Humanized mouse model of glucose 6-phosphate dehydrogenase deficiency for in vivo assessment of hemolytic toxicity

Abstract

Individuals with glucose 6-phosphate dehydrogenase (G6PD) deficiency are at risk for the development of hemolytic anemia when given 8-aminoquinolines (8-AQs), an important class of antimalarial/antiinfective therapeutics. However, there is no suitable animal model that can predict the clinical hemolytic potential of drugs. We developed and validated a human (hu)RBC-SCID mouse model by giving nonobese diabetic/SCID mice daily transfusions of huRBCs from G6PD-deficient donors. Treatment of SCID mice engrafted with G6PD-deficient huRBCs with primaquine, an 8-AQ, resulted in a dose-dependent selective loss of huRBCs. To validate the specificity of this model, we tested known nonhemolytic antimalarial drugs: mefloquine, chloroquine, doxycycline, and pyrimethamine. No significant loss of G6PD-deficient huRBCs was observed. Treatment with drugs known to cause hemolytic toxicity (pamaquine, sitamaquine, tafenoquine, and dapsone) resulted in loss of G6PD-deficient huRBCs comparable to primaquine. This mouse model provides an important tool to test drugs for their potential to cause hemolytic toxicity in G6PD-deficient populations.

Fig. 1.

Engraftment of Med-, A-, and normal huRBCs in NOD/SCID mice. (A) Mice were engrafted with huRBCs from Med-, A-, and normal donors, n = 99, 242, and 40, respectively in 5, 12, and 2 separate experiments. Mice received 5 × 10⁹ red blood cells i.p. for 14 d and were assessed for levels of huRBCs at day 14 by FACS staining using an anti-CD235a FITC-conjugated monoclonal antibody. The graph shows the engraftment efficiency after 14 d in Med-, A-, and normal mice. Values are % mean huRBCs ± SEM. (B) Comparison of total RBCs and hematocrit in huRBC SCID mice. The number of RBCs per μL of blood was determined, as well as the hematocrit in NOD/SCID mice (unengrafted) and in NOD/SCID given huRBCs for 14 d (n = 5 and 4, respectively). Dots represent individual mice. (C) Stability of engrafted Med-, A-, and normal huRBCs in NOD/SCID mice was assessed over 7 d after the last injection of huRBCs. n = 18, 43, and 10 mice from Med-, A-, and normal huRBC SCID mice from 5, 12, and 2 independent experiments, respectively. Data points shown are the mean ± SEM.

Fig. 2.

Treatment with PQ induces hemolysis in G6PD-deficient huRBC NOD/SCID mice. Human RBCs from A-, Med- G6PD-deficient or G6PD normal donors were engrafted i.p. into NOD/SCID mice. Mice were then treated for 7 d with 12.5 mg/kg per day of PQ or with vehicle control as a split dose, 8 h apart. Blood was assessed for the percentage of huRBCs and muRetics by FACS every 24 h after first treatment dose through termination of experiment at 7 d. Results are for four to five mice per group. The kinetics of (A) huRBC and (B) muRetic levels are shown. (C) Spleen and liver weight were also assessed at the termination of experiment and are presented as normalized data relative to total body weight.

Fig. 3.

Evaluation of dose of PQ and G6PD levels on hemolytic response. A- huRBC SCID mice were treated with PQ p.o. daily for 7 d with indicated doses of PQ (25, 12.5, 6.25, or 3.125 mg/kg per day) or given PBS as a vehicle control and assessed for percentage huRBCs (A) and muRetics (B) at day 7 by FACS analysis. Each treatment group consisted of 4 mice. *Statistically significant differences (one-way ANOVA with Bonferroni posttest, P < 0.001). (C) G6PD levels were measured at start of engraftment and correlated with percentage of huRBCs at 7 d after treatment with PQ (25 mg/kg per day, p.o., 7 d) using Spearman rank correlation (P = 0.0005, r = 0.7373). Data pooled from three experiments with three donors. Each dot represents an individual mouse.

Fig. 4.

Qualification of the G6PD-deficient huRBC SCID mouse model with other antimalarial drugs. (A) A- huRBC SCID mice were treated p.o. with PQ (25 mg/kg per day for 7 d), vehicle control (PBS for 7 d), pamaquine (75 or 50 mg/kg per day for 7 d), sitamaquine (40 mg/kg per day for 7 d), tafenoquine (2.5 mg/kg per day for 3 d), chloroquine (25 mg/kg per day for 7 d), mefloquine (40 mg/kg per day for 3 d), or doxycycline (60 mg/kg per day for 7 d). The percentage of huRBCs as measured by flow cytometry at day 7 of treatment was subtracted from the percentage of huRBCs at start of treatment and then divided by percentage of huRBCs at start of treatment to determine the percent loss of huRBCs. n = 15 for PQ and vehicle control, n = 4 for other groups. (B) A- huRBC SCID mice were treated p.o. with PQ (25 mg/kg per day for 3 d), dapsone (20 mg/kg per day for 3 d), pyrimethamine (50 mg/kg per day for 3 d), or vehicle control. Percentages of huRBCs were assessed at posttreatment day 0, day 4, and day 7.