title: Enantioselective metabolism of primaquine by human CYP2D6.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.forename: Pius S
authors.initials: PS
authors.lastname: Fasinu
authors.forename: Babu L
authors.initials: BL
authors.lastname: Tekwani
authors.forename: N P Dhammika
authors.initials: NP
authors.lastname: Nanayakkara
authors.forename: Bharathi
authors.initials: B
authors.lastname: Avula
authors.forename: H M T Bandara
authors.initials: HM
authors.lastname: Herath
authors.forename: Yan-Hong
authors.initials: YH
authors.lastname: Wang
authors.forename: Vijender R
authors.initials: VR
authors.lastname: Adelli
authors.forename: Mahmoud A
authors.initials: MA
authors.lastname: Elsohly
authors.forename: Shabana I
authors.initials: SI
authors.lastname: Khan
authors.forename: Ikhlas A
authors.initials: IA
authors.lastname: Khan
authors.forename: Brandon S
authors.initials: BS
authors.lastname: Pybus
authors.forename: Sean R
authors.initials: SR
authors.lastname: Marcsisin
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Reichard
authors.forename: James D
authors.initials: JD
authors.lastname: McChesney
authors.affiliation: The National Center for Natural Products Research, University of Mississippi, University, MS 38677, USA. lwalker@olemiss.edu.
authors.forename: Larry A
authors.initials: LA
authors.lastname: Walker
mesh_terms: D000962:Antimalarials / Q000737:chemistry / Q000378:metabolism*; D002853:Chromatography, Liquid; D019389:Cytochrome P-450 CYP2D6 / Q000378:metabolism*; D006801:Humans; D007553:Isotope Labeling; D007700:Kinetics; D010966:Plasmodium vivax; D011319:Primaquine / Q000737:chemistry / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D000962:Antimalarials; D019389:Cytochrome P-450 CYP2D6; D011319:Primaquine