An in vivo drug screening model using glucose-6-phosphate dehydrogenase deficient mice to predict the hemolytic toxicity of 8-aminoquinolines

Abstract

Anti-malarial 8-aminoquinolines drugs cause acute hemolytic anemia in individuals with glucose-6-phosphate dehydrogenase deficiency (G6PDD). Efforts to develop non-hemolytic 8-aminoquinolines have been severely limited caused by the lack of a predictive in vivo animal model of hemolytic potential that would allow screening of candidate compounds. This report describes a G6PDD mouse model with a phenotype closely resembling the G6PDD phenotype found in the African A-type G6PDD human. These G6PDD mice, given different doses of primaquine, which used as a reference hemolytic drug, display a full array of hemolytic anemia parameters, consistently and reproducibly. The hemolytic and therapeutic indexes were generated for evaluation of hemotoxicity of drugs. This model demonstrated a complete hemolytic toxicity response to another known hemolytic antimalarial drug, pamaquine, but no response to non-hemolytic drugs, chloroquine and mefloquine. These results suggest that this model is suitable for evaluation of selected 8-AQ type candidate antimalarial drugs for their hemolytic potential.

Figure 1.

Hemolytic course of primaquine in G6PDD mice. G6PDD mice were administered primaquine orally at 6.6 mg/kg/day for 30 consecutive days. Hemolytic parameters were determined on the day before starting primaquine treatment (Day zero) and at about weekly intervals until 5 weeks. Data points are mean ± SE from 5 animals in each group. **Statistically significant difference as compared with Day zero in the same treatment group (P < 0.01).

Figure 2.

Primaquine and chloroquine effects on total and reduced glutathione (GSH) levels in wild-type and G6PDD mice. Groups (N = 7) of wild-type and G6PDD mice were administered primaquine orally at 25 mg/kg/d for 5 consecutive days, or administered chloroquine orally at 25 mg/kg/d for 7 consecutive days. Total and reduced GSH levels were determined on the day before drug administration (Day zero) and on Day 7. (A) Changes in total GSH level caused by primaquine, (B) Changes in reduced GSH level caused by primaquine, (C) Changes in total GSH level caused by chloroquine, (D) Changes in reduced GSH level caused by chloroquine. Data points are mean ± SE. Statistically significant difference as compared with Day zero in the same treatment group, and between wild-type and G6PDD mice in the same day of dosing (**P < 0.01).

Figure 3.

Hemolytic index calculation. A scatter plot was created with the moles of drug/kg (Log₂) of test drugs on the x axis, and percentage of red block cell (RBC) hemolysis on the y axis. The percentage of RBC loss induced by primaquine given at ED₁₀₀ was defined as 100% hemolysis. The percent RBC hemolysis observed in animals administered test compounds was direct compared with a standard curve of primaquine at equimolar doses. •, Primaquine; □, pamaquine; ⋄, chloroquine; △, mefloquine.

Figure 4.

Therapeutic index calculation. A scatter plot was created with effective dose (ED) doses (Log₂) of test drugs on the x axis, and percent of red blood cell (RBC) hemolysis on the y axis. The percentage of RBC loss induced by primaquine given at ED₁₀₀ was defined as 100% hemolysis. The Therapeutic Index was calculated as HD₅₀ over ED₅₀.