Metabolism of primaquine in normal human volunteers: investigation of phase I and phase II metabolites from plasma and urine using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry

Abstract

BackgroundPrimaquine (PQ), an 8-aminoquinoline, is the only drug approved by the United States Food and Drug Administration for radical cure and prevention of relapse in Plasmodium vivax infections. Knowledge of the metabolism of PQ is critical for understanding the therapeutic efficacy and hemolytic toxicity of this drug. Recent in vitro studies with primary human hepatocytes have been useful for developing the ultra high-performance liquid chromatography coupled with high-resolution mass spectrometric (UHPLC-QToF-MS) methods for simultaneous determination of PQ and its metabolites generated through phase I and phase II pathways for drug metabolism.MethodsThese methods were further optimized and applied for phenotyping PQ metabolites from plasma and urine from healthy human volunteers treated with single 45 mg dose of PQ. Identity of the metabolites was predicted by MetaboLynx using LC–MS/MS fragmentation patterns. Selected metabolites were confirmed with appropriate standards.ResultsBesides PQ and carboxy PQ (cPQ), the major plasma metabolite, thirty-four additional metabolites were identified in human plasma and urine. Based on these metabolites, PQ is viewed as metabolized in humans via three pathways. Pathway 1 involves direct glucuronide/glucose/carbamate/acetate conjugation of PQ. Pathway 2 involves hydroxylation (likely cytochrome P450-mediated) at different positions on the quinoline ring, with mono-, di-, or even tri-hydroxylations possible, and subsequent glucuronide conjugation of the hydroxylated metabolites. Pathway 3 involves the monoamine oxidase catalyzed oxidative deamination of PQ resulting in formation of PQ-aldehyde, PQ alcohol and cPQ, which are further metabolized through additional phase I hydroxylations and/or phase II glucuronide conjugations.ConclusionThis approach and these findings augment our understanding and provide comprehensive view of pathways for PQ metabolism in humans. These will advance the clinical studies of PQ metabolism in different populations for different therapeutic regimens and an understanding of the role these play in PQ efficacy and safety outcomes, and their possible relation to metabolizing enzyme polymorphisms.Electronic supplementary materialThe online version of this article (10.1186/s12936-018-2433-z) contains supplementary material, which is available to authorized users.

Determination of Cytochrome P450 Isoenzyme 2D6 (CYP2D6) Genotypes and Pharmacogenomic Impact on Primaquine Metabolism in an Active-Duty US Military Population

“…Carboxyprimaquine, which is produced through the monoamine oxidase pathway [13] and not thought to have antimalarial activity, had a lower C max and AUC ∞ in the NM group, perhaps reflecting its increased metabolism through the CYP2D6 pathway. Levels of the 2-, 3- and 4-hydroxyprimaquine metabolites and 5,6-ortho-quinone were not appreciable in plasma specimens, unlike past murine studies at WRAIR [10], although levels were similar to those from a recent PK study in 7 healthy volunteers in the United States [12]. The inherent instability of the compounds, loss during processing and freezing, or location in other compartments (eg, erythrocytes and tissues) represent possible explanations.…”

“…By using recombinant CYP2D6, in vitro experiments have been able to detect phenolic metabolites of PQ [8], and in vivo studies in mice have confirmed CYP2D metabolism in the PQ pathway by measuring metabolites such as 2-, 3-, and 4-hydroxyprimaquine and 5,6-ortho-quinone in the blood [9–11]. Recently, a human PQ pharmacokinetic (PK) study detected 5,6-ortho-quinone in the urine, although the CYP2D6 genotype of these 7 healthy adult volunteers was not determined [12]. Although the 5,6-ortho-quinone is not active itself, it is a stable surrogate marker for the presence of 5-hydroxyprimaquine, the unstable PQ metabolite generated through 2D6, which is believed to be necessary for radical cure through the generation of oxidative stress from redox cycling [13].…”

Chloroquine Potentiates Primaquine Activity against Active and Latent Hepatic Plasmodia Ex Vivo : Potentials and Pitfalls

“…More positively, such variability offers hitherto unavailable opportunities for demonstrating hepatocyte factor(s) acting as defining determinants of anti-parasitic activity of PQ. That understanding, in turn, may directly inform strategies for elucidating the molecular basis of its potentiation by CQ.Detailed analysis in human volunteers revealed complex pathways that generate numerous PQ metabolites(14), with the production of some but not others by ex vivo culturedon November 1, 2020 by guest http://aac.asm.org/ Downloaded from 6 hepatocytes influenced by the concurrent presence of CQ (15). The decrease of the CYP2D6dependent PQ metabolites by CQ may favour the production of other active metabolites.…”

8-Aminoquinoline Therapy for Latent Malaria

“…Mean maximal daily plasma concentrations with 60-mg doses varied between 54 and 2,924 g/liter on the third day and 89 and 1,400 g/liter on the sixth day, with the lower and higher values representing the values for the same subjects on each day. This interindividual variability in plasma levels appeared with all of the experimental 8-aminoquinolines evaluated (17) and may reflect the then unknown importance of inherent variability in the catalytic activity of the cytochrome P-450 2D6 isozyme in the metabolism of 8-aminoquinolines (79)(80)(81). In 1981, Carson et al (17) pointed out the seemingly paradoxical inverse relationship between the plasma levels of the parent drug and therapeutic activity, perhaps explained by the extent of conversion of the parent drug to the active metabolite(s).…”

“…Among the many metabolic derivatives known, the 5-hydroxylated species (with perhaps variable modifications to the 8-amino alkyl chain) emerge as probably the most relevant (76,77). These highly redox-reactive derivatives appear to be generated by the 2D6 isozyme of cytochrome P-450 (CYP2D6), and their relatively minute amounts and instability impose technical challenges in studying them (78)(79)(80). The most widely studied metabolite, a carboxylated species of the parent drug (carboxyprimaquine) produced by reaction with monoamine oxidase, also happens to be the most abundant, stable, slowly eliminated, and easily detected, but it is without apparent therapeutic or toxic activity (81,82).…”