In vitro metabolism of piperaquine is primarily mediated by CYP3A4

Abstract

Piperaquine (PQ) is part of a first-line treatment regimen for Plasmodium falciparum malaria recommended by the World Health Organization (WHO). We aimed to determine the major metabolic pathway(s) of PQ in vitro. A reliable, validated tandem mass spectrometry method was developed. Concentrations of PQ were measured after incubation with both human liver microsomes (HLMs) and expressed cytochrome P450 enzymes (P450s). In pooled HLMs, incubations with an initial PQ concentration of 0.3 µM resulted in a 34.8 ± 4.9% loss of substrate over 60 min, corresponding to a turnover rate of 0.009 min(-1) (r(2) = 0.9223). Miconazole, at nonspecific P450 inhibitory concentrations, resulted in almost complete inhibition of PQ metabolism. The greatest inhibition was demonstrated with selective CYP3A4 (100%) and CYP2C8 (66%) inhibitors. Using a mixture of recombinant P450 enzymes, turnover for PQ metabolism was estimated as 0.0099 min(-1); recombinant CYP3A4 had a higher metabolic rate (0.017 min(-1)) than recombinant CYP2C8 (p < .0001). Inhibition of CYP3A4-mediated PQ loss was greatest using the selective inhibitor ketoconazole (9.1 ± 3.5% loss with ketoconazole vs 60.7 ± 5.9% with no inhibitor, p < .0001). In summary, the extent of inhibition of in vitro metabolism with ketoconazole (83%) denotes that PQ appears to be primarily catalyzed by CYP3A4. Further studies to support these findings through the identification and characterization of PQ metabolites are planned.

Figure 1

Chemical structures of piperaquine and internal standard used in these studies.

Figure 2

Log transformed piperaquine depletion curves from human liver microsome studies. The initial piperaquine concentration was 0.27 µM and data shown are means ± SD of triplicate determinations. Nonlinear regression fitting yielded an estimated rate constant of 0.009 min⁻¹.

Figure 3

The effect of isoform selective P450 inhibitors on piperaquine metabolism. The data is expressed as percent piperaquine remaining (mean ± SD of triplicate determinations) as a function of time following human liver microsome incubations in the presence of isoform selective P450 inhibitors. The starting concentration of piperaquine was 0.6 µM. The following concentrations of inhibitors were used: 1 µM ketoconazole (CYP3A4), 5 µM ticlopidine (CYP2C19), 20 µM furafylline (CYP1A2), 20 µM sulfaphenazole (2C9), and 25 µM quercetin (CYP2C8).

Figure 4

Log transformed piperaquine depletion curves from incubation with a mixture of recombinant P450 isoforms. Data shown are means ± SD of triplicate determinations. Nonlinear regression fitting yielded estimated rate constants of 0.0037 min⁻¹ and 0.0099 min⁻¹ for 2 and 0.2 µM piperaquine initial concentrations, respectively.

Figure 5

Log transformed piperaquine depletion curves from incubation with recombinant CYP3A4 or CYP2C8. Data shown are means ± SD of triplicate determinations. Nonlinear regression fitting yielded estimated rate constants for CYP2C8 and CYP3A4 metabolism of 0.001 and 0.017 min⁻¹ respectively.

Figure 6

The effect of ketoconazole on piperaquine metabolism. The data is expressed as percent piperaquine remaining (mean ± SD of triplicate determinations) as a function of time following incubations with recombinant CYP3A4 in the presence or absence of ketoconazole (1 µM).