IVM was originally discovered from organisms that were isolated from soil samples collected from the woods nearby to Kitasato Institute in Kawana, Japan. Fermentation products released by a bacterium from the soil, which was later classified as Streptomyces acermitilis, appeared to exhibit antiparasitic activity (specifically against Nematospiroides dubious). Purification and isolation of the bioactive compounds showed naturally occurring macrocyclic lactones, and these were subsequently named avermectins. Avermectins are made up of four compounds, which exist as two variants: A1, A2, B1, and B2. Variants ‘A’ and ‘B’ indicate the presence of methoxy or hydroxyl groups, respectively, at the C5 position. Number ‘1’ describes the double bond between C22 and C23. On the other hand, number ‘2’ indicates the presence of hydrogen at C22 and a hydroxyl group at C23. B1 avermectins were proven to be most active on oral administration, and on this basis, IVM was chemically derived. IVM contains an 80:20 combination of 22,23-dihydro-avermectin B1a and 22,23-dihydro-avermectin B1b. Its antiparasitic effects are primarily caused by high-affinity irreversible binding to glutamate-gated chloride (Cl-) channels located on nerve and muscle cells of nematode, which leads to hyperpolarization (). Ultimately, the increased permeability to Cl- results in paralysis and death of the nematode ().
As of yet, IVM has treated hundreds of millions of people with onchocerciasis, most commonly given at 150-200 μg/kg of body weight for one dose initially, and repeated at 6-12 monthly intervals as appropriate (). Its use extends to a broad spectrum of parasitic nematodes on both oral and parenteral administration and is also effective against arthropods, including lice ().
Importantly, IVM was approved by the FDA for human use in 1987 (). Its low toxicity and safety are attributed to the fact that its human target receptors are ‘secluded’ in the CNS, and IVM has not been shown to cross the blood-brain barrier. In addition, IVM displays a 100-fold greater affinity for parasitic Cl- channels compared to the human homologs (). Moreover, severe detrimental effects in humans were shown only in those who over-dosed using approximately 15.4 mg/kg body weight IVM, which is 77 times above the prescribed dose. This corroborates the advantage of repurposing drugs, as these medications have already been tested arduously and extensively to confirm their efficacy and safety, thereby decreasing the transit time from shelf to intake.
Screening for Inhibitors of Nuclear Import
The potential of IVM as an inhibitor of nuclear transport of viral proteins was initially suggested in 2011. Initially, Wagstaff and colleagues screened for nuclear import inhibitors, which block the interaction between IMPs and potential target cellular proteins (). They randomly selected 480 compounds from LOPAC1280 (Library of Pharmacologically Active Compounds; Sigma, St. Louis, MO). IVM surfaced as a drug that generally inhibits IMP activity (). A year later, they confirmed that this apparent activity of IVM also inhibits nuclear transport of viral proteins HIV and Dengue virus in HeLa cells (36). Specifically, it was shown that GFP-tagged IMP was significantly reduced in the nucleus of HeLa cells after 3 hrs. of co-incubation with IVM (). Moreover, the effect was unique to IMPα/β interactions and did not affect proteins bound only to IMPβ1. The importance of blocking the nuclear import of viral proteins emerged when it was later shown that IVM also prevented replication of HIV (). As such, it surfaced as a possible repurposed drug, capable of preventing viral cargo from interacting with IMPα/β for nuclear import, with the potential to result in viral ‘death’ ().
Soon after, the effect of IVM against the nuclear import of viral proteins was further validated. For example, IVM prevented nuclear translocation of nsp5 in Dengue virus, West Nile virus, and influenza and inhibited transport of large tumor antigen (Tag) in simian virus (). The Wagstaff et al., 2011 and 2012 studies were pivotal in providing much of the initial rationale for the recent consideration of IVM as a SARS-CoV-2 antiviral agent. It was the same researchers who nine years later, in 2020, demonstrated that IVM inhibits SARS-CoV-2 in vitro replication ().
Given its efficacy in inhibiting nuclear import of other viral proteins, the anti-viral effect of IVM against SARS-CoV-2 was evaluated shortly after the pandemic erupted (). Specifically, Vero/hSLAM cells were inoculated with SARS-CoV-2 isolate for 2 hrs., followed up with supplementation of 5 μM of IVM. Within 24 hrs. after treatment, there was a 93% reduction of viral RNA in the supernatant and a 99.8% reduction of cellular viral RNA, compared to controls. After 48 hrs., there was a further 5000-fold reduction of viral RNA in the supernatant as well as the cell pellets, indicating that cells were essentially ‘cleared’ of SARS-CoV-2 (). Although IVM possessed a potent antiviral activity (IC50= ~2μM), no cytotoxicity was detected at any time points in this study ().
IVM Specifically Interacts With IMPα
IVM was shown to specifically inhibit IMP α/β mediated nuclear import required for replication of HIV-1 and Dengue virus, and therefore it was proposed as the potential mechanism by which it inhibits SARS-CoV-2 (). Indeed, this baton was passed on, and a subsequent study verified the IVM-IMPα interaction in host cells (). It was shown that IVM not only inhibits IMPα association with IMPβ but can even dissociate IMPα/β heterodimers (38). The specific binding target of IVM was identified, using CD spectroscopy, to be the alpha-helical rich ‘armadillo’ (ARM) domain of IMPα. Moreover, as concentrations of IVM increased, alpha-helices in the ARM domain became increasingly destabilized. No changes were detected in the structure of IMPβ. They further verified that this observed effect on IMPα impaired its binding to NLS-containing nsp5 from Dengue Virus (38). As such, preventing N interaction with IMPα is a likely mechanism that contributes to IVM’s ability to hinder SARS-CoV-2 in vitro replication (Figure 2).
Proposed mechanism of action of Ivermectin against SARS-CoV-2. IVM has previously been established as a nuclear import inhibitor by binding to and antagonizing the ability of the importin (IMPα) to bind to its target cargo. Because the nucleocapsid (N) protein contains a nuclear localization signal, IVM is expected to prevent the binding of IMPα to the N binding site. Consequently, N would not perform its nuclear activity which is thought to suppress the host immune response and sequester ribosomal subunits, mechanisms that are thought to abrogate sufficient viral replication. In addition, the expression of two major cytokines, TNFα and IL-6 which drive the detrimental cytokine storm in COVID-19 patients were also shown to be dampened in the presence of IVM. As of yet, these two major mechanisms which involve viral replication and immune response suppression appear to characterize the main activities of IVM against SARS-CoV-2.
The Implications of Disrupting IMPα Activity for the Host Cells
Because IVM emerged as a general inhibitor of IMPα-dependent nuclear cargo, it is important to consider the implications this may have on host cell proteins and functions. However, any effect would likely be non-detrimental given the safety record of IVM over the past 50 years and its transient prescription for an acute disease ().
Notably, expression levels of IMPα vary in a cell and developmental specific manner, particularly during differentiation processes (). Animal knock-out studies for impα highlighted its essential role in reproductive organ development. Specifically, impa-/- mice developed lower reproductive organ function in females, including insufficient follicles’ growth during the maturation stage in the ovaries, incomplete uterus construction, and reduced serum progesterone (). Moreover, estrogen-responsive genes were also not efficiently expressed, indicating IMPα may be involved in hormonal regulation. Other cells like muscle stem cells underwent apoptosis and depletion ().
IVM was also shown to disrupt the oxygen regulatory mechanisms (). Hypoxia-induced transcription factors (HIFs) regulate cellular adaptation to decreased oxygenation within the cell. Hypoxia renders the HIF subunit, HIFα stable and causes it to accumulate within the nucleus where it induces transcription of genes that may readjust oxygen levels. HIFα translocation into the nucleus requires nuclear import in an NLS-IMPα/β dependent manner. Indeed, it was shown that IVM results in a decreased association between HIFα and IMPα, preventing its path into the nucleus. Subsequently, nuclear HIFα and transcription of target oxygen-regulatory genes were reduced ().
Pharmacokinetic studies conducted by MERCK show that IVM plasma concentrations peak after 4 hours, following 12 mg doses in healthy human volunteers (). Subsequently, it is metabolized in the liver and its breakdown products are mainly excreted in the feces over 12 days. Its half-life is around 18 hrs. Moreover, it was shown that it does not bind permanently to its target proteins.
Other Possible Modes of Antiviral Activity by IVM
A recent molecular docking study demonstrated that in addition to IMPα, IVM showed high binding affinity to the viral RNA-dependent RNA polymerase (RdRp) complexed with RNA helicase compared to the other 10 viral targets included in the analysis (). However, it was later shown that IVM does not bind to viral RdRp in both Zika virus (Z.V.) and Dengue virus (). It remains to be identified if IVM may bind to RdRp in coronaviruses.
Other mechanisms of IVM action have also been identified (Figure 2). For example, it previously was shown to suppress the production of Interleukin-6 (IL-6) and Tumor Necrosis Factor-alpha (TNFα), two major components of the detrimental cytokine storm induced by SARS-CoV-2 (). Moreover, a study in Syrian hamsters showed that IVM did not affect SARS-CoV-2 viral load but overall dramatically reduced IL-6/IL-10 ratio and modulated infection outcomes (). Specifically, hamsters that were inoculated with SARS-CoV-2 were subcutaneously injected with IVM (0.4 mg/kg body weight). IVM reduced the severity of clinical symptoms in males but eliminated symptoms in females, which suggests a gender-specific effect of this drug and a factor that should be considered in clinical trials. The gender-specific modulation of IVM on cytokines was also apparent. While females displayed lower levels of cytokines such as IL-6, INFγ, and TNFα, males on the other hand developed an enhanced production of INFγ. Notably, viral load in nasal and lung tissues, as well as viral replication rate were not altered in either gender after administration of IVM (). This is in contrast to the finding that IVM significantly blocks viral replication in vitro and it may be attributed to the much higher dose of IVM that was used (). However, it is important to note that the dose of IVM that was used on the cells (5 μM) is approximately 50-fold higher than the normal Cmax associated with one dose of IVM (200 μg/kg) (46, 47). Therefore, it is important to establish a dose-dependent effect of IVM on viral load and safety in human COVID-19 patients at various doses.
Further, IVM was shown to induce an elevated level of IL-6 and TNFα in onchocerciasis patients, two days after a single dose (150 μg/kg body weight) (48). However, this was attributed to the destruction of the parasite microfilariae, which would usually not be a factor in COVID-19 patients.
Thus far, studies on IVM highlight that it remains important to identify the specific dose of IVM that may reduce viral load, without adverse effects, in humans and understand if it will differentially affect male and female COVID-19 patients.
Adverse Effects Reported in Animals and Humans in Previous Studies Using Ivermectin
The direct toxic effects of IVM were first identified in animal studies, mainly as an antiparasitic treatment. The vast amount of evidence around the use of IVM exists using dose regimens of 150-200 μg/kg of body weight. Hence, the risks and associated side effects are mostly reported at these doses. Studies suggest the common adverse effects are rash, headache, nausea, and dizziness, while transient tachycardia is rare and self-limiting (49). Other effects include ataxia, sweating, tremors, and in some cases, coma and death.
A retrospective study looking at residents of an extended care facility showed increased rates of death in patients treated with IVM for resistant scabies. These study results were criticized due to some significant limitations of the study, and therefore, the deaths of these residents could not be reliably attributed to the IVM. For example, there was no control of the lasting previous drugs used to treat scabies, some of which are known for their toxic effects. Importantly, IVM’s toxic effects are short-term and are usually resolved ().
Studies exploring the adverse event profiles of patients on high doses of IVM have also been conducted. Higher dose levels (300-1000 μg/kg) were administered to healthy individuals with head lice, as part of a double-blind and randomized trial, with adverse events reported as having no clinical or biochemical significance ).
TheEffect of Ivermectin on SARS-CoV-2 Patients
Soon after IVM emerged as a potential therapeutic agent, clinical trials on COVID-19 patients ensued. However, the available published data and ongoing clinical trials, which are summarized in Table 1, do not provide a clear and uniform understanding of the effect of IVM on COVID-19 patients. This is mainly due to small sample sizes (n=12-203) and the lack of information specifying when exactly IVM is administered after testing positive for SARS-CoV-2 (. It is important to highlight how soon after testing positive the patient receives IVM, in addition to the degree of COVID-19 severity, to understand if the effect of the drug is dependent on time and symptom severity. Additionally, several studies are retrospective in which investigators examined past COVID-19 patients who were prescribed IVM, without proper placebo control groups. Moreover, most of the studies utilize the antiparasitic effective dose for IVM (0.2 mg/kg body weight), which is substantially less than the equivalent in vitro dose of IVM used against SARS-CoV-2 (, 53, 54). Nevertheless, the available data does indicate that IVM may be effective against COVID-19.
Credited to Front. Immunol
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