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.

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. P rimaquine (PQ) is the only FDA-approved drug for treatment of relapsing infections with malarial strains, including Plasmodium vivax and P. ovale (1-3). PQ belongs to the 8-aminoquinoline (8AQ) class of antimalarial compounds, among which several molecules, including PQ, have potent antihypnozoite activity (2, 4, 5). Low doses of PQ are also recommended for malaria transmission-blocking efforts due to PQ's gametocidal activity (6, 7). PQ's utility in malaria treatment and potential use in malarial transmission reduction and malaria eradication efforts require an understanding of the molecular species involved in its mechanism of action.Recent reports have shown that PQ requires metabolic activation by the cytochrome P450 (CYP) 2D isoenzymes for liver-stage antimalarial activity in both mouse studies (CYP 2D) and human studies (CYP 2D6) (8-11). Pybus et al. demonstrated that PQ was active only in mice capable of metabolizing CYP 2D6 substrates. Deletion of the mouse enzyme closest to human CYP 2D6 (mouse CYP 2D22 via deletion of the CYP 2D gene cluster) in mice completely blocked liver-stage antimalarial activity in vivo (10). The study by Bennett et al. demonstrated a direct link between CYP 2D6 metabolizer status and PQ efficacy for P. vivax treatment in several human subjects (8). PQ therapy is of significant importance for P. vivax radical cure, presumptive antirelapse therapy (PART), and malaria eradication efforts, and the requirement of CYP 2D6 metabolism for PQ efficacy is problematic because CYP 2D6 is highly polymorp...

Closed-head concussive injury is one of the most common causes of traumatic brain injury (TBI). Isolated concussions frequently produce acute neurological impairments, and individuals typically recover spontaneously within a short time frame. In contrast, brain injuries resulting from multiple concussions can result in cumulative damage and elevated risk of developing chronic brain pathologies. Increased attention has focused on identification of diagnostic markers that can prognostically serve as indices of brain health after injury, revealing the temporal profile of vulnerability to a second insult. Such markers may demarcate adequate recovery periods before concussed patients can return to required activities. We developed a noninvasive closed-head impact model that captures the hallmark symptoms of concussion in the absence of gross tissue damage. Animals were subjected to single or repeated concussive impact and examined using a battery of neurological, vestibular, sensorimotor, and molecular metrics. A single concussion induced transient, but marked, acute neurological impairment, gait alterations, neuronal death, and increased glial fibrillary acidic protein (GFAP) expression in brain tissue. As expected, repeated concussions exacerbated sensorimotor dysfunction, prolonged gait abnormalities, induced neuroinflammation, and upregulated GFAP and tau. These animals also exhibited chronic functional neurological impairments with sustained astrogliosis and white matter thinning. Acute changes in molecular signatures correlated with behavioral impairments, whereas increased times to regaining consciousness and balance impairments were associated with higher GFAP and neuroinflammation. Overall, behavioral consequences of either single or repeated concussive impact injuries appeared to resolve more quickly than the underlying molecular, metabolic, and neuropathological abnormalities. This observation, which is supported by similar studies in other mTBI models, underscores the critical need to develop more objective prognostic measures for guiding return-to-play decisions.

dCytochrome 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 CYP mouse 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.T he 8-aminoquinoline class of antimalarial compounds is the only molecular scaffold with proven efficacy against relapsing strains of malaria (1-5). Currently, the only FDA-approved drug from this class that is available for clinical use is primaquine. Primaquine has been utilized for over 6 decades in treating malaria (6). Despite the long history of primaquine therapy for malaria treatment, primaquine has several disadvantages, including the hemolytic toxicity associated with glucose-6-phosphate dehydrogenase (G6PD) deficiency; its relatively short elimination half-life in humans, which requires daily administration; and the potential requirement for cytochrome P450 (CYP) 2D6-mediated activation for radical curative activity (7-11). The 8-aminoquinoline molecule tafenoquine, currently under late-stage clinical development, has a significantly longer elimination half-life than primaquine (12-16) and has single-dose radical curative activity in humans (3). Tafenoquine is also being developed as a chemoprophylactic agent and has demonstrated efficacy against Plasmodium vivax and Plasmodium falciparum (17)(18)(19)(20)(21). Despite the pharmacological advantages of tafenoquine over primaquine, both molecules seem to have the same pharmacogenomic liability of CYP 2D6-mediated activation for liver-stage antimalarial activity (7,10,22,23) and are not free of hemolytic liability in G6PD def...

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