Enhanced Ca2+-Dependent SK-Channel Gating and Membrane Trafficking in Human Atrial Fibrillation.

BACKGROUND: Small-conductance Ca2+-activated K+ (SK)-channel inhibitors have antiarrhythmic effects in animal models of atrial fibrillation (AF), presenting a potential novel antiarrhythmic option. However, the regulation of SK-channels in human atrial cardiomyocytes and its modification in patients with AF are poorly understood and were the object of this study. METHODS: Apamin-sensitive SK-channel current (ISK) and action potentials were recorded in human right-atrial cardiomyocytes from sinus rhythm control (Ctl) patients or patients with (long-standing persistent) chronic AF (cAF). RESULTS: ISK was significantly higher, and apamin caused larger action potential prolongation in cAF- versus Ctl-cardiomyocytes. Sensitivity analyses in an in silico human atrial cardiomyocyte model identified IK1 and ISK as major regulators of repolarization. Increased ISK in cAF was not associated with increases in mRNA/protein levels of SK-channel subunits in either right- or left-atrial tissue homogenates or right-atrial cardiomyocytes, but the abundance of SK2 at the sarcolemma was larger in cAF versus Ctl in both tissue-slices and cardiomyocytes. Latrunculin-A and primaquine (anterograde and retrograde protein-trafficking inhibitors) eliminated the differences in SK2 membrane levels and ISK between Ctl- and cAF-cardiomyocytes. In addition, the phosphatase-inhibitor okadaic acid reduced ISK amplitude and abolished the difference between Ctl- and cAF-cardiomyocytes, indicating that reduced calmodulin-Thr80 phosphorylation due to increased protein phosphatase-2A levels in the SK-channel complex likely contribute to the greater ISK in cAF-cardiomyocytes. Finally, rapid electrical activation (5 Hz, 10 minutes) of Ctl-cardiomyocytes promoted SK2 membrane-localization, increased ISK and reduced action potential duration, effects greatly attenuated by apamin. Latrunculin-A or primaquine prevented the 5-Hz-induced ISK-upregulation. CONCLUSIONS: ISK is upregulated in patients with cAF due to enhanced channel function, mediated by phosphatase-2A-dependent calmodulin-Thr80 dephosphorylation and tachycardia-dependent enhanced trafficking and targeting of SK-channel subunits to the sarcolemma. The observed AF-associated increases in ISK, which promote reentry-stabilizing action potential duration shortening, suggest an important role for SK-channels in AF auto-promotion and provide a rationale for pursuing the antiarrhythmic effects of SK-channel inhibition in humans.

Comparative single dose pharmacokinetics and metabolism of racemic primaquine and its enantiomers in human volunteers.

Primaquine (PQ) is a racemic drug used in treatment of malaria for six decades. Recent studies suggest that the two enantiomers of PQ are differentially metabolized in animals, and this results in different pharmacological and toxicological profiles. The current study characterizes the pharmacokinetic (PK) properties, metabolism and tolerability of the individual enantiomers of PQ in healthy human volunteers with normal glucose-6-phosphate dehydrogenase (G6PD) activity. Two cohorts (at two dose levels), each with 18 subjects, participated in three study arms in a crossover fashion: a single dose of the (-)-R enantiomer (RPQ), a single dose of the (+)-S enantiomer (SPQ), and a single dose of racemic PQ (RSPQ). PQ and its key metabolites carboxyprimaquine (cPQ) and PQ-N-carbamoyl glucuronide (PQ-N-CG) were analyzed. Clear differences were observed in PK and metabolism of the two enantiomers. Relative PQ exposure was higher with SPQ as compared to RPQ. PQ maximum plasma concentration (Cmax) and area under the plasma concentration-time curve were higher for SPQ, while the apparent volume of distribution and total body clearance were higher for RPQ. Metabolism of the two enantiomers showed dramatic differences: plasma PQ-N-CG was derived solely from SPQ, while RPQ was much more efficiently converted to cPQ than was SPQ. Cmax of cPQ and PQ-N-CG were 10 and 2 times higher, respectively, than the parent drugs. The study demonstrates that the PK properties of PQ enantiomers show clear differences, and metabolism is highly enantioselective. Such differences in metabolism suggest potentially distinct toxicity profiles in multi-dose regimens, especially in G6PD-deficient subjects.

Combination of transdermal patches and solid microneedles for improved transdermal delivery of primaquine.

Malaria caused by various types of Plasmodium has become a global health problem. One of the drugs used as the first line of malaria therapy is primaquine (PMQ). PMQ is generally administered through the oral route. However, the use of PMQ orally could potentially cause some side effects and undergo the first-pass metabolism in the liver, reducing its effectiveness. Therefore, it is necessary to develop another drug administration route to avoid this effect. In this study, for the first time, PMQ was formulated into a transdermal patch for transdermal delivery, combined with solid microneedles, Dermaroller®. Following several optimizations, HPMC and glycerin were used as the main polymer and plasticizer, respectively. Specifically, the concentration of PEG 400 as a permeation enhancer was also optimized. The transdermal patches were evaluated for weight uniformity, thickness, surface pH, folding endurance, moisture content, moisture absorption ability, bioadhesive evaluation, and drug content recovery. PMQ release and permeation were also investigated through in vitro and ex vivo tests on rats' skin tissue. Importantly, the safety of the transdermal patch was also evaluated through in vitro hemolytic and in vivo irritation tests which were confirmed by histopathological examinations. The results showed that all formulations showed desired physical and bioadhesive properties with a folding endurance of >300 folds. The results exhibited that 31.31 ± 5.25% and 22.55 ± 4.35% of primaquine were released from transdermal patches following the in vitro and the ex vivo permeation studies. Combined with Dermaroller®, the ex vivo permeation study showed an improved permeation profile with 45.89 ± 5.00% of primaquine permeated after 24 h with a zero-order kinetic during the first 8 h. Hemolysis percentage was found to be <5%, indicating the non-toxic of this approach. Finally, the histopathology study showed that there was no severe tissue damage following the administration of our approach. Further in vivo evaluations should be performed.

Several Plasmodium vivax relapses after correct primaquine treatment in a patient with impaired cytochrome P450 2D6 function.

BACKGROUND: Plasmodium vivax malaria is characterized by the presence of dormant liver-stage parasites, called hypnozoites, which can cause malaria relapses after an initial attack. Primaquine, which targets liver hypnozoites, must be used in combination with a schizonticidal agent to get the radical cure. However, relapses can sometimes occur in spite of correct treatment, due to different factors such as a diminished metabolization of primaquine. CASE PRESENTATION: In January 2019, a 21 years old woman with residence in Madrid, returning from a trip to Venezuela with clinical symptoms compatible with malaria infection, was diagnosed with vivax malaria. Chloroquine for 3 days plus primaquine for 14 days was the elected treatment. Two months later and after a second trip to Venezuela, the patient presented a second P. vivax infection, which was treated as the previous one. A third P. vivax malaria episode was diagnosed 2 months later, after returning from a trip to Morocco, receiving chloroquine for 3 days but increasing to 28 days the primaquine regimen, and with no more relapses after 6 months of follow up. The genotyping of P. vivax in the three malaria episodes revealed that the same strain was present in the different relapses. Upon confirmation of correct adherence to the treatment, non-description of resistance in the infection area and the highly unlikely re-infection on subsequent trips or stays in Spain, a possible metabolic failure was considered. CYP2D6 encodes the human cytochrome P450 isoenzyme 2D6 (CYP2D6), responsible for primaquine activation. The patient was found to have a CYP2D6*4/*1 genotype, which turns out in an intermediate metabolizer phenotype, which has been related to P. vivax relapses. CONCLUSIONS: The impairment in CYP2D6 enzyme could be the most likely cause of P. vivax relapses in this patient. This highlights the importance of considering the analysis of CYP2D6 gene polymorphisms in cases of P. vivax relapses after a correct treatment and, especially, it should be considered in any study of dosage and duration of primaquine treatment.

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

BACKGROUND: Plasmodium vivax malaria requires a 2-week course of primaquine (PQ) for radical cure. Evidence suggests that the hepatic isoenzyme cytochrome P450 2D6 (CYP2D6) is the key enzyme required to convert PQ into its active metabolite. METHODS: CYP2D6 genotypes and phenotypes of 550 service personnel were determined, and the pharmacokinetics (PK) of a 30-mg oral dose of PQ was measured in 45 volunteers. Blood and urine samples were collected, with PQ and metabolites were measured using ultraperformance liquid chromatography with mass spectrometry. RESULTS: Seventy-six CYP2D6 genotypes were characterized for 530 service personnel. Of the 515 personnel for whom a single phenotype was predicted, 58% had a normal metabolizer (NM) phenotype, 35% had an intermediate metabolizer (IM) phenotype, 5% had a poor metabolizer (PM) phenotype, and 2% had an ultrametabolizer phenotype. The median PQ area under the concentration time curve from 0 to ∞ was lower for the NM phenotype as compared to the IM or PM phenotypes. The novel 5,6-ortho-quinone was detected in urine but not plasma from all personnel with the NM phenotype. CONCLUSION: The plasma PK profile suggests PQ metabolism is decreased in personnel with the IM or PM phenotypes as compared to those with the NM phenotype. The finding of 5,6-ortho-quinone, the stable surrogate for the unstable 5-hydroxyprimaquine metabolite, almost exclusively in personnel with the NM phenotype, compared with sporadic or no production in those with the IM or PM phenotypes, provides further evidence for the role of CYP2D6 in radical cure. CLINICAL TRIALS REGISTRATION: NCT02960568.

Antimalarial activity of primaquine operates via a two-step biochemical relay.

Primaquine (PQ) is an essential antimalarial drug but despite being developed over 70 years ago, its mode of action is unclear. Here, we demonstrate that hydroxylated-PQ metabolites (OH-PQm) are responsible for efficacy against liver and sexual transmission stages of Plasmodium falciparum. The antimalarial activity of PQ against liver stages depends on host CYP2D6 status, whilst OH-PQm display direct, CYP2D6-independent, activity. PQ requires hepatic metabolism to exert activity against gametocyte stages. OH-PQm exert modest antimalarial efficacy against parasite gametocytes; however, potency is enhanced ca.1000 fold in the presence of cytochrome P450 NADPH:oxidoreductase (CPR) from the liver and bone marrow. Enhancement of OH-PQm efficacy is due to the direct reduction of quinoneimine metabolites by CPR with the concomitant and excessive generation of H2O2, leading to parasite killing. This detailed understanding of the mechanism paves the way to rationally re-designed 8-aminoquinolines with improved pharmacological profiles.

Formation primaquine-5,6-orthoquinone, the putative active and toxic metabolite of primaquine via direct oxidation in human erythrocytes.

BACKGROUND: The activity and haemolytic toxicity associated with primaquine has been linked to its reactive metabolites. The reactive metabolites are thought to be primarily formed through the action of cytochrome P450-mediated pathways. Human erythrocytes generally are not considered a significant contributor to drug biotransformation. As erythrocytes are the target of primaquine toxicity, the ability of erythrocytes to mediate the formation of reactive oxidative primaquine metabolites in the absence of hepatic enzymes, was evaluated. METHODS: Primaquine and its enantiomers were incubated separately with human red blood cells and haemoglobin. Post-incubation analysis was performed with UPLC-MS/MS to identify products of biotransformation. RESULTS: The major metabolite detected was identified as primaquine-5,6-orthoquinone, reflecting the pathway yielding putative active and haematotoxic metabolites of primaquine, which was formed by oxidative demethylation of 5-hydroxyprimaquine. Incubation of primaquine with haemoglobin in a cell-free system yielded similar results. It appears that the observed biotransformation is due to non-enzymatic processes, perhaps due to reactive oxygen species (ROS) present in erythrocytes or in the haemoglobin incubates. CONCLUSION: This study presents new evidence that primaquine-5,6-orthoquinone, the metabolite of primaquine reflecting the oxidative biotransformation pathway, is generated in erythrocytes, probably by non-enzymatic means, and may not require transport from the liver or other tissues.

Effects of MAO-A and CYP450 on primaquine metabolism in healthy volunteers.

Eliminating the Plasmodium vivax malaria parasite infection remains challenging. One of the main problems is its capacity to form hypnozoites that potentially lead to recurrent infections. At present, primaquine is the only drug used for the management of hypnozoites. However, the effects of primaquine may differ from one individual to another. The aim of this work is to determine new measures to reduce P. vivax recurrence, through primaquine metabolism and host genetics. A genetic study of MAO-A, CYP2D6, CYP1A2 and CYP2C19 and their roles in primaquine metabolism was undertaken of healthy volunteers (n = 53). The elimination rate constant (Ke) and the metabolite-to-parent drug concentration ratio (Cm/Cp) were obtained to assess primaquine metabolism. Allelic and genotypic analysis showed that polymorphisms MAO-A (rs6323, 891G>T), CYP2D6 (rs1065852, 100C>T) and CYP2C19 (rs4244285, 19154G>A) significantly influenced primaquine metabolism. CYP1A2 (rs762551, -163C>A) did not influence primaquine metabolism. In haplotypic analysis, significant differences in Ke (p = 0.00) and Cm/Cp (p = 0.05) were observed between individuals with polymorphisms, GG-MAO-A (891G>T), CT-CYP2D6 (100C>T) and GG-CYP2C19 (19154G>A), and individuals with polymorphisms, TT-MAO-A (891G>T), TT-CYP2D6 (100C>T) and AA-CYP2C19 (19154G>A), as well as polymorphisms, GG-MAO-A (891G>T), TT-CYP2D6 (100C>T) and GA-CYP2C19 (19154G>A). Thus, individuals with CYP2D6 polymorphisms had slower primaquine metabolism activity. The potential significance of genetic roles in primaquine metabolism and exploration of these might help to further optimise the management of P. vivax infection.

Functional characterization of 50 CYP2D6 allelic variants by assessing primaquine 5-hydroxylation.

Cytochrome P450 2D6 (CYP2D6) is responsible for the metabolic activation of primaquine, an antimalarial drug. CYP2D6 is genetically polymorphic, and these polymorphisms are associated with interindividual variations observed in the therapeutic efficacy of primaquine. To further understand this association, we performed in vitro enzymatic analyses of the wild-type CYP2D6.1 and 49 CYP2D6 allelic variants, which were expressed in 293FT cells, using primaquine as a substrate. The concentrations of CYP2D6 variant holoenzymes were measured by using carbon monoxide (CO)-reduced difference spectroscopy, and the wild type and 27 variants showed a peak at 450 nm. The kinetic parameters Km, Vmax, and intrinsic clearance (Vmax/Km) of primaquine 5-hydroxylation were characterized. The kinetic parameters of the wild type and 16 variants were measured, but the values for the remaining 33 variants could not be determined because of low metabolite concentrations. Among the variants, six (i.e., CYP2D6.17, .18, .35, .39, .53, and .70) showed significantly reduced intrinsic clearance compared with that of CYP2D6.1. Three-dimensional structural modeling analysis was performed to elucidate the mechanism of changes in the kinetics of CYP2D6 variants. Our findings provide insights into the allele-specific activity of CYP2D6 for primaquine, which could be clinically useful for malaria treatment and eradication efforts.

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.

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.

Primaquine Pharmacokinetics in Lactating Women and Breastfed Infant Exposures.

Background: Primaquine is the only drug providing radical cure of Plasmodium vivax malaria. It is not recommended for breastfeeding women as it causes hemolysis in glucose-6-phosphate dehydrogenase (G6PD)-deficient individuals, and breast milk excretion and thus infant exposure are not known. Methods: Healthy G6PD-normal breastfeeding women with previous P. vivax infection and their healthy G6PD-normal infants between 28 days and 2 years old were enrolled. Mothers took primaquine 0.5 mg/kg/day for 14 days. Primaquine and carboxyprimaquine concentrations were measured in maternal venous plasma, capillary plasma, and breast milk samples and infant capillary plasma samples taken on days 0, 3, 7, and 13. Results: In 20 mother-infant pairs, primaquine concentrations were below measurement thresholds in all but 1 infant capillary plasma sample (that contained primaquine 2.6 ng/mL), and carboxyprimaquine was likewise unmeasurable in the majority of infant samples (maximum value 25.8 ng/mL). The estimated primaquine dose received by infants, based on measured breast milk levels, was 2.98 µg/kg/day (ie, ~0.6% of a hypothetical infant daily dose of 0.5 mg/kg). There was no evidence of drug-related hemolysis in the infants. Maternal levels were comparable to levels in nonlactating patients, and adverse events in mothers were mild. Conclusions: The concentrations of primaquine in breast milk are very low and therefore very unlikely to cause adverse effects in the breastfeeding infant. Primaquine should not be withheld from mothers breastfeeding infants or young children. More information is needed in neonates. Clinical Trials Registration: NCT01780753.

Synthesis, characterization, and cellular localization of a fluorescent probe of the antimalarial 8-aminoquinoline primaquine.

Primaquine (PQ) is the only commercially available drug that clears dormant liver stages of malaria and blocks transmission to mosquito vectors. Although an old drug, much remains to be known about the mechanism(s) of action. Herein we develop a fluorescent tagged PQ to discover cellular localization in the human malaria parasite, Plasmodium falciparum. Successful synthesis and characterization of a primaquine-coumarin fluorescent probe (PQCP) demonstrated potency equivalent to the parent drug and the probe was not cytotoxic to HepG2 carcinoma cells. Cellular localization was found primarily in the cytosol of the asexual erythrocytic and gametocyte stages of parasite development.

Analysis of Plasmodium vivax Chloroquine Resistance Transporter Mutant Isoforms.

Chloroquine (CQ) resistance (CQR) in Plasmodium falciparum malaria is widespread and has limited the use of CQ in many regions of the globe. Malaria caused by the related human parasite P. vivax is as widespread as is P. falciparum malaria and has been treated with CQ as extensively as has P. falciparum, suggesting that P. vivax parasites have been selected with CQ as profoundly as have P. falciparum parasites. Indeed, a growing number of clinical reports have presented data suggesting increased P. vivax CQR. Cytostatic (growth inhibitory) CQR for P. falciparum is caused by Plasmodium falciparum chloroquine resistance transporter (PfCRT) mutations, and it has been proposed that mutations in the PvCRT orthologue may simliarly cause P. vivax CQR via increasing CQ transport from the P. vivax digestive vacuole. Here we report the first quantitative analysis of drug transport mediated by all known mutant isoforms of Plasmodium vivax chloroquine resistance transporter (PvCRT) in order to test the protein's potential link to growing P. vivax CQR phenomena. Small, but statistically significant, differences in the transport of CQ and other quinoline antimalarial drugs were found for multiple PvCRT isoforms, relative to wild type PvCRT, suggesting that mutations in PvCRT can contribute to P. vivax CQR and other examples of quinoline antimalarial drug resistance.

Pathway-specific inhibition of primaquine metabolism by chloroquine/quinine.

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.

Variation in Human Cytochrome P-450 Drug-Metabolism Genes: A Gateway to the Understanding of Plasmodium vivax Relapses.

Although Plasmodium vivax relapses are classically associated with hypnozoite activation, it has been proposed that a proportion of these cases are due to primaquine (PQ) treatment failure caused by polymorphisms in cytochrome P-450 2D6 (CYP2D6). Here, we present evidence that CYP2D6 polymorphisms are implicated in PQ failure, which was reinforced by findings in genetically similar parasites, and may explain a number of vivax relapses. Using a computational approach, these polymorphisms were predicted to affect the activity of CYP2D6 through changes in the structural stability that could lead to disruption of the PQ-enzyme interactions. Furthermore, because PQ is co-administered with chloroquine (CQ), we investigated whether CQ-impaired metabolism by cytochrome P-450 2C8 (CYP2C8) could also contribute to vivax recurrences. Our results show that CYP2C8-mutated patients frequently relapsed early (<42 days) and had a higher proportion of genetically similar parasites, suggesting the possibility of recrudescence due to CQ therapeutic failure. These results highlight the importance of pharmacogenetic studies as a tool to monitor the efficacy of antimalarial therapy.

Pre-clinical evaluation of CYP 2D6 dependent drug-drug interactions between primaquine and SSRI/SNRI antidepressants.

BACKGROUND: The liver-stage anti-malarial activity of primaquine and other 8-aminoquinoline molecules has been linked to bio-activation through CYP 2D6 metabolism. Factors such as CYP 2D6 poor metabolizer status and/or co-administration of drugs that inhibit/interact with CYP 2D6 could alter the pharmacological properties of primaquine. METHODS: In the present study, the inhibitory potential of the selective serotonin reuptake inhibitor (SSRI) and serotonin norepinephrine reuptake inhibitor (SNRI) classes of antidepressants for CYP 2D6-mediated primaquine metabolism was assessed using in vitro drug metabolism and in vivo pharmacological assays. RESULTS: The SSRI/SNRI classes of drug displayed a range of inhibitory activities on CYP 2D6-mediated metabolism of primaquine in vitro (IC50 1-94 µM). Fluoxetine and paroxetine were the most potent inhibitors (IC50 ~1 µM) of CYP 2D6-mediated primaquine metabolism, while desvenlafaxine was the least potent (IC50 ~94 µM). The most potent CYP 2D6 inhibitor, fluoxetine, was chosen to investigate the potential pharmacological consequences of co-administration with primaquine in vivo. The pharmacokinetics of a CYP 2D6-dependent primaquine metabolite were altered upon co-administration with fluoxetine. Additionally, in a mouse malaria model, co-administration of fluoxetine with primaquine reduced primaquine anti-malarial efficacy. CONCLUSIONS: These results are the first from controlled pre-clinical experiments that indicate that primaquine pharmacological properties can be modulated upon co-incubation/administration with drugs that are known to interact with CYP 2D6. These results highlight the potential for CYP 2D6-mediated drug-drug interactions with primaquine and indicate that the SSRI/SNRI antidepressants could be used as probe molecules to address the primaquine-CYP 2D6 DDI link in clinical studies. Additionally, CYP 2D6-mediated drug-drug interactions can be considered when examining the possible causes of human primaquine therapy failures.

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

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.

Primaquine pharmacology in the context of CYP 2D6 pharmacogenomics: Current state of the art.

Primaquine is the only antimalarial drug available to clinicians for the treatment of relapsing forms of malaria. Primaquine development and usage dates back to the 1940s and has been administered to millions of individuals to treat and eliminate malaria infections. Primaquine therapy is not without disadvantages, however, as it can cause life threatening hemolysis in humans with glucose-6-phosphate dehydrogenase (G6PD) deficiency. In addition, the efficacy of primaquine against relapsing malaria was recently linked to CYP 2D6 mediated activation to an active metabolite, the structure of which has escaped definitive identification for over 75years. CYP 2D6 is highly polymorphic among various human populations adding further complexity to a comprehensive understanding of primaquine pharmacology. This review aims to discuss primaquine pharmacology in the context of state of the art understanding of CYP 2D6 mediated 8-aminoquinoline metabolic activation, and shed light on the current knowledge gaps of 8-aminoquinoline mechanistic understanding against relapsing malaria.

Enantioselective metabolism of primaquine by human CYP2D6.

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.

An LC-MS based study of the metabolic profile of primaquine, an 8-aminoquinoline antiparasitic drug, with an in vitro primary human hepatocyte culture model.

The 8-aminoquinoline drug primaquine (PQ) is currently the only drug in use against the persistent malaria caused by the hypnozoite-forming strains P. vivax and P. ovale. However, despite decades of research, its complete metabolic profile is still poorly understood. In the present study, the metabolism of PQ was evaluated by incubating the drug with pooled human hepatocytes cultured in vitro as well as with recombinant cytochrome P450 (CYP) iso- enzymes, monoamine oxidases (MAO), and flavin-containing monooxygenases (FMO). Targeted LC-MS/MS analysis of hepatocyte incubations using chemical inhibitors indicated that PQ was predominantly metabolized by CYPs 3A4, 1A2 and 2D6, MAO-A, -B and FMO-3. Confirmation of these results was sought by incubation of PQ with the corresponding recombinant enzymes. Small amounts of carboxyprimaquine (CPQ), the major observed PQ metabolite in vivo, were detected in recombinant MAO-A incubations along with another peak at m/z 261, and no significant formation of CPQ with any other recombinant enzymes was observed. Incubations with all recombinant enzymes identified as potentially active towards PQ from the hepatocyte-based assay resulted in significant parent loss over the course of 1 h. These results suggest that several enzymes, including CYPs in combination with FMOs and MAOs, play a role in the overall metabolism of PQ and indicate a major role for MAO-A. Future studies to elucidate the potential role in cytotoxicity and/or efficacy of the PQ metabolite observed at m/z 261, as observed in MAO-A isoenzyme studies, are needed.