Differential kinetic profiles and metabolism of primaquine enantiomers by human hepatocytes

Abstract

Background: The clinical utility of primaquine (PQ), used as a racemic mixture of two enantiomers, is limited due to metabolism-linked hemolytic toxicity in individuals with genetic deficiency in glucose-6-phosphate dehydrogenase. The current study investigated differential metabolism of PQ enantiomers in light of the suggestions that toxicity and efficacy might be largely enantioselective.

Methods: Stable isotope (13)C-labelled primaquine and its two enantiomers (+)-PQ, (-)-PQ were separately incubated with cryopreserved human hepatocytes. Time-tracked substrate depletion and metabolite production were monitored via UHPLC-MS/MS.

Results: The initial half-life of 217 and 65 min; elimination rate constants (λ) of 0.19 and 0.64 h(-1); intrinsic clearance (Clint) of 2.55 and 8.49 (µL/min)/million cells, which when up-scaled yielded Clint of 6.49 and 21.6 (mL/min)/kg body mass was obtained respectively for (+)- and (-)-PQ. The extrapolation of in vitro intrinsic clearance to in vivo human hepatic blood clearance, performed using the well-stirred liver model, showed that the rate of hepatic clearance of (+)-PQ was only 45 % that of (-)-PQ. Two major primary routes of metabolism were observed-oxidative deamination of the terminal amine and hydroxylations on the quinoline moiety of PQ. The major deaminated metabolite, carboxyprimaquine (CPQ) was preferentially generated from the (-)-PQ. Other deaminated metabolites including PQ terminal alcohol (m/z 261), a cyclized side chain derivative from the aldehyde (m/z 241), cyclized carboxylic acid derivative (m/z 257), a quinone-imine product of hydroxylated CPQ (m/z 289), CPQ glucuronide (m/z 451) and the glucuronide of PQ alcohol (m/z 437) were all preferentially generated from the (-)-PQ. The major quinoline oxidation product (m/z 274) was preferentially generated from (+)-PQ. In addition to the products of the two metabolic pathways, two other major metabolites were observed: a prominent glycosylated conjugate of PQ on the terminal amine (m/z 422), peaking by 30 min and preferentially generated by (+)-PQ; and the carbamoyl glucuronide of PQ (m/z 480) exclusively generated from (+)-PQ.

Conclusion: Metabolism of PQ showed enantioselectivity. These findings may provide important information in establishing clinical differences in PQ enantiomers.

Keywords: Enantioselectivity; Hepatocytes; Malaria; Metabolism; Primaquine enantiomers; Primaquine metabolites.

Figures

Fig. 1

a Hepatocyte viability time course determined through cell counts in the presence and absence of (±)-primaquine and its (+)- and (−)-enantiomers; b differential depletion of the 20 µM racemic (±)-primaquine and its (+)-, and (−)-enantiomers in primary human hepatocytes (1 million cells/mL) after 2 h incubation. Each point represents values mean ±SD (n = 4)

Fig. 2

Putative identities and predicted structures of primaquine metabolites generated in human hepatocytes, as determined through MS/MS fragmentation, twin peak detection on the UHPLC chromatogram and prediction by Waters’ Metabolynx® software package

Fig. 3

The relative concentrations of the major metabolites generated by primaquine and its enantiomers at 1 h incubation time point in primary human hepatocytes

Fig. 4

A time-course analysis of the in vitro generation of a carboxyprimaquine (1), b primaquine alcohol (2) and c, d their glucuronide conjugates (5, 6), from (+)-, (−)- and (±)-primaquine in human hepatocytes. Each point represents value mean ±SD of four observations

Fig. 5

A time-course analysis of a cyclized PQ aldehyde (m/z 241) (3), b cyclized carboxyprimaquine (m/z 257) (4), c hydroxylated CPQ quinone-imine (m/z 289) (7) and d hydroxyprimaquine quinone-imine (m/z 274) (8) differentially generated from (+)-, (−)- and (±)-primaquine in vitro in human hepatocytes. Each point represents value mean ±SD of four observations

Fig. 6

A time-course analysis of a glycosylated primaquine (9) generated through the activity of human hepatocytes; b non-enzymatic generation of primaquine-glucose conjugates observed following the incubation of primaquine and its metabolites in cell-free hepatocyte media and; c primaquine carbamoyl-glucuronide (10) differentially generated from (+)-, (−)-and (±)-primaquine in vitro in human hepatocytes. Each point represents value mean ±SD of four observations