Ivermectin postmortem: PAINful experiences with a good drug

Ivermectin is a miracle drug. It cures infections caused by several types of parasitic roundworms, including those that cause river blindness and elephantiasis, two highly unpleasant diseases. Half of the 2015 Nobel Price for Physiology or Medicine was awarded to researchers who discovered the drug.

 

More recently, ivermectin was touted as a treatment for SARS-CoV-2. Unfortunately, after more than 90 human clinical trials, the preponderance of evidence shows that it is ineffective against COVID-19. A new (open-access) Perspective in J. Med. Chem. by Olaf Andersen, Jayme Dahlin, and collaborators at Weill Cornell Medicine, the National Institutes of Health, University of North Carolina Chapel Hill, and University of California San Francisco explores why it proved so misleading, with lessons for other drug repurposing efforts.

 

In 2011, ivermectin was one of 480 compounds tested in a biochemical assay at 50 µM and appeared to disrupt the interaction between HIV integrase and a mammalian protein involved in viral trafficking. In April 2020, low micromolar concentrations of ivermectin were reported to have anti-SARS-CoV-2 activity in a cellular assay. Given a terrifying new disease with no treatments, an approved drug that showed even tenuous activity looked like a lifeline. Researchers around the world began studying ivermectin. PubMed citations jumped from 459 in 2019 to 734 in 2021.

 

The new paper examines some of this past work and dives deeply into the biological effects of ivermectin. The molecule is poorly soluble in water (around 1-2 µM) and partitions into cellular membranes, where it activates a chloride channel receptor in worms, paralyzing them. At this it is quite potent: when taken as directed, human plasma concentrations of ivermectin are around 60 nM, but because the drug is highly protein bound the free concentration is only around 6 nM.

 

The astute reader may notice that the apparent antiviral activity was observed at low micromolar concentrations of ivermectin, three orders of magnitude higher than physiologically relevant, and the initial biochemical assay was conducted at an even higher concentration. In fact, that work was done using an AlphaScreen, which is notoriously sensitive to artifacts. The new paper demonstrates that ivermectin forms aggregates at low micromolar concentrations, and that these aggregates interfere with the AlphaScreen. (We previously wrote about how many of the early reports of compounds active against SARS-CoV-2 proteins were in fact aggregators.)

 

If ivermectin perturbs membrane proteins in worms, what does it do in human cells? The researchers tested the drug against panels of G protein-coupled receptors (GPCRs) and found that in one assay format it inhibited more than a quarter of 168 GPCRs at 10 µM, much higher than physiologically relevant but comparable to the in vitro experiments with SARS-CoV-2. Further studies revealed that ivermectin changes the properties of membranes at low micromolar concentrations, as assessed by multiple methods including electrophysiological assays. Several ivermectin analogs were also tested and found to have similar activity, consistent with nonspecific effects. Impressively, these experiments were done blinded to the experimenter.

 

You might think that messing with membranes would not be good for cells, and you would be right. The researchers found that ivermectin decreased cell viability at low micromolar concentrations in a variety of assay formats through multiple mechanisms, both cytotoxic and apoptotic. Importantly, the concentrations at which ivermectin was active against cells were similar to the concentrations where it showed activity against SARS-CoV-2. The researchers also analyzed 766 PubChem assays and found that ivermectin is active in nearly a third of those assessing cellular toxicity. Killing a host cell is certainly one way of killing a virus, but likely not a useful one.

 

In summary, the original data suggesting that ivermectin is a developable antiviral agent was flawed. The researchers describe this as “a saga of the damage that can be done by assay interference compounds” and a “cautionary tale for the dangers of ‘pandemic exceptionalism.’” They continue:

 

The fact that a repurposed drug is well-characterized clinically, or that there is an ongoing pandemic, may justify performing clinical and mechanistic experiments in parallel, but not skipping mechanistic studies, where the key experiments could have been done in a matter of a few weeks/months.

 

This J. Med. Chem. paper is a meticulous, comprehensive study; with 71 pages of supporting information there is far more to cover than I can do justice to in a blog post. The paper also includes a useful flowchart for derisking nonspecific membrane perturbation. It is well worth reading, particularly for those new to drug discovery. As Richard Feynman warned, “the first principle is that you must not fool yourself -- and you are the easiest person to fool.”