Abstract
BackgroundPrimaquine, 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.MethodsStable 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.ResultsThe 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.ConclusionThe 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.
Highlights
Primaquine, currently the only approved drug for the treatment and radical cure of Plasmodium vivax malaria, is still used as a racemic mixture
This was based on the observed delay in anticatalase activity in an individual dosed with the drug; it was postulated that redox-cycling of the metabolite 6-demethyl-5-hydroxyprimaquine and its corresponding orthoquinone caused oxidative stress [8]
Comparative kinetics for metabolism of primaquine enantiomers Varying concentrations of primaquine and its enantiomers were subjected to human CYP2D6-catalyzed metabolism
Summary
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. The aim of the current study was to investigate the differential generation of the CYP2D6 metabolites by racemic primaquine and its individual enantiomers. Despite the unique therapeutic indications of primaquine, the widespread prevalence of G6PD deficiency across populations in malaria-endemic areas has limited its clinical use [6,7]. In 1962, Tarlov and co-workers suggested that a metabolite of primaquine, rather than primaquine itself, might be responsible for the haematotoxicity of primaquine [8] This was based on the observed delay in anticatalase activity in an individual dosed with the drug; it was postulated that redox-cycling of the metabolite 6-demethyl-5-hydroxyprimaquine and its corresponding orthoquinone caused oxidative stress [8]. Monoamine oxidase (MAO) has been shown as responsible for the formation of carboxyprimaquine [17], whereas CYP enzymes, especially CYP2D6, were found to produce several ringhydroxylated primaquine metabolites [18]
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