Usefulness of a murine model of hepatic cystic echinococcosis for preclinical drug trials: Efficacy of albendazole vs albendazole nanocrystals.

Cystic echinococcosis is a zoonotic infection caused by the larval stage of Echinococcus granulosus sensu lato. The disease is characterized by the long-term growth of cysts, most commonly in the liver and lungs. Although an ideal model of cystic echinococcosis should induce the development of cysts in the liver and imitate the natural infection route, the murine model of intraperitoneal is still widely used in the field of experimental theraphy. The aim of the present work was to evaluate the usefulness of the murine model of hepatic CE for preclinical drug trials. The effectiveness of albendazole could also be assessed by measuring the diameter of the hepatic cyst. The albendazole significantly reduced the size of the cysts. The ultrastructural alterations of the germinal layer of hepatic cysts provoked by albendazole coincided with those observed in the intraperitoneal model. Similar results were obtained with both albendazole doses. Therefore, the efficacy of albendazole nanocrystals in the murine model of hepatic cystic echinococcosis was carried out at albendazole doses of 25 mg/kg. The abdominal ultrasound allows us to assess the response of cysts to drugs only in a qualitative manner. Although the size of cysts in the albendazole nanocrystal group was not significantly lower than that observed with albendazole, at the ultrastructural level, a greater extent of damage was observed. The murine model of hepatic cystic echinococcosis can be effectively used for assessing the effect of novel formulations or compounds. The main advantage of this model is that cysts are located in the orthotopic organ, which resembles the location most commonly found in human cases. In future studies, the usefulness of the model for pharmacokinetics studies in hepatic cysts will be evaluated.

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.

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.

Albendazole-lipid nanocapsules: Optimization, characterization and chemoprophylactic efficacy in mice infected with Echinococcus granulosus.

Cystic echinococcosis (CE), which is caused during the metacestode larval stage of Echinococcus granulosus, is a life-threatening disease and is very difficult to treat. At present, the FDA-approved antihelmintic drugs are mebendazole (MBZ), albendazole (ABZ) and its principal metabolite ABZ sulfoxide (ABZSO), but as these have a therapeutic efficacy over 50%, underlining the need for new drug delivery systems. The aim of this work was the optimization and characterization of previously developed ABZ lipid nanocapsules (ABZ-LNCs) and evaluate their efficacy in mice infected with E. granulosus. LNCs were prepared by the phase inversion technique and characterized in terms of size, surface charge, drug loading, and in vitro stability followed by an in vivo proof-of-concept using a murine model infected with E. granulosus. Stable particle dispersions with a narrow size distribution and high efficiency of encapsulation (≥90%) were obtained. ABZ-LNCs showed a greater chemoprophylactic efficacy than ABZ suspension administered by the oral route as 4 out of the 10 ABZ-LNCs treated mice did not develop any cysts, whereas the infection progressed in all mice from the ABZ suspension group. Regarding the ultrastructural studies of cysts, mice treated with ABZ-LNCs or ABZ suspension revealed changes in the germinal layer. However, the extent of the damage appeared to be greater after ABZ-LNC administration compared to the suspension treatment. These results suggest that ABZ-LNCs could be a promising novel candidate for ABZ delivery to treat CE.

Albendazole nanocrystals in experimental alveolar echinococcosis: Enhanced chemoprophylactic and clinical efficacy in infected mice.

Human alveolar echinococcosis is caused by the fox tapeworm Echinococcus multilocularis and is usually fatal if left untreated. Medical treatment with albendazole (ABZ) remains an effective option. However, due to its low aqueous solubility, ABZ is poorly and erratically absorbed following oral administration resulting in low drug levels in plasma and liver distribution. Thus, there arises the need to find a simple, efficient and scalable method to produce new ABZ formulations with increased bioavailability. Bearing this in mind, ABZ nanocrystals (ABZ-NCs) appears to be a useful tool to achieve this goal. The aim of the current study was to investigate the chemoprophylactic and clinical efficacy of an ABZ-NC formulation on mice infected with E. multilocularis. In the chemoprophylactic efficacy study, mean weight of the cysts recovered from the ABZ-NC group was 50% lower than that recorded from untreated mice, whereas the treatment with ABZ suspension did not show preventive effect. The viability of protoscoleces isolated from ABZ-NC treated mice was significantly lower than control groups. In the clinical efficacy studies, both ABZ formulations resulted in a reduction in the mean weight of the cysts obtained from mice, however only the treatment with the nanosuspension revealed significant differences (P < 0.05) compared to the control groups. Treatment with ABZ-NCs reduced the weight of the cysts by 77% and the viability of their protoscoleces to 34%. All these results coincided with the tissue damage determined at the ultrastructural level. The enhanced chemoprophylactic and clinical efficacy of ABZ-NCs observed in this study could be attributed to an increase in the oral bioavailability of the drug. In a next step, we will characterize the cyst concentration profile after the administration of ABZ-NCs in mice infected with E. multilocularis.

Experimental cystic echinococcosis therapy: In vitro and in vivo combined 5-fluorouracil/albendazole treatment.

Human cystic echinococcosis is a zoonosis caused by the larval stage of the tapeworm Echinococcus granulosus sensu lato (s. l.). Although benzimidazole compounds such as albendazole (ABZ) and mebendazole have been the cornerstone of chemotherapy for the disease, there is often no complete recovery after treatment. Hence, new strategies are required to improve treatment of human cystic echinococcosis. The goals of the current study were as follows: (i) to evaluate the in vitro efficacy of the 5-fluorouracil (5-FU) and ABZ combination against E. granulosus s. l. protoscoleces and cysts, (ii) to compare the clinical efficacy of 5-FU alone or in combination with ABZ in infected mice. The combination of 5-FU+ABZ had a stronger in vitro effect against larval stage than that did both drugs alone. Even at the lowest concentration of 5-FU+ABZ combination (1µg/ml), the reduction of the viability of protoscoleces and cysts was greater than that observed with drugs alone at 10µg/ml. The results were confirmed at the ultrastructural level by scanning electron microscopy. These data helped to justify the in vivo investigations assessing the therapeutic potential of the combination of 5-FU and ABZ suspension in CF-1 mice infected with E. granulosus sensu stricto (s. s.) metacestodes. Treatment with 5-FU (10mg/kg) or 5-FU (10mg/kg) + ABZ suspension (5mg/kg) reduced the weight of cysts recovered from mice compared with control groups. Interestingly, the effect of 5-FU given weekly for 5 consecutive weeks was comparable to that observed with ABZ suspension under a daily schedule during 30days. Co-administration of 5-FU with ABZ did not enhance the in vivo efficacy of drugs alone calculated in relation to cysts weights. However, the combination provoked greater ultrastructural alterations compared to the monotherapy. In conclusion, we demonstrated the efficacy of 5-FU either alone or co-administrated with ABZ against murine experimental cystic echinococcosis. Since 5-FU treatments did not cause toxic effect in mice, further in vivo studies will be performed by adjusting the dosage and the frequency of treatments.

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.

Could thymol have effectiveness on scolices and germinal layer of hydatid cysts?

Scolicidal solutions remain indispensable in the treatment of hydatid cyst disease. Properties of an ideal solution would be inexpensiveness and the promotion of a rapid and complete scolicidal effect with an absence of local and systemic side effects. From this point of view, no ideal solution and agents have been described yet. The aim of the present work was to determine the in vitro effect of high concentrations of thymol against protoscoleces, microcyst and cyst of Echinococcus granulosus and to evaluate its possible role as a scolicidal agent during surgery or PAIR. After short exposure times, a rapid effect was observed depending on the parasitic material. After 2 min of exposure to thymol, viability of protoscoleces was approximately 1.3% at a concentration of 250 µg/ml. The protoscolicidal effect is dose and time dependent. The results of the in vitro treatment with thymol were similar in both microcysts and secondary murine cysts. The employment of SEM and TEM allowed us to examine, at an ultrastructural level, the effects induced by thymol on E. granulosus protoscoleces, microcysts and murine cysts. In conclusion, the data obtained clearly demonstrated that thymol caused severe damages to the parasite even after short incubation times. This fact and the lack of toxicity at the evaluated concentrations, allow us to propose it as a possible scolicidal agent during hydatid cysts surgery and/or PAIR.

Epidemiological surveillance of cystic echinococcosis in rural population of Tierra del Fuego, Argentina, 1997-2006.

Cystic echinococcosis (CE) is the most prevalent zoonosis in Tierra del Fuego. In 1997, ulrasonography (US) was selected as the method of choice for the development of population surveys for epidemiological surveillance and early diagnosis in rural population. The aim of this work was to present the results of the epidemiological surveillance of CE by means of US in rural population of Tierra del Fuego, Argentina between 1997 and 2006. The ultrasonographic diagnostic was realized once a year. The population was stratified in children (4 to 17 years) and adults. From each individual, name, age, sex, actual residence and origin were registered. The images compatible with cysts were graded according to location, number and characteristics. A total of 1400 rural inhabitants were examined for CE. From the total of studied individuals, 27 (1.9%) exhibited images compatible with cysts on the abdominal ultrasound scan. Thirteen of these persons were finally diagnosed as having CE. The overall prevalence of CE was 0.9%. This value is in accordance with the decrease in the prevalence observed in the definitive host and the intermediate hosts (sheep and cattle). The absence of cases in children during the studied period, evidence no transmission of the disease to humans in the recent past.

[Epidemiology of dog bite lesions in Tierra del Fuego, Argentina]. / Epidemiología de las lesiones por mordedura de perro en Tierra del Fuego, Argentina.

The coexistence between man and dog has resulted in mutual benefits during thousands of years, nevertheless some problems have recently arisen where bite injuries have an important role. The aim of this work was to describe the epidemiological characteristics of dog bite lesions which occurred during a year in Tierra del Fuego. A descriptive observational, transversal study of patients who were admitted with dog bite injuries to clinic and pediatric emergency services of hospitals and health centers was carried out between 3/1/05 and 3/1/06. A total of 382 records were made. The children group represents 49.5%. The group between 5 and 9 year-old was the most affected (44.4%; IC95% 37.2-51.8). In the adult group, that between 15 and 24 year-old was the most affected (29.5%; IC95% 23.2-36.5). More lesions were registered in men (57.6%) than in women (42.4%) (chi2 = 8.6, p = 0.003). During spring and summer months, 56.5% of the lesions were registered. A 72.8% of the incidents occurred on the public highway. Dogs of big size were responsible of 49.7% (IC95% 44.6-54.9) of the injuries. The 89.8% of the incidents were caused by another person's dog. Of the lesions, 55.8% (IC95% 50.6-60.8) were registered in lower extremities and 11% (IC95% 8.1-14.7) in head and neck. These data show that dog bite lesions affect the health and impact in the quality of life of the population of Tierra del Fuego, Argentina.

Epidemiología de las lesiones por mordedura de perro en Tierra del Fuego, Argentina / Epidemiology of dog bite lesions in Tierra del Fuego, Argentina

La convivencia del hombre y el perro ha resultado en beneficios mutuos durante miles de años, si bien en el último tiempo han surgido una serie de inconvenientes donde las lesiones por mordeduras ocupan un lugar destacado. El objetivo del presente trabajo fue describir las características epidemiológicas de las lesiones por mordedura de perro ocurridas a lo largo de un año en Tierra del Fuego. Se realizó un estudio observacional descriptivo, transversal, de los pacientes que ingresaron con lesiones por mordedura de perro al Servicio de Guardia Clínica y Pediátrica de los Hospitales y Centros de Salud desde el 1/3/05 hasta el 1/3/06. Se confeccionaron 382 planillas. El 49.5% fueron niños resultando más afectado el grupo de 5-9 años (44.4%; IC95% 37.2-51.8). En adultos correspondió al grupo de 15-24 años (29.5%; IC95% 23.2-36.5). Se registraron más lesiones en varones (57.6%) que en mujeres (42.4%) (x² = 8.6, p= 0.003). En los meses primavera y verano se registró el 56.5% de las lesiones, en la vía pública el 72.8% de ellas. Los perros de tamaño grande causaron el 49.7% (IC95% 44.6-54.9) de las lesiones. El 89.8% de los ataques fueron causados por perros ajenos. El 55.8% (IC95% 50.6-60.8) se registró en miembros inferiores y el 11% (IC95% 8.1-14.7) en cabeza y cuello. Los datos obtenidos demuestran que en Tierra del Fuego las lesiones por mordedura de perro afectan en forma permanente la salud e impactan en la calidad de vida de la población.

The coexistence between man and dog has resulted in mutual benefits during thousands of years, nevertheless some problems have recently arisen where bite injuries have an important role. The aim of this work was to describe the epidemiological characteristics of dog bite lesions which occurred during a year in Tierra del Fuego. A descriptive observational, transversal study of patients who were admitted with dog bite injuries to clinic and pediatric emergency services of hospitals and health centers was carried out between 3/1/05 and 3/1/06. A total of 382 records were made. The children group represents 49.5%. The group between 5 and 9 year-old was the most affected (44.4%; IC95% 37.2-51.8). In the adult group, that between 15 and 24 year-old was the most affected (29.5%; IC95% 23.2- 36.5). More lesions were registered in men (57.6%) than in women (42.4%) (x² = 8.6, p= 0.003). During spring and summer months, 56.5% of the lesions were registered. A 72.8% of the incidents occurred on the public highway. Dogs of big size were responsible of 49.7% (IC95% 44.6-54.9) of the injuries. The 89.8% of the incidents were caused by another person´s dog. Of the lesions, 55.8% (IC95% 50.6-60.8) were registered in lower extremities and 11% (IC95% 8.1-14.7) in head and neck. These data show that dog bite lesions affect the health and impact in the quality of life of the population of Tierra del Fuego, Argentina.