Lack of antiviral activity of probenecid in Vero E6 cells and Syrian golden hamsters: a need for better understanding of inter-lab differences in preclinical assays

  1. Helen Box
  2. Shaun H Pennington
  3. Edyta Kijak
  4. Lee Tatham
  5. Claire H Caygill
  6. Rose C Lopeman
  7. Laura N Jeffreys
  8. Joanne Herriott
  9. Joanne Sharp
  10. Megan Neary
  11. Anthony Valentijn
  12. Henry Pertinez
  13. Paul Curley
  14. Usman Arshad
  15. Rajith KR Rajoli
  16. Steve Rannard
  17. James P. Stewart
  18. Giancarlo A Biagini
  19. Andrew Owen

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Abstract

Antiviral interventions are urgently required to support vaccination programmes and reduce the global burden of COVID-19. Prior to initiation of large-scale clinical trials, robust preclinical data in support of candidate plausibility are required. The speed at which preclinical models have been developed during the pandemic are unprecedented but there is a vital need for standardisation and assessment of the Critical Quality Attributes. This work provides cross-validation for the recent report demonstrating potent antiviral activity of probenecid against SARS-CoV-2 in preclinical models (1). Vero E6 cells were pre-incubated with probenecid, across a 7-point concentration range, or control media for 2 hours before infection with SARS-CoV-2 (SARS-CoV-2/Human/Liverpool/REMRQ0001/2020, Pango B; MOI 0.05). Probenecid or control media was then reapplied and plates incubated for 48 hours. Cells were fixed with 4% v/v paraformaldehyde, stained with crystal violet and cytopathic activity quantified by spectrophotometry at 590 nm. Syrian golden hamsters (n=5 per group) were intranasally inoculated with virus (SARS-CoV-2 Delta variant B.1.617.2; 103 PFU/hamster) for 24 hours prior to treatment. Hamsters were treated with probenecid or vehicle for 4 doses. Hamsters were ethically euthanised before quantification of total and sub-genomic pulmonary viral RNAs. No inhibition of cytopathic activity was observed for probenecid at any concentration in Vero E6 cells. Furthermore, no reduction in either total or sub-genomic RNA was observed in terminal lung samples from hamsters on day 3 (P > 0.05). Body weight of uninfected hamsters remained stable throughout the course of the experiment whereas both probenecid- (6 - 9% over 3 days) and vehicle-treated (5 - 10% over 3 days) infected hamsters lost body weight which was comparable in magnitude (P > 0.5). The presented data do not support probenecid as a SARS-CoV-2 antiviral. These data do not support use of probenecid in COVID-19 and further analysis is required prior to initiation of clinical trials to investigate the potential utility of this drug.

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    SciScore for 10.1101/2022.03.03.482788: (What is this?)

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

    Table 1: Rigor

    EthicsField Sample Permit: All animal studies were conducted in accordance with UK Home Office Animals Scientific Procedures Act (ASPA, 1986).
    IACUC: Additionally, all studies were approved by the local University of Liverpool Animal Welfare and Ethical Review Body and performed under UK Home Office Project Licence PP4715265.
    Euthanasia Agents: In all cases, animal sacrifice was conducted via a lethal intraperitoneal injection of pentobarbitone, followed by cardiac puncture and immediate exsanguination of blood from the heart.
    Sex as a biological variableMale Syrian golden hamsters were purchased from Janvier Labs.
    RandomizationHamsters were randomly assigned into three groups of five and acclimatised for 7 days.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    In vitro Vero E6 cell assay: 7-point concentration-effect analysis was performed with probenecid in 96-well plates using VERO E6 cells.
    VERO E6
    suggested: RRID:CVCL_XD71)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    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.
    • No funding statement was detected.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  2. Version published to 10.1101/2022.03.03.482788v1 on bioRxiv