An in vitro toolbox to accelerate anti-malarial drug discovery and development.

BACKGROUND: Modelling and simulation are being increasingly utilized to support the discovery and development of new anti-malarial drugs. These approaches require reliable in vitro data for physicochemical properties, permeability, binding, intrinsic clearance and cytochrome P450 inhibition. This work was conducted to generate an in vitro data toolbox using standardized methods for a set of 45 anti-malarial drugs and to assess changes in physicochemical properties in relation to changing target product and candidate profiles. METHODS: Ionization constants were determined by potentiometric titration and partition coefficients were measured using a shake-flask method. Solubility was assessed in biorelevant media and permeability coefficients and efflux ratios were determined using Caco-2 cell monolayers. Binding to plasma and media proteins was measured using either ultracentrifugation or rapid equilibrium dialysis. Metabolic stability and cytochrome P450 inhibition were assessed using human liver microsomes. Sample analysis was conducted by LC-MS/MS. RESULTS: Both solubility and fraction unbound decreased, and permeability and unbound intrinsic clearance increased, with increasing Log D7.4. In general, development compounds were somewhat more lipophilic than legacy drugs. For many compounds, permeability and protein binding were challenging to assess and both required the use of experimental conditions that minimized the impact of non-specific binding. Intrinsic clearance in human liver microsomes was varied across the data set and several compounds exhibited no measurable substrate loss under the conditions used. Inhibition of cytochrome P450 enzymes was minimal for most compounds. CONCLUSIONS: This is the first data set to describe in vitro properties for 45 legacy and development anti-malarial drugs. The studies identified several practical methodological issues common to many of the more lipophilic compounds and highlighted areas which require more work to customize experimental conditions for compounds being designed to meet the new target product profiles. The dataset will be a valuable tool for malaria researchers aiming to develop PBPK models for the prediction of human PK properties and/or drug-drug interactions. Furthermore, generation of this comprehensive data set within a single laboratory allows direct comparison of properties across a large dataset and evaluation of changing property trends that have occurred over time with changing target product and candidate profiles.

Potential Diagnostic Imaging of Alzheimer's Disease with Copper-64 Complexes That Bind to Amyloid-ß Plaques.

Amyloid-ß plaques, consisting of aggregated amyloid-ß peptides, are one of the pathological hallmarks of Alzheimer's disease. Copper complexes formed using positron-emitting copper radionuclides that cross the blood-brain barrier and bind to specific molecular targets offer the possibility of noninvasive diagnostic imaging using positron emission tomography. New thiosemicarbazone-pyridylhydrazone based ligands that incorporate pyridyl-benzofuran functional groups designed to bind amyloid-ß plaques have been synthesized. The ligands form stable complexes with copper(II) ( Kd = 10-18 M) and can be radiolabeled with copper-64 at room temperature. Subtle changes to the periphery of the ligand backbone alter the metabolic stability of the complexes in mouse and human liver microsomes, and influenced the ability of the complexes to cross the blood-brain barrier in mice. A lead complex was selected based on possessing the best metabolic stability and brain uptake in mice. Synthesis of this lead complex with isotopically enriched copper-65 allowed us to show that the complex bound to amyloid-ß plaques present in post-mortem human brain tissue using laser ablation-inductively coupled plasma-mass spectrometry. This work provides insight into strategies to target metal complexes to amyloid-ß plaques, and how small modifications to ligands can dramatically alter the metabolic stability of metal complexes as well as their ability to cross the blood-brain barrier.

Using Human Plasma as an Assay Medium in Caco-2 Studies Improves Mass Balance for Lipophilic Compounds.

PURPOSE: To examine the utility of human plasma as an assay medium in Caco-2 permeability studies to overcome poor mass balance and inadequate sink conditions frequently encountered with lipophilic compounds. METHODS: Caco-2 permeability was assessed for reference compounds with known transport mechanisms using either pH 7.4 buffer or human plasma as the assay medium in both the apical and basolateral chambers. When using plasma, Papp values were corrected for the unbound fraction in the donor chamber. The utility of the approach was assessed by measuring the permeability of selected antimalarial compounds using the two assay media. RESULTS: Caco-2 cell monolayer integrity and P-gp transporter function were unaffected by the presence of human plasma in the donor and acceptor chambers. For many of the reference compounds having good mass balance with buffer as the medium, higher Papp values were observed with plasma, likely due to improved acceptor sink conditions. The lipophilic antimalarial compounds exhibited low mass balance with buffer, however the use of plasma markedly improved mass balance allowing the determination of more reliable Papp values. CONCLUSIONS: The results support the utility of human plasma as an alternate Caco-2 assay medium to improve mass balance and permeability measurements for lipophilic compounds.

Evidence for the in vitro bioactivation of aminopyrazole derivatives: trapping reactive aminopyrazole intermediates using glutathione ethyl ester in human liver microsomes.

Drug-induced toxicity is a leading cause of drug withdrawal from clinical development and clinical use and represents a major impediment to the development of new drugs. The mechanisms underlying drug-induced toxicities are varied; however, metabolic bioactivation to form reactive metabolites has been identified as a major contributor.1,2 These electrophilic species can covalently modify important biological macromolecules and thereby increase the risk of adverse drug reactions or idiosyncratic toxicity. Consequently, screening compounds for their propensity to form reactive metabolites has become an integral part of drug discovery programs. This screening process typically involves identification of structural alerts as well as the generation of reactive metabolites in vitro in subcellular hepatic fractions, followed by trapping the reactive species with nucleophiles and characterization via LC-MS. This article presents evidence for the bioactivation of a series of aminopyrazole derivatives via LC-MS detection of glutathione ethyl ester-trapped reactive intermediates formed in human liver microsomal incubations. These results indicate that the aminopyrazole motif, within specific contexts, may be considered a new structural alert for the potential formation of reactive metabolites.

Synthetic ozonide drug candidate OZ439 offers new hope for a single-dose cure of uncomplicated malaria.

Ozonide OZ439 is a synthetic peroxide antimalarial drug candidate designed to provide a single-dose oral cure in humans. OZ439 has successfully completed Phase I clinical trials, where it was shown to be safe at doses up to 1,600 mg and is currently undergoing Phase IIa trials in malaria patients. Herein, we describe the discovery of OZ439 and the exceptional antimalarial and pharmacokinetic properties that led to its selection as a clinical drug development candidate. In vitro, OZ439 is fast-acting against all asexual erythrocytic Plasmodium falciparum stages with IC(50) values comparable to those for the clinically used artemisinin derivatives. Unlike all other synthetic peroxides and semisynthetic artemisinin derivatives, OZ439 completely cures Plasmodium berghei-infected mice with a single oral dose of 20 mg/kg and exhibits prophylactic activity superior to that of the benchmark chemoprophylactic agent, mefloquine. Compared with other peroxide-containing antimalarial agents, such as the artemisinin derivatives and the first-generation ozonide OZ277, OZ439 exhibits a substantial increase in the pharmacokinetic half-life and blood concentration versus time profile in three preclinical species. The outstanding efficacy and prolonged blood concentrations of OZ439 are the result of a design strategy that stabilizes the intrinsically unstable pharmacophoric peroxide bond, thereby reducing clearance yet maintaining the necessary Fe(II)-reactivity to elicit parasite death.

Enduring musician advantage among former musicians in prosodic pitch perception.

Musical training has been associated with various cognitive benefits, one of which is enhanced speech perception. However, most findings have been based on musicians taking part in ongoing music lessons and practice. This study thus sought to determine whether the musician advantage in pitch perception in the language domain extends to individuals who have ceased musical training and practice. To this end, adult active musicians (n = 22), former musicians (n = 27), and non-musicians (n = 47) were presented with sentences spoken in a native language, English, and a foreign language, French. The final words of the sentences were either prosodically congruous (spoken at normal pitch height), weakly incongruous (pitch was increased by 25%), or strongly incongruous (pitch was increased by 110%). Results of the pitch discrimination task revealed that although active musicians outperformed former musicians, former musicians outperformed non-musicians in the weakly incongruous condition. The findings suggest that the musician advantage in pitch perception in speech is retained to some extent even after musical training and practice is discontinued.

Native Language Perceptual Sensitivity Predicts Nonnative Speech Perception Differently in Younger and Older Singaporean Bilinguals.

PURPOSE: We investigate in this study how individual variability in native language speech perception (termed Perceptual Sensitivity) influences nonnative speech perception in Singaporean Tamil-English bilinguals. Further, we assess if and how contextual and demographic factors influence Perceptual Sensitivity in the acquired languages and if the influence of Perceptual Sensitivity on nonnative speech perception is different across younger and older bilinguals. METHOD: Perceptual Sensitivity in the native languages was examined by implementing Tamil and English gating tasks in 87 Singaporean Tamil-English bilinguals from two age groups (younger: 19-33 years; older: 55-70 years). Mandarin lexical tone discrimination was implemented as a measure of nonnative speech perception. RESULTS: There was a wide range of variability in Perceptual Sensitivity scores in both languages across both age groups. Perceptual Sensitivity in the first native language (L1 Tamil) was a robust predictor of nonnative speech perception across both age groups, especially for the older bilinguals. However, general intelligence emerged as a stronger predictor than Tamil Perceptual Sensitivity in younger bilinguals. The influence of Tamil Perceptual Sensitivity on lexical tone perception was not tone-specific, supporting a general enhancement of lexical tone perception with better Tamil Perceptual Sensitivity. There was an influence of demographic factors on English Perceptual Sensitivity in older bilinguals, but not for Tamil and not in younger bilinguals. CONCLUSIONS: Our findings corroborate with previous studies in showing that native language Perceptual Sensitivity is positively associated with and predicts nonnative speech perception in younger and older adulthood regardless of language similarity but to varying degrees. Specifically, the influence of Perceptual Sensitivity on nonnative speech perception is stronger in older adulthood, suggesting a possible shift in reliance on crystallized language knowledge with age. Proficiency and use, among other demographic and language variables, do not appear to influence L1 Perceptual Sensitivity in a lesser used language (Tamil) as significantly as previously assumed.

Effects of Auditory Training in Older Adults.

PURPOSE: This study examines the effects of an auditory training program on the auditory and cognitive abilities of older adults. Auditory rehabilitation programs are generally designed for hearing aid users, and studies have demonstrated benefits for them. In this study, we seek to understand whether such a training program can also benefit older adults who do not wear hearing aids. We also examined if cognitive benefits can indeed be observed as a result of the training. METHOD: Sixty-four older adults were recruited and assigned into three groups: the experimental group (n = 20), the active control group (n = 21), and the no-training control group (n = 23). The experimental group underwent an auditory training program (Listening and Communication Enhancement [LACE]) during the training phase. Meanwhile, the active control group listened to short audio clips and the no-training control group did not participate in any program. An auditory test (Quick Speech-in-Noise [QuickSIN]) and a battery of cognitive tests were conducted before and after the training to examine the participants' performance on auditory ability, short-term memory, and attention. RESULTS: The results showed improvements in auditory and cognitive abilities during the training period. When assessing the training effects by comparing the pre- and the posttraining performances, a significant improvement on the QuickSIN task was found in the training group but not in the other two groups. However, other cognitive tests did not show any significant improvement. That is, the LACE training did not benefit short-term memory and attention. The improved performance on short-term memory during the training was not maintained in the posttraining session. CONCLUSION: Overall, the study has extended the auditory benefit from the LACE training to the typical aging population in terms of improved communication ability, but the effect of training on auditory abilities did not transfer to gains in cognitive abilities.

White matter anisotropy in the ventral language pathway predicts sound-to-word learning success.

According to the dual stream model of auditory language processing, the dorsal stream is responsible for mapping sound to articulation and the ventral stream plays the role of mapping sound to meaning. Most researchers agree that the arcuate fasciculus (AF) is the neuroanatomical correlate of the dorsal steam; however, less is known about what constitutes the ventral one. Nevertheless, two hypotheses exist: one suggests that the segment of the AF that terminates in middle temporal gyrus corresponds to the ventral stream, and the other suggests that it is the extreme capsule that underlies this sound-to-meaning pathway. The goal of this study was to evaluate these two competing hypotheses. We trained participants with a sound-to-word learning paradigm in which they learned to use a foreign phonetic contrast for signaling word meaning. Using diffusion tensor imaging, a brain-imaging tool to investigate white matter connectivity in humans, we found that fractional anisotropy in the left parietal-temporal region positively correlated with the performance in sound-to-word learning. In addition, fiber tracking revealed a ventral pathway, composed of the extreme capsule and the inferior longitudinal fasciculus, that mediated auditory comprehension. Our findings provide converging evidence supporting the importance of the ventral steam, an extreme capsule system, in the frontal-temporal language network. Implications for current models of speech processing are also discussed.

Individual differences in nonnative lexical tone perception: Effects of tone language repertoire and musical experience.

This study sought to understand the effects of tone language repertoire and musical experience on nonnative lexical tone perception and production. Thirty-one participants completed a tone discrimination task, an imitation task, and a musical abilities task. Results showed that a larger tone language repertoire and musical experience both enhanced tone discrimination performance. However, the effects were not additive, as musical experience was associated with tone discrimination performance for single-tone language speakers, but such association was not seen for dual-tone language speakers. Furthermore, among single-tone language speakers, but not among dual-tone language speakers, musical experience and musical aptitude positively correlated with tone discrimination accuracy. It is thus concluded that individuals with varying extents of tone language experience may adopt different strategies when performing tone discrimination tasks; single-tone language speakers may draw on their musical expertise while dual-tone language speakers may rely on their extensive tone language experience instead.

Metabolic, Pharmacokinetic, and Activity Profile of the Liver Stage Antimalarial (RC-12).

The catechol derivative RC-12 (WR 27653) (1) is one of the few non-8-aminoquinolines with good activity against hypnozoites in the gold-standard Plasmodium cynomolgi-rhesus monkey (Macaca mulatta) model, but in a small clinical trial, it had no efficacy against Plasmodium vivax hypnozoites. In an attempt to better understand the pharmacokinetic and pharmacodynamic profile of 1 and to identify potential active metabolites, we now describe the phase I metabolism, rat pharmacokinetics, and in vitro liver-stage activity of 1 and its metabolites. Compound 1 had a distinct metabolic profile in human vs monkey liver microsomes, and the data suggested that the O-desmethyl, combined O-desmethyl/N-desethyl, and N,N-didesethyl metabolites (or a combination thereof) could potentially account for the superior liver stage antimalarial efficacy of 1 in rhesus monkeys vs that seen in humans. Indeed, the rate of metabolism was considerably lower in human liver microsomes in comparison to rhesus monkey microsomes, as was the formation of the combined O-desmethyl/N-desethyl metabolite, which was the only metabolite tested that had any activity against liver-stage P. vivax; however, it was not consistently active against liver-stage P. cynomolgi. As 1 and all but one of its identified Phase I metabolites had no in vitro activity against P. vivax or P. cynomolgi liver-stage malaria parasites, we suggest that there may be additional unidentified active metabolites of 1 or that the exposure of 1 achieved in the reported unsuccessful clinical trial of this drug candidate was insufficient to kill the P. vivax hypnozoites.

Effect of spirometry and medical review on asthma control in patients in general practice: a randomized controlled trial.

BACKGROUND AND OBJECTIVE: Although guidelines for asthma emphasize the importance of spirometry for continuity and evaluation of care, it is underused in general practice. The objective of this study was to investigate the effect of spirometry and medical review on asthma control in general practice over 12 months. METHODS: Patients were recruited through 31 practices, which were randomly allocated to one of three groups: Group A had 3-monthly spirometry with medical review, Group B spirometry only before and after the trial, and Group C usual care. Asthma control data were analysed by intention to treat using non-parametric tests and logistic regression models fitted to allow for confounders, repeated measures and clustering by practice. RESULTS: The trial was completed by 195 patients (Group A 69, Group B 78, Group C 48). Asthma control improved in all groups during the 12 months trial, most impressively in Group A (odds ratio per 3 months = 1.27, 95% confidence interval: 1.08-1.49, P = 0.004), but the difference between the groups' respective 3-monthly changes was not significant. At 6 months, asthma control in Group A had increased more from baseline than in Groups B + C (P = 0.006). CONCLUSIONS: Regular spirometry with medical review was associated with improved asthma control in general practice patients, while there was less improvement in either the spirometry only or usual care group. The mechanisms of this improvement may include appropriate adjustment of medication and improved compliance.

CpG methylation potentiates pixantrone and doxorubicin-induced DNA damage and is a marker of drug sensitivity.

DNA methylation is an epigenetic modification of the mammalian genome that occurs predominantly at cytosine residues of the CpG dinucleotide. Following formaldehyde activation, pixantrone alkylates DNA and particularly favours the CpG motif. Aberrations in CpG methylation patterns are a feature of most cancer types, a characteristic that may determine their susceptibility to specific drug treatments. Given their common target, DNA methylation may modulate the DNA damage induced by formaldehyde-activated pixantrone. In vitro transcription, mass spectrometry and oligonucleotide band shift assays were utilized to establish that pixantrone-DNA adduct formation was consistently enhanced 2-5-fold at discrete methylated CpG doublets. The methylation-mediated enhancement was exquisitely sensitive to the position of the methyl substituent since methylation at neighboring cytosine residues failed to confer an increase in pixantrone-DNA alkylation. Covalent modification of DNA by formaldehyde-activated doxorubicin, but not cisplatin, was augmented by neighbouring CpG methylation, indicating that modulation of binding by CpG methylation is not a general feature of all alkylators. HCT116 colon cancer cells vastly deficient in CpG methylation were 12- and 10-fold more resistant to pixantrone and doxorubicin relative to the wild-type line, suggesting that these drugs may selectively recognize the aberrant CpG methylation profiles characteristic of most tumour types.

Cytochrome P450-Mediated Metabolism and CYP Inhibition for the Synthetic Peroxide Antimalarial OZ439.

OZ439 is a potent synthetic ozonide evaluated for the treatment of uncomplicated malaria. The metabolite profile of OZ439 was characterized in vitro using human liver microsomes combined with LC/MS-MS, chemical derivatization, and metabolite synthesis. The primary biotransformations were monohydroxylation at the three distal carbon atoms of the spiroadamantane substructure, with minor contributions from N-oxidation of the morpholine nitrogen and deethylation cleavage of the morpholine ring. Secondary transformations resulted in the formation of dihydroxylation metabolites and metabolites containing both monohydroxylation and morpholine N-oxidation. With the exception of two minor metabolites, none of the other metabolites had appreciable antimalarial activity. Reaction phenotyping indicated that CYP3A4 is the enzyme responsible for the metabolism of OZ439, and it was found to inhibit CYP3A via both direct and mechanism-based inhibition. Elucidation of the metabolic pathways and kinetics will assist with efforts to predict potential metabolic drug-drug interactions and support physiologically based pharmacokinetic (PBPK) modeling.

Discovery and Structure-Activity Relationships of Quinazolinone-2-carboxamide Derivatives as Novel Orally Efficacious Antimalarials.

A phenotypic high-throughput screen allowed discovery of quinazolinone-2-carboxamide derivatives as a novel antimalarial scaffold. Structure-activity relationship studies led to identification of a potent inhibitor 19f, 95-fold more potent than the original hit compound, active against laboratory-resistant strains of malaria. Profiling of 19f suggested a fast in vitro killing profile. In vivo activity in a murine model of human malaria in a dose-dependent manner constitutes a concomitant benefit.

The comparative antimalarial properties of weak base and neutral synthetic ozonides.

Thirty-three N-acyl 1,2,4-dispiro trioxolanes (secondary ozonides) were synthesized. For these ozonides, weak base functional groups were not required for high antimalarial potency against Plasmodium falciparum in vitro, but were necessary for high antimalarial efficacy in Plasmodium berghei-infected mice. A wide range of LogP/D(pH)(7.4) values were tolerated, although more lipophilic ozonides tended to be less metabolically stable.

Identification of an antimalarial synthetic trioxolane drug development candidate.

The discovery of artemisinin more than 30 years ago provided a completely new antimalarial structural prototype; that is, a molecule with a pharmacophoric peroxide bond in a unique 1,2,4-trioxane heterocycle. Available evidence suggests that artemisinin and related peroxidic antimalarial drugs exert their parasiticidal activity subsequent to reductive activation by haem, released as a result of haemoglobin digestion by the malaria-causing parasite. This irreversible redox reaction produces carbon-centred free radicals, leading to alkylation of haem and proteins (enzymes), one of which--the sarcoplasmic-endoplasmic reticulum ATPase PfATP6 (ref. 7)--may be critical to parasite survival. Notably, there is no evidence of drug resistance to any member of the artemisinin family of drugs. The chemotherapy of malaria has benefited greatly from the semi-synthetic artemisinins artemether and artesunate as they rapidly reduce parasite burden, have good therapeutic indices and provide for successful treatment outcomes. However, as a drug class, the artemisinins suffer from chemical (semi-synthetic availability, purity and cost), biopharmaceutical (poor bioavailability and limiting pharmacokinetics) and treatment (non-compliance with long treatment regimens and recrudescence) issues that limit their therapeutic potential. Here we describe how a synthetic peroxide antimalarial drug development candidate was identified in a collaborative drug discovery project.

Structure-Activity Relationship of Antischistosomal Ozonide Carboxylic Acids.

Semisynthetic artemisinins and other bioactive peroxides are best known for their powerful antimalarial activities, and they also show substantial activity against schistosomes-another hemoglobin-degrading pathogen. Building on this discovery, we now describe the initial structure-activity relationship (SAR) of antischistosomal ozonide carboxylic acids OZ418 (2) and OZ165 (3). Irrespective of lipophilicity, these ozonide weak acids have relatively low aqueous solubilities and high protein binding values. Ozonides with para-substituted carboxymethoxy and N-benzylglycine substituents had high antischistosomal efficacies. It was possible to increase solubility, decrease protein binding, and maintain the high antischistosomal activity in mice infected with juvenile and adult Schistosoma mansoni by incorporating a weak base functional group in these compounds. In some cases, adding polar functional groups and heteroatoms to the spiroadamantane substructure increased the solubility and metabolic stability, but in all cases decreased the antischistosomal activity.

Lead Optimization of a Pyrrole-Based Dihydroorotate Dehydrogenase Inhibitor Series for the Treatment of Malaria.

Malaria puts at risk nearly half the world's population and causes high mortality in sub-Saharan Africa, while drug resistance threatens current therapies. The pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH) is a validated target for malaria treatment based on our finding that triazolopyrimidine DSM265 (1) showed efficacy in clinical studies. Herein, we describe optimization of a pyrrole-based series identified using a target-based DHODH screen. Compounds with nanomolar potency versus Plasmodium DHODH and Plasmodium parasites were identified with good pharmacological properties. X-ray studies showed that the pyrroles bind an alternative enzyme conformation from 1 leading to improved species selectivity versus mammalian enzymes and equivalent activity on Plasmodium falciparum and Plasmodium vivax DHODH. The best lead DSM502 (37) showed in vivo efficacy at similar levels of blood exposure to 1, although metabolic stability was reduced. Overall, the pyrrole-based DHODH inhibitors provide an attractive alternative scaffold for the development of new antimalarial compounds.

Stability of peroxide antimalarials in the presence of human hemoglobin.

Peroxide antimalarials, including artemisinin, are important for the treatment of multidrug-resistant malaria. These peroxides are known to react with iron or heme to produce reactive intermediates that are thought to be responsible for their antimalarial activities. This study investigated the potential interaction of selected peroxide antimalarials with oxyhemoglobin, the most abundant form of iron in the human body. The observed stability of artemisinin derivatives and 1,2,4-trioxolanes in the presence of oxyhemoglobin was in contrast to previous reports in the literature. Spectroscopic analysis of hemoglobin found it to be unstable under the conditions used for previous studies, and it appears likely that the artemisinin reactivity reported in these studies may be attributed to free heme released by protein denaturation. The stability of peroxide antimalarials with intact oxyhemoglobin, and reactivity with free heme, may explain the selective toxicity of these antimalarials toward infected, but not healthy, erythrocytes.