Repositioning Ivermectin for Covid-19 treatment: Molecular mechanisms of action...

Repositioning Ivermectin for Covid-19 treatment: Molecular mechanisms of action against SARS-CoV-2 replication


Abstract

Ivermectin (IVM) is an FDA-approved macrocyclic lactone compound traditionally used to treat parasitic infestations and has shown to have antiviral potential from previous in-vitro studies. Currently, IVM is commercially available as a veterinary drug but has also been applied in humans to treat onchocerciasis (river blindness - a parasitic worm infection) and strongyloidiasis (a roundworm/nematode infection). In light of the recent pandemic, the repurposing of IVM to combat SARS-CoV-2 has acquired significant attention. Recently, IVM has been proven effective in numerous in-silico and molecular biology experiments against infection in mammalian cells and human cohort studies. One promising study had reported a marked reduction of 93% of the released virion and 99.98% unreleased virion levels upon administration of IVM to Vero-hSLAM cells. IVM's mode of action centers around the inhibition of the cytoplasmic-nuclear shuttling of viral proteins by disrupting the Importin heterodimer complex (IMPα/β1) and downregulating STAT3, thereby effectively reducing the cytokine storm. Furthermore, the ability of IVM to block the active sites of viral 3CLpro and S protein disrupts important machinery such as viral replication and attachment. This review compiles all the molecular evidence to date, in the review of the antiviral characteristics exhibited by IVM. Thereafter, we discuss IVM's mechanism and highlight the clinical advantages that could potentially contribute towards disabling the viral replication of SARS-CoV-2. In summary, the collective review of recent efforts suggests that IVM has a prophylactic effect and would be a strong candidate for clinical trials to treat SARS-CoV-2.



1. Introduction

The recent pandemic of Covid-19 has been acknowledged as a global health crisis that has threatened public health and safety. This phenomenon has raised awareness and conflict towards the healthcare sector for their negligence and absence of alternative measures that would have prevented or successfully treated novel causative viral agents. Approximately 233,000,000 individuals have been affected during this pandemic, and 4,750,000 associated deaths were correlated worldwide. Currently, the cases of Covid-19 are still growing rapidly on a global scale [1]. The etiological agent that contributed to this disastrous episode is known to be the SARS Coronavirus – 2 (SARS-CoV-2). To date, the origin of SARS-CoV-2 is still debated though evidence suggests bats as the source of the virus, akin to the SARS-CoV outbreak identified during 2002 [2]. Although SARS-CoV-2 carries a 3.4% mortality rate, which is significantly lower than its predecessors (SARS-CoV and MERS-CoV), it is more infectious. Of note, up to 15% of SARS-CoV-2 cases led to several critical complications such as pneumonia, heart arrhythmia, septic shock, multiple organ failure, and the more pronounced acute respiratory distress syndrome (ARDS) [3]. Thus, emphasizing the need for effective antiviral therapy. In this urgent time of need, drug repositioning attempts are crucial to address the immediate demand for a fast and effective antiviral therapy against COVID-19. Drug repositioning, also known as drug repurposing or drug recycling, is an alternative approach to finding new use of an established drug to treat other diseases aside from the intended ones. A repositioned drug has often gone through all the rigorous safety and pharmacokinetic profiling studies with well-established ADMET (absorption, distribution, metabolism, excretion, toxicity) data [4]. The benefits of utilizing repositioned drugs are the omission of critical and time-consuming drug development stages, which significantly reduces the time needed to produce an effective antiviral drug. In this paradigm, we discuss the numerous drug candidates already proposed for SARS-CoV-2 treatment, such as Remdesivir, Lopinavir-Ritonavir, Oseltamivir, Saquinavir, Tenofovir, and ciclesonide [5]. Firstly, Lopinavir-Ritonavir and Saquinavir have been proven effective in inhibiting the replication of SARS-CoV-2 via forming a stable complex with its main protease (Mopar or 3CLpro), which oversees the regulation of replicase polyprotein proteolytic activity and viral replication [6], [7]. Conversely, tenofovir as a nucleotide analog for HIV treatment was revealed to interact with papain-like protease (PLpro) and Mopar. This interferes with the binding of SARS-CoV-2 S protein to the host ACE2 receptor, subsequently reducing the production of non-structural protein (NSP) required for viral replication [8]. Additionally, tenofovir also binds to SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), an important replicase for viral replication, effectively bringing down the viral load [8]. Ciclesonide is a corticosteroid was reported to have targeted the nsp15 or also known as the Indo viral RNA endoribonuclease uridylate-specific (NendoU) enzyme in SARS-CoV-2. This protein interferes with the human innate immune response, contributing to its immune-evasive properties in COVID-19 patients [9].


2. Antiparasitic action of Ivermectin

As aforementioned, IVM was chemically derived from AVMs, a group of 16-membered macrocyclic lactone compounds. IVM possesses two variants as A or B, which are differentiated by the presence of methoxy or hydroxyl groups at C5, respectively. AVMs naturally occur as mixtures of eight compounds A1a, A1b, A2a, A2b, B1a, B1b, B2a and B2b, from which, B1 is administered orally while B2 is parenterally administered [23]. The subscript ‘1’ following the variants indicates the presence of double bonds between C22 and C23 whereas, the subscript of ‘2’ describes the presence of hydrogen and hydroxyl groups at C22 and C23, respectively [23]. As such, IVM is a semi-synthetic derivative of Avermectin B1 where it consists of two homologues, 22, 23-dihydro-avermectin B1a and 22, 23-dihydro-avermectin B1b in the ratio of 80:20 [24]. IVM is capable of affecting the motility, feeding, and reproduction of parasites through high-affinity binding to the γ-aminobutyric acid (GABA)-regulated or glutamate-gated chloride channels. In response, the activities of these channels are enhanced, leading to hyperpolarization of the cell membrane and influx of chloride ions. Subsequently, this inhibits the regulatory light chain of myosin II phosphorylation via p21 activated kinase 1 (PAK1), causing muscle paralysis and eventually, parasite death [25]. Apart from its original function, IVM has been proven effective in numerous antiviral treatments via the inhibition of the nuclear import of viral nucleoproteins. This encompasses HIV-1, West Nile Virus (WNV), tick-borne encephalitis, Zika Virus (ZKV), Venezuelan equine encephalitis virus, Chikungunya virus, Pseudorabies virus, Adenovirus, Influenza virus, SARS-CoV-1, and most recently in SARS-CoV-2 [26], [27], [28]. The mode of inhibition of the aforementioned viruses were described in detail below.




3. Antiviral action of Ivermectin

Several studies in the past have revealed the possible role of SARS-CoV-1 ORF6 interacting with the Karyopherin-α2 (KPNA2), retaining the IMPα/β1 of the Golgi membrane. Thereafter, inhibiting the STAT1 nuclear transport antagonizes antiviral activity and downplays the host's antiviral response [28], [29], [30]. Given the role of importin in many viruses, especially SARS-CoV-1, it is of great interest to explore the mechanism of action of IVM for its potential in viral inhibition. We briefly describe the antiviral action of IVM for each of the viruses listed above and postulate its role in the SARS-CoV-2 infection cycle.


4. General morphology of SARS-CoV-2

The general morphology of SARS-CoV-2 closely resembles other beta-coronaviruses, such as the SARS-CoV and MERS-CoV [44]. The SARS-CoV-2 is a non-segmented positive-sense RNA virus with a diameter of 65-125 nm [45]. The typical layout for the SARS-CoV-2 genome is denoted as follows [5′‑leader-UTR-replicase-S-E-M-N-3′-UTR-poly (A) tail], with accessory genes scattered between the structural genes (S-E-M-N) at the 3′ end (38×). Under the envelope, it consists of a ~29.9 kilobase (kb) RNA genome with 2/3rd of the genome containing the main open reading frame 1a and 1b (ORF1ab) replicase gene from the 5′-end, encoding for the non-structural proteins (NSP 1–16) while the remaining 1/3rd genome encodes for the structural proteins (spike protein (S), envelope protein (E), membrane protein (M) and nucleocapsid protein (N)) [46]. The NSPs play an important role in viral replication in infected host cells. NSP 1 and 3 are known to inhibit IFN signalling, interrupting the translation of RNA and innate immune responses [47]. NSP 3 and 5 promote cytokine expression and viral protein cleavage [47]. NSP-12 is an RNA-dependent RNA polymerase (RdRp) and is inhibited by IVM in studies on SARS-CoV and MERS-CoV [48], [49]. This discovery discloses strong possibilities for SARS-CoV-2. RdRp, also known as RNA replicase, is a vital enzyme in the life cycle for RNA viruses since it primarily functions to catalyse the replication of RNA from an existing RNA template in the virus thereby initiating viral replication.

5. The molecular action of Ivermectin on SARS-CoV-2

The SARS-CoV accessory protein ORF6 has been shown to sequester IMPα/β1 on the rough endoplasmic reticulum which antagonizes the STAT1 transcription factor, resulting in an antiviral potential [50]. The genomic similarity between SARS-CoV-2 and the previous SARS-CoV may reveal the role of the importin heterodimer complex (IMPα/β1) for viral protein (NSP12-RdRp) shuttling between the nucleus and cytoplasm upon infection. Currently, there is only one RdRp inhibitor approved by the FDA for Covid-19, namely Remdesivir [51]. However, a recent discovery from Monash University, Australia reported that IVM could inhibit SARS-CoV-2 within a 48 h post-infection, drawing much attention worldwide [22]. IVM, a non-specific inhibitor of IMPα/β1-dependent nuclear import, now shows great potential in reducing SARS-CoV-2 viral replication via different modes. Apart from disrupting the importin heterodimer complex IMPα/β1, IVM also prevents cytokine storm via STAT3 regulation. Besides this, IVM also inhibits the viral entry via the ACE2 receptor and is capable of disrupting the viral 3-chymotrypsin-like enzyme in SARS-CoV-2 [52], [53]. We describe each of the proposed molecular mechanisms below.


6. Clinical efficacies of IVM

Numerous clinical studies are underway to study the efficacy of IVM. Ivermectin has illustrated great potency towards asymptomatic SARS-CoV-2-positive subjects in a randomized trial in Lebanon [90]. In that study, the IVM group (n = 50) has an increased cycle-threshold (Ct) value from 15.13 to 30.14 whereas the control group (n = 50) has Ct values from 14.20 to 18.96 at 72 h [90]. A higher Ct-value denotes an insignificant viral remnant or non-viable virus, from which the values of 30 and above are to be considered as negative [91]. Additionally, the subjects from the IVM group developed fewer symptoms compared to the non-IVM group from the reported incidence of fever (2% vs 22%), ageusia (6% vs 24%), anosmia (6% vs 32%), and myalgia (0% vs 18%) [90]. Other notable studies on the positive effects of IVM on COVID-19 patients are as follows. In a study conducted by Elgazzar et al., a randomized 200 healthcare and household contacts with COVID-19 patients consisting of two groups of 100 patients were each given a high dose of 0.4 mg/kg IVM on day 1 followed by a subsequent dose on day 7, or without treatment. The study had reported a significant reduction in PCR contacts testing at 2% on IVM compared to 10% on the non-treatment group [92], [93]. Besides that, Elgazzar et al. had also shown a remarkable outcome of IVM in a randomized controlled trial among 400 hospitalized patients. Their study consisted of four groups with a sample size of 100 patients each. Group 1 was treated with a single IVM dose of 0.4 mg/kg + standard-of-care (SOC); Group 2 was treated with 400 mg of hydroxychloroquine twice during the 1st day followed by a twice-daily dose of 200 mg for the 5 consecutive days + SOC; Group 3 and Group 4 obeyed the same treatment plan from Group 1, and Group 2 respectively, with the specific exception of severely ill patients [92]. The outcomes were statistically significant as a lower rate of progression in the Ivermectin Group 1 and 3 vs hydroxychloroquine Group 2 and 4 (1% and 4% vs 22% and 30%), respectively [92]. These results were accompanied by 0 death count and 2% mortality in IVM Group 1 and 3 respectively, followed by 4 death count and 20% mortality for the Group 2 and 4 hydroxychloroquine settings, respectively [92].


7. Contradictions

Despite numerous positive outcomes for IVM in SARS-CoV-2 treatment, there are several contradictions. For instance, a study has reviewed that the standard dosage of single-dose IVM (200μg/kg) showed no significant clinical and microbiological outcomes compared to the patients that did not receive any IVM [95]. Albeit there was no significant difference, a smaller proportion of the patients from the IVM group required intensive care as compared to the non-IVM group (69% vs 38%), postulating the need for a higher effective dose [95]. Another study pointed no differences in the viral load, outcomes of adverse events, and the laboratory parameters between the IVM treated group (30 participants) and control group (15 participants) from the baseline until day 5 [96]. However, there were positive correlations where the higher IVM plasma concentrations resulted in a greater reduction of viral load in nasopharyngeal secretions and viral decay rate, also suggesting the need for a higher dose [96]. A double-blind randomized trial conducted in Cali, Columbia has also shown similar results where the duration of symptoms and time to resolution in 400 patients (200 with IVM, and 200 controls) showed no statistical difference over a 5-day observation [97]. Besides that, another study suggests larger dose (150–200 μg/kg) of IVM may trigger several side effects ranging across symptoms such as rashes, headaches, nausea, ataxia, sweating, tremors, and more severe tachycardia, coma, and death. Thus, implying the need for more controlled trials and safety and efficacies studies for the mass public [98]. Another report showed that IVM alone has a low probability of success in treating COVID-19 as the approved dose or dose of 10× higher than the approved is unlikely to reach the concentration needed for 50% inhibition (IC50) in the lungs after single-dose oral administration, making it less ideal for COVID-19 treatment [99].


8. Conclusion

The rapid emergence of SARS-CoV-2 has put drug repositioning in a critically important position. IVM, an FDA approved antiparasitic drug for onchocerciasis and strongyloidiasis, now sees great opportunity as an interim antiviral drug for SARS-CoV-2. IVM has already been proven effective in numerous in-vitro viral studies such as HIV-1, Flaviviruses, Influenza A virus and many more. The main mode of antiviral action for IVM revolves around the disruption of the Importin heterodimer complex (IMPα/β1), which is a vital complex that shuttles the viral protein (NS12) into the nucleus from the cytoplasm via the NPC upon receiving NLS. In addition, IVM sees a huge potential in inhibiting cytokine storm in Covid-19 patients. This is due to IVM downregulating STAT3, a key protein in the JAK-STAT signalling pathway that contributes to enhanced inflammatory IL-6 production in infected patients. Furthermore, IVM can bind to several residues in the S protein of SARS-CoV-2, concomitantly hindering the attachment of S protein to host ACE-2 receptors for viral entry, effectively reducing the viral load. Finally, IVM binds on the active sites of 3CLpro, a vital protease, for the production of functional NSPs for viral replication. Owing to its multifarious antiviral machinery described above, IVM could be warranted as an interim solution to delay the rapid progression of SARS-CoV-2 in infected patients while significantly reducing the severity of disease by bringing down the viral load and downregulating the post-Covid-19 cytokine storm in recovering patients. There is an urgent demand for more relevant and comprehensive clinical data to accurately define the pharmacokinetic profile of IVM thereby paving the path for its use against Covid-19. buy ivermectin | buy ivermectin India | buy ivermectin | buy ivermectin India | ivermectin tablet for humans | ivermectin tablet price ||ivermectin 12 mg tablet price in India | ivermectin buy online | where to buy ivermectin for humans | ivermectin dosage | where to buy ivermectin UK | ivermectin uses | ivermectin | Stromectol |buy ivermectin online | buy ivermectin online UK | buy ivermectin online NZ | buy ivermectin online south Africa | buy ivermectin online Malaysia | Buy Stromectol (ivermectin) Online at Lowest Price | Buy Ivermectin for Covid 19 Over the Counter | Buy Ivermectin for Humans and Ivermectin 3mg | Ivermectin Online Prescription | Buy Ivermectin Online (@buyivermectin) | order/ Buy Ivermectin Online Nz | No Prescription ivermectin | Ivermectol 12mg Tablet 2'S - Buy Medicines online |


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