Effect of sample size and P-value filtering techniques on the detection of transcriptional changes induced in rat neuroblastoma (NG108) cells by mefloquine

BACKGROUND: There is no known biochemical basis for the adverse neurological events attributed to mefloquine. Identification of genes modulated by toxic agents using microarrays may provide sufficient information to generate hypotheses regarding their mode of action. However, this utility may be compromised if sample sizes are too low or the filtering methods used to identify differentially expressed genes are inappropriate. METHODS: The transcriptional changes induced in rat neuroblastoma cells by a physiological dose of mefloquine (10 micro-molar) were investigated using Affymetrix arrays. A large sample size was used (total of 16 arrays). Genes were ranked by P-value (t-test). RT-PCR was used to confirm (or reject) the expression changes of several of the genes with the lowest P-values. Different P-value filtering methods were compared in terms of their ability to detect these differentially expressed genes. A retrospective power analysis was then performed to determine whether the use of lower sample sizes might also have detected those genes with altered transcription. RESULTS: Based on RT-PCR, mefloquine upregulated cJun, IkappaB and GADD153. Reverse Holm-Bonferroni P-value filtering was superior to other methods in terms of maximizing detection of differentially expressed genes but not those with unaltered expression. Reduction of total microarray sample size (< 10) impaired the capacity to detect differentially expressed genes. CONCLUSIONS: Adequate sample sizes and appropriate selection of P-value filtering methods are essential for the reliable detection of differentially expressed genes. The changes in gene expression induced by mefloquine suggest that the ER might be a neuronal target of the drug

Clinical development of new prophylactic antimalarial drugs after the 5th Amendment to the Declaration of Helsinki

Malaria is of continuing concern in nonimmune traveling populations. Traditionally, antimalarial drugs have been developed as agents for dual indications (treatment and prophylaxis). However, since 2000, when the 5th Amendment to the Declaration of Helsinki (DH2000) was adopted, development of new malaria prophylaxis drugs in this manner has ceased. As a consequence, there may not be any new drugs licensed for this indication in the foreseeable future. Major pharmaceutical companies have interpreted DH2000 to mean that the traditional development paradigm may be considered unethical because of doubt over the likelihood of benefit to endemic populations participating in clinical studies, the use of placebo, and the sustainability of post-trial access to study medications. In this article, we explore the basis of these concerns and suggest that the traditional development paradigm remains ethical under certain circumstances. We also consider alternative approaches that may be more attractive to sponsors as they either do not use placebo, or utilize populations in endemic countries who may unambiguously benefit. These approaches represent the way forward in the future, but are at present unproven in clinical practice, and face numerous regulatory, logistical and technical challenges. Consequently, in the short term, we argue that the traditional clinical development paradigm remains the most feasible approach and is ethical and consistent with the spirit of DH2000 under the appropriate circumstances

Altered drug susceptibility during host adaptation of a <i>Plasmodium falciparum</i> strain in a non-human primate model

Infections with Plasmodium falciparum, the most pathogenic of the Plasmodium species affecting man, have been reduced in part due to artemisinin-based combination therapies. However, artemisinin resistant parasites have recently emerged in South-East Asia. Novel intervention strategies are therefore urgently needed to maintain the current momentum for control and elimination of this disease. In the present study we characterize the phenotypic and genetic properties of the multi drug resistant (MDR) P. falciparum Thai C2A parasite strain in the non-human Aotus primate model, and across multiple passages. Aotus infections with C2A failed to clear upon oral artesunate and mefloquine treatment alone or in combination, and ex vivo drug assays demonstrated reduction in drug susceptibility profiles in later Aotus passages. Further analysis revealed mutations in the pfcrt and pfdhfr loci and increased parasite multiplication rate (PMR) across passages, despite elevated pfmdr1 copy number. Altogether our experiments suggest alterations in parasite population structure and increased fitness during Aotus adaptation. We also present data of early treatment failures with an oral artemisinin combination therapy in a pre-artemisinin resistant P. falciparum Thai isolate in this animal model

Radical curative efficacy of tafenoquine combination regimens in Plasmodium cynomolgi-infected Rhesus monkeys (Macaca mulatta)

<p>Abstract</p> <p>Background</p> <p>Tafenoquine is an 8-aminoquinoline being developed for radical cure (blood and liver stage elimination) of <it>Plasmodium vivax</it>. During monotherapy treatment, the compound exhibits slow parasite and fever clearance times, and toxicity in glucose-6-phosphate dehydrogenase (G6PD) deficiency is a concern. Combination with other antimalarials may mitigate these concerns.</p> <p>Methods</p> <p>In 2005, the radical curative efficacy of tafenoquine combinations was investigated in <it>Plasmodium cynomolgi</it>-infected naïve Indian-origin Rhesus monkeys. In the first cohort, groups of two monkeys were treated with a three-day regimen of tafenoquine at different doses alone and in combination with a three-day chloroquine regimen to determine the minimum curative dose (MCD). In the second cohort, the radical curative efficacy of a single-day regimen of tafenoquine-mefloquine was compared to that of two three-day regimens comprising tafenoquine at its MCD with chloroquine or artemether-lumefantrine in groups of six monkeys. In a final cohort, the efficacy of the MCD of tafenoquine against hypnozoites alone and in combination with chloroquine was investigated in groups of six monkeys after quinine pre-treatment to eliminate asexual parasites. Plasma tafenoquine, chloroquine and desethylchloroquine concentrations were determined by LC-MS in order to compare doses of the drugs to those used clinically in humans.</p> <p>Results</p> <p>The total MCD of tafenoquine required in combination regimens for radical cure was ten-fold lower (1.8 mg/kg <it>versus </it>18 mg/kg) than for monotherapy. This regimen (1.8 mg/kg) was equally efficacious as monotherapy or in combination with chloroquine after quinine pre-treatment to eliminate asexual stages. The same dose of (1.8 mg/kg) was radically curative in combination with artemether-lumefantrine. Tafenoquine was also radically curative when combined with mefloquine. The MCD of tafenoquine monotherapy for radical cure (18 mg/kg) appears to be biologically equivalent to a 600-1200 mg dose in humans. At its MCD in combination with blood schizonticidal drugs (1.8 mg/kg), the maximum observed plasma concentrations were substantially lower than (20-84 <it>versus </it>550-1,100 ng/ml) after administration of 1, 200 mg in clinical studies.</p> <p>Conclusions</p> <p>Ten-fold lower clinical doses of tafenoquine than used in prior studies may be effective against <it>P. vivax </it>hypnozoites if the drug is deployed in combination with effective blood-schizonticidal drugs.</p

Healing in the Sámi North

There is a special emphasis today on integrating traditional healing within health services. However, most areas in which there is a system of traditional healing have undergone colonization and a number of pressures suppressing tradition for hundreds of years. The question arises as to how one can understand today’s tradition in light of earlier traditions. This article is based on material collected in Sámi areas of Finnmark and Nord-Troms Norway; it compares local healing traditions with what is known of earlier shamanic traditions in the area. The study is based on 27 interviews among healers and their patients. The findings suggest that although local healing traditions among the Sámi in northern Norway have undergone major transformations during the last several hundred years, they may be considered an extension of a long-standing tradition with deep roots in the region. Of special interest are also the new forms tradition may take in today’s changing global society

Comparative Gene Expression Profiling of P. falciparum Malaria Parasites Exposed to Three Different Histone Deacetylase Inhibitors

Histone deacetylase (HDAC) inhibitors are being intensively pursued as potential new drugs for a range of diseases, including malaria. HDAC inhibitors are also important tools for the study of epigenetic mechanisms, transcriptional control, and other important cellular processes. In this study the effects of three structurally related antimalarial HDAC inhibitors on P. falciparum malaria parasite gene expression were compared. The three hydroxamate-based compounds, trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA; Vorinostat®) and a 2-aminosuberic acid derivative (2-ASA-9), all caused profound transcriptional effects, with ∼2–21% of genes having >2-fold altered expression following 2 h exposure to the compounds. Only two genes, alpha tubulin II and a hydrolase, were up-regulated by all three compounds after 2 h exposure in all biological replicates examined. The transcriptional changes observed after 2 h exposure to HDAC inhibitors were found to be largely transitory, with only 1–5% of genes being regulated after removing the compounds and culturing for a further 2 h. Despite some structural similarity, the three inhibitors caused quite diverse transcriptional effects, possibly reflecting subtle differences in mode of action or cellular distribution. This dataset represents an important contribution to our understanding of how HDAC inhibitors act on malaria parasites and identifies alpha tubulin II as a potential transcriptional marker of HDAC inhibition in malaria parasites that may be able to be exploited for future development of HDAC inhibitors as new antimalarial agents

The theory of the firm and its critics: a stocktaking and assessment

Includes bibliographical references."Prepared for Jean-Michel Glachant and Eric Brousseau, eds. New Institutional Economics: A Textbook, Cambridge, Cambridge University Press.""This version: August 22, 2005."Since its emergence in the 1970s the modern economic or Coasian theory of the firm has been discussed and challenged by sociologists, heterodox economists, management scholars, and other critics. This chapter reviews and assesses these critiques, focusing on behavioral issues (bounded rationality and motivation), process (including path dependence and the selection argument), entrepreneurship, and the challenge from knowledge-based theories of the firm

Development and application of a rat malaria model to investigate the pharmacology of microtubule antagonists as antimalarials

Malaria, caused by Plasmodium spp. is a disease responsible for significant morbidity and mortality around the world. Resistance of malaria parasites to conventional antimalarial drugs is rapidly leading to a lack of useful antimalarial compounds. There is an urgent need to develop new antimalarial drugs which exploit novel parasite targets. Microtubules represent a potential drug target in malaria because of their essential role in maintenance of cellular integrity and parasite replication. Albendazole is a benzimidazole anthelmintic used in human and veterinary medicine whilst trifluralin is a dinitroaniline herbicide. Both compounds are microtubule antagonists, and have activity against malaria parasites in vitro. The purpose of the present study was to evaluate their potential usefulness as antimalarial drugs using a P. berghei-rat model of malaria. This approach enabled simultaneous analysis of toxicology, pharmacokinetics, in vitro and in vivo efficacy data with respect to the same host-parasite system. In in vivo studies, albendazole exhibited antimalarial activity against P. berghei in rats after oral administration to rats at higher dose rates than those used in humans. Pharmacokinetic and in vitro drug screening studies indicated that albendazole and its key metabolites exhibited no activity against P. berghei at concentrations detectable in blood. Toxicology studies found that at effective dose rates, albendazole treatment impaired haematopoietic responses to malaria and depleted young red blood cells essential for P. berghei proliferation. These data suggested an indirect mode of action for albendazole against P. berghei. Trifluralin exhibited negligible in vivo activity against P. berghei after oral administration to rats, even when given at the maximum dose rates tolerated by the host. However in vitro studies indicated that trifluralin and related dinitroaniline herbicides exhibited activity against P. berghei at high nanomolar to low micromolar concentrations. The discrepancy between these data was resolved following a pharmacokinetic evaluation, in which plasma trifluralin concentrations were found to be below those necessary for antimalarial activity in vitro. Due to the indirect mode of action of albendazole, and the poor absorption of trifluralin it is unlikely that these compounds will be used clinically as antimalarials. Other benzimidazole derivatives currently in use are less potent antimalarial agents than albendazole and are thus not realistic candidates. However dinitroaniline herbicides such as oryzalin retain their antimalarial activity in vitro and have significantly better pharmacokinetics than trifluralin and may prove to be more useful antimalarials in vivo