Enantiomers of Chloroquine and Hydroxychloroquine Exhibit Different Activities Against SARS-CoV-2 in vitro , Evidencing S -Hydroxychloroquine as a Potentially Superior Drug for COVID-19

  1. Guanguan Li
  2. Jing Sun
  3. Yi-You Huang
  4. Yingjun Li
  5. Yongjie Shi
  6. Zhe Li
  7. Xiang Li
  8. Feng Hua Yang
  9. Jincun Zhao
  10. Hai-Bin Luo
  11. Tony Y. Zhang
  12. Xumu Zhang

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Abstract

In all of the clinical trials for COVID-19 conducted thus far and among those ongoing involving chloroquine or hydroxychloroquine, the drug substance used has invariably been chloroquine (CQ) diphosphate or hydroxychloroquine (HCQ) sulfate, i.e., the phosphoric or sulfuric acid salt of a racemic mixture of R - and S -enantiomer (50/50), respectively. As a result, the clinical outcome from previous CQ or HCQ trials were, in fact, the collective manifestation of both R and S- enantiomers with inherent different pharmacodynamic and pharmacokinetic properties, and toxicity liabilities. Our data for the first time demonstrated the stereoselective difference of CQ and HCQ against live SARS-CoV-2 virus in a Biosafety Level 3 laboratory. S -chloroquine ( S -CQ) and S -hydroxychloroquine ( S -HCQ) significantly more active against SARS-CoV-2, as compared to R -CQ and R -HCQ, respectively. In addition, M pro , as one of the critical enzymes for viral transcription and replication, also exhibited an enantioselective binding affinity toward the S -enantiomers. The most significant finding from this study is the pronounced difference of the two enantiomers of CQ and HCQ observed in hERG inhibition assay. The IC 50 value of S -HCQ was higher than 20 μM against hERG channel, which was much less active over all tested CQ and HCQ compounds. Moreover, S -HCQ alone did not prolong QT interval in guinea pigs after 3 days and 6 days of administration, indicating a much lower cardiac toxicity potential. With these and previous findings on the enantio-differentiated metabolism, we recommend that future clinical studies should employ S -HCQ, substantially free of the R -enantiomer, to potentially improve the therapeutic index for the treatment of COVID-19 over the racemic CQ and HCQ.

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  1. Version published to 10.1101/2020.05.26.114033v4 on bioRxiv
  2. Version published to 10.1101/2020.05.26.114033v3 on bioRxiv
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    SciScore for 10.1101/2020.05.26.114033: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: The experiments were approved by the Institutional Animal Care and Use Committee (No. IACUC2020125).
    RandomizationQT prolongation assay in vivo: Specific pathogen-free (SPF) Hartley guinea pigs, weighing about 300 g were randomly divided into three groups with 8 in each group: control group (Con), S-hydroxychloroquine sulfate group (S-HCQ) and S-hydroxychloroquine sulfate + Azimycin group (S-HCQ + AZM).
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    After blocking with 5% bovine serum albumin (BSA) at room temperature for 1 h, the rabbit polyclonal antibody against SARS Coronavirus Nucleoprotein (NP) (1:1000) as the primary antibody was used to incubate the cells, and then Alexa 488 labeled secondary antibody (Donkey Anti-Rabbit IgG;1:500; Jackson) was added.
    antibody against SARS Coronavirus Nucleoprotein ( NP )
    suggested: None
    Anti-Rabbit IgG;1:500
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    A clinical isolate SARS-CoV-2 virus (Genebank accession no. MT123290.1) was propagated in Vero E6 cells and viral titer was determined by plaque assay.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Software and Algorithms
    SentencesResources
    The IC50 value for each compound was calculated using GraphPad Prism software (version 7.0) using a non-linear regression equation. hERG inhibition assay: Automated QPatch recording: The hERG current recording in the whole-cell patch-clamp configuration on QPatch 16X applied single-hole QPlate.
    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: 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.
    • 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

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  4. Version published to 10.1101/2020.05.26.114033v2 on bioRxiv
  5. Version published to 10.1101/2020.05.26.114033v1 on bioRxiv