Antiviral Research 178 (2020) 104787

Contents lists available at ScienceDirect

Antiviral Research
journal homepage: www.elsevier.com/locate/antiviral

The FDA-approved drug ivermectin inhibits the replication of SARS-CoV-2 in T

vitro
Leon Calya, Julian D. Drucea, Mike G. Cattona, David A. Jansb, Kylie M. Wagstaffb,∗
a
Victorian Infectious Diseases Reference Laboratory, Royal Melbourne Hospital, At the Peter Doherty Institute for Infection and Immunity, Victoria, 3000, Australia
b
Biomedicine Discovery Institute, Monash University, Clayton, Vic, 3800, Australia

A B S T R A C T

Although several clinical trials are now underway to test possible therapies, the worldwide response to the COVID-19 outbreak has been largely limited to mon-
itoring/containment. We report here that Ivermectin, an FDA-approved anti-parasitic previously shown to have broad-spectrum anti-viral activity in vitro, is an
inhibitor of the causative virus (SARS-CoV-2), with a single addition to Vero-hSLAM cells 2 h post infection with SARS-CoV-2 able to effect ~5000-fold reduction in
viral RNA at 48 h. Ivermectin therefore warrants further investigation for possible benefits in humans.

1. Introduction
Ivermectin is an FDA-approved broad spectrum anti-parasitic agent
(Gonzalez Canga et al., 2008) that in recent years we, along with other
groups, have shown to have anti-viral activity against a broad range of
viruses (Gotz et al., 2016; Lundberg et al., 2013; Tay et al., 2013;
Wagstaff et al., 2012) in vitro. Originally identified as an inhibitor of
interaction between the human immunodeficiency virus-1 (HIV-1) in-
tegrase protein (IN) and the importin (IMP) α/β1 heterodimer re-
sponsible for IN nuclear import (Wagstaff et al., 2011), Ivermectin has
since been confirmed to inhibit IN nuclear import and HIV-1 replication
(Wagstaff et al., 2012). Other actions of ivermectin have been reported
(Mastrangelo et al., 2012), but ivermectin has been shown to inhibit
nuclear import of host (eg. (Kosyna et al., 2015; van der Watt et al.,
2016)) and viral proteins, including simian virus SV40 large tumour
antigen (T-ag) and dengue virus (DENV) non-structural protein 5
(Wagstaff et al., 2012, Wagstaff et al., 2011). Importantly, it has been
demonstrated to limit infection by RNA viruses such as DENV 1-4 (Tay
et al., 2013), West Nile Virus (Yang et al., 2020), Venezuelan equine
encephalitis virus (VEEV) (Lundberg et al., 2013) and influenza (Gotz
et al., 2016), with this broad spectrum activity believed to be due to the
reliance by many different RNA viruses on IMPα/β1 during infection
(Caly et al., 2012; Jans et al., 2019). Ivermectin has similarly been
shown to be effective against the DNA virus pseudorabies virus (PRV)
both in vitro and in vivo, with ivermectin treatment shown to increase
survival in PRV-infected mice (Lv et al., 2018). Efficacy was not
observed for ivermectin against Zika virus (ZIKV) in mice, but the au-
thors acknowledged that study limitations justified re-evaluation of
ivermectin's anti-ZIKV activity (Ketkar et al., 2019). Finally, ivermectin
was the focus of a phase III clinical trial in Thailand in 2014–2017,
against DENV infection, in which a single daily oral dose was observed
to be safe and resulted in a significant reduction in serum levels of viral
NS1 protein, but no change in viremia or clinical benefit was observed
(see below) (Yamasmith et al., 2018).
The causative agent of the current COVID-19 pandemic, SARS-CoV-
2, is a single stranded positive sense RNA virus that is closely related to
severe acute respiratory syndrome coronavirus (SARS-CoV). Studies on
SARS-CoV proteins have revealed a potential role for IMPα/β1 during
infection in signal-dependent nucleocytoplasmic shutting of the SARS-
CoV Nucleocapsid protein (Rowland et al., 2005; Timani et al., 2005;
Wulan et al., 2015), that may impact host cell division (Hiscox et al.,
2001; Wurm et al., 2001). In addition, the SARS-CoV accessory protein
ORF6 has been shown to antagonize the antiviral activity of the STAT1
transcription factor by sequestering IMPα/β1 on the rough ER/Golgi
membrane (Frieman et al., 2007). Taken together, these reports sug-
gested that ivermectin's nuclear transport inhibitory activity may be
effective against SARS-CoV-2.
To test the antiviral activity of ivermectin towards SARS-CoV-2, we
infected Vero/hSLAM cells with SARS-CoV-2 isolate Australia/VIC01/
2020 at an MOI of 0.1 for 2 h, followed by the addition of 5 μM iver-
mectin. Supernatant and cell pellets were harvested at days 0–3 and
analysed by RT-PCR for the replication of SARS-CoV-2 RNA (Fig. 1A/B).

The authors would like readers to be aware of the following letter issued by the FDA titled: “Do Not Use Ivermectin Intended for Animals as Treatment for COVID-
19 in Humans” at https://www.fda.gov/animal-veterinary/product-safety-information/fda-letter-stakeholders-do-not-use-ivermectin-intended-animals-treatment-
covid-19-humans.
∗
Corresponding author.
E-mail address: kylie.wagstaff@monash.edu (K.M. Wagstaff).

https://doi.org/10.1016/j.antiviral.2020.104787
Received 18 March 2020; Received in revised form 27 March 2020; Accepted 29 March 2020
Available online 03 April 2020
0166-3542/ © 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
L. Caly, et al. Antiviral Research 178 (2020) 104787

(caption on next page)

2
L. Caly, et al. Antiviral Research 178 (2020) 104787

Fig. 1. Ivermectin is a potent inhibitor of the SARS-CoV-2 clinical isolate Australia/VIC01/2020. Vero/hSLAM cells were in infected with SARS-CoV-2 clinical
isolate Australia/VIC01/2020 (MOI = 0.1) for 2 h prior to addition of vehicle (DMSO) or Ivermectin at the indicated concentrations. Samples were taken at 0–3 days
post infection for quantitation of viral load using real-time PCR of cell associated virus (A) or supernatant (B). IC50 values were determined in subsequent experiments
at 48 h post infection using the indicated concentrations of Ivermectin (treated at 2 h post infection as per A/B). Triplicate real-time PCR analysis was performed on
cell associated virus (C/E) or supernatant (D/F) using probes against either the SARS-CoV-2 E (C/D) or RdRp (E/F) genes. Results represent mean ± SD (n = 3). 3
parameter dose response curves were fitted using GraphPad prism to determine IC50 values (indicated). G. Schematic of ivermectin's proposed antiviral action on
coronavirus. IMPα/β1 binds to the coronavirus cargo protein in the cytoplasm (top) and translocates it through the nuclear pore complex (NPC) into the nucleus
where the complex falls apart and the viral cargo can reduce the host cell's antiviral response, leading to enhanced infection. Ivermectin binds to and destabilises the
Impα/β1 heterodimer thereby preventing Impα/β1 from binding to the viral protein (bottom) and preventing it from entering the nucleus. This likely results in
reduced inhibition of the antiviral responses, leading to a normal, more efficient antiviral response.

At 24 h, there was a 93% reduction in viral RNA present in the su-
pernatant (indicative of released virions) of samples treated with iver-
mectin compared to the vehicle DMSO. Similarly a 99.8% reduction in
cell-associated viral RNA (indicative of unreleased and unpackaged
virions) was observed with ivermectin treatment. By 48 h this effect
increased to an ~5000-fold reduction of viral RNA in ivermectin-
treated compared to control samples, indicating that ivermectin treat-
ment resulted in the effective loss of essentially all viral material by
48 h. Consistent with this idea, no further reduction in viral RNA was
observed at 72 h. As we have observed previously (Lundberg et al.,
2013; Tay et al., 2013; Wagstaff et al., 2012), no toxicity of ivermectin
was observed at any of the timepoints tested, in either the sample wells
or in parallel tested drug alone samples.
To further determine the effectiveness of ivemectin, cells infected
with SARS-CoV-2 were treated with serial dilutions of ivermectin 2 h
post infection and supernatant and cell pellets collected for real-time
RT-PCR at 48 h (Fig. 1C/D). As above, a > 5000 reduction in viral RNA
was observed in both supernatant and cell pellets from samples treated
with 5 μM ivermectin at 48 h, equating to a 99.98% reduction in viral
RNA in these samples. Again, no toxicity was observed with ivermectin
at any of the concentrations tested. The IC50 of ivermectin treatment
was determined to be ~2 μM under these conditions. Underlining the
fact that the assay indeed specifically detected SARS-CoV-2, RT-PCR
experiments were repeated using primers specific for the viral RdRp
gene (Fig. 1E/F) rather than the E gene (above), with nearly identical
results observed for both released (supernatant) and cell-associated
virus.
Taken together these results demonstrate that ivermectin has anti-
viral action against the SARS-CoV-2 clinical isolate in vitro, with a single
dose able to control viral replication within 24–48 h in our system. We
hypothesise that this is likely through inhibiting IMPα/β1-mediated
nuclear import of viral proteins (Fig. 1G), as shown for other RNA
viruses (Tay et al., 2013; Wagstaff et al., 2012; Yang et al., 2020);
confirmation of this mechanism in the case of SARS-CoV-2, and iden-
tification of the specific SARS-CoV-2 and/or host component(s) im-
pacted (see (Yang et al., 2020)) is an important focus future work in this
laboratory. Ultimately, development of an effective anti-viral for SARS-
CoV-2, if given to patients early in infection, could help to limit the
viral load, prevent severe disease progression and limit person-person
transmission. Benchmarking testing of ivermectin against other poten-
tial antivirals for SARS-CoV-2 with alternative mechanisms of action
(Dong et al., 2020; Elfiky, 2020; Gordon et al., 2020; Li and De Clercq,
2020; Wang et al., 2020) would thus be important as soon as practic-
able. This Brief Report raises the possibility that ivermectin could be a
useful antiviral to limit SARS-CoV-2, in similar fashion to those already
reported (Dong et al., 2020; Elfiky, 2020; Gordon et al., 2020; Li and De
Clercq, 2020; Wang et al., 2020); until one of these is proven to be
beneficial in a clinical setting, all should be pursued as rapidly as
possible.
Ivermectin has an established safety profile for human use
(Gonzalez Canga et al., 2008; Jans et al., 2019; Buonfrate et al., 2019),
and is FDA-approved for a number of parasitic infections (Gonzalez
Canga et al., 2008; Buonfrate et al., 2019). Importantly, recent reviews
and meta-analysis indicate that high dose ivermectin has comparable
safety as the standard low-dose treatment, although there is not enough
evidence to make conclusions about the safety profile in pregnancy
(Navarro et al., 2020; Nicolas et al., 2020). The critical next step in
further evaluation for possible benefit in COVID-19 patients will be to
examine a multiple addition dosing regimen that mimics the current
approved usage of ivermectin in humans. As noted, ivermectin was the
focus of a recent phase III clinical trial in dengue patients in Thailand,
in which a single daily dose was found to be safe but did not produce
any clinical benefit. However, the investigators noted that an improved
dosing regimen might be developed, based on pharmacokinetic data
(Yamasmith et al., 2018). Although DENV is clearly very different to
SARS-CoV-2, this trial design should inform future work going forward.
Altogether the current report, combined with a known-safety profile,
demonstrates that ivermectin is worthy of further consideration as a
possible SARS-CoV-2 antiviral.
2. Methods
2.1. Cell culture, viral infection and drug treatment
Vero/hSLAM cells (Ono et al., 2001) were maintained in Earle's
Minimum Essential Medium (EMEM) containing 7% Fetal Bovine
Serum (FBS) (Bovogen Biologicals, Keilor East, AUS) 2 mM L-Gluta-
mine, 1 mM Sodium pyruvate, 1500 mg/L sodium bicarbonate, 15 mM
HEPES and 0.4 mg/ml geneticin at 37 °C, 5% CO2. Cells were seeded
into 12-well tissue culture plates 24 h prior to infection with SARS-CoV-
2 (Australia/VIC01/2020 isolate) at an MOI of 0.1 in infection media
(as per maintenance media but containing only 2% FBS) for 2 h. Media
containing inoculum was removed and replaced with 1 mL fresh media
(2% FBS) containing Ivermectin at the indicated concentrations or
DMSO alone and incubated as indicated for 0–3 days. At the appro-
priate timepoint, cell supernatant was collected and spun for 10 min at
6,000 g to remove debris and the supernatant transferred to fresh col-
lection tubes. The cell monolayers were collected by scraping and re-
suspension into 1 mL fresh media (2% FBS). Toxicity controls were set
up in parallel in every experiment on uninfected cells.
2.2. Generation of SARS-CoV-2 cDNA
RNA was extracted from 200 μL aliquots of sample supernatant or
cell suspension using the QIAamp 96 Virus QIAcube HT Kit (Qiagen,
Hilden, Germany) and eluted in 60 μl. Reverse transcription was per-
formed using the BioLine SensiFAST cDNA kit (Bioline, London, United
Kingdom), total reaction mixture (20 μl), containing 10 μL of RNA
extract, 4 μl of 5x TransAmp buffer, 1 μl of Reverse Transcriptase and
5 μl of Nuclease free water. The reactions were incubated at 25 °C for
10 min, 42 °C for 15 min and 85 °C for 5 min.
2.3. Detection of SARS-CoV-2 using a TaqMan Real-time RT-PCR assay
TaqMan RT-PCR assay were performed using 2.5 μl cDNA, 10 μl
Primer Design PrecisonPLUS qPCR Master Mix 1 μM Forward (5′- AAA
TTC TAT GGT GGT TGG CAC AAC ATG TT-3′), 1 μM Reverse (5′- TAG
GCA TAG CTC TRT CAC AYT T-3′) primers and 0.2 μM probe (5′-FAM-
TGG GTT GGG ATT ATC-MGBNFQ-3′) targeting the BetaCoV RdRp
(RNA-dependent RNA polymerase) gene or Forward (5′-ACA GGT ACG

3
L. Caly, et al.
TTA ATA GTT AAT AGC GT -3′), 1 μM Reverse (5′-ATA TTG CAG CAG
TAC GCA CAC A-3′) primers and 0.2 μM probe (5′-FAM-ACA CTA GCC
ATC CTT ACT GCG CTT CG-286 NFQ-3′) targeting the BetaCoV E-gene
(Corman et al., 2020). Real-time RT-PCR assays were performed on an
Applied Biosystems ABI 7500 Fast real-time PCR machine (Applied
Biosystems, Foster City, CA, USA) using cycling conditions of 95 °C for
2 min, 95 °C for 5 s, 60 °C for 24 s. SARS-CoV-2 cDNA (Ct~28) was used
as a positive control. Calculated Ct values were converted to fold-re-
duction of treated samples compared to control using the ΔCt method
(fold changed in viral RNA = 2^ΔCt) and expressed as % of DMSO alone
sample. IC50 values were fitted using 3 parameter dose response curves
in GraphPad prism.
Funding
This work was supported by a National Breast Cancer Foundation
Fellowship, Australia (ECF-17-007) for KMW and an National Health
and Medical Research Council (NHMRC), Australia Senior Prinicple
Research Fellow (SPRF) (APP1103050) for DAJ.
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 Antiviral Research 178 (2020) 104805
Contents lists available at ScienceDirect
Antiviral Research
journal homepage: www.elsevier.com/locate/antiviral
Ivermectin and COVID-19: A report in Antiviral Research, widespread interest, an FDA warning,
two letters to the editor and the authors' responses
Caly et al. at Monash University in Australia recently published a
paper in Antiviral Research, reporting that ivermectin, a medication
widely used for the treatment of certain parasitic diseases in humans
and livestock animals, inhibits the replication of SARS-CoV-2 in cell
culture (Caly et al., 2020). Despite the authors' cautious conclusion that
ivermectin "warrants further investigation for possible benefits in hu-
mans," the paper has excited widespread interest on medical and ve-
terinary websites, which often incorrectly describe the drug as a
treatment or cure for COVID-19. These inappropriate statements led to
a warning by the US FDA, that ivermectin in veterinary products should
not be used for human therapy,
https://www.fda.gov/animal-veterinary/product-safety-
information/fda-letter-stakeholders-do-not-use-ivermectin-intended-
animals-treatment-covid-19-humans.
The FDA message also explains that in vitro studies such as the re-
port in AVR are "commonly used in the early stages of drug develop-
ment."
The paper by Caly et al. has also elicited two letters to the editor,
which are printed below, followed by the authors' response to both
letters. Readers should be aware that neither the letters nor the re-
sponse has been peer-reviewed, so appropriate caution should be used
in quoting or citing them.
Mike Bray, MD
Editor-in-chief
Antiviral Research
Caly L, Druce JD, Catton MG, Jans DA, Wagstaff KM, 2020. The
FDA-approved Drug Ivermectin inhibits the replication of SARS-CoV-2
in vitro. Antiviral Res. Apr 3:104787.
Craig R. Rayner, Karen Yeo, David Wesche, Lisa Almond,
Michael Dodds, Patrick F. Smith, Mark Sullivan
Certara, Inc, 100 Overlook Center Princeton, NJ, 08540, USA
To the Editor
Recently Caly et al. reported in vitro activity of ivermectin
against SARS-CoV-2 following a single addition to Vero-hSLAM
cells, and suggest that these data “demonstrate that ivermectin is
worthy of further consideration as a possible SARS-CoV-2 anti-
viral” (Caly et al., 2020). In isolation, these in vitro data are robust
and encouraging but the report does not include a correlation of
the in vitro findings with clinically achievable plasma and, more
relevantly, lung concentrations that would permit the determi-
nation of whether the macrocyclic lactones (and specifically in
this case ivermectin) are genuine therapeutic options.
Caly et al. bathed Vero-hSLAM cells with ivermectin at a
https://doi.org/10.1016/j.antiviral.2020.104805
Available online 21 April 2020
0166-3542/ © 2020 Published by Elsevier B.V.
T
concentration of 5μM from 2 hours post-infection SARS-CoV-2
isolate Australia/VIC01/2020 until the conclusion of the experi-
ment. SARS-CoV-2 RNA was determined by RT-PCR at Days 0–3
in both supernatant and cell pellet experiments. The authors
noted 93–99.8% reduction in viral RNA for ivermectin versus
DMSO control at 24h in supernatant (released virions) and cell
associated viral RNA (total virus) respectively. They also describe
by 48 hours a ∼5000-fold reduction of viral RNA and main-
tenance of effect at 72 hours. Additional experiments were con-
ducted with serial dilutions of ivermectin to establish the con-
centration-response profile, and the authors describe ivermectin
as a potent inhibitor of SARS-CoV-2, with an IC50 determined to
be approximately 2 μM under these conditions.
We sought to examine the clinical relevance of the con-
centrations evaluated in these in vitro experiments to those that
may be achieved with ivermectin dosing in practice, in order to
assist in prioritizing ongoing efforts with finding therapeutics that
may be effective in COVID-19.
Ivermectin is one of humanity's most important medicines
(Crump and Omura, 2011) and is extensively used for 5 neglected
tropical diseases at single oral doses of 150–200 μg/kg, resulting
in the mean peak plasma concentrations of approximately 30–47
ng/mL (Merck, 2009). In Phase I studies, doses up to 2000 μg/kg
(Guzzo et al., 2002) have been administered in a fasted state or up
to 600 μg/kg following a standard high-fat meal. Smit et al.
(2019) report that ivermectin 600 μg/kg administered orally re-
sulted in a maximum median concentrations (Cmax) in plasma of
118.9 ng/mL (p5-p95: 45.2–455.1ng/mL), with relatively rapid
clearance and a half-life of approximately 3–5 hours.
Similar to Yao et al. who proposed the potential for hydro-
xychloroquine for treating COVID-19, (Yao et al., 2020) we ap-
plied a physiologic-based pharmacokinetic (PBPK) model of
ivermectin using the Simcyp platform to explore the plasma and
lung concentrations relative to the IC50 values against SARS-CoV-
2 determined in vitro. The ivermectin PBPK model was initially
developed to facilitate drug development for parasitic diseases
including onchocerciasis and is a full model that allows predic-
tion of tissue drug concentrations. The model has been in-
dependently verified. The predicted versus observed plasma pro-
files for ivermectin across clinical studies in the Mectizan NDA
were well aligned, Merck (1996) indicating the base model is well
defined. Furthermore, the PBPK model was able to predict iver-
mectin exposures in plasma, adipose and skin to within 1.3-fold of
observed data in patients infected with Onchocerca volvulus
(Baraka et al., 1996)
Simulations were performed using the Simcyp Simulator
Version 19 Release 1. Ten virtual trials of 10 subjects aged 18–75
years (50% female) were simulated using the Sim-NEurCaucasion
population. In the simulation, high dose ivermectin (600 μg/kg)
was administered orally, daily for 3 days and the virtual study

carried on to 9 days. Dosing was in the Fed state and fraction
unbound was 0.07 (plasma) and 0.13 (lung). Simulations for
mean systemic plasma and lung tissue concentrations are shown
in Fig. 1.
Fig. 1. Simulated mean concentration-time profile of ivermectin in plasma
(black line) and lung tissue (blue line) following 600 μg/kg dose daily for 3
days. The 5th and 95th percentiles are also shown. The red-line is the IC50
(2μM) against SARS-CoV-2 determined in vitro by Caly et al. (2020).
Pharmacodynamic response is generally achieved by ensuring
an appropriate duration of exposure above the minimum ther-
apeutic concentration at the site of action. Even with most gen-
erous assumptions for clinical translation, the in vitro IC50 is > 9-
fold and >21-fold higher than the day 3 plasma and lung tissue
simulated Cmax respectively, following a high dose ivermectin
regimen of 600 μg/kg dose daily for 3 days. (Smit et al., 2019)
This dose scenario, which ignores consistent exposure, exceeds
the highest regulatory approved dose of ivermectin, being a 200
μg/kg single dose for the treatment of Strongyloidiasis (Merck,
2009).
Caly et al. cite the importance of regulatory approval of
ivermectin as a key part of the rationale for further evaluation
against SARS-CoV-2. However, the rigorous data review and re-
assurance of a stringent regulatory authority review only applies
to currently approved doses – clinical pharmacology and tox-
icology margins (including pre-and post-natal and carcinogeni-
city studies) would, therefore, need to be recalculated. In reality,
the resultant unravelling of the supporting package of data could
result in lengthy delays while supporting data are revised and re-
run.
It is understandable that, faced with a devastating pandemic
and a medical and societal imperative, there is great enthusiasm
for promising news of treatments. Picking and supporting the best
therapies and preventions to tackle the COVID-19 pandemic head
on is one of the scientific community's most urgent priorities. To
assist this process, the clinical pharmacological relevance of in
vitro or in vivo findings should be included. In vitro promise leads
to clinical failure in the vast majority of cases, and in the volatile
environment of the current pandemic, it is critical that we are
sensitive to the implications of our communication and apply our
resources to compounds most likely to succeed. A small window
exists for the current data to have relevance for humans: we need
to confirm the effective concentrations, assess if the class of
macrocyclic lactones has similar target interactions, and under-
stand the relevance of the concentrations used in vitro against
SARS-CoV-2 to those likely to be achieved at the site of action,
within a dose range considered to be well tolerated. Alternative
routes could also be considered, although these present new
formulation and safety challenges. Modeling and simulation
2
Antiviral Research 178 (2020) 104805
approaches integrate in vitro findings with the in vivo situation
and may serve to prioritize existing drugs that are candidates for
repurposing.
Declaration of competing interest
KY, DW, LA, MD, PS and CR work for Certara, a consulting
firm in integrated drug development and have directly consulted
with a variety of not-for-profit global health organizations,
biotechnology and pharmaceutical companies and governments
with an interest in medical countermeasures against respiratory
virus infections. MS works for Medicines Development for Global
Health and the Kirby Institute and has no conflicts of interest to
declare.
Funding information
No funding was provided to write this short communication.
References
Baraka, O.Z., et al., 1996. Ivermectin distribution in the plasma and tissues of
patients infected with Onchocerca volvulus. Eur. J. Clin. Pharmacol. 50 (5),
407–410.
Caly, L., et al., 2020. The FDA-approved drug ivermectin inhibits the replication of
SARS-CoV-2 in vitro. Antivir. Res. https://doi.org/10.1016/j.antiviral.2020.
104787.
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perspective. Proc. Jpn. Acad. Ser. B Phys. Biol. Sci. 87 (2), 13–28.
Guzzo, C.A., et al., 2002. Safety, tolerability, and pharmacokinetics of escalating
high doses of ivermectin in healthy adult subjects. J. Clin. Pharmacol. 42 (10),
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Smit, M.R., et al., 2019. Pharmacokinetics-pharmacodynamics of high-dose
ivermectin with dihydroartemisinin-piperaquine on mosquitocidal activity
and QT-prolongation (IVERMAL). Clin. Pharmacol. Ther. 105 (2), 388–401.
Yao, X., et al., 2020. In vitro antiviral activity and projection of optimized dosing
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syndrome coronavirus 2 (SARS-CoV-2). Clin. Infect. Dis.
François Noël
Laboratory of Biochemical and Molecular Pharmacology, Institute of
Biomedical Sciences, Federal University of Rio de Janeiro, Rio de Janeiro,
Brazil
To the Editor,
In the context of repositioning/repurposing strategy for ur-
gent unmet medical needs, various drugs are being proposed for
the treatment of COVID-19, the pandemic disease caused by
SARS-CoV-2 (Noël and Lima, 2020). This is the case for the broad-
spectrum macrocyclic lactone ivermectin, as reported by Caly et
al. (2020) based on their data showing that ivermectin inhibits
the replication of SARS-CoV-2 in vitro. However, this in vitro ac-
tivity occurred at much higher concentrations (IC50 ≈ 2–3 μM)
than the very low (nanomolar) concentrations effective against
many nematode species (Geary, 2005), obtained after a usual
dose of 200 μg/kg. This micromolar concentration is also higher
than the therapeutic peak plasma concentration (about 40 nM)
measured in humans treated for onchocerciasis control with a
standard dose of 150 μg/kg (Apud Shu et al., 2000) and even after
a high daily dose (600 μg/kg) where Cmax of 105–119 ng/ml
(0.12–0.14 μM) has been obtained by PK/PD modeling (Smit et
al., 2019).
As we previously showed (Pimenta et al., 2010) that iver-
mectin is a nonselective inhibitor of three important mammalian
P-type ATPases (SERCA, Na+/K+-ATPase and H+/K+-ATPase) at
similar micromolar concentrations (IC50 ≈ 6–17 μM), we have to
be concerned with putative important adverse effects that this

drug could produce at the higher than usual doses that would be
necessary for treating COVID-19 patients. As a result, a phase 1
study is absolutely needed before using ivermectin since a recent
meta-analysis concluded that there are not enough data to sup-
port a recommendation for its use in higher-than-approved doses
(Navarro et al., 2020).
Declaration of competing interest
No conflict.
References
Caly, L., Druce, J.D., Catton, M.G., Jans, D.A., Wagstaff, K.M., 2020c. The
FDA-approved Drug Ivermectin inhibits the replication of SARS-CoV-2 in
vitro. Antivir. Res. https://doi.org/10.1016/j.antiviral.2020.104787.
Navarro, M., Camprubí, D., Requena-Méndez, A., Buonfrate, D.,
Giorli, G., Kamgno, J., et al., 2020. Safety of high-dose ivermectin: a
systematic review and meta-analysis. J. Antimicrob. Chemother. 75 (4),
827–834.
Noël, F., Lima, J., 2020. Pharmacological aspects and clues for the rationale use of
Chloroquine/Hydroxychloroquine facing the therapeutic challenges of
COVID-19 pandemic. Lat. Am. J. Clin. Sci. Med. Technol. 2, 28–34.
Pimenta, P.H.C., Silva, C.L.M., Noël, F., 2010. Ivermectin is a nonselective
inhibitor of mammalian P-type ATPases. Naunyn-Schmied Arch. Pharmacol.
381, 147–152.
Smit, M.R., Ochomo, E.O., Waterhouse, D., Kwambai, T.K., Abong'o, B.O.,
Bousema, T., et al., 2019. Pharmacokinetics-pharmacodynamics of high dose
ivermectin with Dihydroartemisinin-piperaquine on mosquitocidal activity
and QT-prolongation (IVERMAL). Clin. Pharmacol. Ther. 105 (2), 388–401.
David A. Jans, Kylie M. Wagstaff
Nuclear Signalling Laboratory, Monash Biomedicine Discovery Institute,
Department of Biochemistry and Molecular Biology, Monash University,
Clayton, Victoria, 3800, Australia
Response of the authors
To the Editor
Yeo et al. and Noël aptly point out that published studies show
that blood levels of ivermectin achieved during standard therapy
are much lower than the concentrations we reported as inhibitory
for SARS-CoV-2 in cell culture (Caly et al., 2020). Yeo et al.
(2020) further explore the question via pharmacokinetic mod-
eling (from Certara Inc.), while Noël and Lima (2020) voices
concern that if high concentrations of ivermectin could be
achieved, this would likely be toxic. These points are well made,
and we are in agreement, but they do not address the reported
mechanism of action of ivermectin (Yang et al., 2020), and
thereby fail to highlight a further vitally important reason to be
very cautious in considering ivermectin as a therapeutic for viral
infection in human patients.
Ivermectin's key direct target in mammalian cells is a not a
Mike Bray∗, Craig Rayner, François Noël, David Jans, Kylie Wagstaff,
viral component, but a host protein important in intracellular
transport (Yang et al., 2020); the fact that it is a host-directed
agent (HDA) is almost certainly the basis of its broad-spectrum
∗
Corresponding author.
Antiviral Research 178 (2020) 104805
activity against a number of different RNA viruses in vitro (Tay et
al., 2013; Yang et al., 2020). The way a HDA can reduce viral load
is by inhibiting a key cellular process that the virus hijacks to
enhance infection by suppressing the host antiviral response.
Reducing viral load by even a modest amount by using a HDA at
low dose early in infection can be the key to enabling the body's
immune system to begin to mount the full antiviral response
before the infection takes control.
Pharmaceutical research efforts are currently underway to
refine liquid formulations for intravenous administration of long-
acting ivermectin, develop aerosol administration, and consider
using ivermectin in combination with other agents to enhance
efficacy at low doses. However, it is important to urge great
caution in approaching the use of ivermectin in this simplistic
way, precisely because ivermectin is a HDA. Because it targets a
host component, it cannot be assumed that even doses lower than
those discussed by Yeo et al. (2020) and Noël and Lima (2020)
are safe in the context of a burgeoning viral infection, where a
measured immune response is key to recovery. Clinical testing of
ivermectin at any dose in the fight against viral infection must include
intensive monitoring of patient well-being, to pre-empt any im-
munosuppressive or other adverse reactions as early as possible.
Finally, it is critically important to remember that ivermectin
as an antiviral is in a very early phase – under no circumstances
should self-medication be considered without the guidance of a qua-
lified physician, and especially not using therapeutics designed for
veterinary purposes!
Declaration of competing interest
Authors have no conflict of interest, with no link to any
pharma company.
Funding information
No funding supported this letter to the editor.
References
Caly, L., Druce, J.D., Catton, M.G., Jans, D.A., Wagstaff, K.M., 2020b. The
FDA-approved Drug Ivermectin inhibits the replication of SARS-CoV-2 in
vitro. Antiviral Res. Apr 3, 104787.
Tay, M.Y., Fraser, J.E., Chan, W.K., Moreland, N.J., Rathore, A.P., Wang, C.,
Vasudevan, S.G., Jans, D.A., 2013. Nuclear localization of dengue virus
(DENV) 1-4 non-structural protein 5; protection against all 4 DENV serotypes
by the inhibitor Ivermectin. Antivir. Res. 99 (3), 301–306. https://doi.org/10.
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Yang, S.N.Y., Atkinson, S.C., Wang, C., Lee, A., Bogoyevitch, M.A., Borg, N.A.,
Jans, D.A., 2020. The broad spectrum antiviral ivermectin targets the host
nuclear transport importin α/β1 heterodimer. Antiviral Res. 2, 104760 2020
Mar.
E-mail address: mikebrayavr@gmail.com (M. Bray).