The superior efficacy of chloroquine over buparvaquone in reducing the chronic cerebral Toxoplasma gondii cysts load and improving the ultrastructural pathology in an immunocompromised murine model

@#Toxoplasma gondii, the etiologic agent of toxoplasmosis, infects about 30 – 50% of the world population. The currently available anti-Toxoplasma agents have serious limitations. The present study aimed to investigate the effects of two antimalarials; buparvaquone (BPQ) and chloroquine (CQ), on immunocompromised mice with chronic cerebral toxoplasmosis, using spiramycin as a reference drug. The assessed parameters included the estimation of mortality rates (MR) among mice of the different study groups, in addition to the examination of the ultrastructural changes in the brain tissues by transmission electron microscopy. The results showed that only CQ treatment could decrease the MR significantly with zero deaths, while both spiramycin and BPQ caused an insignificant reduction of MR compared to the infected non-treated group. All the used drugs decreased the number of mature ruptured cysts significantly compared to the infected non-treated group, while only CQ increased the number of atrophic and necrotic cysts significantly. Furthermore, both spiramycin and BPQ improved the microvasculopathy and neurodegeneration accompanying the infection with different degrees of reactive astrocytosis and neuronal damage with the best results regarding the repair of the microvascular damage with less active glial cells, and normal neurons in the CQ-treated group. In conclusion, this study sheds light on CQ and its excellent impact on treating chronic cerebral toxoplasmosis in an immunocompromised mouse model.

Efficacy of Arteether Against Theileriosis in Cattle

The present study was done with the objective of comparative efficacy of arteether and buparvaquone against theileriosis in cattle. Total 67 cattle suspected for theileriosis were screened on the basis of clinical and blood smear examination. Group I (n=6) was treated with Inj. buparvaquone @ 2.5 mg/kg body weight once and Group II (n=6) was treated with Inj. arteether @5 mg/kg body weight intramuscularly for three consecutive days. Rectal temperature, heart rate and respiration rate were significantly increased before treatment and in both the groups after treatment showed significant improvement. Haematological parameters showed significant decreased in Hb, PCV, TEC, TLC and neutrophil and significant increase in lymphocytes, monocytes and eosinophil. Non significant difference in basophil count was observed. After treatment, significant improvement was observed in mean hemoglobin, PCV, TEC, TLC, neutrophil, lymphocyte and eosinophil. Non significant improvement was observed in monocyte and basophil count. Present study revealed percent efficacy of arteether was 66.66% and buparvaquone 100%

Buparvaquone nanostructured lipid carrier development: physicochemical and in vitro leishmanicidal performances / Desenvolvimento de carreadores lipídicos nanoestruturados contendo buparvaquona: caracterização físico-química e avaliação da atividade leishmanicida in vitro

Leishmaniases is a group of diseases caused by parasites of the genus Leishmania. The estimated number of deaths from visceral leishmaniases ranges from 20,000 to 50,000 annually. The most common treatment over the past 60 years has been pentavalent antimonials. Besides the doubtful effectiveness, they present several disadvantages such as the need for parenteral administration, large doses, long treatment, severe toxicity and parasite resistance. Buparvaquone (BPQ), a drug used for veterinary treatment of theileriosis, showed promising activity against Leishmania spp. However, due to its low aqueous solubility and bioavailability, it has failed in in vivo tests. The use of nanotechnologies has the potential to overcome these drawbacks due to the following advantages: increase in drug water-solubility, increase in therapeutic efficacy and treatment toxicity reduction. Therefore, the present work aimed the development, optimization, physical-chemical evaluation and in vitro performances of nanostructured lipid carriers (NLC) for BPQ encapsulation. The NLC preparation was performed by high pressure homogenization, and surface response and factorial design were applied to formulation optimization. In vitro dissolution profiles were evaluated in phosphate buffer pH 7.4 with tween 80 0.07% w/v or sodium dodecyl sulfate 1% w/v and simulated body fluid pH 7.4. Cytotoxicity was evaluated in mouse peritoneal macrophages and leishmanicidal activity in L. infantum amastigotes. Six optimized NCL were prepared and they showed solubility improvement from 1.5- fold to 611-fold when compared with free BPQ, depending on the formulation and medium. Dissolution profiles showed the NLC formulation suitability for BPQ regarding oral administration, the release could reach 83.29% of a 4mg dose in 30 minutes for formulation of 175.1 nm, while the free drug could be dissolved only 2.89% of the same dose after 4 hours. Moreover, formulation of 230.7 nm showed 81.42% of drug release in in phosphate buffer pH 7.4 with dodecyl sulfate 1.0% w/v after 30 minutes, while BPQ did not dissolved. Cytotoxicity assay showed the safety of all formulations. The iv CC50 values were close to 500 µM, while the IC50 against amastigotes was only 456.5 nM for free BPQ. Developed NLCs showed an increase in IC50 from 2.0 to 3.1-fold when compared to free drug in the in vitro leishmanicidal evaluation. Therefore, the NLC containing BPQ are a promising alternative for the treatment of leishmaniases as oral and parenteral drug dosage forms. Additionally, they have a potential use for lymphatic targeted drug delivery, which can be an innovative approach for this neglected disease.

Leishmanioses são um grupo de doenças causadas por parasitas do gênero Leishmania. O número estimado de óbitos por leishmaniose visceral varia entre 20.000 e 50.000 por ano. O tratamento mais comum nos últimos 60 anos tem sido os antimônios pentavalentes. Além da eficácia duvidosa, eles apresentam várias desvantagens, como a necessidade de administração parenteral, altas doses, tratamento prolongado, toxicidade severa e resistência parasitária. Buparvaquona (BPQ), um fármaco usado para tratamento veterinário da teileriose, mostrou atividade promissora contra Leishmania donovani. No entanto, devido à sua baixa solubilidade e biodisponibilidade aquosa, falhou em testes in vivo. O uso das nanotecnologias tem o potencial de superar esses obstáculos devido às seguintes vantagens: aumento da solubilidade em água, aumento da eficácia terapêutica e redução da toxicidade do tratamento. Portanto, o presente trabalho objetivou o desenvolvimento, otimização, avaliação físico-química e avaliação do desempenho in vitro de carreadores lipídicos nanoestruturados (NLC) para o encapsulação da BPQ. A preparação do NLC foi realizada por homogeneização de alta pressão e superfície de resposta e planejamento fatorial foram aplicados à otimização das formulações. Os perfis de dissolução in vitro foram avaliados em tampão fosfato pH 7.4 com tween 80 a 0.07% p/v ou dodecilsulfato de sódio 1.0% p/v e fluido corporal simulado pH 7.4. A citotoxicidade foi avaliada em macrófagos peritoneais de camundongos e atividade leishmanicida em amastigotas de L. infantum. Foram preparados quatro NCL otimizados e mostraram melhora da solubilidade de 1,5 a 611 vezes quando comparado com a BPQ livre, dependendo da formulação e do meio. Os perfis de dissolução mostraram a adequação da formulação NLC para BPQ em relação à administração oral. A dissolução pode atingir 83,29% de uma dose de 4.0 mg em 30 minutos para a formulação de 175,1 nm, enquanto o fármaco livre dissolveu apenas vi 2,89% da mesma dose após 4 horas. Além disso, a formulação de 230,7 nm mostrou 81,42% de liberação do fármaco em tampão fosfato pH 7.4 com dodecil sulfato de sódio 1.0% p/v após 30 minutos, enquanto o BPQ não se dissolveu. O teste de citotoxicidade mostrou a segurança de todas as formulações. Os valores CC50 foram próximos de 500 µM, enquanto o IC50 em amastigotas foi de apenas 456,5 nM para BPQ livre. Os NLC desenvolvidos mostraram um aumento no IC50 de 2,0 a 3,1 vezes quando comparado ao;fármaco livre na avaliação leishmanicida in vitro. Logo, as NLC contendo BPQ são uma alternativa promissora para o tratamento de leishmanioses como formas farmacêuticas oral e parenteral. Além disso, eles têm um uso potencial para a sítio-específico ao sistema linfático, o que pode ser uma abordagem inovadora para esta doença negligenciada.

In vitro Antitheilerial Activity of Paluther (Artemether 80) Against Theileria lestoquardi.

Aim: The aim of this study was to screen Artemether 80 for activity against Theileria lestoquardi (Apicomplexa: Theileridae) using buparvaquone as a standard drug. Study Design: In vitro study under laboratories conditions. Place and Duration of Study: Veterinary Research Institute, between 2006 and 2008. Methodology: Artemether 80 was screened for the first time to investigate activity against T. lestoquardi at different concentrations. Blood was collected separately from normal sheep and sheep infected naturally with Theileria. Normal lymphocyte cells and lymphocyte cells infected with Theileria were isolated from heparinized blood with Ficoll-paque. Isolated cells were grown in Minimum Essential Medium (MEM), supplemented with 20% calf serum and sub cultured. The parasite was identified with indirect fluorescent antibody test (IFA). A volume of 2.7 ml of lymphoblast cell suspension at concentration of 5x104 cell/ ml was distributed in tissue culture plates, and then 0.3 ml of drug at concentrations of 0.1, 1.0, 10 and 100 mg/L was added separately. A volume of 0.3 ml MEM was added to infected untreated control. Results: The in vitro antitheilerial activity of Artemether 80 against T. lestoquardi 48 h after exposure was 0%, 14%, 30% and 45% at concentrations of 0.01, 0.1, 1.0 and 10 mg/L, respectively as compared with activity of buparvaquone at the same concentrations being 74%, 83%, 92% and 100%, respectively. Both Artemether 80 and buparvaquone caused in vitro partial cytotoxic effect at the highest concentrations. Activity and/ or partial cytotoxic effect of both drugs caused changes in the morphology of macroscizonts and host lymphoblast cells, decreased the number of macroschizonts/cell, mean number of dividing cells, increased the number of cells with extra cellular macroschizonts. Conclusion: It was concluded that Artemether 80 is slightly effective in vitro against T. lestoquardi.

Densitometric and Spectrometric Determinations of Florfenicol and Buparvaquone.

Three new accurate and sensitive methods were developed for the determination of two veterinary drugs. Florfenicol was analyzed by a stability-indicating TLC/densitometric method in presence of its amine degradation product obtained via acid hydrolysis. It was based on the difference in Rf values of the drug and its degradation product at 254 nm on silica gel 60 GF254 plates using chloroform–methanol (7:3 v/v) as developing solvent (Method A). However, buparvaquone was analyzed through reduction with NaBH4 in methanolto yielda red colored product measured spectrophotometrically at 489 nm (Method B). This reduction product was also measured spectrofluorometrically at 450 nm after excitation at 334 nm (Method C). Good linearity was obtained in the range of 10-100 μg/spot for florfenicol in method A and 10-120 or 0.3-2.4 μg mL-1 for buparvaquone by methods B and C with mean accuracy of 99.94%±0.89, 99.99%±0.99 or 99.66%±1.80, respectively. The densitometric method A proved to be stability-indicating in presence of 10-90% of buparvaquone amine degradation product. The three proposed methods were validated according to ICH guidelines and successfully applied for the assay of the cited drugs in their pharmaceutical formulations.