The clinical pharmacology of ribavirin: Intracellular effects on endogenous nucleotides and hemolytic anemia

Hepatitis C virus (HCV) is one of the leading causes of liver disease in the world with approximately 130-150 million people infected according to the World Health Organization. For more than twenty years, HCV was treated with the combination of interferon (IFN) and ribavirin (RBV). IFN has been replaced with new direct acting antiviral (DAA) agents, but RBV is still important in the treatment of certain patient populations including those with advanced liver disease, with pre-existing viral mutations, and those requiring retreatment after DAA failure. As an adenosine and guanosine analog, RBV is metabolized intracellulary to a mono- (RMP), di- (RDP), and tri-phosphate (RTP). Phosphorylated forms of RBV have been associated with antiviral/toxic effects in vitro, but its precise mechanism(s) of action in vivo is unknown. While RBV has been extremely useful for increasing the cure rate of HCV when combined with other drugs, it has an unfortunate side effect of hemolytic anemia. This may be because RTP causes a decrease of adenosine triphosphate (ATP) inside red blood cells (RBCs) leading to oxidative stress and membrane damage, but this is not well studied in humans. Single nucleotide polymorphisms (SNPs) found in the gene encoding inosine triphosphatase (ITPA) result in protection against hemolytic anemia, but the mechanism behind this is also not clear in humans. The purpose of this work was to determine the effect of RBV treatment and RTP concentrations on endogenous nucleotides and how this relates specifically to toxicity in human subjects. To do this, the first step was to develop sophisticated analytical methods to measure RMP, RDP, RTP, and endogenous nucleotides in RBCs, peripheral blood mononuclear cells (PBMCs), and dried blood spots (DBS) collected from subjects undergoing RBV treatment. Three LC-MS/MS methods were developed and validated according to FDA guidelines to achieve this. The second part of this work was to determine factors that influence intracellular RTP concentrations (including ITPA activity phenotype) in vivo. Finally, the impact of RBV treatment on endogenous purine concentrations and associations with changes in endogenous purines was determined. This enhanced our understanding of how RBV works in humans and generated important knowledge on the mechanisms of RBV toxicity, understanding of the intracellular pharmacology of RBV, and pharmacokinetic-pharmacodynamic-pharmacogenomic (PK-PD-PG) associations for this drug which can be used to improve treatment with RBV.