Development of an ectopic huLiver model for Plasmodium liver stage infection.

Early Plasmodium falciparum and P. vivax infection requires parasite replication within host hepatocytes, referred to as liver stage (LS). However, limited understanding of infection dynamics in human LS exists due to species-specificity challenges. Reported here is a reproducible, easy-to-manipulate, and moderate-cost in vivo model to study human Plasmodium LS in mice; the ectopic huLiver model. Ectopic huLiver tumors were generated through subcutaneous injection of the HC-04 cell line and shown to be infectible by both freshly dissected sporozoites and through the bite of infected mosquitoes. Evidence for complete LS development was supported by the transition to blood-stage infection in mice engrafted with human erythrocytes. Additionally, this model was successfully evaluated for its utility in testing antimalarial therapeutics, as supported by primaquine acting as a causal prophylactic against P. falciparum. Presented here is a new platform for the study of human Plasmodium infection with the potential to aid in drug discovery.

Mechanisms of 8-aminoquinoline induced haemolytic toxicity in a G6PDd humanized mouse model.

Primaquine (PQ) and Tafenoquine (TQ) are clinically important 8-aminoquinolines (8-AQ) used for radical cure treatment of P. vivax infection, known to target hepatic hypnozoites. 8-AQs can trigger haemolytic anaemia in individuals with glucose-6-phosphate dehydrogenase deficiency (G6PDd), yet the mechanisms of haemolytic toxicity remain unknown. To address this issue, we used a humanized mouse model known to predict haemolytic toxicity responses in G6PDd human red blood cells (huRBCs). To evaluate the markers of eryptosis, huRBCs were isolated from mice 24-48 h post-treatment and analysed for effects on phosphatidylserine (PS), intracellular reactive oxygen species (ROS) and autofluorescence. Urinalysis was performed to evaluate the occurrence of intravascular and extravascular haemolysis. Spleen and liver tissue harvested at 24 h and 5-7 days post-treatment were stained for the presence of CD169+ macrophages, F4/80+ macrophages, Ter119+ mouse RBCs, glycophorin A+ huRBCs and murine reticulocytes (muRetics). G6PDd-huRBCs from PQ/TQ treated mice showed increased markers for eryptosis as early as 24 h post-treatment. This coincided with an early rise in levels of muRetics. Urinalysis revealed concurrent intravascular and extravascular haemolysis in response to PQ/TQ. Splenic CD169+ macrophages, present in all groups at day 1 post-dosing were eliminated by days 5-7 in PQ/TQ treated mice only, while liver F4/80 macrophages and iron deposits increased. Collectively, our data suggest 8-AQ treated G6PDd-huRBCs have early physiological responses to treatment, including increased markers for eryptosis indicative of oxidative stress, resulting in extramedullary haematopoiesis and loss of splenic CD169+ macrophages, prompting the liver to act as the primary site of clearance.

Measurements of 5,6-Orthoquinone, a Surrogate for the Presumed Active Primaquine Metabolite 5-Hydroxyprimaquine, in the Urine of Cambodian Adults.

The active metabolites of primaquine, in particular 5-hydroxyprimaquine, likely responsible for the clearance of dormant hypnozoites, are produced through the hepatic CYP450 2D6 (CYP2D6) enzymatic pathway. With the inherent instability of 5-hydroxyprimaquine, a stable surrogate, 5,6-orthoquinone, can now be detected and measured in the urine as part of primaquine pharmacokinetic studies. This study performed CYP450 2D6 genotyping and primaquine pharmacokinetic testing, to include urine 5,6-orthoquinone, in 27 healthy adult Cambodians, as a preliminary step to prepare for future clinical studies assessing primaquine efficacy for Plasmodium vivax infections. The CYP2D6 *10 reduced activity allele was found in 57% of volunteers, and the CYP2D6 genotypes were dominated by *1/*10 (33%) and *10/*10 (30%). Predicted phenotypes were evenly split between Normal Metabolizer (NM) and Intermediate Metabolizer (IM) except for one volunteer with a gene duplication and unclear phenotype, classifying as either IM or NM. Median plasma primaquine (PQ) area under the curve (AUC) was lower in the NM group (460 h*ng/mL) compared to the IM group (561 h*ng/mL), although not statistically significant. Similar to what has been found in the US study, no 5,6-orthoquinone was detected in the plasma. The urine creatinine-corrected 5,6-orthoquinone AUC in the NM group was almost three times higher than in the IM group, with peak measurements (Tmax) at 4 h. Although there is variation among individuals, future studies examining the relationship between the levels of urine 5,6-orthoquinone and primaquine radical cure efficacy could result in a metabolism biomarker predictive of radical cure.

Safety, Pharmacokinetics, and Activity of High-Dose Ivermectin and Chloroquine against the Liver Stage of Plasmodium cynomolgi Infection in Rhesus Macaques.

Previously, ivermectin (1 to 10 mg/kg of body weight) was shown to inhibit the liver-stage development of Plasmodium berghei in orally dosed mice. Here, ivermectin showed inhibition of the in vitro development of Plasmodium cynomolgi schizonts (50% inhibitory concentration [IC50], 10.42 µM) and hypnozoites (IC50, 29.24 µM) in primary macaque hepatocytes when administered as a high dose prophylactically but not when administered in radical cure mode. The safety, pharmacokinetics, and efficacy of oral ivermectin (0.3, 0.6, and 1.2 mg/kg) with and without chloroquine (10 mg/kg) administered for 7 consecutive days were evaluated for prophylaxis or radical cure of P. cynomolgi liver stages in rhesus macaques. No inhibition or delay to blood-stage P. cynomolgi parasitemia was observed at any ivermectin dose (0.3, 0.6, and 1.2 mg/kg). Ivermectin (0.6 and 1.2 mg/kg) and chloroquine (10 mg/kg) in combination were well-tolerated with no adverse events and no significant pharmacokinetic drug-drug interactions observed. Repeated daily ivermectin administration for 7 days did not inhibit ivermectin bioavailability. It was recently demonstrated that both ivermectin and chloroquine inhibit replication of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro Further ivermectin and chloroquine trials in humans are warranted to evaluate their role in Plasmodium vivax control and as adjunctive therapies against COVID-19 infections.

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.

Updating the modified Thompson test by using whole-body bioluminescence imaging to replace traditional efficacy testing in experimental models of murine malaria.

BACKGROUND: Rodent malaria models are extensively used to predict treatment outcomes in human infections. There is a constant need to improve and refine these models by innovating ways to apply new scientific findings and cutting edge technologies. In addition, and in accordance with the three R's of animal use in research, in vivo studies should be constantly refined to avoid unnecessary pain and distress to the experimental animals by using preemptive euthanasia as soon as the main scientific study objective has been accomplished. METHODS: The new methodology described in this manuscript uses the whole-body bioluminescence signal emitted by transgenic, luciferase-expressing Plasmodium berghei parasites to assess the parasite load predicted parasitaemia (PLPP) in drug and control treated female ICR-CD1 mice infected with 1 × 105 luciferase-expressing P. berghei (ANKA strain) infected erythrocytes. This methodology can replace other time-consuming and expensive methods that are routinely used to measure parasitaemia in infected animals, such as Giemsa-stained thin blood smears and flow cytometry. RESULTS: There is a good correlation between whole-body bioluminescence signal and parasitaemia measured using Giemsa-stained thin blood smears and flow cytometry respectively in donor and study mice in the modified Thompson test. The algebraic formulas which represent these correlations can be successfully used to assess PLPP in donor and study mice. In addition, the new methodology can pinpoint sick animals 2-8 days before they would have been otherwise diagnosed based on behavioural or any other signs of malaria disease. CONCLUSIONS: The new method for predicting parasitaemia in the modified Thompson test is simple, precise, objective, and minimizes false positive results that can lead to the premature removal of animals from study. Furthermore, from the animal welfare perspective of replace, reduce, and refine, this new method facilitates early removal of sick animals from study as soon as the study objective has been achieved, in many cases well before the clinical signs of disease are present.

Single-Dose Primaquine in a Preclinical Model of Glucose-6-Phosphate Dehydrogenase Deficiency: Implications for Use in Malaria Transmission-Blocking Programs.

Individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency (G6PDd) are at risk for developing hemolytic anemia when given the antimalarial drug primaquine (PQ). The WHO Evidence Review Group released a report suggesting that mass administration of a single dose of PQ at 0.25 mg of base/kg of body weight (mpk) (mouse equivalent of 3.125 mpk) could potentially reduce malaria transmission based on its gametocytocidal activity and could be safely administered to G6PD-deficient individuals, but there are limited safety data available confirming the optimum single dose of PQ. A single-dose administration of PQ was therefore assessed in our huRBC-SCID mouse model used to predict hemolytic toxicity with respect to G6PD deficiency. In this model, nonobese diabetic (NOD)/SCID mice are engrafted with human red blood cells (huRBC) from donors with the African or Mediterranean variant of G6PDd (A-G6PDd or Med-G6PDd, respectively) and demonstrate dose-dependent sensitivity to PQ. In mice engrafted with A-G6PD-deficient huRBC, single-dose PQ at 3.125, 6.25, or 12.5 mpk had no significant loss of huRBC compared to the vehicle control group. In contrast, in mice engrafted with Med-G6PDd huRBC, a single dose of PQ at 3.125, 6.25, or 12.5 mpk resulted in a significant, dose-dependent loss of huRBC compared to the value for the vehicle control group. Our data suggest that administration of a single low dose of 0.25 mpk of PQ could induce hemolytic anemia in Med-G6PDd individuals but that use of single-dose PQ at 0.25 mpk as a gametocytocidal drug to block transmission would be safe in areas where A-G6PDd predominates.

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.

Differential cytochrome P450 2D metabolism alters tafenoquine pharmacokinetics.

Cytochrome P450 (CYP) 2D metabolism is required for the liver-stage antimalarial efficacy of the 8-aminoquinoline molecule tafenoquine in mice. This could be problematic for Plasmodium vivax radical cure, as the human CYP 2D ortholog (2D6) is highly polymorphic. Diminished CYP 2D6 enzyme activity, as in the poor-metabolizer phenotype, could compromise radical curative efficacy in humans. Despite the importance of CYP 2D metabolism for tafenoquine liver-stage efficacy, the exact role that CYP 2D metabolism plays in the metabolism and pharmacokinetics of tafenoquine and other 8-aminoquinoline molecules has not been extensively studied. In this study, a series of tafenoquine pharmacokinetic experiments were conducted in mice with different CYP 2D metabolism statuses, including wild-type (WT) (reflecting extensive metabolizers for CYP 2D6 substrates) and CYPmouse 2D knockout (KO) (reflecting poor metabolizers for CYP 2D6 substrates) mice. Plasma and liver pharmacokinetic profiles from a single 20-mg/kg of body weight dose of tafenoquine differed between the strains; however, the differences were less striking than previous results obtained for primaquine in the same model. Additionally, the presence of a 5,6-ortho-quinone tafenoquine metabolite was examined in both mouse strains. The 5,6-ortho-quinone species of tafenoquine was observed, and concentrations of the metabolite were highest in the WT extensive-metabolizer phenotype. Altogether, this study indicates that CYP 2D metabolism in mice affects tafenoquine pharmacokinetics and could have implications for human tafenoquine pharmacokinetics in polymorphic CYP 2D6 human populations.

Differential CYP 2D6 metabolism alters primaquine pharmacokinetics.

Primaquine (PQ) metabolism by the cytochrome P450 (CYP) 2D family of enzymes is required for antimalarial activity in both humans (2D6) and mice (2D). Human CYP 2D6 is highly polymorphic, and decreased CYP 2D6 enzyme activity has been linked to decreased PQ antimalarial activity. Despite the importance of CYP 2D metabolism in PQ efficacy, the exact role that these enzymes play in PQ metabolism and pharmacokinetics has not been extensively studied in vivo. In this study, a series of PQ pharmacokinetic experiments were conducted in mice with differential CYP 2D metabolism characteristics, including wild-type (WT), CYP 2D knockout (KO), and humanized CYP 2D6 (KO/knock-in [KO/KI]) mice. Plasma and liver pharmacokinetic profiles from a single PQ dose (20 mg/kg of body weight) differed significantly among the strains for PQ and carboxy-PQ. Additionally, due to the suspected role of phenolic metabolites in PQ efficacy, these were probed using reference standards. Levels of phenolic metabolites were highest in mice capable of metabolizing CYP 2D6 substrates (WT and KO/KI 2D6 mice). PQ phenolic metabolites were present in different quantities in the two strains, illustrating species-specific differences in PQ metabolism between the human and mouse enzymes. Taking the data together, this report furthers understanding of PQ pharmacokinetics in the context of differential CYP 2D metabolism and has important implications for PQ administration in humans with different levels of CYP 2D6 enzyme activity.

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.

Scalable preparation and differential pharmacologic and toxicologic profiles of primaquine enantiomers.

Hematotoxicity in individuals genetically deficient in glucose-6-phosphate dehydrogenase (G6PD) activity is the major limitation of primaquine (PQ), the only antimalarial drug in clinical use for treatment of relapsing Plasmodium vivax malaria. PQ is currently clinically used in its racemic form. A scalable procedure was developed to resolve racemic PQ, thus providing pure enantiomers for the first time for detailed preclinical evaluation and potentially for clinical use. These enantiomers were compared for antiparasitic activity using several mouse models and also for general and hematological toxicities in mice and dogs. (+)-(S)-PQ showed better suppressive and causal prophylactic activity than (-)-(R)-PQ in mice infected with Plasmodium berghei. Similarly, (+)-(S)-PQ was a more potent suppressive agent than (-)-(R)-PQ in a mouse model of Pneumocystis carinii pneumonia. However, at higher doses, (+)-(S)-PQ also showed more systemic toxicity for mice. In beagle dogs, (+)-(S)-PQ caused more methemoglobinemia and was toxic at 5 mg/kg of body weight/day given orally for 3 days, while (-)-(R)-PQ was well tolerated. In a novel mouse model of hemolytic anemia associated with human G6PD deficiency, it was also demonstrated that (-)-(R)-PQ was less hemolytic than (+)-(S)-PQ for the G6PD-deficient human red cells engrafted in the NOD-SCID mice. All these data suggest that while (+)-(S)-PQ shows greater potency in terms of antiparasitic efficacy in rodents, it is also more hematotoxic than (-)-(R)-PQ in mice and dogs. Activity and toxicity differences of PQ enantiomers in different species can be attributed to their different pharmacokinetic and metabolic profiles. Taken together, these studies suggest that (-)-(R)-PQ may have a better safety margin than the racemate in human.

Tafenoquine and NPC-1161B require CYP 2D metabolism for anti-malarial activity: implications for the 8-aminoquinoline class of anti-malarial compounds.

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.

CYP450 phenotyping and metabolite identification of quinine by accurate mass UPLC-MS analysis: a possible metabolic link to blackwater fever.

BACKGROUND: The naturally occurring alkaloid drug, quinine is commonly used for the treatment of severe malaria. Despite centuries of use, its metabolism is still not fully understood, and may play a role in the haemolytic disorders associated with the drug. METHODS: Incubations of quinine with CYPs 1A2, 2C9, 2C19, 2D6, and 3A4 were conducted, and the metabolites were characterized by accurate mass UPLC-MS(E) analysis. Reactive oxygen species generation was also measured in human erythrocytes incubated in the presence of quinine with and without microsomes. RESULTS: The metabolites 3-hydroxyquinine, 2'-oxoquininone, and O-desmethylquinine were observed after incubation with CYPs 3A4 (3-hydroxyquinine and 2'-oxoquininone) and 2D6 (O-desmethylquinine). In addition, multiple hydroxylations were observed both on the quinoline core and the quinuclidine ring system. Of the five primary abundance CYPs tested, 3A4, 2D6, 2C9, and 2C19 all demonstrated activity toward quinine, while 1A2 did not. Further, quinine produced robust dose-dependent oxidative stress in human erythrocytes in the presence of microsomes. CONCLUSIONS: Taken in context, these data suggest a CYP-mediated link between quinine metabolism and the poorly understood haemolytic condition known as blackwater fever, often associated with quinine ingestion.

The metabolism of primaquine to its active metabolite is dependent on CYP 2D6.

BACKGROUND: The efficacy of the 8-aminoquinoline (8AQ) drug primaquine (PQ) has been historically linked to CYP-mediated metabolism. Although to date no clear evidence exists in the literature that unambiguously assigns the metabolic pathway or specific metabolites necessary for activity, recent literature suggests a role for CYP 2D6 in the generation of redox active metabolites. METHODS: In the present study, the specific CYP 2D6 inhibitor paroxetine was used to assess its effects on the production of specific phenolic metabolites thought to be involved in PQ efficacy. Further, PQ causal prophylactic (developing liver stage) efficacy against Plasmodium berghei in CYP 2D knockout mice was assessed in comparison with a normal C57 background and with humanized CYP 2D6 mice to determine the direct effects of CYP 2D6 metabolism on PQ activity. RESULTS: PQ exhibited no activity at 20 or 40 mg/kg in CYP 2D knockout mice, compared to 5/5 cures in normal mice at 20 mg/kg. The activity against developing liver stages was partially restored in humanized CYP 2D6 mice. CONCLUSIONS: These results unambiguously demonstrate that metabolism of PQ by CYP 2D6 is essential for anti-malarial causal prophylaxis efficacy.

CYP450 phenotyping and accurate mass identification of metabolites of the 8-aminoquinoline, anti-malarial drug primaquine.

BACKGROUND: The 8-aminoquinoline (8AQ) drug primaquine (PQ) is currently the only approved drug effective against the persistent liver stage of the hypnozoite forming strains Plasmodium vivax and Plasmodium ovale as well as Stage V gametocytes of Plasmodium falciparum. To date, several groups have investigated the toxicity observed in the 8AQ class, however, exact mechanisms and/or metabolic species responsible for PQ's haemotoxic and anti-malarial properties are not fully understood. METHODS: In the present study, the metabolism of PQ was evaluated using in vitro recombinant metabolic enzymes from the cytochrome P450 (CYP) and mono-amine oxidase (MAO) families. Based on this information, metabolite identification experiments were performed using nominal and accurate mass measurements. RESULTS: Relative activity factor (RAF)-weighted intrinsic clearance values show the relative role of each enzyme to be MAO-A, 2C19, 3A4, and 2D6, with 76.1, 17.0, 5.2, and 1.7% contributions to PQ metabolism, respectively. CYP 2D6 was shown to produce at least six different oxidative metabolites along with demethylations, while MAO-A products derived from the PQ aldehyde, a pre-cursor to carboxy PQ. CYPs 2C19 and 3A4 produced only trace levels of hydroxylated species. CONCLUSIONS: As a result of this work, CYP 2D6 and MAO-A have been implicated as the key enzymes associated with PQ metabolism, and metabolites previously identified as potentially playing a role in efficacy and haemolytic toxicity have been attributed to production via CYP 2D6 mediated pathways.

Discovery of a series of potent, orally active α,α-disubstituted piperidine NK(1) antagonists.

Modification of prototype NK(1) antagonist 2 resulted in the synthesis of a series of simple amides 6 and retroamides 9. These compounds were shown to be potent and orally active NK(1) antagonists.