New method for screening anti-Leishmania compounds in plants extracts by HPTLC-bioautography.

Leishmania genus is responsible for leishmaniasis, a group of diseases affecting 12 million people in the tropical and subtropical zone. Currently, the few drugs that are available to treat this disease are expensive and cause many side effects. Searching for new therapeutics from plant species seems to be a promising path. This work proposes an original HPTLC test against parasites, in particular on Leishmania infantum, to screen new molecules from plant extracts. The technique uses protozoa transformed to express the luciferase gene to observe the bioautogram in bioluminescence. We have developed two different test protocols based on the two dimorphic stages of the parasite. The free promastigote stage, and an intracellular stage parasitizing macrophage cells called the amastigote stage. These two stages only survive under extremely different conditions which required the development of two very different test protocols. For the promastigote free stage of the protozoa, the direct bioautography technique was chosen while for the intracellular amastigote stage, bioautography by immersion (agar overlay) was required. Amphotericine B was chosen as the reference compound for this assay. The development of each of these two tests made it possible to clearly detect areas of activity on the bioautogram, allowing a rapid and inexpensive screening of the antiparasitic properties of molecules in natural extracts.

Natural trypanocidal product produced by endophytic fungi through co-culturing.

Endophytic fungi live inside vegetal tissues without causing damage to the host plant and may provide lead compounds for drug discovery. The co-culture of two or more endophytic fungi can trigger silent gene clusters, which could lead to the isolation of bioactive compounds. In this study, two endophytic strains isolated from Handroanthus impetiginosus leaves, identified as Talaromyces purpurogenus H4 and Phanerochaete sp. H2, were grown in mixed and axenic cultures. The meroterpenoid austin was detected only in the extracts from the mixed culture. Once isolated, austin displayed very interesting trypanocidal activity, with an IC50 value of 36.6 ± 1.2 µg/mL against Trypanosoma cruzi in the epimastigote form. The results obtained highlight the importance of the co-culturing of endophytic fungi to obtain natural bioactive products. The findings also enhance our understanding of the ecological relationships between endophytic fungi.

Negligible exposure to nifurtimox through breast milk during maternal treatment for Chagas Disease.

BACKGROUND: Treatment with nifurtimox (NF) for Chagas disease is discouraged during breast-feeding because no information on NF transfer into breast milk is available. NF is safe and effective for paediatric and adult Chagas disease. We evaluated the degree of NF transfer into breast milk in lactating women with Chagas disease. PATIENTS AND METHODS: Prospective study of a cohort of lactating women with Chagas disease. Patients were treated with NF for 1 month. NF was measured in plasma and milk by high performance liquid chromatography (HPLC). Breastfed infants were evaluated at admission, 7th and 30th day of treatment (and monthly thereafter, for 6 months). RESULTS: Lactating women with chronic Chagas disease (N = 10) were enrolled (median age 28 years, range 17-36). Median NF dose was 9.75 mg/kg/day three times a day (TID). Six mothers had mild adverse drug reactions (ADRs), but no ADRs were observed in any of the breastfed infants. No interruption of breastfeeding was observed. Median NF concentrations were 2.15 mg/L (Inter quartil range (IQR) 1.32-4.55) in milk and 0.30 mg/L (IQR 0.20-0.95) in plasma. Median NF milk/plasma ratio was 16 (range 8.75-30.25). Median relative infant NF dose (assuming a daily breastmilk intake of 150 mL/kg/day) was 6.7% of the maternal dose/kg/day (IQR 2.35-7.19%). CONCLUSIONS: The low concentrations of NF in breast milk and the normal clinical evaluation of the breastfed babies imply that maternal NF treatment for Chagas disease during breastfeeding is unlikely to lead to clinically relevant exposures in the breastfed infants. TRIAL REGISTRATION: Clinical trial registry name and registration number: ClinicalTrials.gov NCT01744405.

Accelerating Drug Discovery Efforts for Trypanosomatidic Infections Using an Integrated Transnational Academic Drug Discovery Platform.

According to the World Health Organization, more than 1 billion people are at risk of or are affected by neglected tropical diseases. Examples of such diseases include trypanosomiasis, which causes sleeping sickness; leishmaniasis; and Chagas disease, all of which are prevalent in Africa, South America, and India. Our aim within the New Medicines for Trypanosomatidic Infections project was to use (1) synthetic and natural product libraries, (2) screening, and (3) a preclinical absorption, distribution, metabolism, and excretion-toxicity (ADME-Tox) profiling platform to identify compounds that can enter the trypanosomatidic drug discovery value chain. The synthetic compound libraries originated from multiple scaffolds with known antiparasitic activity and natural products from the Hypha Discovery MycoDiverse natural products library. Our focus was first to employ target-based screening to identify inhibitors of the protozoan Trypanosoma brucei pteridine reductase 1 ( TbPTR1) and second to use a Trypanosoma brucei phenotypic assay that made use of the T. brucei brucei parasite to identify compounds that inhibited cell growth and caused death. Some of the compounds underwent structure-activity relationship expansion and, when appropriate, were evaluated in a preclinical ADME-Tox assay panel. This preclinical platform has led to the identification of lead-like compounds as well as validated hits in the trypanosomatidic drug discovery value chain.

The individual components of commercial isometamidium do not possess stronger trypanocidal activity than the mixture, nor bypass isometamidium resistance.

The four components present in the trypanocidal treatment Samorin, the commercially available formulation of isometamidium, were separated and purified by column chromatography. These compounds as well as the Samorin mixture and the other phenanthridine trypanocide, homidium, were tested on Trypanosoma congolense and wild type, diamidine- and isometamidium-resistant Trypanosoma brucei brucei strains using an Alamar blue drug sensitivity assay. EC50 values obtained suggest that M&B4180A (2) was the most active of the components, followed by M&B38897 (1) in all the strains tested, whereas M&B4596 (4) was inactive. Samorin was found to be significantly more active than any of the individual components alone, against T. congolense and all three T. b, brucei strains. Samorin and all its active constituents displayed reduced activity against the previously characterised isometamidium-resistant strain ISMR1.

Establishment of a quantitative and qualitative analysis and isolation method for tetracyclic iridoids from Morinda lucida Bentham leaves.

A new high performance liquid chromatography (HPLC) method has been established for quantitative and qualitative analysis of three tetracyclic iridoids: ML-2-3 (1), molucidin (2), and ML-F52 (3), which are responsible for anti-trypanosomal and anti-leishmanial activities of Morinda lucida Bentham leaves. Separation of 1-3 from dried 80% aqueous (aq.) ethanol extract was achieved on a reversed-phase cholester column packed with cholesteryl-bonded silica using an acetonitrile-0.1% aq. formic acid mobile phase system. Ultraviolet-visible (UV-VIS) spectroscopy was employed for detection of compounds, and their contents were determined by measuring absorbance at 254 nm. Depending on the above system, several factors potentially affecting the concentration of tetracyclic iridoids were evaluated resulting in several variation on plant organs, seasonality, variation between individual trees, and branch positions within the trees. Moreover, we developed a simple, quick, and effective method for tetracyclic iridoid isolation from M. lucida leaves that consisted of extraction by sonication into 80% aq. ethanol, basic hydrolysis, acid neutralization, liquid-liquid extraction into an organic solvent, and reverse phase open column chromatography. Employing this method, we have succeeded to obtain 1 as a colorless crystal yielding of 0.23%, which was 28 times higher than that of previous isolation method. Setting up methodology in this paper may be important for future in vitro and in vivo studies of tetracyclic iridoids and moreover for their applications in new drug design and development.

Drug quality analysis of isometamidium chloride hydrochloride and diminazene diaceturate used for the treatment of African animal trypanosomosis in West Africa.

BACKGROUND: Diminazene diaceturate (DA) and isometamidium chloride hydrochloride (ISM) are with homidium bromide, the main molecules used to treat African Animal Trypanosomosis (AAT). These drugs can be purchased from official suppliers but also from unofficial sources like local food markets or street vendors. The sub-standard quality of some of these trypanocides is jeopardizing the efficacy of treatment of sick livestock, leading thus to economic losses for the low-resource farmers and is contributing to the emergence and spread of drug resistance. The objective of this study was to assess the quality of trypanocidal drugs sold in French speaking countries of West Africa. In total, 308 drug samples including 282 of DA and 26 of ISM were purchased from official and unofficial sources in Benin, Burkina Faso, Côte d'Ivoire, Mali, Niger and Togo. All samples were analysed at LACOMEV (Dakar, Senegal), a reference laboratory of the World Organisation for Animal Health, by galenic inspection and high performance liquid chromatography. RESULTS: The results showed that 51.90% of the samples were non-compliant compared to the standards and were containing lower quantity of the active ingredient compared to the indications on the packaging. The non-compliances ranged from 63.27% in Togo to 32.65% in Burkina Faso (61.82% in Benin, 53.84% in Mali, 50% in Côte d'Ivoire, 47.36% in Niger). The rates of non-compliance were not statistically different (P = 0.572) from official or unofficial suppliers and ranged from 30 to 75% and from 0 to 65% respectively. However, the non-compliance was significantly higher for ISM compared to DA (P = 0.028). CONCLUSIONS: The high non-compliance revealed in this study compromises the efficacy of therapeutic strategies against AAT, and is likely to exacerbate chemoresistance in West Africa. Corrective actions against sub-standard trypanocides urgently need to be taken by policy makers and control authorities.

Photo-affinity labelling and biochemical analyses identify the target of trypanocidal simplified natural product analogues.

Current drugs to treat African sleeping sickness are inadequate and new therapies are urgently required. As part of a medicinal chemistry programme based upon the simplification of acetogenin-type ether scaffolds, we previously reported the promising trypanocidal activity of compound 1, a bis-tetrahydropyran 1,4-triazole (B-THP-T) inhibitor. This study aims to identify the protein target(s) of this class of compound in Trypanosoma brucei to understand its mode of action and aid further structural optimisation. We used compound 3, a diazirine- and alkyne-containing bi-functional photo-affinity probe analogue of our lead B-THP-T, compound 1, to identify potential targets of our lead compound in the procyclic form T. brucei. Bi-functional compound 3 was UV cross-linked to its target(s) in vivo and biotin affinity or Cy5.5 reporter tags were subsequently appended by Cu(II)-catalysed azide-alkyne cycloaddition. The biotinylated protein adducts were isolated with streptavidin affinity beads and subsequent LC-MSMS identified the FoF1-ATP synthase (mitochondrial complex V) as a potential target. This target identification was confirmed using various different approaches. We show that (i) compound 1 decreases cellular ATP levels (ii) by inhibiting oxidative phosphorylation (iii) at the FoF1-ATP synthase. Furthermore, the use of GFP-PTP-tagged subunits of the FoF1-ATP synthase, shows that our compounds bind specifically to both the α- and ß-subunits of the ATP synthase. The FoF1-ATP synthase is a target of our simplified acetogenin-type analogues. This mitochondrial complex is essential in both procyclic and bloodstream forms of T. brucei and its identification as our target will enable further inhibitor optimisation towards future drug discovery. Furthermore, the photo-affinity labeling technique described here can be readily applied to other drugs of unknown targets to identify their modes of action and facilitate more broadly therapeutic drug design in any pathogen or disease model.

Increased Body Exposure to New Anti-Trypanosomal Through Nanoencapsulation.

Lychnopholide, a lipophilic sesquiterpene lactone, is efficacious in mice at the acute and chronic phases of Chagas disease. Conventional poly-ε-caprolactone (PCL) and long-circulating poly(D,L-lactide)-block-polyethylene glycol (PLA-PEG) nanocapsules containing lychnopholide were developed and characterized. Lychnopholide presented high association efficiency (>90%) with the nanocapsules. A new, fast and simple HPLC-UV-based bioanalytical method was developed, validated in mouse plasma and applied to lychnopholide quantification in in vitro release kinetics and pharmacokinetics. The nanocapsules had mean hydrodynamic diameters in the range of 100-250 nm, negative zeta potentials (-30 mV to -57 mV), with good physical stability under storage. Atomic force microscopy morphological analysis revealed spherical monodispersed particles and the absence of lychnopholide crystallization or aggregation. Association of lychnopholide to PLA-PEG nanocapsules resulted in a 16-fold increase in body exposure, a 26-fold increase in plasma half-life and a dramatic reduction of the lychnopholide plasma clearance (17-fold) in comparison with free lychnopholide. The improved pharmacokinetic profile of lychnopholide in long-circulating nanocapsules is in agreement with the previously reported improved efficacy observed in Trypanosoma cruzi-infected mice. The present lychnopholide intravenous dosage form showed great potential for further pre-clinical and clinical studies in Chagas disease and cancer therapies.

Bioactive new metabolites from the green alga Udotea orientalis growing on the Gorgonian coral Pseudopterogorgia rigida.

As part of our continued search for bioactive secondary metabolites from marine sources using a bioassay-guided fractionation technique (Cytotoxic and anti-trypanosome activities), we have examined the organic extract of Papua New Guinean collection of the green alga Udotea orientalis growing on the Gorgonian coral Pseudopterogorgia rigida. Successive HPLC investigations resulted in isolation of three new compounds, (+) curcuepoxide A, (+) curcuepoxide B and (+)-10α-hydroxycurcudiol. Analysis of different spectroscopic data e.g. UV, IR, LRMS, HRMS, 1D NMR and 2D NMR on the isolated compounds allowed for construction of the planar structures. Stereochemistry assignment at C-7 and C-10 in the new compounds was discussed. Isolated compounds were found to be active in an in vitro assay of antitrypanosome activity. The isolated compounds were found to have variable cytotoxic activity in human lung cancer cell lines.

Simultaneous determination of benznidazole and itraconazole using spectrophotometry applied to the analysis of mixture: A tool for quality control in the development of formulations.

The aim of this work was the development of an analytical procedure using spectrophotometry for simultaneous determination of benznidazole (BNZ) and itraconazole (ITZ) in a medicine used for the treatment of Chagas disease. In order to achieve this goal, the analysis of mixtures was performed applying the Lambert-Beer law through the absorbances of BNZ and ITZ in the wavelengths 259 and 321 nm, respectively. Diverse tests were carried out for development and validation of the method, which proved to be selective, robust, linear, and precise. The lower limits of detection and quantification demonstrate its sensitivity to quantify small amounts of analytes, enabling its application for various analytical purposes, such as dissolution test and routine assays. In short, the quantification of BNZ and ITZ by analysis of mixtures had shown to be efficient and cost-effective alternative for determination of these drugs in a pharmaceutical dosage form.

Avaliação das atividades antibacteriana, tripanocida e citotóxica do extrato hidroalcóolico das raízes de Tradescantia sillamontana Matuda (Veludo Branco) (Commelinaceae) / Evaluation of antibacterial, trypanocidal and cytotoxical activities of the hydroalcoholic extract from the roots of Tradescantia sillamontana Matuda (Veludo Branco) (Commelinaceae)

RESUMO O objetivo deste estudo foi avaliar o efeito antibacteriano e tripanocida in vitro do extrato hidroalcóolico das raízes de Tradescantia sillamontana Matuda (Commelinaceae), conhecida popularmente como veludo branco. Foi avaliada a atividade antibacteriana in vitro frente às bactérias Streptococcus mitis (CIM = 100 µg/mL; CMB = 150 µg/mL), Streptococcus mutans (CIM = 200 µg/mL; CMB = 220 µg/mL), Streptococcus sanguinis (CIM = 400 µg/mL; CMB = 425 µg/mL), Streptococcus sobrinus (CIM = 400 µg/mL; CMB = 420 µg/mL) e Bacteroides fragilis (CIM = 400 µg/mL; CMB = 430 µg/mL) pelo método de diluição em caldo. Os protozoários da família tripanossomatídeo causam doenças tropicais que costumam ser negligenciadas que costumam ser como a tripanossomíase, para a qual estão disponíveis poucos medicamentos. Neste contexto, o extrato hidroalcóolico das raízes de T. sillamontana também foi avaliado frente às formas tripomastigotas da cepa Y de Trypanosoma cruzi, com promissora atividade frente a este protozoário (IC50 = 2,4 µg/mL). Quando avaliada a atividade citotóxica frente a fibroblastos da linhagem LLCMK2, o extrato apresentou moderada citotoxicidade (CC50 = 480,37 µg/mL). Os resultados ora apresentados para o extrato hidroalcóolico das raízes de Tradescantia sillamontana Matuda demonstraram promissoras atividades antibacteriana e tripanocida, sendo uma fonte alternativa de produtos naturais com atividades contra T. cruzi e algumas bactérias do gênero Streptococcus e Bacteroides.

ABSTRACT The aim of this study was to investigate the in vitro, antibacterial and trypanocidal effect of the hydroalcoholic extract from the roots of Tradescantia sillamontana Matuda (Commelinaceae), commonly known as Veludo branco. The in vitro antibacterial activity against the standard bacteria Streptococcus mitis (MIC = 100µg/mL; MBC = 150 µg/mL), Streptococcus mutans (MIC = 200µg/mL; MBC = 220 µg/mL), Streptococcus sanguinis (MIC = 400µg/mL; MBC = 425 µg/mL), Streptococcus sobrinus (MIC = 400µg/mL; MBC = 420 µg/mL) andBacteroides fragilis (MIC = 400µg/mL; MBC = 430 µg/mL), using microdilution broth methods. Protozoans from the trypanosomatid family cause neglected tropical diseases such as trypanosomiasis, for which few drugs are available. In this context, the hydroalcoholic extract of the Tradescantia sillamontana roots was also investigated with regards to the in vitro effects against the trypomastigote forms of theY strain of Trypanosoma cruzi, showing strong activity against this parasite (IC50 = 2.4 µg/mL). When performing cytotoxic activity against fibroblasts LLCMK2 line, the extract showed moderate cytotoxicity (CC50 = 480.37 mg/mL). The results presented for the hydroalcoholic extract of the roots of Tradescantia sillamontana Matuda demonstrated effective antibacterial and trypanocidal activities and were shown to be an alternative source of natural products with activity against T. cruzi and some bacteria of the genus Streptococcus and Bacteroides.

Development of an ionic-liquid-based dispersive liquid-liquid microextraction method for the determination of antichagasic drugs in human breast milk: Optimization by central composite design.

Chagas disease constitutes a major public health problem in Latin America. Human breast milk is a biological sample of great importance for the analysis of therapeutic drugs, as unwanted exposure through breast milk could result in pharmacological effects in the nursing infant. Thus, the goal of breast milk drug analysis is to inquire to which extent a neonate may be exposed to a drug during lactation. In this work, we developed an analytical technique to quantify benznidazole and nifurtimox (the two antichagasic drugs currently available for medical treatment) in human breast milk, with a simple sample pretreatment followed by an ionic-liquid-based dispersive liquid-liquid microextraction combined with high-performance liquid chromatography and UV detection. For this technique, the ionic liquid 1-octyl-3-methylimidazolium hexafluorophosphate has been used as the "extraction solvent." A central composite design was used to find the optimum values for the significant variables affecting the extraction process: volume of ionic liquid, volume of dispersant solvent, ionic strength, and pH. At the optimum working conditions, the average recoveries were 77.5 and 89.7%, the limits of detection were 0.06 and 0.09 µg/mL and the interday reproducibilities were 6.25 and 5.77% for benznidazole and nifurtimox, respectively. The proposed methodology can be considered sensitive, simple, robust, accurate, and green.

Chromatographic and spectroscopic analysis of the components present in the phenanthridinium trypanocidal agent isometamidium.

The chromatographic isolation and characterisation of the four compounds present in the quaternary phenanthridine veterinary trypanocidal agent, isometamidium chloride hydrochloride (ISM), is reported. The isolated compounds were unambiguously characterised using spectroscopic (NMR, UV, IR and MS) methods as 3-amino-8-[3-(3-carbamimidoyl-phenyl)-triazenyl]-5-ethyl-6-phenylethidium (1a) and related isomers, 8-amino-3-[3-(3-carbamimidoyl-phenyl)-triazenyl]-5-ethyl-6-phenylethidium, 3,-8-diamino-7-[3-(3-carbamimidoyl-phenyl)-triazenyl]-5-ethyl-6-phenylethidium and 3,-8-bis[3-(3-carbamimidoyl-phenyl)-triazenyl]-5-ethyl-6-phenylethidium. During the course of this study, it was realised that the nature of the solvent used in the NMR study was critical as in DMSO-d6 the quaternary group in the compounds was reduced to dihydro forms (e.g. 2a).

A simple and sensitive assay for eflornithine quantification in rat brain using pre-column derivatization and UPLC-MS/MS detection.

Eflornithine (α-difluoromethylornithine) has been used to treat second-stage (or meningoencephalitic-stage) human African trypanosomiasis and currently is under clinical development for cancer prevention. In this study, a new ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)-based assay was developed and validated for the quantification of eflornithine in rat brain. To improve chromatographic retention and MS detection, eflornithine was derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate for 5 min at room temperature prior to injection. Derivatized eflornithine was separated on a reverse-phase C18 UPLC column with a 6-min gradient; elution occurred at approximately 1.5 min. Prior to derivatization, eflornithine was reproducibly extracted from rat brain homogenate by methanol protein precipitation (~70% recovery). Derivatized eflornithine was stable in the autosampler (6 °C) for at least 24 h. This new assay had acceptable intra- and interday accuracy and precision over a wide dynamic range (5000-fold) and excellent sensitivity with a lower limit of quantification of 0.1 µm (18 ng/mL) using only 10 µL of rat brain homogenate. The validated eflornithine assay was applied successfully to determine eflornithine distribution in different regions of rat brain in an in situ rat brain perfusion study.

An image-based algorithm for precise and accurate high throughput assessment of drug activity against the human parasite Trypanosoma cruzi.

We present a customized high content (image-based) and high throughput screening algorithm for the quantification of Trypanosoma cruzi infection in host cells. Based solely on DNA staining and single-channel images, the algorithm precisely segments and identifies the nuclei and cytoplasm of mammalian host cells as well as the intracellular parasites infecting the cells. The algorithm outputs statistical parameters including the total number of cells, number of infected cells and the total number of parasites per image, the average number of parasites per infected cell, and the infection ratio (defined as the number of infected cells divided by the total number of cells). Accurate and precise estimation of these parameters allow for both quantification of compound activity against parasites, as well as the compound cytotoxicity, thus eliminating the need for an additional toxicity-assay, hereby reducing screening costs significantly. We validate the performance of the algorithm using two known drugs against T.cruzi: Benznidazole and Nifurtimox. Also, we have checked the performance of the cell detection with manual inspection of the images. Finally, from the titration of the two compounds, we confirm that the algorithm provides the expected half maximal effective concentration (EC50) of the anti-T. cruzi activity.

Reduced antimony accumulation in ARM58-overexpressing Leishmania infantum.

Antimony-based drugs are still the mainstay of chemotherapy against Leishmania infections in many countries where the parasites are endemic. The efficacy of antimonials has been compromised by increasing numbers of resistant infections, the basis of which is not fully understood and likely involves multiple factors. By using a functional cloning strategy, we recently identified a novel antimony resistance marker, ARM58, from the parasite Leishmania braziliensis that protects the parasites against antimony-based antileishmanial compounds. Here we show that the Leishmania infantum homologue also confers resistance against antimony but not against other antileishmanial drugs and that its function depends critically on one of four conserved domains of unknown function. This critical domain requires at least two hydrophobic amino acids and is predicted to form a transmembrane structure. Overexpression of ARM58 in antimony-exposed parasites reduces the intracellular Sb accumulation by over 70%, indicating a role for ARM58 in Sb extrusion pathways, but without involvement of energy-dependent transporter proteins.

A simple and efficient HPLC method for benznidazole dosage in human breast milk.

BACKGROUND: Due to migration, Chagas disease is a significant public health problem in Latin America, and in other nonendemic regions. The 2 drugs currently available for the treatment, nifurtimox and benznidazole (BNZ), are associated with a high risk of toxicity in therapeutic doses. Excretion of drug into human breast milk is a potential source of unwanted exposure and pharmacologic effects in the nursing infant. However, this phenomenon was not evaluated until now, and measurement techniques for both drugs in milk were not developed. METHODS: In this work, we described the development of a simple and fast method to quantify BNZ in human milk using a pretreatment that involves acid protein precipitation followed by tandem microfiltration, and reverse phase high-performance liquid chromatography/ultraviolet analysis. It is simple because it takes only 3 steps to obtain a clean extracted solution that is ready to inject into the high-performance liquid chromatography equipment. It is fast because a complete analysis of a sample takes only 36 minutes. RESULTS: Although the human breast milk composition is very variable, and lipids are one of the most difficult compounds to clean up on a milk sample, the procedure has proven to be robust and sensitive with a limit of detection of 0.3 µg/mL and quantization of 0.9 µg/mL. Despite a 70% recovery value, which could be considered a relatively low result, this recovery is reproducible (coefficient of variation <10%) and the analytical response under the linear range is very good (r = 0.9969 adjusted). Real samples of human breast milk from patients in treatment with BNZ were dosed to support the validation process of the method. CONCLUSIONS: The method described is fast, specific, accurate, precise, and sufficiently sensitive in the clinical context for the quantification of BNZ in human milk. For all these reasons, it is suitable for clinical risk evaluation studies.

Citotocixidade e atividade antiparasitária de Lygodium venustum SW / Cytotoxic and antiparasitic activity of Lygodium venustum SW

. Doenças parasitárias infecciosas como leishmaniose e doença de Chagas tem se difundido nas últimas décadas a locais onde antes não se observava sua ocorrência. São consideradas negligenciadas por assolarem países pobres e serem marginalizadas farmacologicamente. O tratamento não apresenta muitas opções de fármacos e estes demonstram relevante toxicidade contribuindo para o aparecimento de diversos efeitos colaterais. A pesquisa com produtos naturais tem se mostrado uma interessante alternativa para a procura por novos fármacos. Lygodium venustum é uma samambaia cosmopolita de hábito lianescente encontrada na encosta na Chapada do Araripe, considerada por algumas populações americanas como planta medicinal para o tratamento de dermatoses, infecções, micoses e tricomoníases. Neste estudo foi avaliada sua atividade anti-parasitária contra Leishmania brasiliensis e Trypanosoma cruzi, bem como sua citotoxicidade através de ensaios n vitro. Foram testadas a fração hexânica e o extrato etanólico obtido das folhas de Lygodium venustum em diferentes concentrações. Para os testes in vitro de T. cruzi, foi utilizado o clone CL-B5 e para Leishmania brasiliensis foram utilizadas formas promastigotas. O ensaio de citotoxicidade foi realizado com linhagens de fbroblastos. L. venustum não apresentou atividade antiparasitária clinicamente relevante na forma de extrato etanólico bruto nem como fração hexânica contra Leishmania. A fração hexânica apresentou uma atividade intermediária contra T. cruzi, porém a concentração de efeito moderado possui citotoxicidade máxima tornando-se inviável para aplicação clínica. Entretanto, a citoxicicidade apresentada poderá ser útil em pesquisas sobre atividade antineoplásica em células tumorais.

Infectious and parasitic diseases like leishmaniasis and Chagas disease have spreading recent decades to places not observed before. They are considered neglected by desolating poor countries and marginalized pharmacologically. There are not many options for the treatment and these drugs have shown signifcant toxicity contributing to the appearance of several side effects. Research on natural products has been shown to be an interesting alternative to the search for new drugs. Lygodium venustum is a cosmopolitan fern with latescence habit found on the Chapada do Araripe, considered by some American popula-tions as a medicinal plant for the treatment of skin diseases, infections, fungal infections and trichomoniasis. This study evaluated its antiparasitic activity against Trypanosoma cruzi and Leishmania brasiliensis, as well as its cytotoxicity through trials in vitro. We tested the ethanolic extract and hexane fraction obtained from the leaves of L. venustum at different concentrations. For in vitro tests of T. cruzi, we used the clone CL-B5 and for L. brasiliensis we used promastigotes. The cytotoxicity assay was performed with strains of fbroblasts. L.venustum showed no antiparasitic activity clinically relevant in the form of crude ethanolic extractor as the hexane fraction against Leishmania. The hexane fraction showed an intermediate activity against T.cruzi, but the concentration of moderate effect has maximum cytotoxicity becoming unfeasible for clinical application. However, the cytotoxicity presented may be useful in research on antineoplastic activity in tumor cells.

Anti-protozoal activity of aporphine and protoberberine alkaloids from Annickia kummeriae (Engl. & Diels) Setten & Maas (Annonaceae).

BACKGROUND: Malaria, trypanosomiasis and leishmaniasis have an overwhelming impact in the poorest countries in the world due to their prevalence, virulence and drug resistance ability. Currently, there is inadequate armory of drugs for the treatment of malaria, trypanosomiasis and leishmaniasis. This underscores the continuing need for the discovery and development of new anti-protozoal drugs. Consequently, there is an urgent need for research aimed at the discovery and development of new effective and safe anti-plasmodial, anti-trypanosomal and anti-leishmanial drugs. METHODS: Bioassay-guided chromatographic fractionation was employed for the isolation and purification of antiprotozoal alkaloids. RESULTS: The methanol extract from the leaves of Annickia kummeriae from Tanzania exhibited a strong anti-plasmodial activity against the multi-drug resistant Plasmodium falciparum K1 strain (IC50 0.12 ± 0.01 µg/ml, selectivity index (SI) of 250, moderate activity against Trypanosoma brucei rhodesiense STIB 900 strain (IC50 2.50 ± 0.19 µg/ml, SI 12) and mild activity against Leishmania donovani axenic MHOM-ET-67/82 strain (IC50 9.25 ± 0.54 µg/ml, SI 3.2). Bioassay-guided chromatographic fractionation led to the isolation of four pure alkaloids, lysicamine (1), trivalvone (2), palmatine (3), jatrorrhizine (4) and two sets of mixtures of jatrorrhizine (4) with columbamine (5) and palmatine (3) with (-)-tetrahydropalmatine (6). The alkaloids showed low cytotoxicity activity (CC50 30 - >90 µg/ml), strong to moderate anti-plasmodial activity (IC50 0.08 ± 0.001 - 2.4 ± 0.642 µg/ml, SI 1.5-1,154), moderate to weak anti-trypanosomal (IC50 2.80 ± 0.001 - 14.3 ± 0.001 µg/ml, SI 2.3-28.1) and anti-leishmanial activity IC50 2.7 ± 0.001 - 20.4 ± 0.003 µg/ml, SI 1.7-15.6). CONCLUSION: The strong anti-plasmodial activity makes these alkaloids good lead structures for drug development programs.