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Differential kinetic profiles and metabolism of primaquine enantiomers by human hepatocytes.

Fasinu PS, Avula B, Tekwani BL, Nanayakkara NP, Wang YH, Bandara Herath HM, McChesney JD, Reichard GA, Marcsisin SR, Elsohly MA, Khan SI, Khan IA, Walker LA - Malar. J. (2016)

Bottom Line: The major quinoline oxidation product (m/z 274) was preferentially generated from (+)-PQ.Metabolism of PQ showed enantioselectivity.These findings may provide important information in establishing clinical differences in PQ enantiomers.

View Article: PubMed Central - PubMed

Affiliation: The National Center for Natural Products Research, School of Pharmacy, The University of Mississippi, University, MS, 38677, USA.

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.

No MeSH data available.


Related in: MedlinePlus

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)
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Fig1: 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)

Mentions: PQ was incubated in vitro with human hepatocytes (1 million cells/mL) at a final concentration of 20 µM. Substrate concentrations, as initially titrated, showed optimal metabolism at 20 µM (Km = 37 µM). Although this is higher than expected peak plasma concentrations in humans as we earlier reported (<2 µM) [15], it approximates expected liver concentrations of PQ, which we observed to be—at therapeutic doses about 20 times higher than plasma in our studies in mice [22]. The viable cell counts in the drug-free incubation at the predetermined time-points were similar to those of the PQ-containing incubates, suggesting the absence of PQ-induced direct hepatocellular toxicity (Fig. 1a). Hepatocyte viability at the 2 h termination time was 88 % of the starting value, an indication that the integrity of the hepatocytes was not compromised through the incubation period.Fig. 1


Differential kinetic profiles and metabolism of primaquine enantiomers by human hepatocytes.

Fasinu PS, Avula B, Tekwani BL, Nanayakkara NP, Wang YH, Bandara Herath HM, McChesney JD, Reichard GA, Marcsisin SR, Elsohly MA, Khan SI, Khan IA, Walker LA - Malar. J. (2016)

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)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4837544&req=5

Fig1: 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)
Mentions: PQ was incubated in vitro with human hepatocytes (1 million cells/mL) at a final concentration of 20 µM. Substrate concentrations, as initially titrated, showed optimal metabolism at 20 µM (Km = 37 µM). Although this is higher than expected peak plasma concentrations in humans as we earlier reported (<2 µM) [15], it approximates expected liver concentrations of PQ, which we observed to be—at therapeutic doses about 20 times higher than plasma in our studies in mice [22]. The viable cell counts in the drug-free incubation at the predetermined time-points were similar to those of the PQ-containing incubates, suggesting the absence of PQ-induced direct hepatocellular toxicity (Fig. 1a). Hepatocyte viability at the 2 h termination time was 88 % of the starting value, an indication that the integrity of the hepatocytes was not compromised through the incubation period.Fig. 1

Bottom Line: The major quinoline oxidation product (m/z 274) was preferentially generated from (+)-PQ.Metabolism of PQ showed enantioselectivity.These findings may provide important information in establishing clinical differences in PQ enantiomers.

View Article: PubMed Central - PubMed

Affiliation: The National Center for Natural Products Research, School of Pharmacy, The University of Mississippi, University, MS, 38677, USA.

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.

No MeSH data available.


Related in: MedlinePlus