RNA viruses like SARS-CoV-2, influenza virus, and respiratory syncytial virus (RSV) are dependent on host genes for replication. We investigated if probenecid, an FDA-approved and safe urate-lowering drug that inhibits organic anion transporters (OATs) has prophylactic or therapeutic efficacy to inhibit RSV replication in three epithelial cell lines used in RSV studies, i.e., Vero E6 cells, HEp-2 cells, and in primary normal human bronchoepithelial (NHBE) cells, and in BALB/c mice. The studies showed that nanomolar concentrations of all probenecid regimens prevent RSV strain A and B replication in vitro and RSV strain A in vivo, representing a potential prophylactic and chemotherapeutic for RSV.

Probenecid is an orally bioavailable, uricosuric agent that was first approved in 1951 for the treatment of gout, but was later found to have potent, broad-spectrum antiviral activity against several respiratory viruses including SARS-CoV-2. We conducted a phase 2 randomized, placebo-controlled, single-blind, dose-range finding study in non-hospitalized patients with symptomatic, mild-to-moderate COVID-19. Patients were randomly assigned in a 1:1:1 ratio to receive either 500 mg of probenecid, 1000 mg of probenecid, or a matching placebo every 12 h for five days. The patients’ COVID-19 viral load hospitalization, or death from any cause through day 28, as well as safety, were evaluated. COVID-19-related symptoms were assessed at baseline, and on days 3, 5, 10, 15, and 28. The primary endpoints of the study were time to first negative SARS-CoV-2 viral test (or viral clearance) and the proportion of patients that were symptom-free at day 5. A total of 75 patients were randomized, with 25 patients in each group. All of the patients completed the study as planned with no hospitalizations or deaths being reported. The median time to viral clearance was significantly shorter for the probenecid 1000 mg group than for placebo (7 days vs. 11 days, respectively; p < 0.0001), and for the probenecid 500 mg group versus placebo (9 days vs. 11 days, respectively; p < 0.0001). In addition, the median time to viral clearance was significantly shorter for the probenecid 1000 mg group than for the probenecid 500 mg group (7 days vs. 9 days, respectively; p < 0.0001). All patients reported at least one COVID-19-related symptom on days 3 and 5; however, on day 10, a significantly greater proportion of patients receiving probenecid 1000 mg reported the complete resolution of symptoms versus placebo (68% vs. 20%, respectively; p = 0.0006), as well as for those receiving probenecid 500 mg versus placebo (56% vs. 20%, respectively, p = 0.0087). The incidence of adverse events during treatment was similar across all groups for any adverse event, and was 12%. All events were mild with no serious adverse events reported and no discontinuations due to an adverse event. The treatment of patients with symptomatic, mild-to-moderate COVID-19 with probenecid resulted in a significant, dose-dependent decrease in the time to viral clearance and a significantly higher proportion of patients reporting complete symptom resolution by day 10. (Supported by TrippBio; ClinicalTrials.gov number, NCT05442983 and Clinical Trials Registry India number CTRI/2022/07/043726).

Influenza can cause respiratory infections, leading to significant morbidity and mortality in humans. While current influenza vaccines offer varying levels of protection, there remains a pressing need for effective antiviral drugs to supplement vaccine efforts. Currently, the FDA-approved antiviral drugs for influenza include oseltamivir, zanamivir, peramivir, and baloxavir marboxil. These antivirals primarily target the virus, making them vulnerable to drug resistance. In this study, we evaluated the efficacy of the neuraminidase inhibitor, oseltamivir, against probenecid, which targets the host cells and is less likely to engender resistance. Our results show that probenecid has superior antiviral efficacy compared to oseltamivir in both in vitro replication assays and in vivo mouse models of influenza infection.

We examined the effect of probenecid in regulating the ERK and JNK downstream MAPK pathways affecting respiratory syncytial virus replication. Background: We have previously shown that probenecid inhibits RSV, influenza virus, and SARS-CoV-2 replication in vitro in preclinical animal models and in humans. In a Phase two randomized, placebo-controlled, single-blind, dose range-finding study using probenecid to treat non-hospitalized patients with symptomatic, mild-to-moderate COVID-19, we previously showed that a 1000 mg twice daily treatment for 5 days reduced the median time to viral clearance from 11 to 7 days, and a 500 mg twice daily treatment for 5 days reduced the time to viral clearance from 11 to 9 days more than the placebo. Methods: In this study, we sought to determine the mechanism of action of the probenecid inhibition of RSV replication in human respiratory epithelial (A549) cells. Results: We show that probenecid inhibits the RSV-induced phosphorylation of JNKs and ERKs and the downstream phosphorylation of c-jun, a component of the AP-1 transcription complex needed for virus replication. The inhibition of JNKs by probenecid reversed the repression of transcription factor HNF-4. Conclusion: The probenecid inhibition of JNK and ERK phosphorylation involves the MAPK pathway that precludes virus replication.

Probenecid has long been a versatile drug in pharmacological therapies, primarily known for blocking active tubular secretion in the kidney, affecting both endogenous substances like uric acid and exogenous ones like penicillin. Beyond its renal applications, probenecid has shown capabilities in crossing the blood–brain barrier and modulating the activity of various membrane channels and transporters. This compound has emerged as a potent antiviral agent, demonstrating efficacy against multiple viruses, including influenza, COVID-19, and RSV. Clinical trials with COVID-19 patients have confirmed its antiviral potential, sparking further investigation into its mechanisms of action. This study explores probenecid’s significant anti-inflammatory properties, focusing on its ability to inhibit inflammasome activation. Our study aims to unravel the anti-inflammatory effects of probenecid on the NLRP3 inflammasome and MAPK signaling pathways using murine macrophages as a relevant inflammation model. We reveal that probenecid treatment blocks JNK and ERK signaling without affecting p38 MAPK, suppressing NLRP3 inflammasome activation. Additionally, probenecid does not affect NFκB-directed protein expression, although it efficiently inhibits NLRP3 inflammasome outputs, e.g., IL-1β and pyroptosis. These results indicate probenecid’s potential therapeutic applications.

Sickle cell disease (SCD) induces red blood cell sickling, which causes debilitating symptoms including vaso-occlusion and inflammation. We investigated a food enriched with omega-3 fatty acids to determine its effect on certain factors: blood cell membrane fatty acid composition (including anti-inflammatory elements-docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA)-and the pro-inflammatory, arachidonic acid (AA)); the inflammation biomarker, C-reactive protein (CRP); and vaso-occlusive crisis (VOC) pain. Ten adults with SCD ingested the food, daily, for 28 days. Evaluated measures included blood cell membrane fatty acid ratios (AA vs omega-3 (DHA+EPA)), CRP (mg/L) levels, and Visual Analogue Scale (VAS) scores (a VOC assessment). The food was well tolerated and led to a statistically significant CRP reduction (39%). However, changes in omega-3 fatty acid ratios and VAS scores were not significant. Overall, while the omega-3-enriched food reduced inflammation, larger, blinded studies are needed to assess its effectiveness on other measures.