Triple Combination Nitazoxanide, Ribavirin, and Hydroxychloroquine results in the multiplicative reduction of in vitro SARS-CoV-2 viral replication

  1. Elena Lian
  2. Carley McAlister
  3. Gabriela Ramirez
  4. David N. Chernoff
  5. Gregory Went
  6. Justin Hoopes
  7. Rushika Perera

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Abstract

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.

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  1. ScreenIT

    SciScore for 10.1101/2020.11.25.399055: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Briefly, 12-well plates were seeded with Vero E6 at 6 x 105 cells/well and allowed to adhere overnight.
    Vero E6
    suggested: None
    Software and Algorithms
    SentencesResources
    Statistical Analysis: Mean and standard deviation for virus yield was calculated and plotted using Graphpad Prism Ver.8.3.1.
    Graphpad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    We have hypothesized that combining multiple drugs targeting sequential steps in the replication process of SARS-CoV2 may help overcome the efficacy limitations observed in previous studies. To test this hypothesis, we selected three drugs, two of which (HCQ, RBV) had been previously evaluated and showed efficacy in vitro at physiologically achievable concentrations but failed to show effectiveness in clinical trials when used as monotherapies. Vero E6 cells were selected for this study because SARS-COV-2 produces high titers and CPE with a fast replication rate in this cell line. A large well format with a low multiplicity of infection (MOI) was also employed to maximize the number of rounds of replication and reinfection and apply mathematical modeling to analyze the results. Each drug was shown previously to have a dose-dependent effect on virus yield in VeroE6 cells [21,22]. In this study, drugs were administered at concentrations that corresponded to clinically achievable active metabolite concentrations. Despite being administered at sub-optimal concentrations, all three drugs reduced virus yield at days 1 and 2, with inhibitory effects ranging from 8- to 160-fold. Through day 2, the double combinations of TIZ and HCQ and TIZ and RBV further improved compared with either drug alone, each being approximately multiplicative. In contrast, the combination of RBV and HCQ did not reduce virus yield compared to RBV alone. Finally, the triple combination of TIZ, RBV, and HCQ sh...

    Results from TrialIdentifier: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04605588RecruitingA Triple Combination Antiviral Coronavirus Therapy (TriACT) …
    NCT4563208Trial number did not resolve on clinicaltrials.gov. Is the number correct?NA

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2020.11.25.399055v1 on bioRxiv