Formation primaquine-5,6-orthoquinone, the putative active and toxic metabolite of primaquine via direct oxidation in human erythrocytes

Abstract

Background: The activity and haemolytic toxicity associated with primaquine has been linked to its reactive metabolites. The reactive metabolites are thought to be primarily formed through the action of cytochrome P₄₅₀-mediated pathways. Human erythrocytes generally are not considered a significant contributor to drug biotransformation. As erythrocytes are the target of primaquine toxicity, the ability of erythrocytes to mediate the formation of reactive oxidative primaquine metabolites in the absence of hepatic enzymes, was evaluated.

Methods: Primaquine and its enantiomers were incubated separately with human red blood cells and haemoglobin. Post-incubation analysis was performed with UPLC-MS/MS to identify products of biotransformation.

Results: The major metabolite detected was identified as primaquine-5,6-orthoquinone, reflecting the pathway yielding putative active and haematotoxic metabolites of primaquine, which was formed by oxidative demethylation of 5-hydroxyprimaquine. Incubation of primaquine with haemoglobin in a cell-free system yielded similar results. It appears that the observed biotransformation is due to non-enzymatic processes, perhaps due to reactive oxygen species (ROS) present in erythrocytes or in the haemoglobin incubates.

Conclusion: This study presents new evidence that primaquine-5,6-orthoquinone, the metabolite of primaquine reflecting the oxidative biotransformation pathway, is generated in erythrocytes, probably by non-enzymatic means, and may not require transport from the liver or other tissues.

Fig. 1

Putative pathway for primaquine metabolism to the PQ-5,6-orthoquinone

Fig. 2

LC/MS chromatogram showing orthoquinone and PQ after incubation of human RBCs with PQ containing a mixture of 12C-PQ and 13C-PQ (13C at 6 quinoline core carbons). The peak at retention time 4.65 min corresponds to the orthoquinone standard, while the peak at 14 min corresponds to PQ. The mass spectrometric fragmentation at right shows the expected +6 mu

Fig. 3

The concentration-time profiles of primaquine incubated with human RBC. Whole RBC treatment involved the precipitation and lysing of the cells followed by recovery of the analytes. Alternatively, the incubation mixture was separated into the cellular pellets and the supernatants prior to analyte recovery and analysis

Fig. 4

The concentration-time profile of primaquine 5,6-orthoquinone formed from primaquine incubation in human RBC. Whole RBC treatment involved the precipitation and lysing of the cells followed by recovery of the analytes. Alternatively, the incubation mixture was separated into the cellular pellets and the supernatants prior to analyte recovery and analysis