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Medication for parasite infestations

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Ivermectin is an antiparasitic drug.[6] After its discovery in 1975,[7] its first uses were in veterinary medicine to prevent and treat heartworm and acariasis.[8] Approved for human use in 1987,[9] it is used to treat infestations including head lice, scabies, river blindness (onchocerciasis), strongyloidiasis, trichuriasis, ascariasis and lymphatic filariasis.[8][10][11][12] It works through many mechanisms to kill the targeted parasites,[10] and can be taken by mouth, or applied to the skin for external infestations.[10][13] It belongs to the avermectin family of medications.[10]

William Campbell and Satoshi Ōmura won the 2015 Nobel Prize in Physiology or Medicine for its discovery and applications.[14] It is on the World Health Organization's List of Essential Medicines,[15][16] and is approved by the U.S. Food and Drug Administration as an antiparasitic agent.[17] In 2021, it was the 341st most commonly prescribed medication in the United States, with more than 100,000 prescriptions.[18] It is available as a generic medicine.[19][20]

During the COVID-19 pandemic, misinformation has been widely spread claiming that ivermectin is beneficial for treating and preventing COVID-19.[21][22] Such claims are not backed by credible scientific evidence.[23][24][25] Multiple major health organizations, including the U.S. Food and Drug Administration,[26] the U.S. Centers for Disease Control and Prevention,[27] the European Medicines Agency,[28] and the World Health Organization have stated that ivermectin is not authorized or approved to treat COVID-19.[24][29]

Medical uses

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Ivermectin is used to treat human diseases caused by roundworms and a wide variety of external parasites.[30]

Worm infections

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For river blindness (onchocerciasis) and lymphatic filariasis, ivermectin is typically given as part of mass drug administration campaigns that distribute the drug to all members of a community affected by the disease.[31] Adult worms survive in the skin and eventually recover to produce larval worms again; to keep the worms at bay, ivermectin is given at least once per year for the 10–15-year lifespan of the adult worms.[32]

The World Health Organization (WHO) considers ivermectin the drug of choice for strongyloidiasis.[33] Ivermectin is also the primary treatment for Mansonella ozzardi and cutaneous larva migrans.[34][35] The U.S. Centers for Disease Control and Prevention (CDC) recommends ivermectin, albendazole, or mebendazole as treatments for ascariasis.[36][note 1] Ivermectin is sometimes added to albendazole or mebendazole for whipworm treatment, and is considered a second-line treatment for gnathostomiasis.[35][40]

Mites and insects

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Ivermectin is also used to treat infection with parasitic arthropods. Scabies – infestation with the mite Sarcoptes scabiei – is most commonly treated with topical permethrin or oral ivermectin. A single application of permethrin is more efficacious than a single treatment of ivermectin. For most scabies cases, ivermectin is used in a two dose regimen: a first dose kills the active mites, but not their eggs. Over the next week, the eggs hatch, and a second dose kills the newly hatched mites.[41][42] The two dose regimen of ivermectin has similar efficacy to the single dose permethrin treatment. Ivermectin is, however, more effective than permethrin when used in the mass treatment of endemic scabies.[43]

For severe "crusted scabies", where the parasite burden is orders of magnitude higher than usual, the U.S. Centers for Disease Control and Prevention (CDC) recommends up to seven doses of ivermectin over the course of a month, along with a topical antiparasitic.[42] Both head lice and pubic lice can be treated with oral ivermectin, an ivermectin lotion applied directly to the affected area, or various other insecticides.[44][45] Ivermectin is also used to treat rosacea and blepharitis, both of which can be caused or exacerbated by Demodex folliculorum mites.[46][47]

Contraindications

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The only absolute contraindication to the use of ivermectin is hypersensitivity to the active ingredient or any component of the formulation.[48][49] In children under the age of five or those who weigh less than 15 kilograms (33 pounds),[50] there is limited data regarding the efficacy or safety of ivermectin, though the available data demonstrate few adverse effects.[51] However, the American Academy of Pediatrics cautions against use of ivermectin in such patients, as the blood-brain barrier is less developed, and thus there may be an increased risk of particular CNS side effects such as encephalopathy, ataxia, coma, or death.[52] The American Academy of Family Physicians also recommends against use in these patients, given a lack of sufficient data to prove drug safety.[53] Ivermectin is secreted in very low concentration in breast milk.[54] It remains unclear if ivermectin is safe during pregnancy.[55]

Adverse effects

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Side effects, although uncommon, include fever, itching, and skin rash when taken by mouth;[10] and red eyes, dry skin, and burning skin when used topically for head lice.[56] It is unclear if the drug is safe for use during pregnancy, but it is probably acceptable for use during breastfeeding.[57]

Ivermectin is considered relatively free of toxicity in standard doses (around 300 µg/kg).[58][59] Based on the data drug safety sheet for ivermectin,[a] side effects are uncommon. However, serious adverse events following ivermectin treatment are more common in people with very high burdens of larval Loa loa worms in their blood.[60] Those who have over 30,000 microfilaria per milliliter of blood risk inflammation and capillary blockage due to the rapid death of the microfilaria following ivermectin treatment.[60]

One concern is neurotoxicity after large overdoses, which in most mammalian species may manifest as central nervous system depression,[61] ataxia, coma, and even death,[62][63] as might be expected from potentiation of inhibitory chloride channels.[64]

Since drugs that inhibit the enzyme CYP3A4 often also inhibit P-glycoprotein transport, the risk of increased absorption past the blood-brain barrier exists when ivermectin is administered along with other CYP3A4 inhibitors. These drugs include statins, HIV protease inhibitors, many calcium channel blockers, lidocaine, the benzodiazepines, and glucocorticoids such as dexamethasone.[65]

During the course of a typical treatment, ivermectin can cause minor aminotransferase elevations. In rare cases it can cause mild clinically apparent liver disease.[66]

To provide context for the dosing and toxicity ranges, the LD50 of ivermectin in mice is 25 mg/kg (oral), and 80 mg/kg in dogs, corresponding to an approximated human-equivalent dose LD50 range of 2.02–43.24 mg/kg,[67] which is far in excess of its FDA-approved usage (a single dose of 0.150–0.200 mg/kg to be used for specific parasitic infections).[3] While ivermectin has also been studied for use in COVID-19, and while it has some ability to inhibit SARS-CoV-2 in vitro, achieving 50% inhibition in vitro was found to require an estimated oral dose of 7.0 mg/kg (or 35x the maximum FDA-approved dosage),[68] high enough to be considered ivermectin poisoning.[67] Despite insufficient data to show any safe and effective dosing regimen for ivermectin in COVID-19, doses have been taken far in excess of FDA-approved dosing, leading the CDC to issue a warning of overdose symptoms including nausea, vomiting, diarrhea, hypotension, decreased level of consciousness, confusion, blurred vision, visual hallucinations, loss of coordination and balance, seizures, coma, and death. The CDC advises against consuming doses intended for livestock or doses intended for external use and warns that increasing misuse of ivermectin-containing products is resulting in an increase in harmful overdoses.[69]

Pharmacology

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Ivermectin (IVM) bound to a C. elegans GluClR. IVM molecules interact with a binding pocket formed by the transmembrane domains of adjacent GluClR subunits, "locking" the receptor in an activated (open) conformation that allows unrestricted passage of chloride (Cl−) ions into the cell. (The plasma membrane is represented as a blue–pink gradient.) From ​.

Mechanism of action

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Ivermectin and its related drugs act by interfering with the nerve and muscle functions of helminths and insects.[70] The drug binds to glutamate-gated chloride channels common to invertebrate nerve and muscle cells.[71] The binding pushes the channels open, which increases the flow of chloride ions and hyper-polarizes the cell membranes,[70] paralyzing and killing the invertebrate.[71] Ivermectin is safe for mammals (at the normal therapeutic doses used to cure parasite infections) because mammalian glutamate-gated chloride channels only occur in the brain and spinal cord: the causative avermectins usually do not cross the blood–brain barrier, and are unlikely to bind to other mammalian ligand-gated channels.[71]

Pharmacokinetics

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Ivermectin can be given by mouth, topically, or via injection. Oral doses are absorbed into systemic circulation; the alcoholic solution form is more orally available than tablet and capsule forms. Ivermectin is widely distributed in the body.[72]

Ivermectin does not readily cross the blood–brain barrier of mammals due to the presence of P-glycoprotein (the MDR1 gene mutation affects the function of this protein).[73] Crossing may still become significant if ivermectin is given at high doses, in which case brain levels peak 2–5 hours after administration. In contrast to mammals, ivermectin can cross the blood–brain barrier in tortoises, often with fatal consequences.[74]

Chemistry

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Avermectins produced by fermentation are the chemical starting point for ivermectin

Fermentation of Streptomyces avermitilis yields eight closely related avermectin homologues, of which B1a and B1b form the bulk of the products isolated. In a separate chemical step, the mixture is hydrogenated to give ivermectin, which is an approximately 80:20 mixture of the two 22,23-dihydroavermectin compounds.[75][76][6]

Ivermectin is a macrocyclical lactone.[77]

History

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The avermectin family of compounds was discovered by Satoshi Ōmura of Kitasato University and William Campbell of Merck.[6] In 1970, Ōmura isolated a strain of Streptomyces avermitilis from woodland soil near a golf course along the south east coast of Honshu, Japan.[6] Ōmura sent the bacteria to William Campbell, who showed that the bacterial culture could cure mice infected with the roundworm Heligmosomoides polygyrus.[6] Campbell isolated the active compounds from the bacterial culture, naming them "avermectins" and the bacterium Streptomyces avermitilis for the compounds' ability to clear mice of worms (in Latin: a 'without', vermis 'worms').[6] Of the various avermectins, Campbell's group found the compound "avermectin B1" to be the most potent when taken orally.[6] They synthesized modified forms of avermectin B1 to improve its pharmaceutical properties, eventually choosing a mixture of at least 80% 22,23-dihydroavermectin B1a and up to 20% 22,23-dihydroavermectin B1b, a combination they called "ivermectin".[6][78]

The discovery of ivermectin has been described as a combination of "chance and choice." Merck was looking for a broad-spectrum anthelmintic, which ivermectin is indeed; however, Campbell noted that they "...also found a broad-spectrum agent for the control of ectoparasitic insects and mites."[79]

Merck began marketing ivermectin as a veterinary antiparasitic in 1981.[6] By 1986, ivermectin was registered for use in 46 countries and was administered massively to cattle, sheep and other animals.[80] By the late 1980s, ivermectin was the bestselling veterinary medicine in the world.[6] Following its blockbuster success as a veterinary antiparasitic, another Merck scientist, Mohamed Aziz, collaborated with the World Health Organization to test the safety and efficacy of ivermectin against onchocerciasis in humans.[9] They found it to be highly safe and effective,[81] triggering Merck to register ivermectin for human use as "Mectizan" in France in 1987.[9] A year later, Merck CEO Roy Vagelos agreed that Merck would donate all ivermectin needed to eradicate river blindness.[9] In 1998, that donation would be expanded to include ivermectin used to treat lymphatic filariasis.[9]

Ivermectin earned the title of "wonder drug" for the treatment of nematodes and arthropod parasites.[82] Ivermectin has been used safely by hundreds of millions of people to treat river blindness and lymphatic filariasis.[6]

Half of the 2015 Nobel Prize in Physiology or Medicine was awarded jointly to Campbell and Ōmura for discovering avermectin, "the derivatives of which have radically lowered the incidence of river blindness and lymphatic filariasis, as well as showing efficacy against an expanding number of other parasitic diseases".[14][83]

Society and culture

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COVID-19 misinformation

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These paragraphs are an excerpt from Ivermectin during the COVID-19 pandemic

Early in the COVID-19 pandemic, laboratory research suggested ivermectin might have a role in preventing or treating COVID-19.[84] Online misinformation campaigns and advocacy boosted the drug's profile among the public. While scientists and physicians largely remained skeptical, some nations adopted ivermectin as part of their pandemic-control efforts. Some people, desperate to use ivermectin without a prescription, took veterinary preparations, which led to shortages of supplies of ivermectin for animal treatment. The FDA responded to this situation by saying "You are not a horse" in a Tweet to draw attention to the issue, which they were later sued for.[85][86]

Subsequent research failed to confirm the utility of ivermectin for COVID-19,[87][88] and in 2021 it emerged that many of the studies demonstrating benefit were faulty, misleading, or [89][90] Nevertheless, misinformation about ivermectin continued to be propagated on social media and the drug remained a [91]

Subsequent research failed to confirm the utility of ivermectin for COVID-19,and in 2021 it emerged that many of the studies demonstrating benefit were faulty, misleading, or fraudulent Nevertheless, misinformation about ivermectin continued to be propagated on social media and the drug remained a cause célèbre for anti-vaccinationists and conspiracy theorists

Economics

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The initial price proposed by Merck in 1987 was US$6 per treatment, which was unaffordable for patients who most needed ivermectin.[92] The company donated hundreds of millions of courses of treatments since 1988 in more than 30 countries.[92] Between 1995 and 2010, using donated ivermectin to prevent river blindness, the program is estimated to have prevented seven million years of disability at a cost of US$257 million.[93]

Ivermectin is considered an inexpensive drug.[94] As of 2019, ivermectin tablets (Stromectol) in the United States were the least expensive treatment option for lice in children at approximately US$9.30, while Sklice, an ivermectin lotion, cost around US$300 for 120 mL (4 US fl oz).[95]

As of 2019 , the cost effectiveness of treating scabies and lice with ivermectin has not been studied.[96][97]

Brand names

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It is sold under the brand names Heartgard, Sklice[98] and Stromectol[3] in the United States, Ivomec worldwide by Merial Animal Health, Mectizan in Canada by Merck, Iver-DT[99] in Nepal by Alive Pharmaceutical and Ivexterm in Mexico by Valeant Pharmaceuticals International. In Southeast Asian countries, it is marketed by Delta Pharma Ltd. under the trade name Scabo 6. The formulation for rosacea treatment is sold under the brand name Soolantra.[4] While in development, it was assigned the code MK-933 by Merck.[100]

Research

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Parasitic disease

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Ivermectin has been researched in laboratory animals, as a potential treatment for trichinosis[31] and trypanosomiasis.[101]

Tropical diseases

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As of 2016 ivermectin was studied as a potential antiviral agent against chikungunya and yellow fever.[102] In chikungunya, ivermectin showed a wide in vitro safety margin as an antiviral.[102]

Ivermectin is also of interest in the prevention of malaria, as it is toxic to both the malaria plasmodium itself and the mosquitos that carry it.[103][104] A direct effect on malaria parasites could not be shown in an experimental infection of volunteers with Plasmodium falciparum.[105] Use of ivermectin at higher doses necessary to control malaria is probably safe, though large clinical trials have not yet been done to definitively establish the efficacy or safety of ivermectin for prophylaxis or treatment of malaria.[106][58] Mass drug administration of a population with ivermectin to treat and prevent nematode infestation is effective for eliminating malaria-bearing mosquitos and thereby potentially reducing infection with residual malaria parasites.[107] Whilst effective in killing malaria-bearing mosquitos, a 2021 Cochrane review found that, to date, the evidence shows no significant impact on reducing incidence of malaria transmission from the community administration of ivermectin.[106]

One alternative to ivermectin is moxidectin, which has been approved by the Food and Drug Administration for use in people with river blindness.[108] Moxidectin has a longer half-life than ivermectin and may eventually supplant ivermectin as it is a more potent microfilaricide, but there is a need for additional clinical trials, with long-term follow-up, to assess whether moxidectin is safe and effective for treatment of nematode infection in children and women of childbearing potential.[109][110]

There is tentative evidence that ivermectin kills bedbugs, as part of integrated pest management for bedbug infestations.[111][112][113] However, such use may require a prolonged course of treatment which is of unclear safety.[114]

NAFLD

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In 2013, ivermectin was demonstrated as a novel ligand of the farnesoid X receptor,[115][116] a therapeutic target for nonalcoholic fatty liver disease.[117]

During the COVID-19 pandemic, ivermectin was researched for possible utility in preventing and treating COVID-19, but no good evidence of benefit was found.[118][119]

Veterinary use

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Ivermectin is routinely used to control parasitic worms in the gastrointestinal tract of ruminant animals. These parasites normally enter the animal when it is grazing, pass the bowel, and set and mature in the intestines, after which they produce eggs that leave the animal via its droppings and can infest new pastures. Ivermectin is only effective in killing some of these parasites, this is because of an increase in anthelmintic resistance.[120] This resistance has arisen from the persistent use of the same anthelmintic drugs for the past 40 years.[121][122] Additionally, the use of Ivermectin for livestock has a profound impact on dung beetles, such as T. lusitanicus, as it can lead to acute toxicity within these insects.[123]

In dogs, ivermectin is routinely used as prophylaxis against heartworm.[124] Dogs with defects in the P-glycoprotein gene (MDR1), often collie-like herding dogs, can be severely poisoned by ivermectin. The mnemonic "white feet, don't treat" refers to Scotch collies that are vulnerable to ivermectin.[125] Some other dog breeds (especially the Rough Collie, the Smooth Collie, the Shetland Sheepdog, and the Australian Shepherd), also have a high incidence of mutation within the MDR1 gene (coding for P-glycoprotein) and are sensitive to the toxic effects of ivermectin.[126][127] Clinical evidence suggests 7-week-old kittens are susceptible to ivermectin toxicity.[128] A 0.01% ivermectin topical preparation for treating ear mites in cats is available.[129]

Ivermectin is sometimes used as an acaricide in reptiles, both by injection and as a diluted spray. While this works well in some cases, care must be taken, as several species of reptiles are very sensitive to ivermectin. Use in turtles is particularly contraindicated.[130]

A characteristic of the antinematodal action of ivermectin is its potency: for instance, to combat Dirofilaria immitis in dogs, ivermectin is effective at 0.001 milligram per kilogram of body weight when administered orally.[78]

For dogs, the insecticide spinosad may have the effect of increasing the toxicity of ivermectin.[131][132]

Notes

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1. ^[37] while the textbook Parasitic Diseases recommends albendazole or mebendazole.[38] A 2020 [39]

   This recommendation is not universal. The World Health Organization recommends ascariasis be treated with mebendazole or pyrantel pamoate while the textbookrecommends albendazole or mebendazole.A 2020 Cochrane review concluded that the three drugs are equally safe and effective for treating ascariasis.

1. ^

   New Drug Application Identifier: 50-742/S-022

References

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Associated Data

Supplementary Materials

Supplementary Figure 1: The drug cytotoxicity profiles of H2.35 murine liver cells, and dose-response curves of MHV-infected H2.35 cells. (A) Cytotoxicity profiles of remdesivir and chloroquine on H2.35 cells as measured by MTS assay after 48 hours exposure. Percentage of cell viability was normalized to untreated cells and blank control. Experiments were performed in quadruplicates. (B) Viral inhibitory activities of remdesivir and chloroquine against MHV infection of H2.35 cells. Live coronavirus titers (pfu/ml) were quantified by plaque assays performed in triplicates. The dose-response curves were fitted using the non-linear regression method, and IC50 values were calculated using the Prism 7 software. Error bars represent standard error of the mean (SEM). (C) Mean values of CC50, IC50 and selectivity index (SI = CC50/EC50) of remdesivir and chloroquine treatment of H2.35 cells.

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Supplementary Figure 2: Representative morphological features of RAW264.7 macrophages under different infection and/or treatment conditions. Microscopic images were captured by the EVOS XL microscope at 10× magnification. The scale bar represents 200 μm. (A) Uninfected control cells. (B) MHV-infected control cells. Monotherapy of MHV-infected macrophages using: (C) remdesivir alone, (D) chloroquine alone, (E) ivermectin alone, (F) doxycycline alone. Combination therapy of MHV-infected macrophages using: (G) remdesivir and chloroquine, (H) remdesivir and ivermectin, (J) remdesivir and doxycycline.

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Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Abstract

The recent COVID-19 pandemic has highlighted the urgency to develop effective antiviral therapies against the disease. Murine hepatitis virus (MHV) is a coronavirus that infects mice and shares some sequence identity to SARS-CoV-2. Both viruses belong to the Betacoronavirus genus, and MHV thus serves as a useful and safe surrogate model for SARS-CoV-2 infections. Clinical trials have indicated that remdesivir is a potentially promising antiviral drug against COVID-19. Using an in vitro model of MHV infection of RAW264.7 macrophages, the safety and efficacy of monotherapy of remdesivir, chloroquine, ivermectin, and doxycycline were investigated. Of the four drugs tested, remdesivir monotherapy exerted the strongest inhibition of live virus and viral RNA replication of about 2-log10 and 1-log10, respectively (at 6 µM). Ivermectin treatment showed the highest selectivity index. Combination drug therapy was also evaluated using remdesivir (6 µM) together with chloroquine (15 µM), ivermectin (2 µM) or doxycycline (15 µM) – above their IC50 values and at high macrophage cell viability of over 95%. The combination of remdesivir and ivermectin exhibited highly potent synergism by achieving significant reductions of about 7-log10 of live virus and 2.5-log10 of viral RNA in infected macrophages. This combination also resulted in the lowest cytokine levels of IL-6, TNF-α, and leukemia inhibitory factor. The next best synergistic combination was remdesivir with doxycycline, which decreased levels of live virus by ~3-log10 and viral RNA by ~1.5-log10. These results warrant further studies to explore the mechanisms of action of the combination therapy, as well as future in vivo experiments and clinical trials for the treatment of SARS-CoV-2 infection.

Keywords:

coronavirus, murine hepatitis virus, remdesivir, chloroquine, ivermectin, doxycycline, combination treatment, RAW264.7 macrophage cells

Materials and Methods

Virus and Cell Cultures

All viral infection experiments were conducted using MHV strain A59 (MHV-A59; GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"AY910861","term_id":"60548081","term_text":"AY910861"}}AY910861) (Chiow et al., 2016). The RAW264.7 murine macrophage cell line was maintained at 37°C in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). H2.35 murine liver cells were maintained at 35°C in Dulbecco’s modified Eagle medium (DMEM) with 10% FBS. Cells were seeded at a density of 200,000 cells per well on 24-well plates, and at a density of 15,000 cells per well on 96-well plates. Cells subjected to infection and/or drug treatment were monitored daily for any cytopathic effect (CPE).

Drugs

Remdesivir (HY-104077; MedChemExpress, Monmouth Junction, NJ, USA) was prepared in 100 mM and 10 mM stock solutions in sterile water. Chloroquine (C6628; Sigma-Aldrich, St. Louis, MO, USA) and ivermectin (CAS 70288-86-7; Merck, Burlington, MA, USA) were prepared in 10 mM stock solutions in dimethyl sulfoxide (DMSO). Doxycycline (CAS 24390-14-5; Merck, Burlington, MA, USA) was prepared in 10 mM stock solution in sterile water. For treatment experiments, all drug dilutions were prepared in 0.5% DMSO with the respective cell culture medium.

Cell Viability Assay

Cell viability was assessed using the CellTiter 96 AQueous One Solution Cell Proliferation (MTS) assay (Promega, Madison, WI, USA), according to the manufacturer’s instructions. RAW264.7 cells were incubated in the presence of increasing drug concentrations (0.16 μM, 0.8 μM, 4 μM, 20 μM, 100 μM) for 48 hours. Cell viability was determined using a microplate reader (Tecan, Mannedorf, Switzerland) with values normalized to those of untreated cells.

Evaluation of Antiviral Inhibitory Activities of Monotherapy and Combination Therapy

Sub-confluent monolayers of RAW264.7 cells in 24-well plates were infected with MHV at multiplicity of infection (MOI) of 0.1 for 2 hours. The inoculum was then removed, and the cells were treated with the indicated concentrations of drugs for monotherapy or combination therapy for 48 hours. The cell supernatant was harvested at 48 hours post-infection, and subjected to virus plaque assay using H2.35 mouse liver cells to determine live coronavirus titers, and to real-time PCR to quantify the viral RNA loads. Each experiment was carried out in triplicates.

Live Coronavirus Quantification by Plaque Assay

Live virus was quantified using plaque assay. Sub-confluent H2.35 cells were infected with the diluted supernatant samples for 1 hour. 1.2% Avicel BioPolymer (FMC, Philadelphia, PA, USA) in phosphate-buffered saline (PBS) was added to each well and incubated for 72 hours. The cells were fixed with 4% paraformaldehyde in PBS for 2 hours, and then stained with 1% crystal violet for 15 minutes.

RNA Extraction, Viral RNA Quantification by Real-Time Reverse Transcription- Quantitative PCR

Cell supernatant was harvested from the experiments, and total viral RNA was purified using QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany). RNA concentration was determined using the NanoDrop ND-1000 spectrophotometer. Reverse transcription was carried out at 35°C for 1 hour. Quantification of viral RNA was performed using MHV-NF forward and MHV-NR reverse primers for the specific detection of the N gene of MHV (5′-ACGCTTACATTATCWACTTC-3′ and 5′-GATCTAAATTAGAATTGGTC-3′, respectively). The qPCR was performed with FastStart Essential DNA Green Master using the LightCycler 96 instrument (Roche Diagnostics, Basel, Switzerland). To plot the standard curve, a range of positive controls with known plaque-forming units (PFU) of MHV was included. The thermal cycling conditions were as follows: 95°C for 600 s, followed by 55 cycles of 95°C for 10 sec, 40°C for 5 sec, 72°C for 8 sec, and a final 95°C for 10 sec, 65°C for 60 sec and 97°C for 1 sec.

Determining Cytokine Protein Expression Profiles Using Luminex Multiplex Assay

To determine cytokine expression levels, multiplex assays using the Myokine 5-Plex Mouse ProcartaPlex Panel kit (Thermo Fisher Scientific, Waltham, MA, USA) were conducted according to the manufacturer’s instructions. This 5-plex kit quantified the following cytokines: IL-6, IL-10, IL-15, leukemia inhibitory factor (LIF), and TNF-α. Standard curves and values were measured on Luminex MAGPIX, and calculated using xPONENT 4.2 software for MAGPIX (with 30 beads being set as the detection limit).

Statistical Analyses

Non-linear regression and curve-fitting parameter analyses were performed to calculate inhibitory concentration values using GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA). Statistical significance and P-values were calculated by one-way ANOVA using Dunnett’s multiple comparison test. Each error bar of dose-response curves represents the standard error of mean (SEM) of three technical replicates.

Conclusion

In summary, this in vitro study has provided evidence that the combination of remdesivir and ivermectin was highly synergistic and potent, culminating in striking reduction in live murine coronavirus replication and viral RNA synthesis. Furthermore, the efficacy and beneficial effects of this combination therapy also included immunomodulatory effects via inhibition of cytokine production, and improving cell morphology of infected macrophages.

The limitation of our study is that antiviral activities of drugs were investigated only against MHV in the RAW264.7 macrophage cell culture model. For direct comparison versus MHV, future detailed studies are thus warranted to evaluate the potential utility of this drug combination against SARS-CoV-2 and other coronaviruses (e.g. SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43), such as multi-dose checkerboard experiments to ascertain additive effects of drugs (Bakowski et al., 2021). This caveat is also critically important given the observations of cell line-dependent compound efficacy against SARS-CoV-2 infection reported in many previous SARS-CoV-2 antiviral studies. For example, Vero cells, human Huh-7.5 liver cancer cells and human Calu-3 lung epithelial cells exhibit major differences in sensitivity to drugs with antiviral activity due to the distinct entry pathways utilized by SARS-CoV-2 in these cell lines (Dittmar et al., 2021). Also necessary are in vivo experiments using suitable animal model(s) of COVID-19 before progressing onto clinical trials in SARS-CoV-2 infected patients.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Author Contributions

VC and YT conceptualized and designed the research project, and analyzed the data. All experiments were carried out by YT. All authors contributed to the article and approved the submitted version.

Funding

This study was supported by a research grant of the National University of Singapore.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s Note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Acknowledgments

We thank S. H. Lau, Joe Ong, J. H. Ch’ng, S. Lee, and other staff members of the National University Health System for their technical, statistical, and other assistance.

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcimb.2021.700502/full#supplementary-material

Supplementary Figure 1

The drug cytotoxicity profiles of H2.35 murine liver cells, and dose-response curves of MHV-infected H2.35 cells. (A) Cytotoxicity profiles of remdesivir and chloroquine on H2.35 cells as measured by MTS assay after 48 hours exposure. Percentage of cell viability was normalized to untreated cells and blank control. Experiments were performed in quadruplicates. (B) Viral inhibitory activities of remdesivir and chloroquine against MHV infection of H2.35 cells. Live coronavirus titers (pfu/ml) were quantified by plaque assays performed in triplicates. The dose-response curves were fitted using the non-linear regression method, and IC50 values were calculated using the Prism 7 software. Error bars represent standard error of the mean (SEM). (C) Mean values of CC50, IC50 and selectivity index (SI = CC50/EC50) of remdesivir and chloroquine treatment of H2.35 cells.

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Supplementary Figure 2

Representative morphological features of RAW264.7 macrophages under different infection and/or treatment conditions. Microscopic images were captured by the EVOS XL microscope at 10× magnification. The scale bar represents 200 μm. (A) Uninfected control cells. (B) MHV-infected control cells. Monotherapy of MHV-infected macrophages using: (C) remdesivir alone, (D) chloroquine alone, (E) ivermectin alone, (F) doxycycline alone. Combination therapy of MHV-infected macrophages using: (G) remdesivir and chloroquine, (H) remdesivir and ivermectin, (J) remdesivir and doxycycline.

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