Fosfatriclaben, a prodrug of triclabendazole: Preparation, stability, and fasciolicidal activity of three new intramuscular formulations.

In this study, we present the preparation, stability, and in vivo fasciolicidal activity of three new intramuscular formulations in sheep of a prodrug based on triclabendazole, named fosfatriclaben. The new formulations were ready-to-use aqueous solutions with volumes recommended for intramuscular administration in sheep. The use of poloxamers (P-407 and P-188) and polysorbates (PS-20 and PS-80) in the new formulations improved the aqueous solubility of fosfatriclaben by 8-fold at pH 7.4. High-performance liquid chromatography with UV detection was used to evaluate the stability of fosfatriclaben in the three formulations. High recovery (> 90%) of fosfatriclaben was found for all formulations after exposure at 57 ± 2 °C for 50 h. The three intramuscular formulations showed high fasciolicidal activity at a dose of 6 mg/kg, which was equivalent to the triclabendazole content. The fasciolicidal activity of fosfatriclaben was similar to commercial oral (Fasimec®) and intramuscular (Endovet®) triclabendazole formulations at a dose of 12 mg/kg. In the in vivo experiments, all formulations administered intramuscularly reduced egg excretion by 100%, and formulations F1, F2, and F3 presented fasciolicidal activities of 100%, 100%, and 99.6%, respectively.

Field study on the determination of the effective dose of injectable fosfatriclaben prodrug in sheep naturally infected with Fasciola hepatica.

The aim of the present study was to determine the effective dose of fosfatriclaben through a field study in sheep naturally infected with F. hepatica. Thirty crossbred sheep positive for fluke eggs were selected for inclusion in the trial. On day 0, 5 groups of 6 animals each were formed for treatments: group 1 (G1), G2, and G3 received fosfatriclaben at 4, 6, and 8 mg/kg/IM, respectively. G4 received triclabendazole at 10 mg/kg/PO, and G5 was the untreated control group. Fecal samples of the sheep were analyzed to count the number of fluke eggs to evaluate the percentage of egg reduction. Twenty-one days after treatment, all sheep were humanely euthanized to extract the flukes from the bile ducts. They were counted to assess the percentage of fluke reduction. Fosfatriclaben reduced fluke eggs by 99.6, 99.6, and 100% and flukes by 94.3, 100, and 100%, respectively. Triclabendazole reduced fluke eggs by 95.2% and flukes by 100%. The fosfatriclaben injectable prodrug showed a high fasciolicidal efficacy similar to triclabendazole, with advantages over its predecessor, since only half the dose as compared to triclabendazole was required to remove eggs and flukes in the sheep that were studied.

Effectiveness of an experimental injectable prodrug formulation against Fasciola hepatica of different ages in experimentally infected sheep.

In this work, we present an evaluation of the fasciolicidal efficacy of a new injectable formulation of fosfatriclaben in comparison with the subcutaneous closantel and oral triclabendazole formulations currently used in veterinary practice as fasciolicides. The study was carried out in vivo on Fasciola hepatica at 2, 4, 6 and 8 weeks of age in experimentally infected sheep. To evaluate the formulation, the percent reduction of the parasite load was measured and the number of fluke eggs. Fosfatriclaben was used at 6 mg/kg/IM (dose equivalent to triclabendazole content), closantel at 5% at 10 mg/kg/SC, and triclabendazole at 10 mg/kg/PO; the control group received no treatment. Fosfatriclaben showed fasciolicidal efficacies of 95.5 %, 100 %, 100 % and 100 %, and triclabendazole showed similar efficacies of 97.4 %, 100 %, 100 % and 100 %, at the different treatment weeks (P > 0.05). Closantel showed limited efficacy against 2-, 4- and 6-week-old flukes but 100 % efficacy in adult flukes. All three evaluated formulations eliminated all 8-week-old F. hepatica trematode eggs. Although fosfatriclaben and triclabendazole showed similar fasciolicidal efficacy, the intramuscular administration of fosfatriclaben has several advantages over the oral administration of triclabendazole, such as ease of administration for veterinary use and a reduced risk of accidents for both the operator and the animals. In addition, the dose used in this injectable formulation is only 60 % of the oral dose, which reduces environmental contamination.

Efficacy of a novel topical combination of esafoxolaner, eprinomectin and praziquantel in cats against Toxocara cati and Dipylidium caninum.

NexGard® Combo, a novel topical antiparasitic product for cats, combines the insecticide/acaricide esafoxolaner with the nematocide eprinomectin and cestodicide praziquantel. The efficacy of this combination product was evaluated against two common endoparasites of global occurrence in cats, the nematode Toxocara cati and the cestode Dipylidium caninum, in five controlled studies using naturally or experimentally infected cats with parasites of North American, South African or European origin. Cats evaluated in these studies harbored patent infection of the target parasite confirmed through a pre-treatment fecal examination. In each study, cats were allocated randomly to two groups of equal size (8 or 10 cats per group per study), one group treated with a placebo (mineral oil) and the other with NexGard® Combo. Both treatments were administered once as a spot-on at 0.12 mL per kg body weight to deliver the minimum label dosage (1.44 mg/kg esafoxolaner, 0.48 mg/kg eprinomectin, and 10.0 mg/kg praziquantel) to the NexGard® Combo-treated cats. To determine efficacy, geometric mean parasite counts seven to 12 days after treatment of placebo-treated (control) cats and NexGard® Combo-treated cats were compared. The efficacy of NexGard® Combo was 98.8% and 100% against adult T. cati in two studies; and 98.0%, 98.3% and 93.2% against D. caninum in three studies. No adverse events related to treatment were observed throughout the studies. These studies demonstrate high efficacy against these major feline endoparasites and excellent acceptability of the novel topical antiparasitic combination of esafoxolaner, eprinomectin and praziquantel.

TITLE: Efficacité d'une nouvelle association topique d'esafoxolaner, d'éprinomectine et de praziquantel chez le chat contre Toxocara cati et Dipylidium caninum. ABSTRACT: NexGard® Combo, un nouveau produit antiparasitaire topique pour chats combine l'insecticide/acaricide esafoxolaner avec le nématocide éprinomectine et le cestodicide praziquantel. L'efficacité de ce produit d'association a été évaluée contre deux endoparasites communs d'occurrence mondiale chez le chat, le nématode Toxocara cati et le cestode Dipylidium caninum, dans cinq études contrôlées utilisant des chats naturellement ou expérimentalement infectés par des parasites d'origine nord-américaine, sud-africaine ou européenne. Les chats évalués dans ces études présentaient une infection patente du parasite cible confirmée par un examen fécal avant le traitement. Dans chaque étude, les chats ont été répartis au hasard en deux groupes de taille égale (8 ou 10 chats par groupe et par étude), un groupe traité avec un placebo (huile minérale) et l'autre avec NexGard® Combo. Les deux traitements ont été administrés une fois par spot-on à 0,12 mL par kg de poids corporel pour délivrer la dose minimale indiquée sur l'étiquette (1,44 mg/kg d'esafoxolaner, 0,48 mg/kg d'éprinomectine et 10,0 mg/kg de praziquantel) pour les chats du groupe traité par NexGard® Combo. Pour déterminer l'efficacité, les nombres moyens géométriques de parasites sept à 12 jours après le traitement des chats traités par placebo (témoins) et des chats traités par NexGard® Combo ont été comparés. L'efficacité de NexGard® Combo était de 98,8 % et de 100 % contre T. cati adulte dans deux études, et de 98,0 %, 98,3 % et 93,2 % contre D. caninum dans trois études. Aucun événement indésirable lié au traitement n'a été observé tout au long des études. Ces études démontrent la grande efficacité contre ces principaux endoparasites félins et l'excellente acceptabilité de la nouvelle combinaison antiparasitaire topique d'esafoxolaner, d'éprinomectine et de praziquantel.

Efficacy of purified fractions of Artemisia ludoviciana Nutt. mexicana and ultraestructural damage to newly excysted juveniles of Fasciola hepatica in vitro.

The study aimed to evaluate the fasciolicidal efficacy of extracts and fractions of Artemisia ludoviciana and identify the active substance. Extracts from A. ludoviciana were obtained by using hexane, ethyl acetate and methanol. To test the extracts, newly excysted juveniles of Fasciola hepatica were artificially obtained. The extracts were tested at concentrations of 125, 250, 375 and 500 mg/L. In each test run, an untreated control group and control wells containing triclabendazole sulfoxide were also included. The flukes were examined at 24, 48 and 72 h after treatment. Ethyl acetate extract (ALEAE) showed 100 % efficacy at 48 h of exposure (P < 0.05). Then, this extract was fractionated by column chromatography (CC), and the obtained fractions were evaluated in vitro as previously mentioned. The results indicated that fraction 3 yielded 100 % efficacy at 48 h (P < 0.05). Subsequently, the purification of fraction 3 was performed. New fractions were obtained (A-L), with sub-fraction "J" exhibiting 100 % efficacy at 24 h (P < 0.05). These sub-fractions were submitted to phytochemical analysis, demonstrated the presence of sesquiterpene lactones. Likewise, were analyzed by HPLC/MS/DAD, and the results showed that artemisinin was the main compound. In addition, flukes treated were examined by scanning electron microscopy (SEM) showing areas of inflammation throughout the tegument.

Efficiency comparison of experimental fosfatriclaben with three commercial fasciolicides in experimentally infected sheep.

In this work, we compare and evaluate the efficiency of fosfatriclaben with three commercial fasciolicides in experimentally infected sheep. Fosfatriclaben is a novel prodrug derived from triclabendazole; it is highly water-soluble with excellent aqueous stability at pH 7, properties that make it ideal for developing intramuscular pharmaceutical compositions in the form of solutions. In order to compare, 30 mixed breed sheep, previously diagnosed negative to fluke eggs, were infected with 200 metacercariae of Fasciola hepatica, twice. Five groups of six animals/each were formed for treatments. Group 1 (G1) was treated with closantel 5% injectable at 5 mg/kg subcutaneously, G2 with clorsulon at 2 mg/kg subcutaneously, G3 with triclabendazole at 12 mg/kg per os, G4 with fosfatriclaben at 6 mg/kg intramuscularly (dose equivalent to triclabendazole content), and G5 remained as the non-treated control. On day 110, fecal samples were examined to determine the percentage of egg reduction after treatment, and sheep were humanely euthanized. The livers were collected, the flukes were extracted, measured, and counted. Efficiency in egg reduction was of 86.8, 90.5, 98.4, and 97.3% for closantel, clorsulon, triclabendazole, and fosfatriclaben, respectively, and efficiency against flukes was of 96.2, 91.9, 99.4, and 95.7%, respectively. No statistical differences were found between treatments. It is concluded that fosfatriclaben at 6 mg/kg intramuscularly presented a high fasciolicide efficiency, similar to the best commercial fasciolicides, having advantage over its predecessor since it uses half of the dose required by triclabendazole to remove flukes in sheep under study.

First database of the spatial distribution of Eimeria species of cattle, sheep and goats in Mexico.

Ruminant coccidiosis is a gastrointestinal disease caused by parasites of the genus Eimeria. Environmental and climatic factors are relevant for the development, survival, and transmission of coccidiosis because Eimeria oocysts are able to survive in the environment for several weeks or months in favorable conditions of moderate heat and moisture. The aim of the present study was to georeference, for the first time, the locations of Eimeria occurrences in Mexico from 1961 to 2018. A dataset was created for 3414 reports of Eimeria occurrences in cattle, sheep, and goats in Mexico. Twelve species of Eimeria that infect cattle were recorded, 11 Eimeria species of sheep are present in Mexico, and eight species of goats are geographically distributed in the country. In the current findings, it has been observed that Eimeria colonizes mainly the temperate semihumid, temperate humid, warm humid, and warm semihumid areas during spring and summer in animals younger than 1 year of age. Macroenvironmental variables like temperature and rainfall influence the prevalence of Eimeria in cattle, sheep, and goats, and for some species, the rearing system, facility type, farm size, and altitude affect the occurrence of this parasite. Results may support future studies aimed at reducing the disease prevalence of the parasite in endemic regions of Mexico. The use of recorded cases and climate variables yields a more comprehensive perspective of the epidemiology of eimeriosis, which would be difficult to infer from laboratory studies alone.

Development of the first georeferenced map of Rhipicephalus (Boophilus) spp. in Mexico from 1970 to date and prediction of its spatial distribution.

The tick genus Ripicephalus (Boophilus), particularly R. microplus, is one of the most important ectoparasites that affects livestock health and considered an epidemiological risk because it causes significant economic losses due, mainly, to restrictions in the export of infested animals to several countries. Its spatial distribution has been tied to environmental factors, mainly warm temperatures and high relative humidity. In this work, we integrated a dataset consisting of 5843 records of Rhipicephalus spp., in Mexico covering close to 50 years to know which environmental variables mostly influence this ticks' distribution. Occurrences were georeferenced using the software DIVA-GIS and the potential current distribution was modelled using the maximum entropy method (Maxent). The algorithm generated a map of high predictive capability (Area under the curve = 0.942), providing the various contribution and permutation importance of the tested variables. Precipitation seasonality, particularly in March, and isothermality were found to be the most significant climate variables in determining the probability of spatial distribution of Rhipicephalus spp. in Mexico (15.7%, 36.0% and 11.1%, respectively). Our findings demonstrate that Rhipicephalus has colonized Mexico widely, including areas characterized by different types of climate. We conclude that the Maxent distribution model using Rhipicephalus records and a set of environmental variables can predict the extent of the tick range in this country, information that should support the development of integrated control strategies.

Novel triclabendazole prodrug: A highly water soluble alternative for the treatment of fasciolosis.

In this work we present the synthesis, aqueous solubility and stability, hydrolysis by alkaline phosphatase, and in vivo fasciolicidal activity in sheep of a highly water soluble phosphate salt prodrug of triclabendazole (MFR-5). The aqueous solubility of MFR-5 at pH 7 was 88,000-fold that of triclabendazole. MFR-5 showed excellent aqueous stability (>95% after 26h) at pH 7, making it ideal for developing pharmaceutical compositions in the form of solutions that can easily be hydrolyzed by the enzyme alkaline phosphatase (t=13.6s) to liberate the precursor compound. An aqueous solution of MFR-5 administered intramuscularly to sheep at concentrations of 4, 6 and 8mg/kg presented a fasciolicidal efficiency of 96.5%, 98.4% and 99.2%, respectively. In the in vivo experiments, MFR-5 reduced 100% the excretion of eggs in all of the above concentrations.

In vitro antihelmintic effect of fifteen tropical plant extracts on excysted flukes of Fasciola hepatica.

BACKGROUND: Fasciolosis due to Fasciola hepatica is the most important hepatic disease in veterinary medicine. Its relevance is important because of the major economical losses to the cattle industry such as: reduction in milk, meat and wool production; miscarriages, anemia, liver condemnation and occasionally deaths, are estimated in billons of dollars. The emergence of fluke resistance due to over or under dosing of fasciolides as well as environmental damage produced by the chemicals eliminated in field have stimulated the need for alternative methods to control Fasciola hepatica. The aim of this study was to evaluate the in vitro anthelmintic effect of fifteen tropical plant extracts used in tradicional Mexican medicine, on newly excysted flukes of Fasciola hepatica. RESULTS: The flukes were exposed in triplicate at 500, 250 and 125 mg/L to each extract. The efficacy was assessed as the mortality rate based on the number of live and dead flukes after 24, 48 and 72 h post-exposure. The plants with anthelmintic effect were evaluated once again with a concentration of 375 mg/L in order to confirm the results and to calculate lethal concentrations at 50%, 90% and 99% (LC(50), LC(90), and LC(99)). Plant extracts of Lantana camara, Bocconia frutescens, Piper auritum, Artemisia mexicana and Cajanus cajan had an in vitro anthelmintic effect (P <0.05). The LC(50), LC(90) and LC(99) to A. mexicana, C. cajan and B. frutescens were 92.85, 210.44 and 410.04 mg/L, 382.73, 570.09 and 788.9 mg/L and 369.96, 529.94 and 710.34 mg/L, respectively. CONCLUSION: It is concluded that five tropical plant extracts had promising anthelmintic effects against F. hepatica. Further studies on toxicity and in vivo biological evaluation in ruminant models might help to determine the anthelmintic potential of these plant extracts.

A highly water soluble benzimidazole derivative useful for the treatment of fasciolosis.

This study describes the synthesis of compound (7), a highly hydrosoluble phosphonooxymethyl prodrug of compound alpha (4). Compound (7) improved the aqueous solubility of its precursor compound (4) by 50,000 times and it is stable at neutral pH. The prodrug showed faciolicidal activity when evaluated in vitro against excysted Fasciola hepatica metacercariae. The in vivo evaluation of (7) was carried out via oral, intramuscular and subcutaneous administration in sheep artificially infected with F. hepatica metacercariae. At an intramuscular dose of 4 mg/kg, the activity of (7) was similar to that of compound alpha (4) at an oral dose of 15 mg/kg.

Efficacy of a novel topical fipronil, (S)-methoprene, eprinomectin and praziquantel combination against naturally acquired intestinal nematode and cestode infections in cats.

The efficacy of a novel topical combination formulation of fipronil, (S)-methoprene, eprinomectin and praziquantel against naturally acquired intestinal nematode and cestode infections in cats was evaluated in seven negative control, blinded studies. Cats were selected based on a pre-treatment faecal examination indicating a patent infection with at least hookworms (two studies), Toxocara ascarids (one study), taeniid cestodes (two studies) or Dipylidium cestodes (two studies). In each study, cats were assigned randomly to blocks of two animals each, based on decreasing pre-treatment body weight and were randomly allocated to one of two groups of six to 12 cats: untreated (control) or treated with topical fipronil (8.3%, w/v), (S)-methoprene (10%, w/v), eprinomectin (0.4%, w/v) and praziquantel (8.3%, w/v) (BROADLINE(®), Merial) at 0.12 mL/kg body weight (providing a minimum of 10mg fipronil+12 mg S-methoprene+0.5mg eprinomectin+10mg praziquantel per kg body weight). The topical treatment was administered directly on the skin in the midline of the neck in a single spot once on Day 0. For parasite recovery and count, cats were euthanized humanely and necropsied seven or ten days after treatment. A single treatment with the novel topical combination product provided 91% efficacy against Ancylostoma braziliense, ≥ 99% efficacy against Ancylostoma tubaeforme, and >97% efficacy against Toxocara cati. Similarly, excellent efficacy was established against Taenia taeniaeformis, Dipylidium caninum and Diplopylidium spp. as demonstrated by >97% and up to 100% reductions of cestode counts in the treated cats when compared to the untreated controls (P<0.01). All cats accepted the treatment well based on health observations post-treatment and daily health observations. No adverse experiences or other health problems were observed throughout the studies. The results of this series of controlled studies demonstrated high efficacy and excellent acceptability of the novel topical combination formulation of fipronil, (S)-methoprene, eprinomectin and praziquantel against a broad range of feline intestinal nematode and cestode infections.

Antiprotozoan lead discovery by aligning dry and wet screening: prediction, synthesis, and biological assay of novel quinoxalinones.

Protozoan parasites have been one of the most significant public health problems for centuries and several human infections caused by them have massive global impact. Most of the current drugs used to treat these illnesses have been used for decades and have many limitations such as the emergence of drug resistance, severe side-effects, low-to-medium drug efficacy, administration routes, cost, etc. These drugs have been largely neglected as models for drug development because they are majorly used in countries with limited resources and as a consequence with scarce marketing possibilities. Nowadays, there is a pressing need to identify and develop new drug-based antiprotozoan therapies. In an effort to overcome this problem, the main purpose of this study is to develop a QSARs-based ensemble classifier for antiprotozoan drug-like entities from a heterogeneous compounds collection. Here, we use some of the TOMOCOMD-CARDD molecular descriptors and linear discriminant analysis (LDA) to derive individual linear classification functions in order to discriminate between antiprotozoan and non-antiprotozoan compounds as a way to enable the computational screening of virtual combinatorial datasets and/or drugs already approved. Firstly, we construct a wide-spectrum benchmark database comprising of 680 organic chemicals with great structural variability (254 of them antiprotozoan agents and 426 to drugs having other clinical uses). This series of compounds was processed by a k-means cluster analysis in order to design training and predicting sets. In total, seven discriminant functions were obtained, by using the whole set of atom-based linear indices. All the LDA-based QSAR models show accuracies above 85% in the training set and values of Matthews correlation coefficients (C) vary from 0.70 to 0.86. The external validation set shows rather-good global classifications of around 80% (92.05% for best equation). Later, we developed a multi-agent QSAR classification system, in which the individual QSAR outputs are the inputs of the aforementioned fusion approach. Finally, the fusion model was used for the identification of a novel generation of lead-like antiprotozoan compounds by using ligand-based virtual screening of 'available' small molecules (with synthetic feasibility) in our 'in-house' library. A new molecular subsystem (quinoxalinones) was then theoretically selected as a promising lead series, and its derivatives subsequently synthesized, structurally characterized, and experimentally assayed by using in vitro screening that took into consideration a battery of five parasite-based assays. The chemicals 11(12) and 16 are the most active (hits) against apicomplexa (sporozoa) and mastigophora (flagellata) subphylum parasites, respectively. Both compounds depicted good activity in every protozoan in vitro panel and they did not show unspecific cytotoxicity on the host cells. The described technical framework seems to be a promising QSAR-classifier tool for the molecular discovery and development of novel classes of broad-antiprotozoan-spectrum drugs, which may meet the dual challenges posed by drug-resistant parasites and the rapid progression of protozoan illnesses.

Prevalencia de Giardia intestinalis y predominio de genotipos zoonóticos en ovinos y bovinos de traspatio de cinco estados de la República Mexicana / Prevalence of Giardia intestinalis and zoonotic genotype predominance in small scale sheep and cattle farms in five states of the Mexican Republic

The aim of this paper was to discover the prevalence and assemblages of Giardia intestinalis, harbored in sheep and cows on familiar farms from five states of the Mexican Republic. Stool samples from 265 sheep and 174 cows were analyzed by centrifugation and flotation in zinc sulfate to search for cysts and ova. The samples with Giardia cysts were processed in a Sheather solution in order to isolate them. Afterwards, cultures were established in TYI-S-33, each one of which was the Giardia DNA source. The DNA was obtained and used as a template to amplify a fragment of the glutamate dehydrogenase (gdh) enzyme. The 430 bp amplicons were restricted with Nla IV and Rsa I in order to identify the restriction fragments length polymorphisms (RFLP's) patterns. From the cyst analysis, Giardia cysts in nine cows (5.1%) and 30 sheep (11.3%) were found. Then 10 axenic cultures (5 from sheep and 5 from cows) were set up. From the RFLP's pattern it was found that one cow had assemblage (AI), another two had a mixture of assemblages (AI + BIII) and the other two had (E + BIII). In sheep, it was found that two sheep had assemblage (AI) and the other three had a mixture of assemblages (AI + BIII). This is the first report in which zoonotic assemblages (A-I and BIII) predominance in ruminants from five states of Mexico have been demonstrated. Therefore, it is necessary to carry out further studies aimed at discovering other Giardia genotypes and transmission patterns between animals and humans in Mexico.

Con el fin de determinar la frecuencia y genotipos de Giardia intestinalis en ovinos y bovinos de traspatio de algunos estados de la República Mexicana, en este trabajo se colectaron heces de 265 ovinos y 174 bovinos, para la búsqueda de Giardia mediante coproparasitoscópicos (CPS) de concentración flotación. De las muestras fecales que resultaron positivas se obtuvieron los quistes por el método de Sheather. Los quistes se desenquistaron in vitro y los trofozoítos se mantuvieron en cultivo TYI-S-33 axénico. El ADN de los trofozoítos se obtuvo mediante extracciones fenólicas y se amplificó un segmento de ≈ 430 pb del gen de la enzima glutamato deshidrogenasa (gdh) por medio de la reacción en cadena de la ADN polimerasa (PCR), el producto se restringió con las enzimas Nla IV y Rsa I y se obtuvieron los polimorfismos de los fragmentos de restricción (RFLP). En los CPS se encontró a Giardia en nueve bovinos (5.1%) y 30 ovinos (11.3%). Se establecieron 10 cultivos axénicos (5 de bovinos y 5 de ovinos). En un bovino se encontró el genotipo (AI), dos tuvieron mezcla de los genotipos (AI + BIII) y los otros dos fueron (E + BIII). Un ovino fue genotipo (AI) y tres tuvieron mezcla de los genotipos (AI + BIII). Éste es el primer informe que presenta predominio de genotipos zoonóticos (AI y BIII) en ovinos y bovinos de México. Es necesario investigar los genotipos de Giardia y patrones de transmisión entre animales y humanos en México.

Solubilidad de un compuesto con actividad fasciolicida: evaluación de eficacia in vitro y en ovinos experimentalmente infectados con Fasciola hepatica / Solubility of a compound with fasciolicidal activity: evaluation of efficacy in vitro and experimentally on infected sheep with Fasciola hepatica

Compound alpha (Ca) is a benzimidazolic derívate which has shown a high fasciolicidal efficacy when it is given by oral via. Solubilization of Ca would be a suitable alternative to obtain an injectable formulation. The objectives of the present study were to evaluate the solubility of Ca in order to determine the in vitro fasciolicidal efficacy and experimentally on infected ovines. The assays of solubility quantified by high-performance liquid chromatography (HPLC), showed that beta-cyclodextrin (β-CD) and hydroxi-propylbeta-cyclodextrin (HP-β-CD) solubilized in 0.015% and 1.018% respectively. The solubilization for HP-β-CD was calculated as 3.95% for 5.8 mg; 10.76% for HP-β-CD with methanol, 39.048% for proylene-glycol (PG), 100% for glycerol formal (GF), and 100% for combined PG and GF and water in proportions of 3:2:5 and 3:2:4. In vitro evaluation using immature F. hepatica showed that Ca induced 100% efficacy 24 hours after solubilization with either GF or GF combined with PG and water. The efficacy in sheep was 56.4 and 68.4% at 1 and 2 mg/kg respectively; whereas 1 mg/kg of sulphoxide metabolite induced an efficacy of 73.9%. The HPLC assays performed showed no evidence of the sulphoxide and sulphone metabolites. It is concluded that besides solubility and high in vitro fasciolicidal efficacy, the in vivo results in sheep showed only a moderate efficacy.

El compuesto alfa es un derivado bencimidazólico de gran eficacia fasciolicida, que se ingiere vía oral, su solubilización constituye una alternativa para obtener una formulación inyectable. Los objetivos de este estudio fueron: evaluar la solubilidad del compuesto alfa, determinar la eficacia fasciolicida in vitro y en ovinos experimentalmente infectados. Los ensayos de solubilidad que fueron cuantificados mediante cromatografía de líquidos de alta resolución, mostraron que la betaciclodextrina e hidroxipropil-betaciclodextrina solubilizaron en 0.051% y 1.018%. Esta última se evaluó con cinco concentraciones, mostró que a 5.8 mg la solubilidad fue 3.95%; hidroxipropil-betaciclodextrina y metanol, 10.76%; propilén-glicol, 39.048%; glicerol formal, 100%; y glicerol formal con propilén-glicol y agua en proporciones 3:2:5 y 3:2:4, 100%. La evaluación in vitro con fasciolas inmaduras mostró que el compuesto con glicerol formal y la combinación de vehículos a las 24 horas fueron eficaces en 100%. La eficacia in vivo en ovinos mostró que el compuesto alfa a dosis de 1 mg/kg fue de 56.46%, a 2 mg/kg de 68.4% y con sulfóxido del compuesto alfa a 1 mg/kg de 73.9%. No se encontró el compuesto alfa ni sus metabolitos sulfóxido y sulfona en el suero determinado por medio del cromatógrafo de líquidos de alta resolución. Se concluye que a pesar de obtener solubilidad del compuesto alfa y alta eficacia fasciolicida in vitro, los resultados in vivo en ovinos revelaron eficacia moderada.

New antitrichomonal drug-like chemicals selected by bond (edge)-based TOMOCOMD-CARDD descriptors.

Bond-based quadratic indices, new TOMOCOMD-CARDD molecular descriptors, and linear discriminant analysis (LDA) were used to discover novel lead trichomonacidals. The obtained LDA-based quantitative structure-activity relationships (QSAR) models, using nonstochastic and stochastic indices, were able to classify correctly 87.91% (87.50%) and 89.01% (84.38%) of the chemicals in training (test) sets, respectively. They showed large Matthews correlation coefficients of 0.75 (0.71) and 0.78 (0.65) for the training (test) sets, correspondingly. Later, both models were applied to the virtual screening of 21 chemicals to find new lead antitrichomonal agents. Predictions agreed with experimental results to a great extent because a correct classification for both models of 95.24% (20 of 21) of the chemicals was obtained. Of the 21 compounds that were screened and synthesized, 2 molecules (chemicals G-1, UC-245) showed high to moderate cytocidal activity at the concentration of 10 microg/ml, another 2 compounds (G-0 and CRIS-148) showed high cytocidal activity only at the concentration of 100 microg/ml, and the remaining chemicals (from CRIS-105 to CRIS-153, except CRIS-148) were inactive at these assayed concentrations. Finally, the best candidate, G-1 (cytocidal activity of 100% at 10 microg/ml) was in vivo assayed in ovariectomized Wistar rats achieving promising results as a trichomonacidal drug-like compound.

In vitro fasciolicide activity of some plant extracts against newly excysted flukes.

The aim of the present study was to evaluate, under in vitro conditions, the activity of 20 plant extracts against newly excysted Fasciola hepatica metacercariae. The plants were selected on the basis of anthelmintic activity, traditional use, availability, solubility, and permanence in the liver. For the in vitro evaluation, 24-well Nunc-immunoplates containing RPMI-1640 culture medium bovine sera plus antibiotics were used. Extract concentrations of 500 and 50 mg/mL were placed in triplicate. Approximately 10 newly excysted flukes were placed in each well, and the plates were incubated in a CO(2) incubator at 5% atmosphere for 3 days. In each plate four control wells containing nontreated flukes were included. All procedures were undertaken under an air-flow cabinet. Efficacy was measured on days 1 and 3 by considering the motility of the live or dead trematodes evaluated with the aid of an inverted microscope. Results indicated that, from all extracts evaluated, Tilia mexicana showed an average efficacy for concentrations of 500 and 50 mg/mL, respectively, of 100 and 100%; Eriobotrya japonica, 100 and 95%; Coffea arabica, 100 and 100%; Crataegus mexicana, 95 and 90%; and Ternstroemia pringlei 100 and 100%. From this preliminary evaluation, we conclude that further and more detailed evaluations at lower concentrations should be undertaken to understand the real efficacy of these plant extracts.

In silico fasciolicide activity of three experimental compounds in sheep.

The aim of the present study was to evaluate the fasciolicide activity of three experimental drugs, selected by an in silico system called TOMOCOMD-CARDD, in sheep. Drugs were identified by the computer system, and, after statistical selection, 24 Pelibuey sheep were infected on days 0 and 30, each with 200 metacercariae of Fasciola hepatica. When the infection reached 8 and 4 weeks of age, respectively, four groups of six animals each were formed. Group 1 received thiacetazone 150 mg/animal/p.o. Group 2 was treated with 3,5,5, trimethyloxazolidine 2,4-dione at 450 mg/animal/p.o. G3 received guanabenz acetate at a dose of 1.5 mg/animal/p.o. G4 served as an untreated control. Monitoring of the animals was followed by individual coprological examinations and slaughter of the animals 15 days after treatment to collect and count flukes from the liver. Efficacy was measured as the reduction in the percentage of flukes of treated animals relative to untreated controls. Results indicated an efficacy of 80.0, 43.8, and 100% for 8-week-old flukes and 62.1, 57.9, and 100% for 4-week-old flukes in the three experimental groups, respectively. Even though guanabenz acetate showed a high efficacy, it was highly toxic since two animals died approximately 24 h after being treated. We conclude that further investigations should be conducted to perform computer-aided prediction of drugs aimed to detect fasciolicide activity.

Paramphistomum spp: improved artificial excystment and in vitro culture of immature and adult stages.

The aims of the present study were to establish an adequate excystment method for metacercariae of Paramphistomum spp. and to compare the in vitro survival of paramphistomids in three different culture media. In a preliminary study, 31 different assays were performed solely with the purpose of obtaining the best percentage of metacercariae excystment. Once the best excystment method was selected, 21 further assays using 100 metacercariae/assay were carried out to determine the average percentage of excystment. For the in vitro culture of paramphistomids, the Hedon-Fleig, Rohrbacher, and RPMI-1640 culture media were compared by using 24-well immunoplates containing newly excysted flukes or 50-ml capacity tissue-culture flasks for adult paramphistomids. Results indicated an average of 69.0% excystment, the best culture medium to support the survival of immature and adult stages of the trematode being Hedon-Fleig and Rohrbacher, respectively. The best percentage of survival obtained was on day 4 (80%), with some immature and adult trematodes remaining alive until days 10 and 11, respectively.

Effect of a cysteine protease inhibitor on Fasciola hepatica (liver fluke) fecundity, egg viability, parasite burden, and size in experimentally infected sheep.

Fasciola hepatica secretes proteolytic enzymes during liver invasion. The present study examined the effects of the cysteine protease inhibitor Ep-475 on sheep considering the following variables: serum levels of aspartate aminotransferase, L-lactate dehydrogenase, and gamma-glutamyltransferase, liver fluke fecundity, egg viability, parasite burden, and size. Twenty-four male sheep were randomly allocated in four groups of six animals each as follows: group A was infected with F. hepatica metacercariae and treated with 50 mg/kg of Ep-475, group B was infected and untreated, group C was uninfected and treated, and group D was uninfected and untreated. All animals were euthanized 10 weeks after the experimental infection. Serum activities of enzymes in infected animals were significantly lower in Ep-475-treated sheep than in untreated controls, although liver damage was produced. No significant reduction in total worm burden was observed between treated and untreated sheep. However, there was a significant difference on the average size, structure development, ova counts, and egg viability of liver flukes from these two groups. Results showed that Ep-475 reduces liver damage due to fasciolosis and induces an impairment of liver fluke growth and fecundity. These effects pinpoint liver fluke proteases as potential targets for pharmacological intervention.