key: cord-0845253-h5cewkv8
authors: Lian, Elena; McAlister, Carley; Ramirez, Gabriela; Chernoff, David N.; Went, Gregory; Hoopes, Justin; Perera, Rushika
title: Triple Combination Nitazoxanide, Ribavirin, and Hydroxychloroquine results in the multiplicative reduction of in vitro SARS-CoV-2 viral replication
date: 2020-11-26
journal: bioRxiv
DOI: 10.1101/2020.11.25.399055
sha: 4a3419ab5cd5c8aac6edf86c50bac7e00e64ad1c
doc_id: 845253
cord_uid: h5cewkv8

Background An immediate unmet medical need exists to test and develop existing approved drugs against SARS-COV-2. Despite many efforts, very little progress has been made regarding finding low-cost oral medicines that can be made widely available worldwide to address the global pandemic. Methods We sought to examine if a triple combination of nitazoxanide (using its active metabolite tizoxanide), ribavirin, and hydroxychloroquine would lead to a multiplicative effects on viral replication of SARS-COV-2 resulting in a significant reduction of virus yield using VERO E6 cells as a model of viral replication. Results Virus yield measured in PFU/ml was ~ 2 logs lower with triple combination versus either drug alone, resulting in the prolongation of time to peak cytopathic effects (CPE). The time to produce 50% CPE increased from 2.8 days for viral controls versus 5.3 days for triple combination therapy. Finally, for each 1-log reduction in virus yield 24 hours post-infection, there was an additional 0.7-day delay in onset of CPE. Conclusions A triple combination of tizoxanide, ribavirin, and hydroxychloroquine produced a reduction in SARS-COV-2 viral replication in Vero E6 cells, warranting exploration in additional cell lines as well as human clinical trials.

From its origins in late 2019 in Wuhan, China, infection with severe acute respiratory 47 6

Vero E6 cells were washed with DMEM and treated with either 0.32 mcg/mL TIZ, 0.32 116 mcg/mL HCQ, or 100 µg/mL RBV as single agents as well as in double and triple 117 combination before infection. Each condition was run in triplicate. Drug concentrations 118

were selected based on preliminary experiments and represent concentrations that 119 approximate human physiologic concentrations, with sub-optimal activity as single 120

agents. Cells were washed once with DMEM and then treated for 4 hours with media 121 containing 2% FBS and each drug alone or in combination for 4 hours. Drug pre-122 treatment was extended because RBV uptake takes several hours in Vero E6 cells, 123 after which it is slowly converted by adenosine kinase to the active metabolite RBV-MP 124 [18, 19] . Following pre-treatment, each test well was infected with 10 plaque-forming 7 The stain was then removed, and the number of plaques were counted in each well. 138

The first dilution that resulted in countable plaques was used to calculate the virus yield 139 The reduction seen in viral yield with drugs alone and in combination versus viral 173 controls correlated with a prolongation of time to peak CPE, where 100% CPE was 174 observed at day 3 onward in viral controls versus <5% at day 3 to 60% at day 6 in triple 175 combination groups ( Figure 3) . TIZ, RBV, and HCQ as single agents produced 50% 176 CPE in 3.5 days, 3.9 days, and 3.5 days, respectively. TIZ and HCQ, TIZ and RBV, and 177 RBV and HCQ produced 50% CPE in 4.1, 4.5, and 4.6 days, versus 2.8 days for viral 

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The authors would like to thank Brian J. Geiss, Ph.D., The Office