Differential kinetic profiles and metabolism of primaquine enantiomers by human hepatocytes
Pius S Fasinu, Bharathi Avula, Babu L Tekwani, N P Dhammika Nanayakkara, Yan-Hong Wang, H M T Bandara Herath, James D McChesney, Gregory A Reichard, Sean R Marcsisin, Mahmoud A Elsohly, Shabana I Khan, Ikhlas A Khan, Larry A Walker, Pius S Fasinu, Bharathi Avula, Babu L Tekwani, N P Dhammika Nanayakkara, Yan-Hong Wang, H M T Bandara Herath, James D McChesney, Gregory A Reichard, Sean R Marcsisin, Mahmoud A Elsohly, Shabana I Khan, Ikhlas A Khan, Larry A Walker
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.
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