Abstract

The polar nucleoside drug ribavirin is front-line treatment for chronic hepatitis C virus infection. The human equilibrative nucleoside transporter (ENT) 1 transports ribavirin into erythrocytes where it is phosphorylated. These phosphorylated metabolites accumulate in the erythrocytes and produce dose-limiting hemolytic anemia. Here, we examined the in vitro and ex vivo transport and metabolism of ribavirin by erythrocytes isolated from humans and Ent1-null mice. Ribavirin (2.4 μM) uptake was significantly higher (1044 ± 255 amol/μg/10 s) into erythrocytes from Ent1(+/+) mice compared with that from Ent1(-/-) mice (76.48 ± 11.20 amol/μg/10 s). Our results showed a saturable (K_m of 382 ± 75.1 μM) transport of [³H]ribavirin into erythrocytes from Ent1(+/+) mice. We found that ribavirin concentration rapidly (within 60 s) reached equilibrium in erythrocytes using a time course of [³H]ribavirin transport (2.5 μM) and metabolism in mouse and human erythrocytes for 8 h. However, total radioactivity of ribavirin was predominantly attributed to the phosphorylated metabolites ribavirin monophosphate and ribavirin triphosphate. Our findings allow us to estimate ribavirin transport, diffusion, and metabolic clearance and to predict in vivo accumulation of ribavirin phosphates in erythrocytes of both mice and humans. Our modeling of ribavirin in erythrocytes on long-term administration of ribavirin suggests that the accumulation of ribavirin inside the cells is dependent on ENT1/Ent1 transport and the rates of intracellular phosphorylation and the degradation of the phosphorylated metabolites. We predict that Ent1(+/+) and Ent1(-/-) mice will serve as excellent models to investigate the contribution of Ent1 to the pharmacokinetics and toxicity of ribavirin in vivo.