© 2014 Fasinu et al. Background: Primaquine, currently the only approved drug for the treatment and radical cure of Plasmodium vivax malaria, is still used as a racemic mixture. Clinical use of primaquine has been limited due to haemolytic toxicity in individuals with genetic deficiency in glucose-6-phosphate dehydrogenase. Earlier studies have linked its therapeutic effects to CYP2D6-generated metabolites. The aim of the current study was to investigate the differential generation of the CYP2D6 metabolites by racemic primaquine and its individual enantiomers. Methods: Stable isotope 13C-labelled primaquine and its two enantiomers were incubated with recombinant cytochrome-P450 supersomes containing CYP2D6 under optimized conditions. Metabolite identification and time-point quantitative analysis were performed using LC-MS/MS. UHPLC retention time, twin peaks with a mass difference of 6, MS-MS fragmentation pattern, and relative peak area with respect to parent compound were used for phenotyping and quantitative analysis of metabolites. Results: The rate of metabolism of (+)-(S)-primaquine was significantly higher (50% depletion of 20 μM in 120 min) compared to (-)-(R)-primaquine (30% depletion) when incubated with CYP2D6. The estimated Vmax (μmol/min/mg) were 0.75, 0.98 and 0.42, with Km (μM) of 24.2, 33.1 and 21.6 for (±)-primaquine, (+)-primaquine and (-)-primaquine, respectively. Three stable mono-hydroxylated metabolites, namely, 2-, 3-and 4-hydroxyprimaquine (2-OH-PQ, 3-OH-PQ, and 4-OH-PQ), were identified and quantified. 2-OH-PQ was preferentially formed from (+)-primaquine in a ratio of 4:1 compared to (-)-primaquine. The racemic (±)-primaquine showed a pattern similar to the (-)-primaquine; 2-OH-PQ accounted for about 15-17% of total CYP2D6-mediated conversion of (+)-primaquine. In contrast, 4-OH-PQ was preferentially formed with (-)-primaquine (5:1), accounting for 22% of the total (-)-primaquine conversion. 3-OH-PQ was generated from both enantiomers and racemate. 5-hydroxyprimaquine was unstable. Its orthoquinone degradation product (twice as abundant in (+)-primaquine compared to (-)-primaquine) was identified and accounted for 18-20% of the CYP2D6-mediated conversion of (+)-primaquine. Other minor metabolites included dihydroxyprimaquine species, two quinone-imine products of dihydroxylated primaquine, and a primaquine terminal alcohol with variable generation from the individual enantiomers. Conclusion: The metabolism of primaquine by human CYP2D6 and the generation of its metabolites display enantio-selectivity regarding formation of hydroxylated product profiles. This may partly explain differential pharmacologic and toxicologic properties of primaquine enantiomers.
Fasinu, P. S., Tekwani, B. L., Nanayakkara, N. P. D., Avula, B., Herath, H. M. T. B., Wang, Y. H., … Walker, L. A. (2014). Enantioselective metabolism of primaquine by human CYP2D6. Malaria Journal, 13(1). https://doi.org/10.1186/1475-2875-13-507