Probenecid inhibits SARS-CoV-2 replication in vivo and in vitro

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Link: Probenecid inhibits SARS-CoV-2 replication in vivo and in vitro



Results


After performing RNAi screens in human lung type II epithelial (A549) cells that allowed us to determine and validate host genes required for influenza virus replication6, we chose to evaluate OAT310. Transfection of A549 cells with siRNA targeting the SLC22A8 gene, OAT3, completely blocked influenza A/WSN/33 (H1N1) virus replication, and probenecid treatment reduced OAT3 mRNA and protein levels in vitro and BALB/c mice. As probenecid treatment in vitro or in vivo did not block influenza virus infection but did block virus titers as measured by plaque assay and hemagglutination assay, we determined the in vitro inhibitory effect on SARS-CoV-2 replication in Vero E6 cells, and NHBE cells (Fig. 1). Vero E6 cells and NHBE cells were pretreated with differing probenecid concentrations and the level of infectious virions in the tissue culture supernatant was determined at 48 h after infection by plaque assay. Probenecid treatment reduced SARS-CoV-2 replication by 90% in NHBE cells (A) or 60% in Vero E6 cells (B). The IC50 value for probenecid was shown to be 0.75 μM in Vero E6 cells and 0.0013 μM in NHBE cells. Viability was also assessed over the differing concentrations, demonstrating no cellular toxicity at the highest drug concentration (data not shown). Probenecid treatment was also effective at inhibiting a SARS-CoV-2 variant of concern (VOC), i.e. hCoV-19/USA/CA_CDC_5574/2020, B.1.1.7 (Supplemental Fig. 2). This variant was isolated from a nasopharyngeal swab on December 29, 2020, and after sequencing it to the lineage B.1.1.7 (a.k.a. UK variant). This VOC may have increased transmissibility16.

Figure 1

Probenecid potently inhibits SARS-CoV-2 replication in (A) NHBE cells and (B) Vero E6 cells. IC50 and IC90 values were calculated in NHBE cells and Vero E6 cells after treating with different probenecid concentrations. Cellular toxicity was calculated in NHBE cells and Vero E6 cells following treatment with different concentrations of probenecid and performing a cell viability assay that measured the percent toxicity based on the conversion of a redox dye (resazurin) into a fluorescent end product (resorufin) after 72 h. Figures were prepared using GraphPad Prism, v9.2, https://www.graphpad.com/updates/.
Having shown in vitro efficacy and determined the IC50 values, we next determined the efficacy of probenecid in the hamster model regarded as a preclinical model of SARS-CoV-2 disease with hamsters having self-limiting pneumonia17,18. We examined hamsters infected with SARS-CoV-2 and treated them with probenecid for either 24 h before infection (prophylaxis) or 48 h post-infection (post-treatment). The dosing groups tested were: a) two prophylaxis groups treated with doses of 2 mg/kg or 200 mg/kg, b) two post-treatment groups treated with doses of 2 mg/kg or 200 mg/kg, and two control groups that were SARS-CoV-2 infected or not infected. Disease in hamsters following SARS-CoV-2 infection is transient peaking around day 3–4 post-infection with no clinical signs17. Consistent with this observation, no substantial clinical symptoms or weight loss was evident in any group throughout the study (Supplementary Fig. 1). Hamsters treated with probenecid had dramatically reduced lung virus titers (Fig. 2), i.e. a 4–5-log reduction of virus compared to the PBS controls that were approximately 109logs of virus. At day 7 pi all groups cleared the virus in the lungs. RT-qPCR analysis showed low-to-no virus detection at day 7 pi with Ct = 30 except for 2 mg probenecid treatment which was Ct = 28 (Supplementary Fig. 3).
Figure 2

(A) Lung virus titers from male hamsters. Hamsters were divided into pre-infection or post-infection groups and i.p. treated with probenecid or PBS (n = 6/group). The groups received 200 mg/kg or 2 mg/kg of probenecid prophylactically at 24 h pre-infection or prophylactically at 48 h pi. The hamsters were i.n. infected with 103 PFU of SARS-CoV-2, and at days 3 or 7 pi, the lungs were harvested and virus levels determined by plaque assay. (B) Weight gain in hamsters treated prophylactically or therapeutically with 200 mg/kg or 2 mg/kg probenecid. Uninfected hamsters or PBS-treated and infected hamsters were the controls. No substantial weight loss was evident in any of the groups during this study. Figures were prepared using GraphPad Prism, v9.2, https://www.graphpad.com/updates/.
Finally, a population pharmacokinetics (pop-PK) model was developed to characterize probenecid PK. It had a one-compartment structure with saturable elimination and first-order absorption. We performed simulations using the final pop-PK model to generate probenecid exposure profiles comparing 600 mg twice daily, 900 mg twice daily, or 1800 mg once daily administration (Fig. 3 and Table 1). The doses examined are predicted to provide plasma concentrations exceeding the protein binding adjusted IC90 value at all time points. All doses were below the maximum allowable FDA-approved dose and are generally safe with no significant side effects.
Figure 3

Simulated probenecid concentrations. A population pharmacokinetic model was used to generate probenecid exposure profiles comparing (A) 600 mg twice daily, 900 mg twice daily, or (B) 1800 mg once daily administration for 20 days. The IC90 level corrected for 95% protein binding is 2.08 µg/ml (shown as dashed line). All doses provided exposures well over the IC90 level at all time points. Figures were prepared using GraphPad Prism, v9.2, https://www.graphpad.com/updates/.
Table 1 Steady state concentration and ratio to protein binding adjusted IC90 value after different probenecid doses.


 

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