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.

Development of an improved anesthesia protocol to increase CF1 mice survival in a portal vein infection with Echinococcus granulosus sensu lato protoscoleces.

In order to optimize the survival rate of animals, the purpose of this study was to evaluate an injectable anesthesia protocol for the development of a murine model of hepatic cystic echinococcosis in female CF-1 mice. Three protocols of injectable anesthesia were evaluated during the infection of mice with Echinococcus granulosus sensu lato protoscoleces via the portal vein. The use or not of pre-anesthesia [atropine (0.4 mg/kg) and tramadol (2 mg/kg)] and the incorporation or not of yohimbine (0.5 mg/kg) (a reverser of xylazine) in mice anesthetized with ketamine/xylazine 80/8 mg/kg were evaluated. Most mice treated only with ketamine/xylazine 80/8 mg/kg did not achieve a deep surgical anesthetic plane. All mice treated with pre-anesthetic drugs achieved a deep surgical anesthetic plane after the administration of the anesthetic cocktail. Pre-anesthetic drugs application significantly reduced time induction of animals compared with those that received only anesthetic cocktail. Recovery time was significantly faster in the group that received yohimbine. Mice underwent laparotomy that did not receive yohimbine after surgery had a survival rate of 67%, whereas in the group treated with yohimbine the survival was 100 %. We recommend the protocol that applied pre-anesthetic drugs + ketamine/xylazine 80/8 mg/kg + yohimbine, as safe and reliable for the portal vein infection of mice with protoscoleces of E. granulosus sensu lato.

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.

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.

Enhanced chemoprophylactic and clinical efficacy of albendazole formulated as solid dispersions in experimental cystic echinococcosis.

Cystic echinococcosis is a chronic, complex, and still neglected disease. Although albendazole has demonstrated efficacy, only about one-third of patients experience complete remission or cure and 30-50% of treated patients develop some evidence of a therapeutic response. Different strategies have been developed in order to improve the albendazole water solubility and dissolution rate. The aim of the current work was to investigate the chemoprophylactic and clinical efficacy of an albendazole:poloxamer 188 solid dispersion formulation on mice infected with Echinococcus granulosus metacestodes. Albendazole formulated as solid dispersion had greater chemoprophylactic and clinical efficacy than albendazole alone. The improved in therapeutic efficacy could be a consequence of the increase in the systemic availability of albendazole sulfoxide. The work reported here demonstrates that in vivo treatment with albendazole:poloxamer 188 impairs the development of the hydatid cysts. This new pharmacotechnically based strategy could be a suitable alternative for treating cystic echinococcosis in humans.

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.