Assessment of a pretomanid analogue library for African trypanosomiasis: Hit-to-lead studies on 6-substituted 2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]thiazine 8-oxides.

A 900 compound nitroimidazole-based library derived from our pretomanid backup program with TB Alliance was screened for utility against human African trypanosomiasis (HAT) by the Drugs for Neglected Diseases initiative. Potent hits included 2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]thiazine 8-oxides, which surprisingly displayed good metabolic stability and excellent cell permeability. Following comprehensive mouse pharmacokinetic assessments on four hits and determination of the most active chiral form, a thiazine oxide counterpart of pretomanid (24) was identified as the best lead. With once daily oral dosing, this compound delivered complete cures in an acute infection mouse model of HAT and increased survival times in a stage 2 model, implying the need for more prolonged CNS exposure. In preliminary SAR findings, antitrypanosomal activity was reduced by removal of the benzylic methylene but enhanced through a phenylpyridine-based side chain, providing important direction for future studies.

Antimicrobial Assessment of Resins from Calophyllum Antillanum and Calophyllum Inophyllum.

The Calophyllum genus is well-known for its antimicrobial and cytotoxic activities, and therefore, we analyzed these biological activities for resins of Calophyllum antillanum and Calophyllum inophyllum growing in Cuba. C. antillanum resins showed a potent activity against Plasmodium falciparum (IC50 = 0.3 ± 0.1 µg/mL), while its cytotoxicity against MRC-5 cells was much lower (IC50 = 21.6 ± 1.1 µg/mL). In contrary, the resin of C. inophyllum showed an unspecific activity. The presence of apetalic acid, isoapetalic acid, calolongic acid, pinetoric acid I, pinetoric acid II, isocalolongic acid, pinetoric acid III, and isopinetoric acid III in C. antillanum resins was also confirmed. These results demonstrated for the first time the potential activity of C. antillanum resins against P. falciparum.

Flow cytometric enumeration of bacteria using TO-PRO®-3 iodide as a single-stain viability dye.

Quantification of bacteria using conventional viable plate counting (VPC) is labor-intensive and time-consuming. Flow cytometry (FCM) can be proposed as a faster alternative. This study aimed to develop a flow cytometric, single-stain approach using TO-PRO®-3 iodide (TP3) for the quantification of Staphylococcus aureus, Escherichia coli, and Bacillus subtilis cells. Live or dead bacterial suspensions were stained with TP3 and analyzed using a FACSCalibur flow cytometer. After optimization of staining parameters and instrument settings, an excellent separation of viable and dead cells was achieved for all species. The quantitative performance of the technique was assessed by analyzing serial dilutions of bacterial suspensions using FCM and VPC. A highly linear correlation (r2 > 0.99) was observed between the colony forming units (CFU)/mL as determined by FCM and by VPC over a concentration range of about 104 to 108 CFU/mL. As such, FCM quantification of viable bacteria using TP3 can be considered as an accurate and reliable alternative for VPC. The monostain procedure is easy to apply and cost-effective, and it allows bacterial enumeration in a broad variety of samples.

Assessment of antimicrobial and antiprotozoal activity of the olive oil macerate samples of Hypericum perforatum and their LC-DAD-MS analyses.

Twenty-one samples of traditionally-prepared (home-made) and ready-made (commercial) St. John's Wort olive oil macerates were profiled for their in vitro antimicrobial and antiprotozoal activity. Their cytotoxic potential was evaluated on MRC-5 fibroblasts. In the antiprotozoal assays, ten of the oils inhibited Trypanosoma brucei rhodesiense (IC(50) 15.9-64.5 µg/mL), while only one oil exerted antimicrobial activity towards Staphylococcus aureus (IC(50)=88.7 µg/mL). LC-DAD-MS data revealed the presence of pseudohypericin (0.135-3.280 µg/g) and hypericin (0.277-6.634 µg/g) in all the oils, whereas chlorogenic acid (1.063 µg/g) was detected only in one oil sample. Hyperforin was detected in four (0.977-2.399 µg/g) and adhyperforin in six samples (0.005-3.165 µg/g). Hypericin and pseudohypericin were common in the active oils, whereas hyperforin, adhyperforin, and chlorogenic acid were absent in these samples. Our results indicated that if the correct plant material is used, the infused oils from Hypericum perforatum may contain active components.

Validation of a simple resazurin-based promastigote assay for the routine monitoring of miltefosine susceptibility in clinical isolates of Leishmania donovani.

Simple, cost-effective approach for routine surveillance of parasite susceptibility to antileishmanial drug miltefosine (MIL) is highly desirable for controlling emergence of drug resistance in visceral leishmaniasis (VL). We validated a simple resazurin-based fluorimetric assay using promastigotes to track natural MIL tolerance in Leishmania donovani parasites from VL cases (n = 17) against standard amastigote assay, in two different labs in India. The inter-stage MIL susceptibility correlated strongly (r = 0.70, p = 0.0018) using J774.A.1 macrophage cell line-based amastigote assay and fluorescence-based resazurin assay for promastigotes. Investigation of inter-stage MIL susceptibility for the same set of clinical isolates in another lab also showed a strong correlation (r = 0.72, p = 0.0012) using mouse peritoneal macrophages for amastigote assay and resazurin-based alamar blue assay for promastigotes. Additionally, parasites from post-kala-azar dermal leishmaniasis (PKDL) lesions (n = 7, r = 0.78, p = 0.046) and MIL-induced parasites (r = 0.92, p = 0.0001; n = 3) also exhibited a strongly correlated inter-stage miltefosine susceptibility. Thus, our results support the utility of resazurin assay as a simplified biological tool for MIL susceptibility monitoring in clinical isolates from MIL-treated VL/PKDL patients.

Assessment of the in vitro antiprotozoal and cytotoxic potential of 20 selected medicinal plants from the island of Soqotra.

Malaria, leishmaniasis and human African trypanosomiasis continue to be major public health problems in need of new and more effective drugs. The aim of this study was to evaluate in vitro antiprotozoal activity of twenty endemic medicinal plants collected from the island of Soqotra in the Indian Ocean. The plant materials were extracted with methanol and tested for antiplasmodial activity against erythrocytic schizonts of Plasmodium falciparum, for antileishmanial activity against intracellular amastigotes of Leishmania infantum and for antitrypanosomal activity against intracellular amastigotes of Trypanosoma cruzi and free trypomastigotes of T. brucei. To assess selectivity, cytotoxicity was determined against MRC-5 fibroblasts. Selective activity was obtained for Punica protopunica against Plasmodium (IC50 2.2 µg/mL) while Eureiandra balfourii and Hypoestes pubescens displayed activity against the three kinetoplastid parasites (IC50 < 10 µg/mL). Acridocarpus socotranus showed activity against T. brucei and T. cruzi (IC50 3.5 and 8.4 µg/mL). Ballochia atrovirgata, Dendrosicycos socotrana, Dracaena cinnabari and Euphorbia socotrana displayed non-specific inhibition of the parasites related to high cytotoxicity.

In vitro antimicrobial assessment of Cuban propolis extracts

Propolis is a resinous mixture of different plant exudates collected by honeybees. Currently, propolis is widely used as a food supplement and in folk medicine. We have evaluated 20 Cuban propolis extracts of different chemical types, brown (BCP), red and yellow (YCP), with respect to their in vitro antibacterial, antifungal and antiprotozoal properties. The extracts inhibited the growth of Staphylococcus aureus and Trichophyton rubrum at low µg/mL concentrations, whereas they were not active against Escherichia coli and Candida albicans. The major activity of the extracts was found against the protozoa Leishmania, Trypanosoma and Plasmodium, although cytotoxicity against MRC-5 cells was also observed. The BCP-3, YCP-39 and YCP-60 extracts showed the highest activity against P. falciparum, with 50% of microbial growth (IC50) values of 0.2 µg/mL. A positive correlation between the biological activity and the chemical composition was observed for YCP extracts. The most promising antimicrobial activity corresponds to YCP subtype B, which contains acetyl triterpenes as the main constituents. The present in vitro study highlights the potential of propolis against protozoa, but further research is needed to increase selectivity towards the parasite. The observed chemical composition-activity relationship of propolis can contribute to the identification of the active principles and standardisation of this bee product.

In vitro antimicrobial assessment of Cuban propolis extracts.

Propolis is a resinous mixture of different plant exudates collected by honeybees. Currently, propolis is widely used as a food supplement and in folk medicine. We have evaluated 20 Cuban propolis extracts of different chemical types, brown (BCP), red and yellow (YCP), with respect to their in vitro antibacterial, antifungal and antiprotozoal properties. The extracts inhibited the growth of Staphylococcus aureus and Trichophyton rubrum at low µg/mL concentrations, whereas they were not active against Escherichia coli and Candida albicans. The major activity of the extracts was found against the protozoa Leishmania, Trypanosoma and Plasmodium, although cytotoxicity against MRC-5 cells was also observed. The BCP-3, YCP-39 and YCP-60 extracts showed the highest activity against P. falciparum, with 50% of microbial growth (IC50) values of 0.2 µg/mL. A positive correlation between the biological activity and the chemical composition was observed for YCP extracts. The most promising antimicrobial activity corresponds to YCP subtype B, which contains acetyl triterpenes as the main constituents. The present in vitro study highlights the potential of propolis against protozoa, but further research is needed to increase selectivity towards the parasite. The observed chemical composition-activity relationship of propolis can contribute to the identification of the active principles and standardisation of this bee product.