Abstract: PLOS ONE RESEARCH ARTICLE Synergistic drug combinations designed to fully suppress SARS-CoV-2 in the lung of COVID-19 patients Davide De Forni1☯, Barbara Poddesu1☯, Giulia Cugia1, James Chafouleas1, Julianna Lisziewicz2, Franco Lori1,2* 1 ViroStatics s.r.l., Tramariglio, Italy, 2 Research Institute for Genetic and Human Therapy, Colorado Springs, CO, United States of America a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: De Forni D, Poddesu B, Cugia G, Chafouleas J, Lisziewicz J, Lori F (2022) Synergistic drug combinations designed to fully suppress SARS-CoV-2 in the lung of COVID-19 patients. PLoS ONE 17(11): e0276751. https://doi. org/10.1371/journal.pone.0276751 Editor: Hiroj Bagde, Rama Dental College and Hospital and Research Centre, INDIA Received: March 22, 2022 Accepted: October 12, 2022 Published: November 10, 2022 Copyright: © 2022 De Forni et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: Funds were collected by the not-for-profit Research Institute for Genetic and Human Therapy (RIGHT) through a crowdfunding campaign named GoFundMe "COVID-19: ora serve una Cura" (https://www.gofundme.com/manage/emergenzacoronavirus-ora-serve-una-cura). RIGHT acted as the sponsor of the study and, as institution, had no role in the writing of the manuscript or the decision ☯ These authors contributed equally to this work. * f.lori@virostatics.com Abstract Despite new antivirals are being approved against SARS-CoV-2 they suffer from significant constraints and are not indicated for hospitalized patients, who are left with few antiviral options. Repurposed drugs have previously shown controversial clinical results and it remains difficult to understand why certain trials delivered positive results and other trials failed. Our manuscript contributes to explaining the puzzle: this might have been caused by a suboptimal drug exposure and, consequently, an incomplete virus suppression, also because the drugs have mostly been used as add-on monotherapies. As with other viruses (e.g., HIV and HCV) identifying synergistic combinations among such drugs could overcome monotherapy-related limitations. In a cell culture model for SARS-CoV-2 infection the following stringent criteria were adopted to assess drug combinations: 1) identify robust, synergistic antiviral activity with no increase in cytotoxicity, 2) identify the lowest drug concentration inhibiting the virus by 100% (LIC100) and 3) understand whether the LIC100 could be reached in the lung at clinically indicated drug doses. Among several combinations tested, remdesivir with either azithromycin or ivermectin synergistically increased the antiviral activity with no increase in cytotoxicity, improving the therapeutic index and lowering the LIC100 of every one of the drugs to levels that are expected to be achievable and maintained in the lung for a therapeutically relevant period of time. These results are consistent with recent clinical observations showing that intensive care unit admission was significantly delayed by the combination of AZI and RDV, but not by RDV alone, and could have immediate implications for the treatment of hospitalized patients..