. Thus, NCL has an anti-inflammatory effect that may mitigate hyperinflammatory status and hypercytokinemia during acute SARS-CoV-2 infection and may reduce risk of ARDS [55].

Despite of these beneficial effects of NCL, it may increase of oxidative stress by induction of mitochondrial dysfunction [56]. At this point, NCL may aggravate SARS-CoV-2 infection-induced oxidative stress and may be linked with Covid-19 severity [57]. However, Chen et al. [71] illustrated that NCL-induced oxidative stress could be beneficial in the management of cervical cancer through inhibition of mTOR signaling pathway.

Of interest, male subjects are highly susceptible to the SARS-CoV-2 infection than females due to high androgen level, which enhances SARS-CoV-2 entry through activation of transmembrane protease serine 2 (TMPRSS2) [58]. Liu et al. [59] confirmed that NCL blocks androgen receptors in prostatic carcinoma. Therefore, depending on this evidence, NCL may produce more protection against SARS-CoV-2 infection in men compared to women.

On the other hand, NCL has immune regulatory and modulatory effects on the immune response by inhibiting dendritic cells, T cell proliferation, and antigen response with inhibition of NF-κB /NLRP3 inflammasome and mTOR signaling pathway [60]. Experimental studies illustrated that NCL has a dose-dependent effect in inhibiting macrophage function and secretion of pro-inflammatory cytokines via suppression of SATA-3/ NF-κB signaling pathway [61]. It has been shown by Ruscitti et al. [62] that macrophage activation syndrome (MAS) is linked with the development of cytokine storm, ALI, ARDS, poor clinical outcomes, and high mortality rate in severely affected Covid-19 patients [62]. Thus, it will be expected that using of NCL in Covid-19 patients will produce a more significant protective effect through suppression of MAS.

It has been shown that endothelial dysfunction is associated with severe Covid-19, leading to pulmonary endothelial dysfunction and thrombotic disorders [73]. Experimental study demonstrated that NCL attenuate endothelial dysfunction through activation release of nitric oxide (NO) from vascular endothelium with stimulation of cyclic guanosine monophosphate (cAMP) [74]. Likewise, NCL reduces SARS-CoV-2-induced platelet hyper-reactivity and thrombotic disorders [75].

The net effects of NCL on SARS-CoV-2 infection and associated inflammatory and immunological derangements in Covid-19 are summarized (Fig. 2). This study highlighted the protective role of NCL therapy in Covid-19 from in vitro and docking studies; however, prospective clinical studies and clinical trials are mandatory in this regard to confirm the potential role of NCL in patients with Covid-19 concerning the severity and clinical outcomes.

Fig. 2.

Antiviral mechanisms of niclosamide: the antiviral mechanisms of niclosamide (NCL) are either of PH-dependent, which inhibit endosomal acidification, or PH-independent pathway. NCL inhibits mitochondrial oxidative phosphorylation with subsequent depletion of intracellular ATP, which trigger adenosine monophosphate protein kinase (AMPK) which directly blocks mTOR signaling pathway

Conclusion

NCL has anti-inflammatory and immune regulatory effects by modulating the release of pro-inflammatory cytokines, inhibition of NF-κB /NLRP3 inflammasome and mTOR signaling pathway. NCL has an anti-SARS-CoV-2 effect via interruption of viral life-cycle and/or induction of cytopathic effect. The present findings cannot give a conclusion regarding the ultimate effectiveness of NCL in Covid-19 patients. Prospective clinical studies and clinical trials are required to authorize the potential role of NCL in patients with Covid-19 regarding the severity and clinical outcomes.

Author contributions

Manuscript preparation: HMAK, AIAG, KJA. Manuscript editing: GELSB, AA.

Funding

This research received no external funding.

Declarations

Conflict of interest

The authors declare that they have no competing interests.