Estimation of the comparative hemolytic potential of primaquine and analogs in a humanized nod-scid mouse model of G6PD deficiency

Abstract

8-aminoquinolines (8-AQs), of which primaquine (PQ) is the prototype, are critical to malaria elimination efforts, given their anti-relapse and gametocidal activity. Clinical use of 8-AQs is impeded by hemolysis in glucose-6-phosphate dehydrogenase defcient (G6PDd) subjects. A critical need is animal models in which human G6PDd sensitivity to PQ can be reproduced. We have previously reported a model in which 8-AQs cause hemolysis in NOD-SCID mice engrafted with human G6PDd RBCs. In this model, direct comparison of hemolytic potency of analogs with PQ is feasible, but fails to take account of the comparative effcacy. We therefore developed an approach which frst estimates the effective causal prophylactic (CP) dose in an ICR mouse Plasmodium berghei (ANKA strain) sporozoite challenge model. In the CP model, drugs are dosed for 3 days (-1, 0, and 1) with sporozoite inoculation given on d 0; untreated mice succumb to infection after several days. 8-AQs are highly effective in this model with an effective dose (ED)100 for PQ at 25 mg/kg/d given orally for 3 d. In the NOD-SCID hemolytic model, this CP ED100 dose of PQ yields loss of about 75% of human G6PDd (A- genotype) RBCs by d 7. To generate a normalized PQ hemolytic dose-response curve to compare effcacy and toxicity, we did a hemolytic dose response curve at multiples of the CP ED 100. Thus, a new 8AQ analog's ED100 can be established in CP model, then the hemolytic potential of PQ and the analog can be directly compared in the huRBC SCID model at equi-effective CP doses. NPC1161B, an 8-AQ development candidate which is a pure (-) (R) enantiomer, has a 3-d ED100 in the CP model of 0.5 mg/kg, while at this dose elicits no hemolysis in the huRBC SCID model. In contrast, NPC1161A, the opposite (+) (S) enantiomer, has a 3-d ED100 of 8 mg/kg, and at this dose it is indistinguishable from PQ with regard to hemolysis. This result indicates that hemolytic potential can be reduced without compromising effcacy in 8-AQs.