Beyond the complex: inosine drives the antiviral and epigenetic effects of inosine pranobex.

Inosine pranobex (IP) is a long-used antiviral drug whose mechanism of action remains incompletely understood. However, molecular efficacy has been attributed mainly to immunomodulatory effects. There are data which suggest that the cellular activity of IP stems from its major constituent compound, inosine, known for its pleiotropic roles in purine metabolism, RNA modification, and translation regulation. We investigated whether IP acts as a stable complex or a mixture of its components and compared the biological effects of inosine itself and IP in vitro. The structural composition of IP was analyzed using compositional and microscopic methods. Cytotoxicity, antiviral activity against coxsackievirus B3 (CVB-3), and global DNA methylation changes were evaluated in A549 and HeLa cell lines using MTT, colony formation, plaque reduction, and post-labeling methods, respectively. We found that IP is physically heterogeneous and function as a mixture of components rather than a stable complex. In cell-based assays, inosine exhibited higher antiviral activity than IP, particularly under pre-treatment conditions, where it provided stronger protection against CVB-3-induced cytopathic effects. Neither compound showed significant cytotoxicity within the tested concentration ranges. Both inosine and IP influenced global DNA methylation levels, but inosine induced more pronounced and concentration-dependent changes. The superior antiviral and epigenetic activity of inosine compared with IP suggests that inosine is the main principal active component responsible for IP's biological effects. While IP's immunomodulatory functions were not evaluated here, our findings strongly suggest that inosine contributes substantially to its antiviral efficacy. Further studies, including in vivo models, are warranted to clarify the epigenetic mechanism underlying these observations.