Abstract

Primaquine and tafenoquine are the only approved drugs that can achieve a radical cure for Plasmodium vivax malaria but are contraindicated in patients who are deficient in glucose 6-phosphate dehydrogenase (G6PDd) due to risk of severe hemolysis from reactive oxygen species (ROS) generated by redox cycling of drug metabolites. 5-hydroxyprimaquine and its quinone-imine cause robust redox cycling in red blood cells (RBCs), but are so labile as to not be detected in blood or urine. Rather, the 5,8-quinoneimine is rapidly converted into primaquine-5,6-orthoquinone (5,6-POQ) that is then excreted in the urine. The extent to which 5,6-POQ contributes to hemolysis remains unclear, although some have suggested that it is a minor toxin that should be used predominantly as a surrogate to infer levels of 5-hydroxyprimaquine. In this report, we describe a novel humanized mouse model of the G6PD Mediterranean variant (hG6PD_Med-) that recapitulates the human biology of RBC age dependent enzyme decay, as well as an isogenic matched control mouse with human non-deficient G6PD hG6PD_ND. In vitro challenge of RBCs with 5,6-POQ causes increased generation of superoxide and methemoglobin. Infusion of treated RBCs shows that 5,6-POQ selectively causes in vivo clearance of older hG6PD_Med- RBCs. These findings support the hypothesis that 5,6-POQ directly induces hemolysis and challenges the notion that 5,6-POQ is an inactive metabolic waste product. Indeed, given the extreme lability of 5-hydroxyprimaquine and the relative stability of 5,6-POQ, these data raise the possibility that 5,6-POQ is a major hemolytic primaquine metabolite in vivo.

Significance Statement These findings demonstrate that 5,6-POQ, which has been suggested to be an inert waste product of active primaquine metabolites, directly induces ROS that lead specifically to removal of older G6PDd RBCs from circulation. As 5,6-POQ is relatively stable compared to other active primaquine metabolites, these data support the hypothesis that 5,6-POQ is a major toxin in primaquine induced hemolysis. In addition, a new model of G6PDd is used to show that young G6PDd RBCs are resistant to primaquine induced hemolysis.