Activation of Peripheral Blood Mononuclear Cells by Dengue Virus Infection Depotentiates Balapiravir

Abstract

bIn a recent clinical trial, balapiravir, a prodrug of a cytidine analog (R1479), failed to achieve efficacy (reducing viremia after treatment) in dengue patients, although the plasma trough concentration of R1479 remained above the 50% effective concentration (EC 50 ). Here, we report experimental evidence to explain the discrepancy between the in vitro and in vivo results and its implication for drug development. R1479 lost its potency by 125-fold when balapiravir was used to treat primary human peripheral blood mononuclear cells (PBMCs; one of the major cells targeted for viral replication) that were preinfected with dengue virus. The elevated EC 50 was greater than the plasma trough concentration of R1479 observed in dengue patients treated with balapiravir and could possibly explain the efficacy failure. Mechanistically, dengue virus infection triggered PBMCs to generate cytokines, which decreased their efficiency of conversion of R1479 to its triphosphate form (the active antiviral ingredient), resulting in decreased antiviral potency. In contrast to the cytidine-based compound R1479, the potency of an adenosine-based inhibitor of dengue virus (NITD008) was much less affected. Taken together, our results demonstrate that viral infection in patients before treatment could significantly affect the conversion of the prodrug to its active form; such an effect should be calculated when estimating the dose efficacious for humans. Dengue virus (DENV) is the most prevalent mosquito-borne virus that causes human disease. A recent study estimated that 390 million humans are infected and that 96 million infected humans exhibit disease symptoms annually (1). No licensed vaccine or antiviral for the prevention and treatment of DENV is currently available. Upon transmission by infected mosquitoes, the virus first infects dendritic cells, spreads to lymph nodes, and disseminates to various tissues and organs. Although the sites of DENV replication in natural human infections remain to be conclusively defined, monocytes and macrophages in peripheral blood mononuclear cells (PBMCs) were reported to be major replication sites in patients (2, 3).Nucleoside analogs represent the major class of antiviral drugs in clinical use (4). To exert antiviral effects, nucleoside analogs must be converted to the triphosphate form (by host and/or viral kinases) before being incorporated into the viral DNA/RNA chain by viral polymerase. Balapiravir is an ester prodrug of the cytidine analog 4=-azidocytidine, also known as R1479 (Fig. 1A). It was originally developed as treatment against hepatitis C virus (HCV) infection (5, 6). Although balapiravir exhibited potency in HCV-infected patients, its clinical development was discontinued due to unacceptable toxicity (7). Since R1479 has anti-DENV activity in vitro, balapiravir was repurposed for a phase II trial for treatment of DENV infection. Surprisingly, no viremia reduction was observed in balapiravir-treated dengue patients, even though the maximum concentration in plasma (C max ) of R147...

The search for nucleoside/nucleotide analog inhibitors of dengue virus

“…In contrast to the cytidinebased balapiravir, the potency of an adenosine-based inhibitor of DENV (NITD008) was much less affected by pre-infection of DENV or cytokine treatment. These results demonstrate that viral infection in patient before treatment could significantly affect the prodrug conversion to its active form; such effect is nucleoside class-dependent (Chen et al, 2014). Similarly, HIV infection of PBMCs was also reported to affect both the nucleoside analog triphosphate conversion and the concentration of corresponding natural nucleoside triphosphate, leading to reduced compound efficacy (Gao et al, 1993;García-Lerma et al, 2011).…”

“…In support of the latter point, we recently showed that DENV infection triggered PBMCs to generate cytokines which decreased their efficiency to convert balapiravir to its triphosphate form, resulting in decreased antiviral potency (Chen et al, 2014). In contrast to the cytidinebased balapiravir, the potency of an adenosine-based inhibitor of DENV (NITD008) was much less affected by pre-infection of DENV or cytokine treatment.…”

“…Although multiple reasons may account for the efficacy failure, one explanation could be the decreased potency of balapiravir when treating human PBMC (peripheral blood mononuclear cells) that were pre-infected with DENV (see details in Section 3.4) (Chen et al, 2014).…”

Synergistic Suppression of Dengue Virus Replication Using a Combination of Nucleoside Analogs and Nucleoside Synthesis Inhibitors

“…Nevertheless, these observations may have clinical implications. When patients receive therapy, DENV has already established its replication, triggering an immune response to produce IFN and other cytokines (41,42). If patients are treated with brequinar, its ability to augment IFNinduced ISRE activation should enhance the overall antiviral status of patients.…”

Discovery of Dengue Virus NS4B Inhibitors

“…Most importantly, the current compound has provided an opportunity to answer two paramount questions for dengue therapeutics development: can an antiviral drug reduce viremia in dengue patients (infected with DENV-2 or -3), and, if so, will viremia reduction prevent patients from developing severe hemorrhagic fever and shock? Since compound 14a is not a prodrug and directly inhibits viral NS4B, it is expected to eliminate the complications of nucleoside inhibitors (which require host kinases to convert them to their triphosphate forms before being antiviral active), as evidenced by the efficacy failure of balapiravir in dengue patients (6,33).…”