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Review 1: "Nonspecific Membrane Bilayer Perturbations by Ivermectin Underlie SARS-CoV-2 in Vitro Activity"

The reviewer found the study reliable, providing evidence against the translational potential of ivermectin for COVID-19 treatment. While the experiments were well-designed and controlled, the reviewer noted that the overall impact and novelty were limited.

Published onJul 01, 2024
Review 1: "Nonspecific Membrane Bilayer Perturbations by Ivermectin Underlie SARS-CoV-2 in Vitro Activity"
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Nonspecific membrane bilayer perturbations by ivermectin underlie SARS-CoV-2 in vitro activity
Nonspecific membrane bilayer perturbations by ivermectin underlie SARS-CoV-2 in vitro activity

Abstract Since it was proposed as a potential host-directed antiviral agent for SARS-CoV-2, the antiparasitic drug ivermectin has been investigated thoroughly in clinical trials, which have provided insufficient support for its clinical efficacy. To examine the potential for ivermectin to be repurposed as an antiviral agent, we therefore undertook a series of preclinical studies. Consistent with early reports, ivermectin decreased SARS-CoV-2 viral burden in in vitro models at low micromolar concentrations, five-to ten-fold higher than the reported toxic clinical concentration. At similar concentrations, ivermectin also decreased cell viability and increased biomarkers of cytotoxicity and apoptosis. Further mechanistic and profiling studies revealed that ivermectin nonspecifically perturbs membrane bilayers at the same concentrations where it decreases the SARS-CoV-2 viral burden, resulting in nonspecific modulation of membrane-based targets such as G-protein coupled receptors and ion channels. These results suggest that a primary molecular mechanism for the in vitro antiviral activity of ivermectin may be nonspecific membrane perturbation, indicating that ivermectin is unlikely to be translatable into a safe and effective antiviral agent. These results and experimental workflow provide a useful paradigm for performing preclinical studies on (pandemic-related) drug repurposing candidates.

RR:C19 Evidence Scale rating by reviewer:

  • Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.


Review: The experiments are well designed and well controlled, and the conclusions largely support the data. While the findings of the manuscript provide evidence for the lack of translational potential of ivermectin, this could have been already concluded based on existing data: selectivity index close to 1 and plasma drug concentrations way below EC50 values required to suppress SARS-CoV-2 replication.

The overall impact and novelty are therefore somewhat limited, yet it is still important to communicate these findings to individuals involved in drug discovery and development and in prioritizing compounds for clinical trials as this drug was tested clinically despite these initial limitations.

Several comments to be addressed are:

  1. The last paragraph of the discussion is important but should be edited to convey clearer message and workflow scheme:

    1. High-throughput screens should be performed in a rigorous way and ideally incorporate counter-screens and/or publicly existing data regarding promiscuity of compounds to increase specificity.

    2. For an existing approved drug to be considered as a host-targeted repurposed antiviral candidate it has to meet at minimum the following criteria:

      1. Antiviral activity demonstrated by orthogonal methods in a few human cell lines and ideally primary human cells or human organoids;

      2. No effect on cytotoxicity within the concentration range showing antiviral activity (ideally measured in the infected cells);

      3. A reasonable selectivity index - the threshold should be determined on a case by case basis.

      4. Achieving serum and relevant tissue concentrations at (or below) the approved dose for the original indication (based on existing PK studies) that are sufficiently above the EC50 values measured for the antiviral activity. Ivermectin failed to meet all of the above criteria, and thus there was no rationale to propose its use as an antiviral candidate to begin with. In contrast, for drugs that meet the above criteria, existing data should be thoroughly reviewed and follow up studies considered accordingly. One of the main advantages of repurposing existing drugs is the opportunity to rely on already existing data which typically include safety pharmacology screen data for off-target effects, PK and toxicology in animal models and humans etc. Therefore, if a drug is proposed to be used at a dose that is equivalent or lower to that approved from its original indication, there is no need to conduct items 4 and 5 in the blanket framework proposed by the author.

  2. To increase the specificity of the findings, the authors should consider conducting the gramicidin-based fluorescence assay using the five-component anionic model raft lipid mixture DOPC/DOPG/
    DPPC/DPPG/cholesterol or lipids extracted from A549-ACE2 cells (vs. DC22:1PC).

  3. The number of independent experiments conducted should be stated in each figure legend.

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