key: cord-0902296-k5swhwgo authors: Jeffreys, Laura N; Pennington, Shaun H; Duggan, Jack; Caygill, Claire H; Lopeman, Rose C; Breen, Alastair F; Jinks, Jessica B; Ardrey, Alison; Donnellan, Samantha; Patterson, Edward I; Hughes, Grant L; Hong, David W; O’Neill, Paul M; Aljayyoussi, Ghaith; Owen, Andrew; Ward, Stephen A; Biagini, Giancarlo A title: Remdesivir-ivermectin combination displays synergistic interaction with improved in vitro antiviral activity against SARS-CoV-2 date: 2021-09-01 journal: bioRxiv DOI: 10.1101/2020.12.23.424232 sha: 6fcfe23488313a308a6c6bad21508f78bb121a55 doc_id: 902296 cord_uid: k5swhwgo A key element for the prevention and management of COVID-19 is the development of effective therapeutics. Drug combination strategies of repurposed drugs offer several advantages over monotherapies, including the potential to achieve greater efficacy, the potential to increase the therapeutic index of drugs and the potential to reduce the emergence of drug resistance. Here, we report on the in vitro synergistic interaction between two FDA approved drugs, remdesivir and ivermectin resulting in enhanced antiviral activity against SARS-CoV-2. These findings warrant further investigations into the clinical potential of this combination, together with studies to define the underlying mechanism. At the time of writing, the World Health Organisation (WHO) has reported more than 211 million cases of COVID-19, and more than 4.4 million deaths (1). There remains a clear need for therapeutic strategies with activity against SARS-CoV-2. Potential therapeutic strategies may include the repurposing of existing drugs as well as the discovery of novel therapies. Thousands of clinical trials are currently underway, with therapeutic approaches involving direct-acting antivirals, for the prevention of virus replication, and host-directed therapies aimed at mitigating against the disease pathology (2, 3). Combination therapies can offer several advantages over monotherapies. They have the potential to achieve greater efficacy, to increase the therapeutic index of drugs and to reduce the emergence of drug resistance. Strategies to identify effective combination therapies are emerging, with several laboratories reporting in vitro combination screens (4) and in vivo animal combinations studies (5) . In a recent clinical trial, baricitinib administered in combination with remdesivir was found to be superior, and to elicit fewer adverse effects, compared to either drug in isolation (6) . Importantly, even in the absence of synergistic activity, an additive interaction between two drugs with separate mechanisms of action may profoundly reduce the speed at which drug resistance is established. Both remdesivir and ivermectin have received attention for the treatment of COVID-19. Remdesivir is a prodrug C-adenosine nucleoside analogue that inhibits the viral RNAdependent, RNA polymerase. Remdesivir was shown early in the pandemic to display in vitro antiviral efficacy against SARS-CoV-2 (7). In a double-blind, randomized, placebo-controlled trial, intravenous administration of remdesivir showed superiority relative to placebo in shortening the time to recovery in adults who were hospitalized with COVID-19 (8) . However, other studies have suggested that its impact may be negligible (9) , and on 20 th November 2020 the WHO issued a conditional recommendation against the use of remdesivir in hospitalised patients (irrespective of disease severity) because there is no evidence supporting an improvement in survival or other outcomes in these patients. Ivermectin is an anti-parasitic which is active against a wide range of parasites, including gastrointestinal roundworms, lungworms, mites, lice, hornflies and ticks (10) . Ivermectin is reported to exhibit broad spectrum anti-viral activity against a wide range of RNA and DNA viruses (11) . Recently, ivermectin was also shown to display anti-viral activity against SARS-CoV-2 (12), but approved doses are not expected to be high enough to achieve in vitro-defined target exposures systemically (13) . Several clinical trials are now evaluating the potential of ivermectin for both prophylaxis and treatment of COVID-19, but the low exposures make the anti-inflammatory and/or immunomodulatory mechanisms of action more plausible than a direct antiviral activity of the monotherapy (14) . In particular since studies with SARS-CoV-2 in Syrian Golden Hamsters showed an impact upon disease pathology in the absence of any effect on viral titres (15) . Here, we present the Fractional Inhibitory Concentration Index (FICI), and report a synergistic interaction between remdesivir and ivermectin resulting in improved in vitro antiviral activity against SARS-CoV-2. The data are discussed in the context of current therapeutic efforts against COVID-19. to each well, as appropriate. After 48 hours, 4% v/v paraformaldehyde was added to each well and the plate incubated for 1 hour at room temperature. The medium was removed and cells were stained with crystal violet. Cells were washed three times with water and cytopathic viral activity was determined by measuring absorbance of each well at 590 nm using a Varioskan LUX microplate reader (Thermo Fisher Scientific). Z′ was calculated for each plate using the uninfected/untreated controls and infected/untreated according to equation 1. Where * + and * -represent the standard deviation of the non-viral and viral controls respectively, while ̂ + and ̂ -represent the corresponding means of these controls. Drug activity was expressed as a percentage of inhibition of viral growth relative to the uninfected/untreated control (100% inhibition of viral cytopathic activity) and the infected/untreated control (0% inhibition of viral cytopathic activity) on that plate. Ratio dilutions were performed in a single 2mL deep-well plate, and added in parallel to three 96-well plates for each biological replicate. One additional plate which was not inoculated with virus was included to observe drug toxicity. Compound incubation and viral addition was performed as described above. Interpretation of FICI (FICI<=0.5 = synergy; FICI>4.0 = antagonism; FICI>0.5-4 = no interaction) was based on guidance provided by the Journal of Antimicrobial Chemotherapy (17) . Initial experiments were conducted to determine the anti-SARS-CoV-2 activity of ivermectin and remdesivir in isolation. For each compound a robust 4-parameter fit was generated ( Figure 1 ). For ivermectin the EC50 was 2.4 ± 1.1 µM and for remdesivir the EC50 was 1.3 ± 2.1 µM (geometric mean ± geometric standard deviation, n= 3). We (Figure 2 ). For all biological replicates which did not meet the defined threshold hold of synergy, their activity did consistently exceed the predicted effect assuming a purely additive relationship ( Figure 2 ). Here, we described the synergistic interaction between two FDA approved drugs resulting in enhanced in vitro antiviral activity against SARS-CoV-2. Although combination therapy offers a number of advantages to monotherapy, genuine descriptions of synergy are relatively infrequent (18) . Despite thousands of combination experiments having been performed, there have been very few reports of validated synergistic interactions against SARS-CoV-2 (4, 19) . At this stage, the mechanism underpinning the synergistic interaction between remdesivir and ivermectin is unclear (7, 12) . Based on available pharmacokinetic data, concentrations required to inhibit viral replication will not be achieved in human plasma (13) . However, modelling does indicate that inhibitory concentrations may be achieved in lung (13) , although this has been challenged (20) . Nonetheless, a combination containing ivermectin may offer the opportunity to exploit the antiinflammatory and/or immunomodulatory activity of this agent, while simultaneously augmenting the antiviral activity of RNA polymerase inhibitors. If this synergy is also evident with orally bioavailable polymerase inhibitors such as favipiravir or molnupiravir, the approach may be more widely exploitable for community deployment. Data presented here suggest that remdesivir in combination with ivermectin may enhance antiviral activity, reduce the risk of side effects and reduce the cost of treatment. Further investigation is now required to determine whether the observed synergistic interaction can be replicated in animal models, and if these results are favourable, whether the finding can be translated to the clinic. It remains to be determined whether the observed synergistic remdesivir-ivermectin interaction will result in a favourable shift in the plasma Cmax/EC90 ratio. Nevertheless, the underpinning mechanisms for this synergy warrant further investigation so that this pharmacodynamic phenomenon can be exploited for the development of optimal drug combinations. WHO. 2020. WHO Coronovirus Disease (COVID-19) Dashboard Candidate drugs against SARS-CoV-2 and COVID-19 Pharmacologic Treatments for Coronavirus Disease 2019 (COVID-19): A Review Identification and Tracking of Antiviral Drug Combinations Favipiravir at high doses has potent antiviral activity in SARS-CoV-2-infected hamsters, whereas hydroxychloroquine lacks activity Baricitinib plus Remdesivir for Hospitalized Adults with Covid-19 In vitro evaluation of antiviral activity of single and combined repurposable drugs against SARS-CoV-2 Remdesivir for the Treatment of Covid-19 -Final Report Repurposed Antiviral Drugs for Covid-19 -Interim WHO Solidarity Trial Results Ivermectin, 'wonder drug' from Japan: the human use perspective Ivermectin: a systematic review from antiviral effects to COVID-19 complementary regimen The FDA-approved drug ivermectin inhibits the replication of SARS-CoV-2 in vitro Prioritization of Anti-SARS-Cov-2 Drug Repurposing Opportunities Based on Plasma and Target Site Concentrations Derived from their Established Human Pharmacokinetics Topical Ivermectin 10 mg/g and Oral Doxycycline 40 mg Modified-Release: Current Evidence on the Complementary Use of Anti-Inflammatory Rosacea Treatments Anti-COVID-19 efficacy of ivermectin in the golden hamster A method for testing for synergy with any number of agents Synergy, antagonism, and what the chequerboard puts between them The search for synergy: a critical review from a response surface perspective The Approved Dose of Ivermectin Alone is not the Ideal Dose for the Treatment of COVID-19