Synthesis, antimalarial activity, and preclinical pharmacology of a novel series of 4'-fluoro and 4'-chloro analogues of amodiaquine. Identification of a suitable "back-up" compound for N-tert-butyl isoquine.

On the basis of a mechanistic understanding of the toxicity of the 4-aminoquinoline amodiaquine (1b), three series of amodiaquine analogues have been prepared where the 4-aminophenol "metabolic alert" has been modified by replacement of the 4'-hydroxy group with a hydrogen, fluorine, or chlorine atom. Following antimalarial assessment and studies on mechanism of action, two candidates were selected for detailed ADME studies and in vitro and in vivo toxicological assessment. 4'-Fluoro-N-tert-butylamodiaquine (2k) was subsequently identified as a candidate for further development studies based on potent activity versus chloroquine-sensitive and resistant parasites, moderate to excellent oral bioavailability, low toxicity in in vitro studies, and an acceptable safety profile.

Candidate selection and preclinical evaluation of N-tert-butyl isoquine (GSK369796), an affordable and effective 4-aminoquinoline antimalarial for the 21st century.

N-tert-Butyl isoquine (4) (GSK369796) is a 4-aminoquinoline drug candidate selected and developed as part of a public-private partnership between academics at Liverpool, MMV, and GSK pharmaceuticals. This molecule was rationally designed based on chemical, toxicological, pharmacokinetic, and pharmacodynamic considerations and was selected based on excellent activity against Plasmodium falciparum in vitro and rodent malaria parasites in vivo. The optimized chemistry delivered this novel synthetic quinoline in a two-step procedure from cheap and readily available starting materials. The molecule has a full industry standard preclinical development program allowing first into humans to proceed. Employing chloroquine (1) and amodiaquine (2) as comparator molecules in the preclinical plan, the first preclinical dossier of pharmacokinetic, toxicity, and safety pharmacology has also been established for the 4-aminoquinoline antimalarial class. These studies have revealed preclinical liabilities that have never translated into the human experience. This has resulted in the availability of critical information to other drug development teams interested in developing antimalarials within this class.

Effect of the haematocrit layer geometry on Plasmodium falciparum static thin-layer in vitro cultures.

BACKGROUND: In vitro cultivation of Plasmodium falciparum is usually carried out through the continuous preservation of infected erythrocytes deposited in static thin layers of settled haematocrit. This technique, called the candle-jar method, was first achieved by Trager and Jensen in 1976 and has undergone slight modifications since then. However, no systematic studies concerning the geometry of the haematocrit layer have been carried out. In this work, a thorough investigation of the effects of the geometric culturing conditions on the parasite's development is presented. METHODS: Several experimental trials exploring different settings have been carried out, covering haematocrit layer depths that ranged from 6 mm to 3 mm and separation between the walls of the culturing device that ranged from 7.5 mm to 9 mm. The obtained results have been analysed and compared to different system-level models and to an Individual-Based Model. CONCLUSION: In line with the results, a mechanism governing the propagation of the infection which limits it to the vicinity of the interface between the haematocrit layer and the culture medium is deduced, and the most appropriate configurations are proposed for further experimental assays.

Two-step synthesis of achiral dispiro-1,2,4,5-tetraoxanes with outstanding antimalarial activity, low toxicity, and high-stability profiles.

A rapid, two-step synthesis of a range of dispiro-1,2,4,5-tetraoxanes with potent antimalarial activity both in vitro and in vivo has been achieved. These 1,2,4,5-tetraoxanes have been proven to be superior to 1,2,4-trioxolanes in terms of stability and to be superior to trioxane analogues in terms of both stability and activity. Selected analogues have in vitro nanomolar antimalarial activity and good oral activity and are nontoxic in screens for both cytotoxicity and genotoxicity. The synthesis of a fluorescent 7-nitrobenza-2-oxa-1,3-diazole (NBD) tagged tetraoxane probe and use of laser scanning confocal microscopy techniques have shown that tagged molecules accumulate selectively only in parasite infected erythrocytes and that intraparasitic formation of adducts could be inhibited by co-incubation with the iron chelator desferrioxamine (DFO).

Individual-based model and simulation of Plasmodium falciparum infected erythrocyte in vitro cultures.

Malaria is still one of the most fatal diseases in the world. Development of an effective treatment or vaccine requires the cultivation of the parasite that causes it: Plasmodium falciparum. Several methods for in vitro cultivation of P. falciparum infected erythrocytes have been successfully developed and described in the last 30 years. Some problems arising from the current harvests are the low parasitaemia and daily human supervision requirements. The lack of a suitable model for global culture behavior makes the assay of new methodologies a costly and tenuous task. In this paper we present a model and simulation tool for these systems. We use the INDividual DIScrete SIMulation protocol (INDISIM) to qualitatively reproduce the temporal evolution of the erythrocyte and merozoite populations. Whole system dynamics are inferred by setting the rules of behavior for each individual red blood cell, such as the nutrient uptake, metabolism and infection processes, as well as the properties and rules for the culture medium: composition, diffusion and external manipulation. We set the individual description parameters according to the values in published data, and allow population heterogeneity. Cells are arranged in a three-dimensional grid and the study is focused on the geometric constraints and physical design of experimental sets. Several published experimental cultures have been reproduced with computer simulations of this model, showing that the observed experimental behavior can be explained by means of individual interactions and statistical laws.

Preclinical drug metabolism and pharmacokinetic evaluation of GW844520, a novel anti-malarial mitochondrial electron transport inhibitor.

GW844520 is a potent and selective inhibitor of the cytochrome bc1 complex of mitochondrial electron transport in P. falciparum, the parasite primarily responsible for the mortality associated with malaria worldwide. GW844520 is fully active against the parasite including resistance isolates, showing no cross resistance with agents in use. To evaluate full potential of this development candidate, we conducted drug metabolism and pharmacokinetic studies of this novel anti-malarial. GW844520 had low blood clearance of about 0.5-4% of hepatic blood flow and a steady-state volume of distribution of 2-4 times total body water in mouse, rat, dog, and monkey. Oral bioavailability was high (51-100%). Consistent with the in vivo data, GW844520 had low intrinsic clearance in liver microsomes and hepatocytes of animal and human origin, high passive cellular permeability and was not a P-glycoprotein substrate. GW844520 did not associate appreciably with blood cells but was highly bound to plasma proteins (>99%) in all species. GW844520 was a substrate and inhibitor of human CYP2D6 but not of CYP1A2, 2C9, 2C19, and 3A4. This conjunctive analysis supports continued evaluation of this compound in definitive pre-IND studies and exemplifies our strategy supporting the discovery of novel agents to treat diseases of the developing world.

Computer-assisted enhanced volumetric segmentation magnetic resonance imaging data using a mixture of artificial neural networks.

An accurate computer-assisted method able to perform regional segmentation on 3D single modality images and measure its volume is designed using a mixture of unsupervised and supervised artificial neural networks. Firstly, an unsupervised artificial neural network is used to estimate representative textures that appear in the images. The region of interest of the resultant images is selected by means of a multi-layer perceptron after a training using a single sample slice, which contains a central portion of the 3D region of interest. The method was applied to magnetic resonance imaging data collected from an experimental acute inflammatory model (T(2) weighted) and from a clinical study of human Alzheimer's disease (T(1) weighted) to evaluate the proposed method. In the first case, a high correlation and parallelism was registered between the volumetric measurements, of the injured and healthy tissue, by the proposed method with respect to the manual measurements (r = 0.82 and p < 0.05) and to the histopathological studies (r = 0.87 and p < 0.05). The method was also applied to the clinical studies, and similar results were derived of the manual and semi-automatic volumetric measurement of both hippocampus and the corpus callosum (0.95 and 0.88).

Candida albicans infection enhances immunosuppression induced by cyclophosphamide by selective priming of suppressive myeloid progenitors for NO production.

Systemic infections caused by fungi after cytoreductive therapies are especially difficult to deal with in spite of currently available antimicrobials. However, little is known about the effects of fungi on the immune system of immunosuppressed hosts. We have addressed this by studying the in vitro T cell responses after systemic infection with Candida albicans in cyclophosphamide-treated mice. After cyclophosphamide treatment, a massive splenic colonization of the spleens, but not lymph nodes, by immature myeloid progenitor (Ly-6G(+)CD11b(+))cells is observed. These cells are able to suppress proliferation of T lymphocytes via a nitric oxide (NO)-dependent mechanism. Systemic infection with a sublethal dose of C. albicans did not cause immunosuppression per se but strongly increased NO-dependent suppression in cyclophosphamide-treated mice, by selective priming of suppressive myeloid progenitors (Ly-6G(+)CD11b(+)CD31(+)CD40(+)WGA(+)CD117(low/-)CD34(low/-)) for iNOS protein expression. The results indicate that systemic C. albicans infection can augment the effects of immunosuppressive therapies by promoting functional changes in immunosuppressive cells.

Monitoring acute inflammatory processes in mouse muscle by MR imaging and spectroscopy: a comparison with pathological results.

We have studied an animal model of acute local inflammation in muscle induced by Aspergillus fumigatus by using magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS). We have compared our data to those found using histopathology and segmentation maps obtained by the mathematical processing of three-dimensional T2-weighted MRI data via a neural network. The MRI patterns agreed satisfactorily with the clinical and biological evidence of the phases of acute local infection and its evolution towards chronicity. The MRS results show a statistically significant increase in inorganic phosphate and a significant decrease in phosphocreatine levels in the inflamed region. Image segmentation made with a self-organizing, neural-network map yielded a set of ordered representatives that remained constant for all animals during the inflammatory process, allowing a non-invasive, three-dimensional identification and quantification of the inflamed infected regions by MRI.