29 research outputs found
Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis.
Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades the central nervous system (CNS). In vivo study of the late stage has been limited as traditional methodologies require the removal of the brain to determine the presence of the parasites. Bioluminescence imaging is a non-invasive, highly sensitive form of optical imaging that enables the visualization of a luciferase-transfected pathogen in real-time. By using a transfected trypanosome strain that has the ability to produce late stage disease in mice we are able to study the kinetics of a CNS infection in a single animal throughout the course of infection, as well as observe the movement and dissemination of a systemic infection. Here we describe a robust protocol to study CNS infections using a bioluminescence model of African trypanosomiasis, providing real time non-invasive observations which can be further analyzed with optional downstream approaches
Bioluminescence imaging of chronic Trypanosoma cruzi infections reveals tissue-specific parasite dynamics and heart disease in the absence of locally persistent infection.
Chronic Trypanosoma cruzi infections lead to cardiomyopathy in 20-30% of cases. A causal link between cardiac infection and pathology has been difficult to establish because of a lack of robust methods to detect scarce, focally distributed parasites within tissues. We developed a highly sensitive bioluminescence imaging system based on T. cruzi expressing a novel luciferase that emits tissue-penetrating orange-red light. This enabled long-term serial evaluation of parasite burdens in individual mice with an in vivo limit of detection of significantly less than 1000 parasites. Parasite distributions during chronic infections were highly focal and spatiotemporally dynamic, but did not localize to the heart. End-point ex vivo bioluminescence imaging allowed tissue-specific quantification of parasite loads with minimal sampling bias. During chronic infections, the gastro-intestinal tract, specifically the colon and stomach, was the only site where T. cruzi infection was consistently observed. Quantitative PCR-inferred parasite loads correlated with ex vivo bioluminescence and confirmed the gut as the parasite reservoir. Chronically infected mice developed myocarditis and cardiac fibrosis, despite the absence of locally persistent parasites. These data identify the gut as a permissive niche for long-term T. cruzi infection and show that canonical features of Chagas disease can occur without continual myocardium-specific infection
Design, Synthesis and Antitrypanosomal Activities of 2,6-Disubstituted-4,5,7-Trifluorobenzothiophenes
Current treatments for Human African Trypanosomiasis (HAT) are limited in their application, have undesirable dosing regimens and unsatisfactory toxicities highlighting the need for the development of a safer drug pipeline. Our medicinal chemistry programme in developing rapidly accessible and modifiable heterocyclic scaffolds led to the design and synthesis of novel substituted benzothiophenes, with 6-benzimidazol-1-ylbenzothiophene derivatives demonstrating significant antitrypanosomal activities (IC50 <1 µM) against Trypanosoma brucei rhodesiense and no toxicity towards mammalian cells
Dose-dependent effect and pharmacokinetics of fexinidazole and its metabolites in a mouse model of human African trypanosomiasis.
Human African trypanosomiasis (HAT) is a neglected tropical disease, with a population of 70 million at risk. Current treatment options are limited. In the search for new therapeutics, the repurposing of the broad-spectrum antiprotozoal drug fexinidazole has completed Phase III trials with the anticipation that it will be the first oral treatment for HAT. This study used the recently validated bioluminescence imaging model to assess the dose and rate of kill effect of fexinidazole in infected mice, and the dose-dependent effect of fexinidazole on trypanosome infection. Pharmacokinetics of fexinidazole in plasma and central nervous system (CNS) compartments were similar in both infected and uninfected mice. Drug distribution within the CNS was further examined by microdialysis, showing similar levels in the cortex and hippocampus. However, high variability in drug distribution and exposure was found between mice
Design, synthesis and antitrypanosomal activities of 2,6-disubstituted-4,5,7-Trifluorobenzothiophenes
Current treatments for Human African Trypanosomiasis (HAT) are limited in their application, have undesirable dosing regimens and unsatisfactory toxicities highlighting the need for the development of a safer drug pipeline. Our medicinal chemistry programme in developing rapidly accessible and modifiable heterocyclic scaffolds led to the design and synthesis of novel substituted benzothiophenes, with 6-benzimidazol-1-ylbenzothiophene derivatives demonstrating significant antitrypanosomal activities (IC50 <1 μM) against Trypanosoma brucei rhodesiense and no toxicity towards mammalian cells
Drug reformulation for a neglected disease. The NANOHAT project to develop a safer more effective sleeping sickness drug.
BACKGROUND: Human African trypanosomiasis (HAT or sleeping sickness) is caused by the parasite Trypanosoma brucei sspp. The disease has two stages, a haemolymphatic stage after the bite of an infected tsetse fly, followed by a central nervous system stage where the parasite penetrates the brain, causing death if untreated. Treatment is stage-specific, due to the blood-brain barrier, with less toxic drugs such as pentamidine used to treat stage 1. The objective of our research programme was to develop an intravenous formulation of pentamidine which increases CNS exposure by some 10-100 fold, leading to efficacy against a model of stage 2 HAT. This target candidate profile is in line with drugs for neglected diseases inititative recommendations. METHODOLOGY: To do this, we evaluated the physicochemical and structural characteristics of formulations of pentamidine with Pluronic micelles (triblock-copolymers of polyethylene-oxide and polypropylene oxide), selected candidates for efficacy and toxicity evaluation in vitro, quantified pentamidine CNS delivery of a sub-set of formulations in vitro and in vivo, and progressed one pentamidine-Pluronic formulation for further evaluation using an in vivo single dose brain penetration study. PRINCIPAL FINDINGS: Screening pentamidine against 40 CNS targets did not reveal any major neurotoxicity concerns, however, pentamidine had a high affinity for the imidazoline2 receptor. The reduction in insulin secretion in MIN6 β-cells by pentamidine may be secondary to pentamidine-mediated activation of β-cell imidazoline receptors and impairment of cell viability. Pluronic F68 (0.01%w/v)-pentamidine formulation had a similar inhibitory effect on insulin secretion as pentamidine alone and an additive trypanocidal effect in vitro. However, all Pluronics tested (P85, P105 and F68) did not significantly enhance brain exposure of pentamidine. SIGNIFICANCE: These results are relevant to further developing block-copolymers as nanocarriers, improving BBB drug penetration and understanding the side effects of pentamidine
Design, synthesis and antitrypanosomal activities of 2,6-disubstituted-4,5,7-trifluorobenzothiophenes
A sensitive and reproducible in vivo imaging mouse model for evaluation of drugs against late-stage human African trypanosomiasis
Objectives To optimize the Trypanosoma brucei brucei GVR35 VSL-2 bioluminescent strain as an innovative drug evaluation model for late-stage human African trypanosomiasis.<p></p>
Methods An IVIS® Lumina II imaging system was used to detect bioluminescent T. b. brucei GVR35 parasites in mice to evaluate parasite localization and disease progression. Drug treatment was assessed using qualitative bioluminescence imaging and real-time quantitative PCR (qPCR).<p></p>
Results We have shown that drug dose–response can be evaluated using bioluminescence imaging and confirmed quantification of tissue parasite load using qPCR. The model was also able to detect drug relapse earlier than the traditional blood film detection and even in the absence of any detectable peripheral parasites.<p></p>
Conclusions We have developed and optimized a new, efficient method to evaluate novel anti-trypanosomal drugs in vivo and reduce the current 180 day drug relapse experiment to a 90 day model. The non-invasive in vivo imaging model reduces the time required to assess preclinical efficacy of new anti-trypanosomal drugs.<p></p>
Tb927.10.12940 has opposing effects on apoL1 and NHS sensitivity, and localises to the lysosome.
<p>Representative EC<sub>50</sub> assays carried out in quadruplicate showing the impact of Tb927.10.12940 RNAi knockdown on <i>T</i>. <i>b</i>. <i>brucei</i> sensitivity to (A) apoL1 and (B) NHS. Inset charts show pooled EC<sub>50</sub> data for three independent cell lines. Error bars, standard deviation; <i>P</i>-values derived from paired students t-test. (C) Western blot showing tetracycline (tet)-inducible expression of Tb927.10.12940<sup>GFP</sup>. (D) Immunofluorescence localisation of Tb927.10.12940<sup>GFP</sup> and the lysosomal membrane protein, p67; counter-staining with the DNA intercalating dye, DAPI, reveals the kinetoplast (k) and nucleus (n). A processed merged image of the region of interest shows the co-localisation of Tb927.10.12940<sup>GFP</sup> and p67. Scale bar, 5 μm.</p
TbKIFC1 depletion complements NHS sensitivity of <i>12940</i> null <i>T</i>. <i>b</i>. <i>brucei</i>, but has no additive effect on apoL1 sensitivity or suramin efficacy.
<p>Representative EC<sub>50</sub> assays carried out in quadruplicate showing the impact of TbKIFC1 RNAi knockdown in wild type (left hand panels) and <i>12940</i> null (middle panels) <i>T</i>. <i>b</i>. <i>brucei</i> on (A) apoL1, (B) NHS and (C) suramin sensitivity. Right hand panels, pooled EC<sub>50</sub> data for at least three independent cell lines. Error bars, standard deviation.</p