Ivermectin and HCQ: HEK research
Preamble:
New drugs are typically no longer discovered through ancient lore of herbalists, trial-and-error or happy coincidences. Instead, basic research at the cellular level is used to explore and understand the mechanisms of both diseases and various chemicals that might counteract those diseases. These discoveries hopefully lead to possible ways to interact specifically in a targeted manner, such as designing new or repurposing old chemicals that might become medicines. These potential medications are then studied in Preclinical trials, consisting of "in-vitro" studies, which study the potential medication's effect on living cells in a petri-dish, vial or flask, and also "in-vivo" studies on insects or animals. The goal is to see if the potential medication might work in that specific situation and also to try to understand what dose would be appropriate to have the desired effect with the fewest toxic effects. This is followed by Phase 1-4 Clinical Trials, followed by FDA approval, if the studies continue to be positive.
https://www.fda.gov/patients/learn-about-drug-and-device-approvals/drug-development-process
Cellular or "in-vitro" studies are done at 1. the basic science level, called discovery and development, and also 2. preclinical trials using in-vitro studies. Most medications that were FDA approved prior to the requirement for in-vitro preclinical testing have subsequently undergone extensive biomolecular research. These studies look at the mechanisms of action of the medication on cellular function, such as what cell receptor is used for binding in order to enter the cell, how the drug affects intracellular functioning, electrolytes, and protein production and folding, reasons for side-effects or toxicities, and any other potential uses or benefits.
So even if a drug is already FDA approved prior to preclinical studies using in-vitro research, the repurposing of old medications can potentially involve a corpus of studies with an aborted fetal cell line in both the discovery and development phase or the preclinical phase. Without understanding the molecular mechanism of the drug, and the reason for disease pathology at the molecular level, these medications would not have been considered for preclinical and subsequent clinic trials. And more directly, the preclinical trials directly study the disease and medication on a particular cell line.
A large number of basic discovery research studies have been done using HEK293, which is an immortalized kidney cell of an aborted fetus from the 1970s, although other cell lines have been used as well, such as HeLa, Vero, CHO, etc. Additionally, HEK293 is used directly in some preclinical trials, which are required for subsequent clinical trials.
For both ivermectin (approved 1988 for human use for parasites) and hydroxychloroquine (approved 1998 for malaria), there needed to be a molecular reason to hypothesize that they might be effective in a new non-FDA approved indication, such as for a virus. By contrast, physicians and scientists haven't attempted off-label use of albendazole, metronidazole, mefloquine or paramomycin for COVID-19 because, apparently, there is no current indication from in-vitro studies that there would be clinical benefit as either an antiviral or anti-inflammatory medication. Physicians therefore have not prescribed these other medications and researchers would not be able to obtain IRB approval for a clinical trial without pre-clinical in-vitro studies supporting the study.
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Ivermectin:
As disclaimer, at this time, I do not believe that Ivermectin has good evidence in human clinical trials to suggest benefit as an antiviral medication. There are serious methodological errors in the current observational studies. The doses you would have to take to actually reach the same concentration as the in-vitro studies to suppress viral replication would cause neurotoxicity and other serious side effects. However, I do agree that it does seem to have good evidence of viral suppression at the in-vitro cellular level. It would be a good target to modify the drug for an antiviral if they can figure out exactly how it suppresses viral production at the intracellular or receptor level. Time will tell, and additional clinical trials are being done which should elucidate whether ivermectin has any benefit.
Regarding HEK use, Ivermectin's use as an off-label anti-viral appears essentially unrelated to HEK research. The direct study of SARS-CoV-2 on ivermectin uses Vero (monkey kidney cell line). There are only a couple studies using HEK and ivermectin, but these are not in the setting of covid and do not seem to prominently feature in the preclinical or clinical trials. So I do not think HEK research undergirds ivermectin's proposition as a potential antiviral medication. I don't think any studies suggest anti-inflammatory properties.
This Nature article is an overview of all the antiviral studies using ivermectin. HEK is not mentioned here directly and does not appear to be used in the references. However, a few of the references were not open-access, so I am not 100% sure, and I did not review all of the references to the references. In any case though, HEK does not seem to be implicated in most of the research exploring ivermectin's use on viruses.
The following two are the lone exceptions using HEK293:
This study reviewed a 2004 study which explored ivermectin's action on receptor P2X4 in HEK293. Ivermectin as a potential drug for treatment of covid-19: an in-sync review with clinical and computational attributes.
This study looked at adenovirus suppression with ivermectin and used HEK cells. It has, however, been cited in a number of other studies and papers involving ivermectin and covid this year. However, this study was just one among many supporting ivermectin's potential as an antiviral and I do not think it is the main reason for ivermectin's being considered in an off-label way.
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Hydroxychloroquine
Again, as a disclaimer, I do not find good evidence that this has clinical benefit, although there were certainly high hopes from in-vitro studies. I do think there are serious methodological issues with the studies attempting to support its use in practice.
Regarding HEK use, there is some research involving both chloroquine (CQ) and hydroxychloroquine (HCQ) directly on SARS-CoV-2 using HEK. These studies appear to support further research in clinical trials and lead to the temporary FDA EUA authorization.
CQ was actually studied with the original SARS in 2003-2009 as a antiviral. This is primarily why it was considered again when SARS-CoV-2 emerged. Most of those studies use Vero, not HEK. This review article has several tables that show all the studies involving the anitviral effects of CQ, HCQ, and AQ, including many different viruses, which specific cell line was used for each study, and a number of subsequent clinical trials. They do not use HEK in Table 2, but there is some use of HEK and MRC-5 when studying these drugs effects on other viruses in Table 3.
However, below are a handful of studies showing direct research with HCQ and HEK in the setting of SARS-CoV-2, in no particular order.
Nature: Discovery of SARS-CoV-2 antiviral drugs through large scale compound repurposing (2020) (191 citations)
Identification of Potent and Safe Antiviral Therapeutic Candidates Against SARS-CoV-2 (2020) (8 subsequent citations)
Hydroxychloroquine-mediated inhibition of SARS-CoV-2 entry is attenuated by TMPRSS2 (2021) (38 citations)
If someone were particularly keen on specifically quantifying the 'degree of cooperation' with HEK in the clinical trials studying HCQ and SARS-CoV-2, they would need to start with each clinical trial publication, then weave through each of those references looking at the preclinical trials, usually in the introduction section They would then look for what studies primarily motivated and undergirded the in-vitro justification for the IRB to approve HCQ in the context of a clinical trial in the setting of Covid. There are so many clinical studies looking at various ways of prescribing HCQ (prophylactic, in mild disease, severe disease, with azithromycin, with steroids, etc etc). Some are retracted, others not peer reviewed, and it was just not possible for me to go through them all systematically. So my searches were just variations of "HCQ and 293."
Additionally, CQ and HCQ have also been studied because of the immunomodulatory effects, not just antiviral effects. At least a few of these use HEK but I did not go down the rabbit trail because this was mostly just proposed as a possible or additional mechanism of action based on rheumatological studies. Here is one example though, from Nature.
Finally, HEK was used to study some of the key side effects of HCQ, which later comes up in some of the clinical trials references, especially the cardiac concerns like QT prolongation:
Basic SARS-CoV-2 research using HEK:
The original SARS virus was studied with HEK fairly extensively (search for SARS and '293' and select for 2003-2009), to better understand the Spike Protein's structure and its attachment to the ACE-2 receptor. HEK was also used extensively with research on MERS, another deadly coronavirus (you can search for MERS + HEK). This makes distilling out HEK research difficult because much of the basic understanding of SARS-CoV-2 used this prior research on SARS and MERS (and other coronaviruses). This basic science/discovery research and understanding of the virus and how it affects human cells undergirds much of our molecular knowledge of SARS-CoV-2. This obviously directly affects the research and development of all therapeutics and vaccines.
This 2003 Nature study seems to be one of the earliest studies about the ACE-2 Receptor and SARS with Vero and 293T (HEK).
Others:
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