White and green striate leaves 1, predicted to encode a 16S rRNA processing protein, plays a critical role in the processing of chloroplast ribosomes in maize (Zea mays L.).

Ribosomes play a crucial role in protein biosynthesis and are linked to plant growth and development. The RimM protein has been shown to be involved in the maturation of 30S ribosomal subunits, but its exact function in plants is still unknown. In this study, we discovered a maize mutant with white and green striate leaves (wgsl1) and reduced chlorophyll content. Genetic analysis showed that the wgsl1 mutation was recessive and controlled by a single nuclear gene. Map-based cloning of ZmWGSL1 identified a base substitution (G to A) that generated a missense mutation within the Zm00001d039036 gene in the wgsl1 mutant. Zm00001d039036 encodes a 16S rRNA processing protein containing the RimM motif. Further analysis of transcriptomic data showed that the transcript levels of many ribosomal proteins involved in the small and big ribosomal subunits were dramatically up-regulated in the wgsl1 mutant. Moreover, the level of ribosomal multimers was decreased. This suggests that ZmWGSL1 plays a crucial role in the maturation of the ribosome, leading to abnormal plant growth and development. In addition, subcellular localization results indicate that WGSL1 is localized in chloroplasts. Therefore, we suggest that WGSL1 is a nuclear-encoded protein, is transported to the chloroplast to drive functions, and affects the processing of ribosomes in the chloroplast. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01407-y.

Genome assembly of the Chinese maize elite inbred line RP125 and its EMS mutant collection provide new resources for maize genetics research and crop improvement.

Maize is an important crop worldwide, as well as a valuable model with vast genetic diversity. Accurate genome and annotation information for a wide range of inbred lines would provide valuable resources for crop improvement and pan-genome characterization. In this study, we generated a high-quality de novo genome assembly (contig N50 of 15.43 Mb) of the Chinese elite inbred line RP125 using Nanopore long-read sequencing and Hi-C scaffolding, which yield highly contiguous, chromosome-length scaffolds. Global comparison of the RP125 genome with those of B73, W22, and Mo17 revealed a large number of structural variations. To create new germplasm for maize research and crop improvement, we carried out an EMS mutagenesis screen on RP125. In total, we obtained 5818 independent M2 families, with 946 mutants showing heritable phenotypes. Taking advantage of the high-quality RP125 genome, we successfully cloned 10 mutants from the EMS library, including the novel kernel mutant qk1 (quekou: "missing a small part" in Chinese), which exhibited partial loss of endosperm and a starch accumulation defect. QK1 encodes a predicted metal tolerance protein, which is specifically required for Fe transport. Increased accumulation of Fe and reactive oxygen species as well as ferroptosis-like cell death were detected in qk1 endosperm. Our study provides the community with a high-quality genome sequence and a large collection of mutant germplasm.

Identification and Fine Mapping of the Recessive Gene BK-5, Which Affects Cell Wall Biosynthesis and Plant Brittleness in Maize.

The cellulose of the plant cell wall indirectly affects the cell shape and straw stiffness of the plant. Here, the novel brittleness mutant brittle stalk-5 (bk-5) of the maize inbred line RP125 was characterized. We found that the mutant displayed brittleness of the stalk and even the whole plant, and that the brittleness phenotype existed during the whole growth period from germination to senescence. The compressive strength was reduced, the cell wall was thinner, and the cellulose content was decreased compared to that of the wild type. Genetic analysis and map-based cloning indicated that bk-5 was controlled by a single recessive nuclear gene and that it was located in a 90.2-Kb region on chromosome 3 that covers three open reading frames (ORFs). Sequence analysis revealed a single non-synonymous missense mutation, T-to-A, in the last exon of Zm00001d043477 (B73: version 4, named BK-5) that caused the 951th amino acid to go from leucine to histidine. BK-5 encodes a cellulose synthase catalytic subunit (CesA), which is involved with cellulose synthesis. We found that BK-5 was constitutively expressed in all tissues of the germinating stage and silking stage, and highly expressed in the leaf, auricula, and root of the silking stage and the 2-cm root and bud of the germinating stage. We found that BK-5 mainly localized to the Golgi apparatus, suggesting that the protein might move to the plasma membrane with the aid of Golgi in maize. According to RNA-seq data, bk-5 had more downregulated genes than upregulated genes, and many of the downregulated genes were enzymes and transcription factors related to cellulose, hemicellulose, and lignin biosynthesis of the secondary cell wall. The other differentially expressed genes were related to metabolic and cellular processes, and were significantly enriched in hormone signal transduction, starch and sucrose metabolism, and the plant-pathogen interaction pathway. Taken together, we propose that the mutation of gene BK-5 causes the brittle stalk phenotype and provides important insights into the regulatory mechanism of cellulose biosynthesis and cell wall development in maize.

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.

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.

Cytochrome P450 2D-mediated metabolism is not necessary for tafenoquine and primaquine to eradicate the erythrocytic stages of Plasmodium berghei.

BACKGROUND: Due to the ability of the 8-aminoquinolines (8AQs) to kill different stages of the malaria parasite, primaquine (PQ) and tafenoquine (TQ) are vital for causal prophylaxis and the eradication of erythrocytic Plasmodium sp. parasites. Recognizing the potential role of cytochrome (CYP) 450 2D6 in the metabolism and subsequent hepatic efficacy of 8-aminoquinolines, studies were designed to explore whether CYP2D-mediated metabolism was related to the ability of single-dose PQ and TQ to eliminate the asexual and sexual erythrocytic stages of Plasmodium berghei. METHODS: An IV P. berghei sporozoite murine challenge model was utilized to directly compare causal prophylactic and erythrocytic activity (asexual and sexual parasite stages) dose-response relationships in C57BL/6 wild-type (WT) mice and subsequently compare the erythrocytic activity of PQ and TQ in WT and CYP2D knock-out (KO) mice. RESULTS: Single-dose administration of either 25 mg/kg TQ or 40 mg/kg PQ eradicated the erythrocytic stages (asexual and sexual) of P. berghei in C57BL WT and CYP2D KO mice. In WT animals, the apparent elimination of hepatic infections occurs at lower doses of PQ than are required to eliminate erythrocytic infections. In contrast, the minimally effective dose of TQ needed to achieve causal prophylaxis and to eradicate erythrocytic parasites was analogous. CONCLUSION: The genetic deletion of the CYP2D cluster does not affect the ability of PQ or TQ to eradicate the blood stages (asexual and sexual) of P. berghei after single-dose administration.

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.

Efficacy of intravenous methylene blue, intravenous artesunate, and their combination in preclinical models of malaria.

BACKGROUND: Intravenous artesunate (IV AS) is the present treatment of choice for severe malaria, but development of artemisinin resistance indicates that a further agent will be needed. Methylene blue (MB) is an approved human agent for IV and oral use, and is already being investigated for oral treatment of uncomplicated malaria. To initiate investigation of IV MB for severe malaria, the efficacy of IV MB was compared to IV AS and to their combination in rat and non-human primate malaria models. METHODS: IV MB was compared to IV AS and to their combination in the Plasmodium berghei-infected rat, a self-curing model; the Plasmodium falciparum-infected Aotus monkey, a fatal model; and the Plasmodium cynomolgi-infected rhesus monkey, a fatal model. Key endpoints were clearance of all parasites from the blood and cure (clearance without recrudescence). RESULTS: In rats, the minimal dose of individual drugs and their combination that cleared parasites from all animals was 20 mg IV MB/kg/day, 60 mg IV AS/kg/day and 10 mg IV MB/kg/day plus 30 mg IV AS/kg/day. In Aotus, 8 mg IV MB/kg/day and 8 mg IV AS/kg/day each cured two of three monkeys by one day after therapy, and the third monkey in each group was cured two days later. The combination of both drugs did not result in superior efficacy. In rhesus, 8 mg IV MB/kg/day and 8 mg IV AS/kg/day performed comparably: parasite clearance occurred by day 3 of therapy, although only one of four animals in each dose group cured. Eight mg/kg/day of both drugs in combination was 100% successful: all four of four animals cured. CONCLUSIONS: In each of the three animal models, the efficacy of IV MB was approximately equal to that of standard of care IV AS. In the rat and rhesus models, the combination was more effective than either single agent. This preclinical data suggests that IV MB, alone or in combination with IV AS, is effective against Plasmodium spp. and can be evaluated in severe malaria models.

Anti-relapse activity of mirincamycin in the Plasmodium cynomolgi sporozoite-infected Rhesus monkey model.

BACKGROUND: Mirincamycin is a close analog of the drug clindamycin used to treat Plasmodium falciparum blood stages. The clinical need to treat Plasmodium vivax dormant liver stages and prevent relapse with a drug other than primaquine led to the evaluation of mirinicamycin against liver stages in animals. METHODS: cis-mirinicamycin and trans-mirinicamycin were evaluated as prophylaxis against early liver stages of Plasmodium berghei in mice and as antirelapse hypnozoiticides against Plasmodium cynomolgi in the Rhesus monkey (Macaca mulatta). RESULTS: Mirincamycin was very effective against early liver stages of P. berghei in mice: both cis and trans enantiomers were 90-100% causally prophylactic at 3.3 mg/kg/day for 3 days orally. Both cis and trans mirincamycin, however, failed to kill dormant liver stages (hypnozoites) in the P. cynomolgi infected Rhesus monkey, the only preclinical hypnozoite model. Mirincamycin enantiomers at 80 mg/kg/day for 7 days orally, a dose that generated exposures comparable to that seen clinically, did not prevent relapse in any of four monkeys. CONCLUSIONS: Although efficacy against early liver stages of P. berghei was thought to correlate with anti-hypnozoite activity in primates, for mirincamycin, at least, there was no correlation. The negative P. cynomolgi hypnozoite data from Rhesus monkeys indicates that mirincamycin is unlikely to have potential as a clinical anti-relapse agent.

Assessment of the prophylactic activity and pharmacokinetic profile of oral tafenoquine compared to primaquine for inhibition of liver stage malaria infections.

BACKGROUND: As anti-malarial drug resistance escalates, new safe and effective medications are necessary to prevent and treat malaria infections. The US Army is developing tafenoquine (TQ), an analogue of primaquine (PQ), which is expected to be more effective in preventing malaria in deployed military personnel. METHODS: To compare the prophylactic efficacy of TQ and PQ, a transgenic Plasmodium berghei parasite expressing the bioluminescent reporter protein luciferase was utilized to visualize and quantify parasite development in C57BL/6 albino mice treated with PQ and TQ in single or multiple regimens using a real-time in vivo imaging system (IVIS). As an additional endpoint, blood stage parasitaemia was monitored by flow cytometry. Comparative pharmacokinetic (PK) and liver distribution studies of oral and intravenous PQ and TQ were also performed. RESULTS: Mice treated orally with three doses of TQ at 5 mg/kg three doses of PQ at 25 mg/kg demonstrated no bioluminescence liver signal and no blood stage parasitaemia was observed suggesting both drugs showed 100% causal activity at the doses tested. Single dose oral treatment with 5 mg TQ or 25 mg of PQ, however, yielded different results as only TQ treatment resulted in causal prophylaxis in P. berghei sporozoite-infected mice. TQ is highly effective for causal prophylaxis in mice at a minimal curative single oral dose of 5 mg/kg, which is a five-fold improvement in potency versus PQ. PK studies of the two drugs administered orally to mice showed that the absolute bioavailability of oral TQ was 3.5-fold higher than PQ, and the AUC of oral TQ was 94-fold higher than oral PQ. The elimination half-life of oral TQ in mice was 28 times longer than PQ, and the liver tissue distribution of TQ revealed an AUC that was 188-fold higher than PQ. CONCLUSIONS: The increased drug exposure levels and longer exposure time of oral TQ in the plasma and livers of mice highlight the lead quality attributes that explain the much improved efficacy of TQ when compared to PQ.

Pharmacokinetic evaluation of intravenous artesunate in adults with uncomplicated falciparum malaria in Kenya: a phase II study.

BACKGROUND: Alternatives to treatment for malaria treatment of travellers are needed in the USA and in Europe for travellers who return with severe malaria infections. The objective of this study is to show the pharmacokinetic (PK) profile of intravenous artesunate (AS), which was manufactured under good manufacturing practice (GMP) conditions, in adults with uncomplicated falciparum malaria in Kenya. METHODS: The PK parameters of intravenous AS manufactured under current cGMP were evaluated after a single dose of drug at 2.4 mg/kg infused over 2 min in 28 adults with uncomplicated Plasmodium falciparum malaria. Plasma concentrations of AS and dihydroartemisinin (DHA) were measured using a validated liquid chromatography-mass spectrometry (LC-MS/MS) methodology. Pharmacokinetic data were analysed with a compartmental analysis for AS and DHA. RESULTS: The results suggest there were no drug-related adverse events in any of the patients. After intravenous infusion, the concentration of the parent drug rapidly declined, and the AS was converted to DHA. AS and DHA showed mean elimination half-lives of 0.17 hours and 1.30 hours, respectively. The high mean peak concentration (Cmax) of AS was shown to be 28,558 ng/mL while the Cmax of DHA was determined to be 2,932 ng/mL. Significant variability was noted in the PK profiles of the 28 patients tested. For example, Cmax values of AS were calculated to range from 3,362 to 55,873 ng/mL, and the Cmax value of DHA was noted to vary from 1,493 to 5,569 ng/mL. The mean area under the curve (AUC) of AS was shown to be approximately half that of DHA (1,878 ng · h/mL vs 3,543 ng · h/mL). The DHA/AS ratio observed was 1.94 during the one-day single treatment, and the AUC and half- life measured for DHA were significantly larger and longer than for AS. CONCLUSIONS: Intravenous AS can provide much higher peak concentrations of AS when compared to concentrations achieved with oral therapy; this may be crucial for the rapid elimination of parasites in patients with severe malaria. Given the much longer half-life of DHA compared to the short half-life of AS, DHA also plays a significant role in treatment of severe malaria.

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.

Nanoparticle formulations of decoquinate increase antimalarial efficacy against liver stage Plasmodium infections in mice.

Decoquinate has potent activity against both Plasmodium hepatic development and red cell replication when tested in vitro. Decoquinate, however, is practically insoluble in water. To achieve its maximal in vivo efficacy, we generated nanoparticle formulations of decoquinate with a mean particle size less than 400 nm. Three separate preparations at doses of decoquinate 0.5-5 mg/kg were examined in mice infected with Plasmodium berghei. Oral administration of nanoparticle decoquinate at a dose of 1.25 mg/kg effectively inhibited the liver-stage parasite growth and provided complete causal prophylactic protection. This efficacy is 15 fold greater than that observed for microparticle decoquinate, which requires minimal dose of 20 mg/kg for the same inhibitory effect. Further in vitro studies utilizing dose-response assays revealed that decoquinate nanoformulation was substantially more potent than decoquinate microsuspension in killing both liver and blood stage malarial parasites, proving its potential for therapeutic development. FROM THE CLINICAL EDITOR: In this study, a nanoparticle formulation of decoquinate is shown to have superior bioavailability and efficacy in a mouse model of malaria, paving the way to the development of novel, potentially less toxic and more effective therapeutics to combat a disease that still has an enormous impact on a global scale despite the available partially effective therapies.

The use of a prodrug approach to minimize potential CNS exposure of next generation quinoline methanols while maintaining efficacy in in vivo animal models.

The use of mefloquine (MQ) for antimalarial treatment and prophylaxis has diminished largely in response to concerns about its neurologic side effects. An analog campaign designed to maintain the efficacy of MQ while minimizing blood-brain barrier (BBB) penetration has resulted in the synthesis of a prodrug with comparable-to-superior in vivo efficacy versus mefloquine in a P. berghei mouse model while exhibiting a sixfold reduction in CNS drug levels. The prodrug, WR319670, performed poorly compared to MQ in in vitro efficacy assays, but had promising in vitro permeability in an MDCK-MDR1 cell line BBB permeability screen. Its metabolite, WR308245, exhibited high predicted BBB penetration with excellent in vitro efficacy. Both WR319670 and WR308245 cured 5/5 animals in separate in vivo efficacy studies. The in vivo efficacy of WR319670 was thought to be due to the formation of a more active metabolite, specifically WR308245. This was supported by pharmacokinetics studies in non-infected mice, which showed that both IV and oral administration of WR319670 produced essentially identical levels of WR319670 and WR308245 in both plasma and brain samples at all time points. In these studies, the levels of WR308245 in the brain were 1/4 and 1/6 that of MQ in similar IV and oral studies, respectively. These data show that the use of WR319670 as an antimalarial prodrug was able to maintain efficacy in in vivo efficacy screens, while significantly lowering overall penetration of drug and metabolites across the BBB.

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.

Pharmacokinetic profiles of artesunate following multiple intravenous doses of 2, 4, and 8 mg/kg in healthy volunteers: phase 1b study.

BACKGROUND: Severe malaria results in over a million deaths every year, most of them in children aged less than five years and living in sub-Saharan Africa. Injectable artesunate (AS) was recommended as initial treatment for severe malaria by WHO in 2006. The Walter Reed Army Institute of Research (WRAIR) has been developing a novel good manufacturing practice (GMP) injection of AS, which was approved by the US FDA for investigational drug use and distribution by the CDC. METHODS: Tolerability and pharmacokinetics of current GMP intravenous AS, as an anti-malarial agent, were evaluated after ascending multiple doses of 2, 4, and 8 mg/kg daily for three days with 2-minute infusion in 24 healthy subjects (divided into three groups) in the Phase 1 clinical trial study. RESULTS: Results showed that there were no dose-dependent increases in any adverse events. Drug concentrations showed no accumulation and no decline of the drug during the three days of treatment. After intravenous injection, parent drug rapidly declined and was converted to dihydroartemisinin (DHA) with overall mean elimination half-lives ranging 0.15-0.23 hr for AS and 1.23-1.63 hr for DHA, but the peak concentration (C(max)) of AS was much higher than that of DHA with a range of 3.08-3.78-folds. In addition, the AUC and C(max) values of AS and DHA were increased proportionally to the AS climbing multiple doses. DISCUSSION: The safety of injectable AS, even at the highest dose of 8 mg/kg increases the probability of therapeutic success of the drug even in patients with large variability of parasitaemia.

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.