The metabolism of primaquine to its active metabolite is dependent on CYP 2D6

Abstract

Background: The efficacy of the 8-aminoquinoline (8AQ) drug primaquine (PQ) has been historically linked to CYP-mediated metabolism. Although to date no clear evidence exists in the literature that unambiguously assigns the metabolic pathway or specific metabolites necessary for activity, recent literature suggests a role for CYP 2D6 in the generation of redox active metabolites.

Methods: In the present study, the specific CYP 2D6 inhibitor paroxetine was used to assess its effects on the production of specific phenolic metabolites thought to be involved in PQ efficacy. Further, PQ causal prophylactic (developing liver stage) efficacy against Plasmodium berghei in CYP 2D knockout mice was assessed in comparison with a normal C57 background and with humanized CYP 2D6 mice to determine the direct effects of CYP 2D6 metabolism on PQ activity.

Results: PQ exhibited no activity at 20 or 40 mg/kg in CYP 2D knockout mice, compared to 5/5 cures in normal mice at 20 mg/kg. The activity against developing liver stages was partially restored in humanized CYP 2D6 mice.

Conclusions: These results unambiguously demonstrate that metabolism of PQ by CYP 2D6 is essential for anti-malarial causal prophylaxis efficacy.

Figures

Figure 1

CYP2D6-mediated hydroxylation of primaquine. A. Shown is the MSE fragmentation spectrum for primaquine. The (M + 1) charge state of primaquine is highlighted in blue. The major fragment ions of primaquine produced upon MSE fragmentation are also shown along with their corresponding m/z values and structures. B. MSE fragmentation of hydroxylated primaquine. Fragment ions utilized for localization of the hydroxylation to the quinoline core are indicated in red. It should be noted that MSE fragmentation localizes this transformation to the quinoline core, and that the assignment shown is tentative.

Figure 2

Primaquine metabolism with paroxetine inhibition of CYP2D6. A. Shown is the relative % primaquine remaining after 60-min incubations with CYP2D6 in the absence or presence of varying concentrations of paroxetine. B. Relative % hydroxylated metabolite present remaining after 60-min incubations with CYP2D6 in the absence or presence of varying concentrations of paroxetine. The structures of primaquine and the tentative hydroxylated metabolite are shown in each corresponding panel. Error bars were calculated from average values obtained from duplicate analyses.

Figure 3

Dissemination of malaria parasites by IVIS imaging of C57BL/6 Wild-type (WT), CYP 2D knockout, and humanized CYP 2D knockout/CYP 2D6 knock-in mice at 24-hr and 72-hr post-inoculation with luciferase expressing Plasmodium berghei. WT animals treated with 20 mg/kg x 3 days of oral PQ exhibited no parasitaemia at 24 or 72 hr. All five CYP 2D knockout mice exhibited liver stage parasitaemia at 24 hr which progressed to systemic infection by 72 hr. PQ efficacy was largely restored in mice in which the deleted CYP 2D cluster was replaced with human CYP 2D6.

Figure 4

Parasite dissemination by IVIS imaging of C57BL/6 WT, CYP 2D knockout, and humanized CYP 2D knockout/CYP 2D6 knock-in mice at 24-hr and 72-hr post-inoculation with luciferase expressing Plasmodium berghei.