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...

Monophosphate prodrug analogs of 2′-deoxy-2′-fluoro-2′-C-methylguanosine have been reported as potent inhibitors of hepatitis C virus (HCV) RNA-dependent RNA polymerase. These prodrugs also display potent anti-dengue activities in cellular assays although their prodrug moieties were designed to produce high levels of triphosphate in the liver. Since peripheral blood mononuclear cells (PBMCs) are one of the major targets of dengue virus, different prodrug moieties were designed to effectively deliver 2′-deoxy-2′-fluoro-2′-C-methylguanosine monophosphate prodrugs and their corresponding triphosphates into PBMCs after oral administration. We identified a cyclic phosphoramidate prodrug 17 demonstrating a well-balanced anti-dengue cellular activity and in vitro stability profiles. We further determined the PBMCs concentration of active triphosphate needed to inhibit 50% virus replication (TP50). Compound 17 was assessed in AG129 mouse model and demonstrated 1.6- and 2.2-log viremia reduction at 100 and 300 mg/kg BID, respectively. At 100 mg/kg BID, the terminal triphosphate concentration in PBMCs reached above TP50, demonstrating TP50 as the target exposure for efficacy. In dogs, oral administration of 17 resulted in high PBMCs triphosphate level, exceeding TP50 at 10 mg/kg. Unfortunately, two-week dog toxicity studies at 30, 100, and 300 mg/kg/day showed that No Observed Adverse Effect Level (NOAEL) could not be achieved due to pulmonary inflammation and hemorrhage. The preclinical safety results suspended further development of 17. Nevertheless, present work has proven the concept that an efficacious monophosphate nucleoside prodrug could be developed for the potential treatment of dengue infection.

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47Monophosphate prodrug analogs of 2'-deoxy-2'-fluoro-2'-C-methylguanosine have been 48 reported as potent inhibitors of hepatitis C virus (HCV) RNA-dependent RNA polymerase. These 49 prodrugs also display potent anti-dengue activities in cellular assays although their prodrug 50 moieties were designed to produce high levels of triphosphate in the liver. Since peripheral blood 51 mononuclear cells (PBMCs) are one of the major targets of dengue virus, different prodrug 52 moieties were designed to effectively deliver 2'-deoxy-2'-fluoro-2'-C-methylguanosine 53 monophosphate prodrugs and their corresponding triphosphates into PBMCs after oral 54 administration. We identified a cyclic phosphoramidate prodrug 17 demonstrating a well-balanced 55 anti-dengue cellular activity and in vitro stability profiles. In dogs, oral administration of 17 56 resulted in high PBMC triphosphate level, exceeding TP50 (the intracellular triphosphate 57 concentration at which 50% of virus replication is inhibited) at 10 mg/kg. Compound 17 58 demonstrated 1.6-and 2.2 log viremia reduction in the dengue mouse model at 100 and 300 mg/kg 59 twice daily, respectively. At 100 mg/kg twice daily, the terminal triphosphate concentration in 60 PBMCs reached above TP50, defining for the first time the minimum efficacious dose for a 61 nucleos(t)ide prodrug. In the two-week dog toxicity studies at 30 to 300 mg/kg/day, no observed 62 adverse effect level (NOAEL) could not be achieved due to pulmonary inflammation and 63 hemorrhage. The preclinical safety results suspended further development of 17. Nevertheless, 64 present work has proven the concept that an efficacious monophosphate nucleoside prodrug could 65 be developed for the potential treatment of dengue infection. 67The mosquito-borne dengue virus is endemic to tropical and sub-tropical regions 68 throughout the world, making dengue fever the most important mosquito-borne viral disease 69 afflicting humans. Its global distribution is comparable to that of malaria, with an estimated 2.5 70 billion people at risk for epidemic transmission (1). There has been steady increases in countries 71 affected and incidence since the 1950s and recent estimates suggest annual rates of 390 million 72 cases accompanied by 20,000 deaths (2). 73 Dengue viruses (DENVs) can be further classified into four different serotypes (DENV-1 74 to -4), all of which can lead to disease symptoms with varying severity. Secondary infection by a 75 different serotype may increase the risk of severe dengue diseases. While diagnosis of dengue 76 infection can be rapid and simple, serotype distinction requires additional instrumentation, usually 77 in a laboratory setting. Thus, the ideal treatment for dengue fever should possess pan-serotype 78 activities (3). Recently, a dengue vaccine was approved in certain countries but is recommended 79 only for individuals with prior DENV exposure. This limits its use as well as necessitating pre-80 vaccination screening (4). No antivirals are currently available for the treatment of d...