key: cord-0740169-aku5atqh authors: Andreani, Julien; Le Bideau, Marion; Duflot, Isabelle; Jardot, Priscilla; Rolland, Clara; Boxberger, Manon; Wurtz, Nathalie; Rolain, Jean-Marc; Colson, Philippe; La Scola, Bernard; Raoult, Didier title: In vitro testing of combined hydroxychloroquine and azithromycin on SARS-CoV-2 shows synergistic effect date: 2020-04-25 journal: Microb Pathog DOI: 10.1016/j.micpath.2020.104228 sha: aa942370307672dd0c2013494e35b610a8d68411 doc_id: 740169 cord_uid: aku5atqh Abstract Human coronaviruses SARS-CoV-2 appeared at the end of 2019 and led to a pandemic with high morbidity and mortality. As there are currently no effective drugs targeting this virus, drug repurposing represents a short-term strategy to treat millions of infected patients at low costs. Hydroxychloroquine showed an antiviral effect in vitro. In vivo it showed efficacy, especially when combined with azithromycin in a preliminary clinical trial. Here we demonstrate that the combination of hydroxychloroquine and azithromycin has a synergistic effect in vitro on SARS-CoV-2 at concentrations compatible with that obtained in human lung. Human coronaviruses SARS-CoV-2 appeared at the end of 2019 and led to a pandemic with 17 high morbidity and mortality. As there are currently no effective drugs targeting this virus, 18 drug repurposing represents a short-term strategy to treat millions of infected patients at low 19 costs. Hydroxychloroquine showed an antiviral effect in vitro. In Since the end of 2019, the world has encountered pandemic conditions attributable to a novel 30 Coronavirus SARS-CoV 2 (1-3). This is the 7 th Coronavirus identified to infect the human 31 population (1;4;5) and the first one that had pandemic potential in non-immune populations in 32 the 21 st century (6). Finding therapeutics is thus crucial, and it is proposed to do so by 33 repurposing existing drugs (7-9). This strategy presents the advantages that safety profiles of 34 such drugs are known and that they could be easily produced at relatively low cost, thus being 35 quicker to deploy than new drugs or a vaccine. Chloroquine, a decades-old antimalarial agent, 36 an analog of quinine, was known to inhibit the acidification of intracellular compartments 37 (10) and has shown in vitro and in vivo (mice models) activity against different subtypes of 38 antibacterial components have also been tested. Teicoplanin, a glycopeptide, was (26;27). Azithromycin (azithromycin dihydrate), a macrolide, N-Methyl-11-aza-10-deoxo-10-54 dihydroerythromycin A, has shown antiviral activity against Zika (28-30) . Azithromycin is a 55 well-known and safe drug, widely prescribed in the US, for example, with 12 million 56 treatment courses in children under 19 years of age alone. (31) . A recent study has identified 57 these two compounds (azithromycin and hydroxychloroquine) among 97 total potentially 58 active agents as possible treatments for this disease (32). 59 In a preliminary clinical study, hydroxychloroquine and, with even greater potency, the 60 combination of hydroxychloroquine and azithromycin were found effective in reducing the 61 SARS-CoV-2 viral load in COVID-19 patients (33). Since the beginning of the epidemic in 62 the Marseille region we isolated numerous strains and we tested one of them, the SARS-CoV-63 2 IHUMI-3, using different concentrations of hydroxychloroquine and azithromycin in 64 combination, with Vero E6 cells. 65 96-well plate. We determined the TCID50 of the strain at 5.10 5 infectious particles per mL. 74 (µM), were 1, 2 or 5 µM for hydroxychloroquine associated with 5 or 10 µM for 79 azithromycin. Each test was done at least in triplicate and repeated two times except 80 conditions with 5 µM for hydroxychloroquine associated with 5 or 10 µM for azithromycin 81 that were repeated a third time. Kruskal-Wallis test was used to compare each combinations against positive controls using 96 ∆Ct between H0 and H60. Then, Dunn's test was used to correct the multiple comparison. All 97 test was used at p=0,05 parameter and were bilateral (two-sides) and significant P-value was 98 indicated on the figure 2. All others conditions was not significant. 99 No cytotoxicity was associated with drugs in combination in all 13 control wells 101 (without viruses). We detected RNA viral production from 25 to 16 cycle-thresholds (Ct, 102 inversely correlated with RNA copy numbers) for the positive control that was associated with cell lyses. In all cases, cell lyses at 60 hours was correlated with viral production as 104 compared to control (Figure 1 ). Combination of azithromycin and hydroxychloroquine led to 105 significant inhibition of viral replication for wells containing hydroxychloroquine at 5 µM in 106 combination with azithromycin at 10 and 5 µM (P-values at 0,0003 for A10H5 and at 0,0004 107 for A5H5) (Figure 2A Highlights: • SARS-CoV 2 emergence and spreading need to found urgently therapeutics • Drug repurposing is the best strategy for quick therapeutic response • Azithromycin and hydroxychloroquine shows synergistic effect on replication. • Concentrations of drugs are more compatible with in vivo concentrations. New insights on the antiviral effects of 191 chloroquine against coronavirus: what to expect for COVID-19? In vitro inhibition of severe acute 194 respiratory syndrome coronavirus by chloroquine Remdesivir and chloroquine 197 effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro Hydroxychloroquine, a less toxic 200 derivative of chloroquine, is effective in inhibiting SARS-CoV-2 infection in vitro Treatment of Q fever endocarditis : comparison of two regimens containing 204 doxycycline and ofloxacin or hydroxychloroquine Bactericidal effect of Doxycycline associated with 207 lysosomotropic agents on Coxiella burnetii in P388D1 cells Antibiotic susceptibility of Tropheryma whipplei in Whipple's disease Simultaneous UHPLC-UV analysis 214 of hydroxychloroquine, minocycline and doxycycline from serum samples for the 215 therapeutic drug monitoring of Q fever and Whipple's disease Breakthrough: Chloroquine phosphate has shown apparent 218 efficacy in treatment of COVID-19 associated pneumonia in clinical studies Teicoplanin inhibits Ebola 221 pseudovirus infection in cell culture Teicoplanin potently blocks the cell 223 entry of 2019-nCoV Zika 225 virus cell tropism in the developing human brain and inhibition by azithromycin Azithromycin Inhibits the Replication of Zika Virus Azithromycin Protects 231 against Zika virus Infection by Upregulating virus-induced Type I and III Interferon