Experimental treatment of cystic echinococcosis: Combination therapy with carvacrol and thymol versus albendazole.

Cystic echinococcosis is a worldwide zoonotic disease caused by Echinococcus granulosus sensu lato (s.l.), which produces serious health and economic problems. For human treatment, chemotherapy with albendazole (ABZ), a derivative of benzimidazoles, is widely used. However, due to its low efficacy and the lack of alternatives to ABZ, novel compounds are urgently needed. Aromatic plants exhibit powerful pharmacological activities, are accessible, have a relatively low cost, and have generally mild toxicities, making them an effective choice to traditional therapies. In particular, the pharmaceutical properties of aromatic plants are partially attributed to essential oils (EOs). The aim of the present study was to assess the in vitro and in vivo effects of the combined carvacrol and thymol against E. granulosus sensu stricto (s.s.). The greatest protoscolicidal effect was observed with the 9:1 and 5:5 (carvacrol:thymol) combinations which caused a marked decrease in viability after 6 days post-incubation, agreeing with the ultrastructural changes obtained. Permeation of the cysts and loss of turgidity was observed with the incubation with the different combinations of carvacrol:thymol. In the clinical efficacy study, the combination of thymol (40 mg/kg) and carvacrol (40 mg/kg) caused a tendency to diminish the weight of the cysts in comparison with the control group. On the other hand, the treatment of infected mice with ABZ, thymol or carvacrol, caused a significant decrease in the weight of the cysts. In conclusion, we here demonstrated the efficacy of different concentrations of combined carvacrol and thymol against E. granulosus s.s. protoscoleces and murine cysts, where short periods of treatment were sufficient to achieve a pharmacological effect. Moreover, we observed a reduction in the weight of the cysts in experimentally infected mice after treatment with carvacrol and thymol. The strategy used has an advantage over synthetic drugs because natural compounds are generally safe and non-toxic. Moreover, the combination of two drugs with different modes of action would cause a reduction in the doses and treatment times. Based on the promising results obtained in vitro, in the future, different doses of the combined drugs will be assayed in vivo to determine the potential of these compounds for the treatment of cystic echinococcosis.

The combination of carvacrol and albendazole enhanced the efficacy of monotherapy in experimental alveolar echinococcosis.

Alveolar echinococcosis is a helminthic zoonosis caused by the larval stage of Echinococcus multilocularis. When surgical resection of the parasite is not feasible, pharmacological treatment with albendazole is the only option. Due to the difficulties in achieving the success of treatment, it is necessary to find new drugs to improve the treatment of this disease. In the present work, the efficacy of carvacrol alone or combined with albendazole was evaluated against E. multilocularis metacestodes. The association of carvacrol with albendazole produced a greater in vitro effect than the compounds incubated separately. The most effective treatment was the combination of 10 µg/ml of carvacrol and 1 µg/ml of albendazole. In the clinical efficacy study, treatment of infected mice with carvacrol (40 mg/kg) and albendazole (25 mg/kg) reduced the weight of metacestodes by 29 % and 50 %, respectively; while the combination of drugs had an efficacy of 83 %. These results coincided with the tissue damage observed at the ultrastructural level. In conclusion, carvacrol and albendazole combination enhanced the efficacy of monotherapy. This strategy would allow to improve the efficacy of the treatment without increasing the doses of albendazole or lengthen the treatment period, reducing the occurrence of adverse effects.

In vitro efficacy study of Cinnamomum zeylanicum essential oil and cinnamaldehyde against the larval stage of Echinococcus granulosus.

Hydatidosis or cystic echinococcosis is a disease caused by the larval stage of Echinococcus granulosus sensu lato. Chemotherapy can be used alone or in combination with surgery or percutaneous treatment. Benzimidazoles are the only agents used and approved for treatment, but their efficacy is extremely variable. Therefore, it is necessary to find new drugs to improve the treatment of this disease. In the last decades, the biological properties of essential oils and their components began to be investigated as alternatives in the treatment of different ailments. The aim of the present work was to evaluate the in vitro efficacy of the essential oil of Cinnamomum zeylanicum (cinnamon) and cinnamaldehyde against protoscoleces and metacestodes of E. granulosus. The essential oil and cinnamaldehyde, its major component, showed a dose and time dependent effect against protoscoleces. However, cinnamaldehyde showed a greater protoscolicidal effect than the essential oil. The maximum protoscolicidal effect was found with 50 µg/mL of cinnamaldehyde. Viability decreased by 1.7 ± 0.8% after 4 days of incubation and reached 0% at 8 days. Interestingly, there were no significant differences between the activity of cinnamaldehyde at the concentrations of 25 and 10 µg/mL and the efficacy observed with the essential oil at 200 and 50 µg/mL, respectively. Cinnamaldehyde also had a strong in vitro effect against murine cysts, while only the higher concentration of the essential oil caused ultrastructural alterations. Working with components instead of with essential oils has some advantages, particularly in relation to the reproducibility of the formulations and their effectiveness. For this reason, the results obtained in this work are promising in the search for pharmaceutical alternatives for the treatment of cystic echinococcosis.

Albendazole solid dispersions against alveolar echinococcosis: a pharmacotechnical strategy to improve the efficacy of the drug.

Alveolar echinococcosis is a neglected parasitic zoonosis caused by Echinococcus multilocularis. The pharmacological treatment is based on albendazole (ABZ). However, the low water solubility of the drug produces a limited dissolution rate, with the consequent failure in the treatment of the disease. Solid dispersions are a successful pharmacotechnical strategy to improve the dissolution profile of poorly water-soluble drugs. The aim of this work was to determine the in vivo efficacy of ABZ solid dispersions using poloxamer 407 as a carrier (ABZ:P407 solid dispersions (SDs)) in the murine intraperitoneal infection model for secondary alveolar echinococcosis. In the chemoprophylactic efficacy study, the ABZ suspension, the ABZ:P407 SDs and the physical mixture of ABZ and poloxamer 407 showed a tendency to decrease the development of murine cysts, causing damage to the germinal layer. In the clinical efficacy study, the ABZ:P407 SDs produced a significant decrease in the weight of murine cysts. In addition, the SDs produced extensive damage to the germinal layer. The increase in the efficacy of ABZ could be due to the improvement of water solubility and wettability of the drug due to the surfactant nature of poloxamer 407. In conclusion, this study is the basis for further research. This pharmacotechnical strategy might in the future offer novel treatment alternatives for human alveolar echinococcosis.

Anti-echinococcal activity of menthol and a novel prodrug, menthol-pentanol, against Echinococcus multilocularis.

Alveolar echinococcosis is one of the most dangerous parasitic zoonoses. This disease, widely distributed in the northern hemisphere, is caused by the metacestode stage of the tapeworm Echinococcus multilocularis. All surgical and non-surgical patients should perform chemotherapy with benzimidazoles, mainly with albendazole. However, the efficacy of albendazole is variable due to its deficient pharmacokinetic properties. Therefore, the need to find new therapeutic alternatives for the treatment of alveolar echinococcosis is evident. Menthol is a natural compound of low toxicity, used in industries such as cosmetics and gastronomy and generally recognized as safe by the Food and Drug Administration. In addition, menthol has important pharmacological effects and is effective against a wide variety of organisms. The development of prodrugs allows improving the pharmacokinetic properties of the parental drug. To improve lipophilicity and therefore the bioavailability of menthol, a novel prodrug called menthol-pentanol was developed by masking the functional polar group of menthol by linking n-pentanol by a carbonate bond. The aim of the current work was to evaluate the in vitro and in vivo efficacy of menthol and menthol-pentanol against E. multilocularis. Menthol-pentanol had a greater protoscolicidal effect than menthol. In addition, the prodrug demonstrated a similar clinical efficacy to albendazole. The increase in lipophilicity of the prodrug with respect to menthol was reflected in an increase in its antiparasitic activity against E. multilocularis. Thus, menthol-pentanol appears as a promising candidate for further evaluation as a potential alternative for the treatment of alveolar echinococcosis.

Do albendazole-loaded lipid nanocapsules enhance the bioavailability of albendazole in the brain of healthy mice?

Neurocysticercosis is a neglected tropical disease that affects the central nervous system and is the most common cause of human epilepsy acquired in developing countries. Therapeutic failures attributed to medical management of neurocysticercosis with albendazole (ABZ) have been primarily linked to the poor drug absorption rate resulting in low drug level in plasma and brain tissue. The aim of the current work was to characterize and compare the brain pharmacokinetic behavior of ABZ formulated as a suspension or lipid nanocapsules (ABZ-LNCs) in healthy mice. The relative availability in brain tissue of the active metabolite ABZ sulphoxide increased 183% when ABZ was administered as LNCs, in relation to ABZ suspension. The parent drug was also detected for a short period of time. The bioavailability of ABZ in ABZ-LNCs treated mice increased more than 2 fold compared with ABZ suspension group. The enhanced drug brain exposure observed after administration of ABZ-LNCs to healthy mice has potential usefulness for the treatment of human neurocysticercosis.

Drug repurposing for the treatment of alveolar echinococcosis: in vitro and in vivo effects of silica nanoparticles modified with dichlorophen.

Alveolar echinococcosis is a neglected parasitic zoonosis caused by the metacestode Echinococcus multilocularis, which grows as a malignant tumour-like infection in the liver of humans. Albendazole (ABZ) is the antiparasitic drug of choice for the treatment of the disease. However, its effectiveness is low, due to its poor absorption from the gastro-intestinal tract. It is also parasitostatic and in some cases produces side-effects. Therefore, an alternative to the treatment of this severe human disease is necessary. In this context, the repositioning of drugs combined with nanotechnology to improve the bioavailability of drugs emerges as a useful, fast and inexpensive tool for the treatment of neglected diseases. The in vitro and in vivo efficacy of dichlorophen (DCP), an antiparasitic agent for intestinal parasites, and silica nanoparticles modified with DCP (NP-DCP) was evaluated against E. multilocularis larval stage. Both formulations showed a time and dose-dependent in vitro effect against protoscoleces. The NP-DCP had a greater in vitro efficacy than the drug alone or ABZ. In vivo studies demonstrated that the NP-DCP (4 mg kg-1) had similar efficacy to ABZ (25 mg kg-1) and greater activity than the free DCP. Therefore, the repurposing of DCP combined with silica nanoparticles could be an alternative for the treatment of echinococcosis.

Comparison of different staining methods for determination of viability on Mesocestoides vogae tetrathyridia.

Mesocestoides vogae is widely employed as a model for studying the biology, differentiation, and experimental chemotherapy of cestodes. Currently, there are few techniques to measure the viability of M. vogae metacestodes during pharmacological experiments. The aim of the present work was to evaluate and compare different staining techniques to determine objectively the viability of M. vogae tetrathyridia. Eosin (0.05% w/v), methylene blue (0.01% w/v), propidium iodide (PI, 2 µg/ml), and fluorescein diacetate (FDA, 0.5 µg/ml) solutions were tested against live, heat-killed (cultivated at 65 °C for 2 h) and thymol-treated tetrathyridia (50 and 250 µg/ml). Parasites were counted under a dissecting microscope or a fluorescence compound microscope, as appropriate. Studies by scanning electron microscope were performed to compare the ultrastructural damage with the viability of parasites. After comparing the performance of different dyes, we chose the eosin staining technique because its simplicity, rapidity, sensitivity, low cost and fidelity.

In vitro and in vivo efficacy of carvacrol against Echinococcus granulosus.

Currently, benzimidazoles are used as chemotherapeutic agents and as a complement to surgery and PAIR in the treatment of cystic echinococcosis (CE). They are generally applied at high doses causing side effects and, 50% of cases do not respond favorably to such chemotherapy. The use of essential oils obtained by distillation from aromatic plants would be an effective alternative or complementary to the synthetic compounds, because would not bring the appearance of side effects. Carvacrol and his isomer thymol are the main phenolic components from essential oils of Origanum vulgare (oregano) and Thymus vulgaris (thyme). The aim of the present work was to evaluate the in vitro and in vivo efficacy of carvacrol against Echinococcus granulosus metacestodes. For the in vitro assay, protoscoleces and cysts of E. granulosus were incubated with carvacrol at the following final concentrations: 10, 5 and 1µg/ml of carvacrol. The maximum protoscolicidal effect was found with 10µg/ml of carvacrol. Results of viability tests were consistent with the structural and ultrastructural damage observed in protoscoleces. Ultrastructural studies revealed that the germinal layer of cysts treated with carvacrol lost the multicellular structure feature. In the clinical efficacy study, a reduction in cyst weight was observed after the administration of 40mg/kg of carvacrol during 20days in mice with cysts developed during 4 months, compared to that of those collected from control mice. Given that the in vivo effect of carvacrol was comparable with the treatment of reference with ABZ and the fact that is a safe compound, we postulated that carvacrol may be an alternative option for treatment of human CE.

Cystic echinococcosis therapy: Albendazole-loaded lipid nanocapsules enhance the oral bioavailability and efficacy in experimentally infected mice.

Therapeutic failures attributed to medical management of cystic echinococcosis (CE) with albendazole (ABZ) have been primarily linked to the poor drug absorption rate resulting in low drug level in plasma and hydatid cysts. Lipid nanocapsules (LNCs) represent nanocarriers designed to encapsulate lipophilic drugs, such as ABZ. The goals of the current work were: (i) to characterize the plasma and cyst drug exposure after the administration of ABZ as ABZ-LNCs or ABZ suspension (ABZ-SUSP) in mice infected with Echinococcus granulosus, and ii) to compare the clinical efficacies of both ABZ formulations. Enhanced ABZ sulphoxide (ABZ-SO) concentration profiles were obtained in plasma and cysts from ABZ-LNC treated animals. ABZSO exposure (AUC0-LOQ) was significantly higher in plasma and cyst after the ABZ-LNC treatments, both orally and subcutaneously, compared to that observed after oral administration of ABZ-SUSP. Additionally, ABZSO concentrations measured in cysts from ABZ-LNC treated mice were 1.7-fold higher than those detected in plasma. This enhanced drug availability correlated with an increased efficacy against secondary CE in mice observed for the ABZ-LNCs, while ABZ-SUSP did not reach differences with the untreated control group. This new pharmacotechnically-based strategy could be a potential alternative to improve the treatment of human CE.