Avalia??o anti-Trypanosoma cruzi in vitro e in vivo de lactona sesquiterp?nica isolada de Lychnophora passerina.

Programa de P?s-Gradua??o em Ci?ncias Farmac?uticas. CIPHARMA, Escola de Farm?cia, Universidade Federal de Ouro Preto.Atualmente, a ?nica droga dispon?vel para tratar a DCh no Brasil ? o benznidazol (BZ), que apresenta v?rias limita??es e efeitos adversos, dessa forma estimulando a busca por novas op??es terap?uticas. Assim, este projeto visa o uso em solu??o da lactona sesquiterp?nica goyazensolida (GZL), previamente ativa contra o Trypanosoma cruzi in vitro, com os seguintes objetivos: (1) Determinar o limiar de citotoxicidade in vitro sobre c?lulas de mam?feros (H9c2) e avaliar sua atividade tripanocida contra amastigotas, estabelecendo seu ?ndice de seletividade; (2) Avaliar os sinais de toxicidade aguda in vivo e sua atividade frente ? infec??o experimental de T. cruzi em modelo murino. Para avaliar a citotoxicidade foram realizados os testes MTT e Vermelho Neutro (VN) e, para verificar a atividade da GZL contra o T. cruzi, c?lulas H9c2 foram infectadas com um isolado resistente da cepa Y de T. cruzi e tratadas durante 24 e 48h. Na avalia??o da toxicidade aguda in vivo, foi determinada a dose m?xima tolerada, feito o hemograma e dosagem de par?metros bioqu?micos para avalia??o da toxicidade hep?tica e renal. Para avaliar a atividade terap?utica da GZL, camundongos Swiss infectados com as cepas Y e CL de T. cruzi foram tratados pelas vias oral e intravenosa (IV) por 20 dias consecutivos nas doses de 1, 5 e 25 mg/kg/dia [via oral e IV, cepa Y (isolado resistente ao BZ) e CL (sens?vel ao BZ)] e 1, 3 e 9 mg/kg/dia IV (cepa CL), paralelamente a grupos controles BZ 100mg/kg/dia e infectado n?o tratados (INT). A parasitemia dos animais foi avaliada diariamente at? consistente negativa??o. A a??o do tratamento foi avaliada ainda por exames parasitol?gicos (hemocultura e PCR) e sorol?gico (ELISA). Os resultados obtidos revelaram a citotoxicidade in vitro da GZL em concentra??es acima de 250 ng/mL nos tratamentos de 24 e 48h avaliadas pelos testes MTT e VN, sendo o ?ndice de seletividade em 24h de 351 calculado a partir da rela??o dos par?metros de MTT e do teste de a??o tripanocida da GZL in vitro. Houve discreta nefrotoxicidade revelada pelo aumento de creatinina observado nos animais tratados com GZL 25 mg/kg IV em rela??o ao grupo CNT. Na avalia??o p?s-tratamento em animais infectados pela cepa Y, foi observada uma diminui??o significativa da parasitemia a partir do 5? dia de tratamento com GZL nas doses de 1,0 mg/kg/dia (IV e oral) e 5,0 mg/kg/dia, via oral e BZ 100 mg/kg/dia, quando comparados ao grupo controle INT. As taxas de sobreviv?ncia dos camundongos tratados com GZL foram de 100% na dose de 5,0 mg/kg/dia oral, seguidos dos tratados com 1,0 mg/kg/dia IV e BZ 100 mg/kg/dia oral, que foram de 87,5%, enquanto o grupo controle INT apresentou 25% de sobrevida. Nenhum dos camundongos foram considerados curados segundo o crit?rio de cura adotado (MS, 2016). Os animais infectados pela cepa CL e tratados com GZL demonstraram no 180? dia p?s-tratamento uma diminui??o significativa da parasitemia a partir do primeiro dia do PP em rela??o ao grupo INT, por?m inferior ao observado no grupo tratado com BZ. A maioria dos grupos tratados n?o apresentou 100% de negatividade na HC e PCR simultaneamente, entretanto o grupo tratado com BZ apresentou 57,1% de cura com sobrevida de 87,5%, seguido por GZL 3 mg/kg/dia IV que curou 12,5% dos animais com sobrevida de 100%. Este resultado, em particular, aliado aos demais, representam uma boa perspectiva do uso da GZL para o tratamento da DCh a ser explorada.Currently, benznidazole (BZ) is the only drug available to treat Chagas disease (CD) in Brazil, which presents several limitations and adverse effects, thus stimulating the search for new therapeutic options. This project aims the use in solution, of a sesquiterpene goyazensolide lactone (GZL), previously active against Trypanosoma cruzi in vitro, with the following objectives: (1) To determine the threshold of in vitro cytotoxicity on mammalian cells (H9c2) and to evaluate its trypanocidal activity against amastigotes for establishment of its index of selectivity; (2) To evaluate the signs of acute toxicity in vivo and its activity against the experimental infection of T. cruzi in the murine model. To evaluate cytotoxicity, the MTT and Neutral Red (NR) tests were performed and, to verify the activity of GZL against T. cruzi, H9c2 cells were infected with T. cruzi Y strain (resistant isolate to BZ) and treated for 24 and 48h. In the evaluation of acute toxicity in vivo, the maximum tolerated dose was determined, in parallel to blood count and measures of biochemical parameters to evaluate hepatic and renal toxicity. To evaluate the therapeutic activity of GZL, Swiss mice, infected with T. cruzi Y \9isolate resistant to BZ) and CL (sensitive to BZ) strains, were treated by oral and intravenous routes for 20 consecutive days at doses of 1, 5 and 25 mg/kg/day (oral and IV, Y and CL strains) and 1, 3 and 9 mg/kg/day IV (CL strain), in parallel to BZ 100mg/kg/day and infected not treated groups (INT). The parasitemia of the animals was evaluated daily until consistent negativation. The treatment action was also evaluated by parasitological tests (blood culture and PCR) and serological (ELISA) tests. The results obtained revealed in vitro toxicity of GZL at doses above 250ng/mL in H9c2 cells treated for 24 and 48h and evaluated by the MTT and NR tests, the selectivity index being 351, calculated from the ratio of MTT/trypanocidal action test of GZL in vitro. It was verified only discrete nephrotoxicity revealed by the slight increase in creatinine observed in the animals treated with GZL 25 mg/kg/day IV in relation to the CNT group. In the post-treatment evaluation in Y-infected animals a significant decrease of the parasitaemia was observed from the 5th day in the GZL treatment at doses of 1.0 mg/kg/ day (IV and oral) and 5.0 mg/kg/day, oral and BZ 100 mg/kg/day, when compared to the INT control group. The survival rates of the GZL-treated mice were 100% at the 5.0 mg/kg/day oral, followed by those treated with 1.0 mg/kg/day IV and 100 mg/kg/day oral BZ, which were of 87.5%, while the control group INT presented 25% of survival. None of the mice were considered cured according to the (MS, 2016) criteria. Animals infected with CL strain and treated with GZL demonstrated 180th days post-treatment a significant decrease in parasitemia since the first day of PP when compared to the INT group, but lower than that observed in the group treated with BZ. The majority of the treated groups were not 100% negative in HC e PCR simultaneously, except GZL 5 mg/kg/day oral and 5 mg/kg/day IV, infected with Y strain. In the treatment of animals infected with the CL strain, BZ presented 57.1% of cure with survival of 87.5%, followed by GZL 3 mg/kg/day IV, which cured 12.5% of the animals with 100% survival. This result, in particular, together to the others, represent a good perspective of the use of GZL for the treatment of CD to be further explored

Efficacy of Lychnopholide polymeric nanocapsules after oral and intravenous administration in murine experimental Chagas disease.

The etiological treatment of Chagas disease remains neglected. The compounds available show several limitations, mainly during the chronic phase. Lychnopholide encapsulated in polymeric nanocapsules (LYC-NC) was efficacious in mice infected with Trypanosoma cruzi and treated by intravenous administration during the acute phase (AP). As the oral route is preferred for treatment of chronic infections, such as Chagas disease, this study evaluated the use of oral LYC-NC in the AP and also compared it with LYC-NC administered to mice by the oral and intravenous routes during the chronic phase (CP). The therapeutic efficacy was evaluated by fresh blood examination, hemoculture, PCR, and enzyme-linked immunosorbent assay (ELISA). The cure rates in the AP and CP were 62.5% and 55.6%, respectively, upon oral administration of LYC?poly(D,L-lactide)?polyethylene glycol nanocapsules (LYC-PLA-PEG-NC) and 57.0% and 30.0%, respectively, with LYC?poly- -caprolactone nanocapsules (LYC-PCL-NC). These cure rates were significantly higher than that of free LYC, which did not cure any animals. LYC-NC formulations administered orally during the AP showed cure rates similar to that of benznidazole, but only LYC-NC cured mice in the CP. Similar results were achieved with intravenous treatment during the CP. The higher cure rates obtained with LYC loaded in PLA-PEG-NC may be due to the smaller particle size of these NC and the presence of PEG, which influence tissue diffusion and the controlled release of LYC. Furthermore, PLA-PEG-NC may improve the stability of the drug in the gastrointestinal tract. This work is the first report of cure of experimental Chagas disease via oral administration during the CP. These findings represent a new and important perspective for oral treatment of Chagas disease

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-?-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

Participation of Central Muscarinic Receptors on the Nervous Form of Chagas Disease in Mice Infected via Intracerebroventricular with Colombian Trypanosoma cruzi Strain

Acute chagasic encephalitis is a clinically severe central nervous system (CNS) manifestation. However, the knowledge of the nervous form of Chagas disease is incomplete. The role of the muscarinic acetylcholine receptor (mAChR) on mice behavior and brain lesions induced by Trypanosoma cruzi (Colombian strain) was herein investigated in mice treated with the mAChR agonist and antagonist (carbachol and atropine), respectively. Immunosuppressed or non-immunosuppressed mice were intracerebroventricularly (icv) or intraperitoneally (ip) infected. All groups were evaluated 15 d.p.i. (days post infection). Intraperitoneally infected animals had subpatent parasitemia. Patent parasitemia occurred only in icv infected mice. The blockade of mAChR increased the parasitemia, parasitism and lesions compared to its activation. Infected not treated (INT ip) mice did not present meningitis and encephalitis, regardless of immunosuppression. INT icv brains presented higher cellularity, discrete signs of cellular degeneration, frequent presence of parasites and focal meningitis. The immunosuppressed atropine + icv mice presented increased intracellular parasitism associated with degenerative parenchymal changes, while carbachol + icv mice presented discrete meningitis, preservation of the cortex and absence of relevant parasitism. Cholinergic receptor blockage increased impairment of coordination vs. receptor activation. Muscarinic cholinergic pathway seems to be involved in immune mediated cell invasion events while its blockade favored infection evolution, brain lesions, and behavioral alterations

Activity of the sesquiterpene lactone goyazensolide against Trypanosoma cruzi in vitro and in vivo.

Background: The current drugs for Chagas disease treatment present several limitations Methods: The sesquiterpene lactone goyazensolide (GZL) was evaluated regarding to cytotoxicity and trypanocidal activity against amastigotes, selectivity index (SI) in vitro, acute toxicity and anti-Trypanosoma cruzi activity in vivo. Results: The in vitro cytotoxicity in H9c2 cells was observed at doses >250 ng mL?1 of GZL and the SI were of 52.82 and 4.85 (24 h) and of 915.00 and 41.00 (48 h) for GZL and BZ, respectively. Nephrotoxicity and hepatotoxicity were not verified. Treatment with GZL of mice infected with CL strain led to a significant decrease of parasitaemia and total survival at doses of 1 and 3 mg kg?1 day?1 by oral and IV, respectively. This last group cured 12.5% of the animals (negativation of HC, PCR, qPCR and ELISA). Animals infected with Y strain showed significant decrease of parasitaemia and higher negativation in all parasitological tests in comparison to BZ and control groups, but were ELISA reactive, as well as the BZ group, but mice treated with 5.0 mg kg?1 day?1 by oral were negative in parasitological tests and survived. Conclusion: GZL was more active against T. cruzi than benznidazole in vitro and presented important therapeutic activity in vivo in both T. cruzi strains

Automatic detection of the parasite Trypanosoma cruzi in blood smears using a machine learning approach applied to mobile phone images

Chagas disease is a life-threatening illness caused by the parasite Trypanosoma cruzi. The diagnosis of the acute form of the disease is performed by trained microscopists who detect parasites in blood smear samples. Since this method requires a dedicated high-resolution camera system attached to the microscope, the diagnostic method is more expensive and often prohibitive for low-income settings. Here, we present a machine learning approach based on a random forest (RF) algorithm for the detection and counting of T. cruzi trypomastigotes in mobile phone images. We analyzed micrographs of blood smear samples that were acquired using a mobile device camera capable of capturing images in a resolution of 12 megapixels. We extracted a set of features that describe morphometric parameters (geometry and curvature), as well as color, and texture measurements of 1,314 parasites. The features were divided into train and test sets (4:1) and classified using the RF algorithm. The values of precision, sensitivity, and area under the receiver operating characteristic (ROC) curve of the proposed method were 87.6%, 90.5%, and 0.942, respectively. Automating image analysis acquired with a mobile device is a viable alternative for reducing costs and gaining efficiency in the use of the optical microscope