Brandon S. Pybus

Primaquine failure and cytochrome P-450 2D6 in Plasmodium vivax malaria.

Primaquine is used to eradicate the hepatic or hypnozoite form of Plasmodium vivax that may lead to relapse of infection. Host genetic factors may play a role in the activity of primaquine therapy.

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

BackgroundThe 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.MethodsIn 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.ResultsRelative 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.ConclusionsAs 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.

Differential CYP 2D6 Metabolism Alters Primaquine Pharmacokinetics

ABSTRACT 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.

Review: Improving the Therapeutic Index of 8-Aminoquinolines by the Use of Drug Combinations: Review of the Literature and Proposal for Future Investigations

Because 8-aminoquinolines affect critical survival stages of Plasmodium parasites, treatment and control of malaria could be markedly improved by more widespread use of these drugs; however, hemolytic toxicity, which is widely prevalent in G6PD-deficient patients, severely constrains this use. Primaquine was approved more than 50 years ago after extensive clinical testing. Review of the mid-20th century literature in the light of present understanding of pharmacokinetics and metabolism suggests that manipulation of these factors might dissociate 8-aminoquinoline efficacy from toxicity and lead to an improved therapeutic index.

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.

Ketotifen is an antimalarial prodrug of norketotifen with blood schizonticidal and liver-stage efficacy

Ketotifen is known to exhibit antimalarial activity in mouse and monkey malaria models. However, the low plasma levels and short half life of the drug do not adequately explain its in vivo efficacy. We synthesized most of the known metabolites of ketotifen and evaluated their antimalarial activity and pharmacokinetics in mice. Norketotifen, the de-methylated metabolite of ketotifen, was a more potent antimalarial in vitro as compared to ketotifen, and exhibited equivalent activity in vivo against asexual blood and developing liver-stage parasites. After ketotifen dosing, norketotifen levels were much higher than ketotifen relative to the IC50s of the compounds against Plasmodium falciparum in vitro. The data support the notion that the antimalarial activity of ketotifen in mice is mediated through norketotifen.

Differential Cytochrome P450 2D Metabolism Alters Tafenoquine Pharmacokinetics

ABSTRACT 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.

Investigation into novel thiophene- and furan-based 4-amino-7-chloroquinolines afforded antimalarials that cure mice.

We herein report the design and synthesis of a novel series of thiophene- and furan-based aminoquinoline derivatives which were found to be potent antimalarials and inhibitors of β-hematin polymerization. Tested compounds were 3-71 times more potent in vitro than CQ against chloroquine-resistant (CQR) W2 strain with benzonitrile 30 being as active as mefloquine (MFQ), and almost all synthesized aminoquinolines (22/27) were more potent than MFQ against multidrug-resistant (MDR) strain C235. In vivo experiments revealed that compound 28 showed clearance with recrudescence at 40 mg/kg/day, while 5/5 mice survived in Thompson test at 160 mg/kg/day.

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

BackgroundThe 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.MethodsIncubations of quinine with CYPs 1A2, 2C9, 2C19, 2D6, and 3A4 were conducted, and the metabolites were characterized by accurate mass UPLC-MSE analysis. Reactive oxygen species generation was also measured in human erythrocytes incubated in the presence of quinine with and without microsomes.ResultsThe 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.ConclusionsTaken 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.

Antimalarial Activities of New Guanidylimidazole and Guanidylimidazoline Derivatives

A series of new guanidylimidazole derivatives was prepared and evaluated in mice and Rhesus monkeys infected with malarial sporozoites. The majority of the new compounds showed poor metabolic stability and weak in vitro activities in three clones of Plasmodium falciparum. Compounds 8a, 8h, 9a, 16a, and 16e cured the mice infected with sporozoites of P. berghei at 160 and 320 mg/kg/day × 3 po. Compounds 8a showed better causal prophylactic activity than primaquine, tafenoquine, and Malarone in the Rhesus test. In the radical curative test, 8a cured one monkey and delayed relapse of another for 74 days at 30 mg/kg/day × 7 by im. By oral dosing, 8a delayed relapse 81 days for one and 32 days for other vs 11-12 days for control monkeys treated with 10 mg/kg of chloroquine by po alone. Compound 8h, which showed superior activity to 8a in mouse test, delayed the relapse of treated monkeys for 21-26 days at 30 mg/kg/day × 7 by oral.