Pharma Focus Asia

Synergistic Drug Combinations Designed to Fully Suppress SARS-CoV-2 in the Lung of COVID-19 Patients

Davide De Forni, Barbara Poddesu, Giulia Cugia, James Chafouleas, Julianna Lisziewicz, Franco LoriDavide De Forni, Barbara Poddesu, Giulia Cugia, James Chafouleas, Julianna Lisziewicz, Franco Lori

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.

Introduction
COVID-19 is still plaguing continents and crowding hospitals. Despite new antivirals such as molnupiravir and PF07321332-ritonavir have been recently approved, they suffer from significant shortcomings: 1) they carry a high pill burden (6 to 8 pills daily); 2) molnupiravir could induce genetic mutations and its efficacy is limited 3) the threat of resistance is particularly severe for both such ‘monotherapies’ that each target only one part of the virus; 4) they have not been authorized for initiation of treatment in patients hospitalized due to COVID-19.

Materials and Methods:

Drugs
Remdesivir (GS-5734, HY-104077) was purchased from MedChemExpress (Monmouth Junction, NJ). Azithromycin (CP-62993, S1835), ivermectin (MK-933, S1351), umifenovir (arbidol, S2120) and nitazoxanide (NSC 697855, S1627), hydroxychloroquine (NSC 4375, S4430), homoharringtonine (CGX-635, S59015), camostat (FOY-305, S2874), darunavir (S5250) and lopinavir (ABT-378, S1380) were purchased from SelleckChem (Houston, TX).

Cell preparation
Vero E6 cells (Cercopithecus aethiops, kidney, ATCC CRL-1586) were seeded at a density of 10,000 cells/well into a 96-well plate in DMEM supplemented with 1% glutamine, 1% penicillin/streptomycin and 10% fetal bovine serum (complete medium, CM) at 37°C and 5% CO2. 24 hours after seeding fetal bovine serum concentration was decreased to 2% to avoid interference with the viral infection.

Discussion
From over 100 publications describing at least one drug potentially able to inhibit SARS-CoV-2, we selected 26 drug candidates with well-documented antiviral activity. Each drug was tested as monotherapy for cytotoxicity and antiviral activity using the 2019-nCoV/Italy-INMI1 SARS-CoV-2 strain and 10 of them were further selected as their TI was above 1.

Acknowledgments
We thank Porto Conte Ricerche (Alghero, Sassari, Italy) for the availability of the BSL-3 facilities. We thank Sjlva Petrocchi for editing the manuscript. We thank the Department of Molecular and Translational Medicine, Section of Microbiology and Virology, University of Brescia Medical School in Brescia, Italy and the Istituto Lazzaro Spallanzani in Rome, Italy for providing the SARS-CoV-2 strains.

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: 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/emergenza-coronavirus-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 to submit it for publication. Nonetheless, two authors, including the corresponding author are affiliated with RIGHT working without any compensation on a pro-bono basis. No author has received funds nor has been paid by a pharmaceutical company or any other agency. The work was also supported by Horizon 2020 Programme (Grant Agreement No. 871029). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

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