Interplay among malnutrition, chemoprevention, and the risk of malaria in young Ugandan children: Longitudinal pharmacodynamic and growth analysis.

African children are at risk of malaria and malnutrition. We quantified relationships between malaria and malnutrition among young Ugandan children in a high malaria transmission region. Data were used from a randomized controlled trial where Ugandan HIV-unexposed (n = 393) and HIV-exposed (n = 186) children were randomized to receive no malaria chemoprevention, monthly sulfadoxine-pyrimethamine, daily trimethoprim-sulfamethoxazole, or monthly dihydroartemisinin-piperaquine (DP) from age 6-24 months, and then were followed off chemoprevention until age 36 months. Monthly height and weight, and time of incident malaria episodes were obtained; 89 children who received DP contributed piperaquine (PQ) concentrations. Malaria hazard was modeled using parametric survival analysis adjusted for repeated events, and height and weight were modeled using a Brody growth model. Among 579 children, stunting (height-for-age z-score [ZHA] < -2) was associated with a 17% increased malaria hazard (95% confidence interval [CI] 10-23%) compared with children with a ZHA of zero. DP was associated with a 35% lower malaria hazard (hazard ratio [HR] [95% CI], 0.65 [0.41-0.97]), compared to no chemoprevention. After accounting for PQ levels, stunted children who received DP had 2.1 times the hazard of malaria (HR [95% CI] 2.1 [1.6-3.0]) compared with children with a ZHA of zero who received DP. Each additional malaria episode was associated with a 0.4% reduced growth rate for height. Better dosing regimens are needed to optimize malaria prevention in malnourished populations, but, importantly, malaria chemoprevention may reduce the burden of malnutrition in early childhood.

Activation of mitochondrial TRAP1 stimulates mitochondria-lysosome crosstalk and correction of lysosomal dysfunction.

Numerous studies have established the involvement of lysosomal and mitochondrial dysfunction in the pathogenesis of neurodegenerative disorders such as Alzheimer's and Parkinson diseases. Building on our previous studies of the neurodegenerative lysosomal lipidosis Niemann-Pick C1 (NPC1), we have unexpectedly discovered that activation of the mitochondrial chaperone tumor necrosis factor receptor-associated protein 1 (TRAP1) leads to the correction of the lysosomal storage phenotype in patient cells from multiple lysosomal storage disorders including NPC1. Using small compound activators specific for TRAP1, we find that activation of this chaperone leads to a generalized restoration of lysosomal and mitochondrial health. Mechanistically, we show that this process includes inhibition of oxidative phosphorylation and reduction of oxidative stress, which results in activation of AMPK and ultimately stimulates lysosome recycling. Thus, TRAP1 participates in lysosomal-mitochondrial crosstalk to maintain cellular homeostasis and could represent a potential therapeutic target for multiple disorders.

Piperaquine-Induced QTc Prolongation Decreases With Repeated Monthly Dihydroartemisinin-Piperaquine Dosing in Pregnant Ugandan Women.

BACKGROUND: Intermittent preventive treatment with monthly dihydroartemisinin-piperaquine (DHA-PQ) is highly effective at preventing both malaria during pregnancy and placental malaria. Piperaquine prolongs the corrected QT interval (QTc), and it is possible that repeated monthly dosing could lead to progressive QTc prolongation. Intensive characterization of the relationship between piperaquine concentration and QTc interval throughout pregnancy can inform effective, safe prevention guidelines. METHODS: Data were collected from a randomized controlled trial, where pregnant Ugandan women received malaria chemoprevention with monthly DHA-PQ (120/960 mg DHA/PQ; n = 373) or sulfadoxine-pyrimethamine (SP; 1500/75 mg; n = 375) during the second and third trimesters of pregnancy. Monthly trough piperaquine samples were collected throughout pregnancy, and pre- and postdose electrocardiograms were recorded at 20, 28, and 36 weeks' gestation in each woman. The pharmacokinetics-QTc relationship for piperaquine and QTc for SP were assessed using nonlinear mixed-effects modeling. RESULTS: A positive linear relationship between piperaquine concentration and Fridericia corrected QTc interval was identified. This relationship progressively decreased from a 4.42 to 3.28 to 2.13 millisecond increase per 100 ng/mL increase in piperaquine concentration at 20, 28, and 36 weeks' gestation, respectively. Furthermore, 61% (n = 183) of women had a smaller change in QTc at week 36 than week 20. Nine women given DHA-PQ had grade 3-4 cardiac adverse events. SP was not associated with any change in QTc. CONCLUSIONS: Repeated DHA-PQ dosing did not result in increased risk of QTc prolongation and the postdose QTc intervals progressively decreased. Monthly dosing of DHA-PQ in pregnant women carries minimal risk of QTc prolongation. CLINICAL TRIALS REGISTRATION: NCT02793622.

Genome Sequencing as an Alternative to Cytogenetic Analysis in Myeloid Cancers.

BACKGROUND: Genomic analysis is essential for risk stratification in patients with acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS). Whole-genome sequencing is a potential replacement for conventional cytogenetic and sequencing approaches, but its accuracy, feasibility, and clinical utility have not been demonstrated. METHODS: We used a streamlined whole-genome sequencing approach to obtain genomic profiles for 263 patients with myeloid cancers, including 235 patients who had undergone successful cytogenetic analysis. We adapted sample preparation, sequencing, and analysis to detect mutations for risk stratification using existing European Leukemia Network (ELN) guidelines and to minimize turnaround time. We analyzed the performance of whole-genome sequencing by comparing our results with findings from cytogenetic analysis and targeted sequencing. RESULTS: Whole-genome sequencing detected all 40 recurrent translocations and 91 copy-number alterations that had been identified by cytogenetic analysis. In addition, we identified new clinically reportable genomic events in 40 of 235 patients (17.0%). Prospective sequencing of samples obtained from 117 consecutive patients was performed in a median of 5 days and provided new genetic information in 29 patients (24.8%), which changed the risk category for 19 patients (16.2%). Standard AML risk groups, as defined by sequencing results instead of cytogenetic analysis, correlated with clinical outcomes. Whole-genome sequencing was also used to stratify patients who had inconclusive results by cytogenetic analysis into risk groups in which clinical outcomes were measurably different. CONCLUSIONS: In our study, we found that whole-genome sequencing provided rapid and accurate genomic profiling in patients with AML or MDS. Such sequencing also provided a greater diagnostic yield than conventional cytogenetic analysis and more efficient risk stratification on the basis of standard risk categories. (Funded by the Siteman Cancer Research Fund and others.).

Piperaquine Exposure Is Altered by Pregnancy, HIV, and Nutritional Status in Ugandan Women.

Dihydroartemisinin-piperaquine (DHA-PQ) provides highly effective therapy and chemoprevention for malaria in pregnant African women. PQ concentrations of >10.3 ng/ml have been associated with reduced maternal parasitemia, placental malaria, and improved birth outcomes. We characterized the population pharmacokinetics (PK) of PQ in a post hoc analysis of human immunodeficiency virus (HIV)-infected and -uninfected pregnant women receiving DHA-PQ as chemoprevention every 4 or 8 weeks. The effects of covariates such as pregnancy, nutritional status (body mass index [BMI]), and efavirenz (EFV)-based antiretroviral therapy were investigated. PQ concentrations from two chemoprevention trials were pooled to create a population PK database from 274 women and 2,218 PK observations. A three-compartment model with an absorption lag best fit the data. Consistent with our prior intensive PK evaluation, pregnancy and EFV use resulted in a 72% and 61% increased PQ clearance, compared to postpartum and HIV-uninfected pregnant women, respectively. Low BMI at 28 weeks of gestation was associated with increased clearance (2% increase per unit decrease in BMI). Low-BMI women given DHA-PQ every 8 weeks had a higher prevalence of parasitemia, malaria infection, and placental malaria compared to women with higher BMIs. The reduced piperaquine exposure in women with low BMI as well as during EFV coadministration, compared to pregnant women with higher BMIs and not taking EFV, suggests that these populations could benefit from weekly instead of monthly dosing for prevention of malaria parasitemia. Simulations indicated that because of the BMI-clearance relationship, weight-based regimens would not improve protection compared to a 2,880 mg fixed-dose regimen when provided monthly. (The clinical trials described in this paper have been registered at ClinicalTrials.gov under identifiers NCT02163447 and NCT02282293.).

Metabolism and pharmacokinetics characterization of metarrestin in multiple species.

PURPOSE: Metarrestin is a first-in-class pyrrolo-pyrimidine-derived small molecule targeting a marker of genome organization associated with metastasis and is currently in preclinical development as an anti-cancer agent. Here, we report the in vitro ADME characteristics and in vivo pharmacokinetic behavior of metarrestin. METHODS: Solubility, permeability, and efflux ratio as well as in vitro metabolism of metarrestin in hepatocytes, liver microsomes and S9 fractions, recombinant cytochrome P450 (CYP) enzymes, and potential for CYP inhibition were evaluated. Single dose pharmacokinetic profiles after intravenous and oral administration in mice, rat, dog, monkey, and mini-pig were obtained. Simple allometric scaling was applied to predict human pharmacokinetics. RESULTS: Metarrestin had an aqueous solubility of 150 µM at pH 7.4, high permeability in PAMPA and moderate efflux ratio in Caco-2 assays. The compound was metabolically stable in liver microsomes, S9 fractions, and hepatocytes from six species, including human. Metarrestin is a CYP3A4 substrate and, in mini-pigs, is also directly glucuronidated. Metarrestin did not show cytochrome P450 inhibitory activity. Plasma concentration-time profiles showed low to moderate clearance, ranging from 0.6 mL/min/kg in monkeys to 48 mL/min/kg in mice and moderate to high volume of distribution, ranging from 1.5 L/kg in monkeys to 17 L/kg in mice. Metarrestin has greater than 80% oral bioavailability in all species tested. The excretion of unchanged parent drug in urine was < 5% in dogs and < 1% in monkeys over collection periods of ≥ 144 h; in bile-duct cannulated rats, the excretion of unchanged drug was < 1% in urine and < 2% in bile over a collection period of 48 h. CONCLUSIONS: Metarrestin is a low clearance compound which has good bioavailability and large biodistribution after oral administration. Biotransformation appears to be the major elimination process for the parent drug. In vitro data suggest a low drug-drug interaction potential on CYP-mediated metabolism. Overall favorable ADME and PK properties support metarrestin's progression to clinical investigation.

High-throughput Chemical Screening Identifies Focal Adhesion Kinase and Aurora Kinase B Inhibition as a Synergistic Treatment Combination in Ewing Sarcoma.

PURPOSE: Ewing sarcoma is an aggressive solid tumor malignancy of childhood. Although current treatment regimens cure approximately 70% of patients with localized disease, they are ineffective for most patients with metastases or relapse. New treatment combinations are necessary for these patients. EXPERIMENTAL DESIGN: Ewing sarcoma cells are dependent on focal adhesion kinase (FAK) for growth. To identify candidate treatment combinations for Ewing sarcoma, we performed a small-molecule library screen to identify compounds synergistic with FAK inhibitors in impairing Ewing cell growth. The activity of a top-scoring class of compounds was then validated across multiple Ewing cell lines in vitro and in multiple xenograft models of Ewing sarcoma. RESULTS: Numerous Aurora kinase inhibitors scored as synergistic with FAK inhibition in this screen. We found that Aurora kinase B inhibitors were synergistic across a larger range of concentrations than Aurora kinase A inhibitors when combined with FAK inhibitors in multiple Ewing cell lines. The combination of AZD-1152, an Aurora kinase B-selective inhibitor, and PF-562271 or VS-4718, FAK-selective inhibitors, induced apoptosis in Ewing sarcoma cells at concentrations that had minimal effects on survival when cells were treated with either drug alone. We also found that the combination significantly impaired tumor progression in multiple xenograft models of Ewing sarcoma. CONCLUSIONS: FAK and Aurora kinase B inhibitors synergistically impair Ewing sarcoma cell viability and significantly inhibit tumor progression. This study provides preclinical support for the consideration of a clinical trial testing the safety and efficacy of this combination for patients with Ewing sarcoma.

Pharmacokinetic evaluation of the PNC disassembler metarrestin in wild-type and Pdx1-Cre;LSL-KrasG12D/+;Tp53R172H/+ (KPC) mice, a genetically engineered model of pancreatic cancer.

PURPOSE: Metarrestin is a first-in-class small molecule clinical candidate capable of disrupting the perinucleolar compartment, a subnuclear structure unique to metastatic cancer cells. This study aims to define the pharmacokinetic (PK) profile of metarrestin and the pharmacokinetic/pharmacodynamic relationship of metarrestin-regulated markers. METHODS: PK studies included the administration of single or multiple dose of metarrestin at 3, 10, or 25 mg/kg via intravenous (IV) injection, gavage (PO) or with chow to wild-type C57BL/6 mice and KPC mice bearing autochthonous pancreatic tumors. Metarrestin concentrations were analyzed by UPLC-MS/MS. Pharmacodynamic assays included mRNA expression profiling by RNA-seq and qRT-PCR for KPC mice. RESULTS: Metarrestin had a moderate plasma clearance of 48 mL/min/kg and a large volume of distribution of 17 L/kg at 3 mg/kg IV in C57BL/6 mice. The oral bioavailability after single-dose (SD) treatment was > 80%. In KPC mice treated with SD 25 mg/kg PO, plasma AUC0-∞ of 14400 ng h/mL, Cmax of 810 ng/mL and half-life (t1/2) of 8.5 h were observed. At 24 h after SD of 25 mg/kg PO, the intratumor concentration of metarrestin was high with a mean value of 6.2 µg/g tissue (or 13 µM), well above the cell-based IC50 of 0.4 µM. At multiple dose (MD) 25 mg/kg/day PO in KPC mice, mean tissue/plasma AUC0-24h ratio for tumor, spleen and liver was 37, 30 and 31, respectively. There was a good linear relationship of dosage to AUC0-24h and C24h. AUC0-24h MD to AUC0-24h SD ratios ranged from two for liver to five for tumor indicating additional accumulation in tumors. Dose-dependent normalization of FOXA1 and FOXO6 mRNA expression was observed in KPC tumors. CONCLUSIONS: Metarrestin is an effective therapeutic candidate with a favorable PK profile achieving excellent intratumor tissue levels in a disease with known poor drug delivery.

Preclinical Pharmacological Development of Chlorcyclizine Derivatives for the Treatment of Hepatitis C Virus Infection.

Hepatitis C virus (HCV) is a small, single-stranded, positive-sense RNA virus that infects more than an estimated 70 million people worldwide. Untreated, persistent HCV infection often results in chronic hepatitis, cirrhosis, or liver failure, with progression to hepatocellular carcinoma. Current anti-HCV regimens comprising direct acting antivirals (DAAs) can provide curative treatment; however, due to high costs there remains a need for effective, shorter-duration, and affordable treatments. Recently, we disclosed anti-HCV activity of the cheap antihistamine chlorcyclizine, targeting viral entry. Following our hit-to-lead optimization campaign, we report evaluation of preclinical in vitro absorption, distribution, metabolism, and excretion properties, and in vivo pharmacokinetic profiles of lead compounds. This led to selection of a new lead compound and evaluation of efficacy in chimeric mice engrafted with primary human hepatocytes infected with HCV. Further development and incorporation of this compound into DAA regimens has the potential to improve treatment efficacy, affordability, and accessibility.

Corrigendum: Mega-evolutionary dynamics of the adaptive radiation of birds.

This corrects the article DOI: 10.1038/nature21074.

The phosphatidylinositol-3-phosphate 5-kinase inhibitor apilimod blocks filoviral entry and infection.

Phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) is a lipid kinase involved in endosome maturation that emerged from a haploid genetic screen as being required for Ebola virus (EBOV) infection. Here we analyzed the effects of apilimod, a PIKfyve inhibitor that was reported to be well tolerated in humans in phase 2 clinical trials, for its effects on entry and infection of EBOV and Marburg virus (MARV). We first found that apilimod blocks infections by EBOV and MARV in Huh 7, Vero E6 and primary human macrophage cells, with notable potency in the macrophages (IC50, 10 nM). We next observed that similar doses of apilimod block EBOV-glycoprotein-virus like particle (VLP) entry and transcription-replication competent VLP infection, suggesting that the primary mode of action of apilimod is as an entry inhibitor, preventing release of the viral genome into the cytoplasm to initiate replication. After providing evidence that the anti-EBOV action of apilimod is via PIKfyve, we showed that it blocks trafficking of EBOV VLPs to endolysosomes containing Niemann-Pick C1 (NPC1), the intracellular receptor for EBOV. Concurrently apilimod caused VLPs to accumulate in early endosome antigen 1-positive endosomes. We did not detect any effects of apilimod on bulk endosome acidification, on the activity of cathepsins B and L, or on cholesterol export from endolysosomes. Hence by antagonizing PIKfyve, apilimod appears to block EBOV trafficking to its site of fusion and entry into the cytoplasm. Given the drug's observed anti-filoviral activity, relatively unexplored mechanism of entry inhibition, and reported tolerability in humans, we propose that apilimod be further explored as part of a therapeutic regimen to treat filoviral infections.

Mega-evolutionary dynamics of the adaptive radiation of birds.

The origin and expansion of biological diversity is regulated by both developmental trajectories and limits on available ecological niches. As lineages diversify, an early and often rapid phase of species and trait proliferation gives way to evolutionary slow-downs as new species pack into ever more densely occupied regions of ecological niche space. Small clades such as Darwin's finches demonstrate that natural selection is the driving force of adaptive radiations, but how microevolutionary processes scale up to shape the expansion of phenotypic diversity over much longer evolutionary timescales is unclear. Here we address this problem on a global scale by analysing a crowdsourced dataset of three-dimensional scanned bill morphology from more than 2,000 species. We find that bill diversity expanded early in extant avian evolutionary history, before transitioning to a phase dominated by packing of morphological space. However, this early phenotypic diversification is decoupled from temporal variation in evolutionary rate: rates of bill evolution vary among lineages but are comparatively stable through time. We find that rare, but major, discontinuities in phenotype emerge from rapid increases in rate along single branches, sometimes leading to depauperate clades with unusual bill morphologies. Despite these jumps between groups, the major axes of within-group bill-shape evolution are remarkably consistent across birds. We reveal that macroevolutionary processes underlying global-scale adaptive radiations support Darwinian and Simpsonian ideas of microevolution within adaptive zones and accelerated evolution between distinct adaptive peaks.

Detection of hepatocellular carcinoma in hepatitis C patients: biomarker discovery by LC-MS.

Hepatocellular carcinoma (HCC) accounts for most cases of liver cancer worldwide; contraction of hepatitis C (HCV) is considered a major risk factor for liver cancer even when individuals have not developed formal cirrhosis. Global, untargeted metabolic profiling methods were applied to serum samples from patients with either HCV alone or HCC (with underlying HCV). The main objective of the study was to identify metabolite based biomarkers associated with cancer risk, with the long term goal of ultimately improving early detection and prognosis. Serum global metabolite profiles from patients with HCC (n=37) and HCV (n=21) were obtained using high performance liquid chromatography-mass spectrometry (HPLC-MS) methods. The selection of statistically significant metabolites for partial least-squares discriminant analysis (PLS-DA) model creation based on biological and statistical significance was contrasted to that of a traditional approach utilizing p-values alone. A PLS-DA model created using the former approach resulted in a model with 92% sensitivity, 95% specificity, and an AUROC of 0.93. A series of PLS-DA models iteratively utilizing three to seven metabolites that were altered significantly (p<0.05) and sufficiently (FC≤0.7 or FC≥1.3) showed good performance using p-values alone; the best of these PLS-DA models was capable of generating 73% sensitivity, 95% specificity, and an AUROC of 0.92. Metabolic profiles derived from LC-MS readily distinguish patients with HCC and HCV from those with HCV only. Differences in the metabolic profiles between high-risk individuals and HCC indicate the possibility of identifying the early development of liver cancer in at risk patients. The use of biological significance as a selection process prior to PLS-DA modeling may offer improved probabilities for translation of newly discovered biomarkers to clinical application.