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Background: Primaquine (PQ) has been used for the radical cure of relapsing Plasmodium vivax malaria for more than 60 years. PQ is also recommended for prophylaxis and prevention of transmission of Plasmodium falciparum. However, clinical utility of PQ has been limited due to toxicity in individuals with genetic deficiencies in glucose 6-phosphate dehydrogenase (G6PD). PQ is currently approved for clinical use as a racemic mixture. Recent studies in animals as well as humans have established differential pharmacological and toxicological properties of the two enantiomers of PQ. This has been attributed to differential metabolism and pharmacokinetics of individual PQ enantiomers. The aim of the current study is to evaluate the comparative pharmacokinetics (PK), tissue distribution and metabolic profiles of the individual enantiomers in mice., Methods: Two groups of 21 male Albino ND4 Swiss mice were dosed orally with 45 mg/kg of S-(+)-PQ and R-(-)PQ respectively. Each of the enantiomers was comprised of a 50:50 mixture of 12 C- and 13 C- stable isotope labelled species (at 6 carbons on the benzene ring of the quinoline core). Three mice were euthanized from each group at different time points (at 0, 0.5, 1, 2, 4, 8, 24 h) and blood was collected by terminal cardiac bleed. Liver, spleen, lungs, kidneys and brain were removed, extracted and analysed using UPLC/MS. The metabolites were profiled by tandem mass (MS/MS) fragmentation profile and fragments with 12 C- 13 C twin peaks. Non-compartmental analysis was performed using the Phoenix WinNonLin PK software module., Results: The plasma AUC 0-last (µg h/mL) (1.6 vs. 0.6), T 1/2 (h) (1.9 vs. 0.45), and T max (h) (1 vs. 0.5) were greater for SPQ as compared to RPQ. Generally, the concentration of SPQ was higher in all tissues. At T max , (0.5-1 h in all tissues), the level of SPQ was 3 times that of RPQ in the liver. Measured C max of SPQ and RPQ in the liver were about 100 and 40 times the C max values in plasma, respectively. Similar observations were recorded in other tissues where the concentration of SPQ was higher compared to RPQ (2× in the spleen, 6× in the kidneys, and 49× in the lungs) than in the plasma. CPQ, the major metabolite, was preferentially generated from RPQ, with higher levels in all tissues (> 10× in the liver, and 3.5× in the plasma) than from SPQ. The PQ-o-quinone was preferentially formed from the SPQ (> 4× compared to RPQ), with higher concentrations in the liver., Conclusion: These studies show that in mice, PQ enantiomers are differentially biodistributed and metabolized, which may contribute to differential pharmacologic and toxicity profiles of PQ enantiomers. The findings on higher levels of PQ-o-quinone in liver and RBCs compared to plasma and preferential generation of this metabolite from SPQ are consistent with the higher anti-malarial efficacy of SPQ observed in the mouse causal prophylaxis test, and higher haemolytic toxicity in the humanized mouse model of G6PD deficiency. Potential relevance of these findings to clinical use of racemic PQ and other 8-aminoquinolines vis-à-vis need for further clinical evaluation of individual enantiomers are discussed., (© 2022. The Author(s).)

Background: Primaquine (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., Methods: These 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., Results: Besides 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., Conclusion: This 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.

Background: There has been some evidence to suggest that the addition of chloroquine (CQ) or quinine (QN) to 8-aminoquinoline (8-AQ) treatment regimens may increase the therapeutic efficacy of the 8-AQ and simultaneously mitigate against its haemolytic toxicity. However, both CQ and QN are considered effective, although perhaps moderate inhibitors of CYP2D6, an enzyme now regarded as necessary for primaquine (PQ) pharmacologic activity. An understanding of the influence of CQ and QN on the metabolism of PQ may shed light on the potential mechanisms of the beneficial interaction., Methods: Differential metabolism of PQ enantiomers by recombinant human CYP2D6, monoamine oxidase A (MAO), and cryopreserved human hepatocytes in the presence/absence of CQ and QN., Results: Both CQ and QN significantly inhibited the activity of CYP2D6. PQ depletion by MAO and human hepatocytes was not affected significantly by the presence of CQ and QN. CYP2D6-mediated hydroxylation was largely suppressed by both CQ and QN. The formation of the primary deaminated metabolites, including carboxyprimaquine (CPQ) and cyclized side chain derivative from the aldehyde (m/z 241), was not sensitive to the presence of CQ and QN. However, the appearance of the glucuronides of CPQ and PQ alcohol were significantly suppressed. CQ and QN also inhibited the appearance of the m/z 257 metabolite with a similar pattern, suggesting that it may be derived from the CPQ conjugate. The apparent quinone-imine of CPQ (m/z 289) was only partially suppressed by both QN and CQ, but with a differential pattern of inhibition for the two drugs. The m/z 274 (quinone-imine of a ring-hydroxylated PQ metabolite) and m/z 422 (an apparent glucose conjugate of PQ) metabolites in hepatocytes were strongly suppressed by both QN and CQ, perhaps a reflection of the 2D6 inhibition by these drugs. The formation of the carbamoyl glucuronide of PQ (m/z 480) was not affected by CQ/QN., Conclusion: The metabolite-specific interactions in the current studies seem at variance with earlier reports of the dependence of PQ on CYP2D6 metabolism, and enhanced PQ anti-malarial activity/reduced toxicity in the presence of CQ/QN. These results suggest a complex picture in which CQ/QN may shift metabolite pathway balances towards a profile that retains efficacy, while reducing the formation or availability of toxic metabolites to erythrocytes. Alternatively, these drugs may alter transport or distribution of PQ metabolites in a fashion that reduces toxicity while maintaining efficacy against the parasite.

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.

Background: Primaquine, currently the only approved drug for the treatment and radical cure of Plasmodium vivax malaria, is still used as a racemic mixture. Clinical use of primaquine has been limited due to haemolytic toxicity in individuals with genetic deficiency in glucose-6-phosphate dehydrogenase. Earlier studies have linked its therapeutic effects to CYP2D6-generated metabolites. The aim of the current study was to investigate the differential generation of the CYP2D6 metabolites by racemic primaquine and its individual enantiomers., Methods: Stable isotope 13C-labelled primaquine and its two enantiomers were incubated with recombinant cytochrome-P450 supersomes containing CYP2D6 under optimized conditions. Metabolite identification and time-point quantitative analysis were performed using LC-MS/MS. UHPLC retention time, twin peaks with a mass difference of 6, MS-MS fragmentation pattern, and relative peak area with respect to parent compound were used for phenotyping and quantitative analysis of metabolites., Results: The rate of metabolism of (+)-(S)-primaquine was significantly higher (50% depletion of 20 μM in 120 min) compared to (-)-(R)-primaquine (30% depletion) when incubated with CYP2D6. The estimated Vmax (μmol/min/mg) were 0.75, 0.98 and 0.42, with Km (μM) of 24.2, 33.1 and 21.6 for (±)-primaquine, (+)-primaquine and (-)-primaquine, respectively. Three stable mono-hydroxylated metabolites, namely, 2-, 3- and 4-hydroxyprimaquine (2-OH-PQ, 3-OH-PQ, and 4-OH-PQ), were identified and quantified. 2-OH-PQ was preferentially formed from (+)-primaquine in a ratio of 4:1 compared to (-)-primaquine. The racemic (±)-primaquine showed a pattern similar to the (-)-primaquine; 2-OH-PQ accounted for about 15-17% of total CYP2D6-mediated conversion of (+)-primaquine. In contrast, 4-OH-PQ was preferentially formed with (-)-primaquine (5:1), accounting for 22% of the total (-)-primaquine conversion. 3-OH-PQ was generated from both enantiomers and racemate. 5-hydroxyprimaquine was unstable. Its orthoquinone degradation product (twice as abundant in (+)-primaquine compared to (-)-primaquine) was identified and accounted for 18-20% of the CYP2D6-mediated conversion of (+)-primaquine. Other minor metabolites included dihydroxyprimaquine species, two quinone-imine products of dihydroxylated primaquine, and a primaquine terminal alcohol with variable generation from the individual enantiomers., Conclusion: The metabolism of primaquine by human CYP2D6 and the generation of its metabolites display enantio-selectivity regarding formation of hydroxylated product profiles. This may partly explain differential pharmacologic and toxicologic properties of primaquine enantiomers.

Background: Tafenoquine (TQ) is an 8-aminoquinoline (8AQ) that has been tested in several Phase II and Phase III clinical studies and is currently in late stage development as an anti-malarial prophylactic agent. NPC-1161B is a promising 8AQ in late preclinical development. It has recently been reported that the 8AQ drug primaquine requires metabolic activation by CYP 2D6 for efficacy in humans and in mice, highlighting the importance of pharmacogenomics in the target population when administering primaquine. A logical follow-up study was to determine whether CYP 2D activation is required for other compounds in the 8AQ structural class., Methods: In the present study, the anti-malarial activities of NPC-1161B and TQ were assessed against luciferase expressing Plasmodium berghei in CYP 2D knock-out mice in comparison with normal C57BL/6 mice (WT) and with humanized/CYP 2D6 knock-in mice by monitoring luminescence with an in vivo imaging system. These experiments were designed to determine the direct effects of CYP 2D metabolic activation on the anti-malarial efficacy of NPC-1161B and TQ., Results: NPC-1161B and TQ exhibited no anti-malarial activity in CYP 2D knock-out mice when dosed at their ED100 values (1 mg/kg and 3 mg/kg, respectively) established in WT mice. TQ anti-malarial activity was partially restored in humanized/CYP 2D6 knock-in mice when tested at two times its ED100., Conclusions: The results reported here strongly suggest that metabolism of NPC-1161B and TQ by the CYP 2D enzyme class is essential for their anti-malarial activity. Furthermore, these results may provide a possible explanation for therapeutic failures for patients who do not respond to 8AQ treatment for relapsing malaria. Because CYP 2D6 is highly polymorphic, variable expression of this enzyme in humans represents a significant pharmacogenomic liability for 8AQs which require CYP 2D metabolic activation for efficacy, particularly for large-scale prophylaxis and eradication campaigns.