Fasciolosis is a zoonotic parasitic disease of worldwide distribution  caused by the liver fluke Fasciola hepatica which affects different kind of animals . Its relevance relies in huge economic losses in cattle production  . Among different drugs used to treat this disease, triclabendazole has shown high efficacy against young and adult stages with a wide margin of safety when applied by the oral route  . Closantel is also a fasciolicide of choice; in addition, it also kills nematodes such as Haemonchus contortus, and the fly Oestrus ovis  .
Recently our research group has synthesized a new water-soluble triclabendazole prodrug, named fosfatriclaben. This new compound increased the aqueous solubility 88,000-fold with respect to its precursor compound (triclabendazole) and allowed the formation of an aqueous solution for IM administration  (Figure 1).
In this work, we present the fasciolicidal efficacy of the fosfatriclaben compared with two commercials well known fasciolicides in naturally infested cattle.
2. Material and Methods
2.1. Study Location
The present study was carried out during the month of march, 2019, in a farm named “Mi Alma” located at approximately 150 km from Mexico City.
2.2. Experimental Animals
Twenty-four cross mixed adult bovines, both sexes, aged between 1 to 3 years, naturally infected with Fasciola hepatica were used. Food was provided as a balanced feed containing oats, alfalfa and ensilage. Water was available ad libitum.
The animals were previously selected on day 8 as positive to fasciola-eggs
Figure 1. Structure of triclabendazole and fosfatriclaben.
using the traditional sedimentation test . Shortly, feces were collected directly from the rectum and thoroughly homogenized to place 5 g in a glass using tap water. The mixture was placed in a beaker and sifted with the aid of a plastic sieve. After 10 min repose, the supernatant was decanted and fulfilled again with tap water. The process was repeated until the sediment was observed clean. The final sediment was observed under stereoscopic microscope to carry out the fluke-egg counting .
The animals were identified by ear tag.
2.3. Conduction of the Study
To apply the treatment, animals were divided into 4 groups of 6 animals each being ranked according to their fecal-egg count from the highest to the lowest number of eggs.
Group 1 (G1) was treated with 6 mg/kg of the experimental water-soluble fosfatriclaben injected once by the IM route. G2 was treated with triclabendazole (Fasimec® Elanco Australasia) given once at 12 mg/kg/PO; G3 was treated with 5% closantel® (Closantil-Chinoin) at 10 mg/kg/SC and G4 was the control group and it was injected with 5 mL of deionized water.
Fecal samples were analyzed by the sedimentation test , on days −8, 0 (day of treatment), 7, 14, 21 and 28 to count the number of eggs present in each one of the experimental animals, following the guide for evaluation of anthelmintics suggested by the World Association for Advancement of Veterinary Parasitology (WAAVP)  .
2.4. Assessment of Efficacy
The efficacy was calculated as the percentage of fluke-egg reduction on the treated groups, relative to the untreated control, according to the following formula .
2.5. Statistical Analysis
The obtained data were compared between the experimental groups using the non-parametric Kruskal-Wallis test. They were analyzed with the statistical IBM SPSS 24.0 (2016) software .
3. Results and Discussion
Table 1 shows the coprological analyses in which the total and mean number of fluke-eggs, observed in the experimental groups.
The percentage of efficacy exerted by fosfatriclaben, triclabendazole and closantel, at 7, 14, 21 and 28 days post-treatment is shown in Figure 2. Fosfatriclaben showed 100% reduction of eggs from day 7 to day 28; triclabendazole dosed orally showed an efficacy of 99.7% at day 7, and 100% from day 14 to day 28.
Table 1. Percentage of fluke-egg reduction before and after treatment with fosfatriclaben compared with two commercial fasciolicides in cattle naturally infected with Fasciola hepatica.
*day of treatment.
Figure 2. Percentage of efficacy of fosfatriclaben compared with two fasciolicides in the experimental groups.
Finally, closantel showed values between 98.8% and 99.7% in the study. No significant differences were observed when the experimental treated groups were compared (P < 0.05).
It is well known that an experimental compound should have high solubility, suitable chemical stability and neutral pH to be adequate for intramuscular application . This could be of great value in veterinary medicine, since it is common to prefer the parenteral treatment application considering the different advantages such as easy of treatment, decreasing stress of the animals, decreasing danger to the operator, the bioavailability of the drug is faster than oral-dosing products. In fact, it is a good advantage since the drug applied intramuscularly reaches the site of action without passing by the gastrointestinal barriers which may affect the drug bioavailability  .
Another important point is that the injectable drugs require less active ingredient to provoke a clinical effect and therefore less quantity of compound .
It is important to know that fosfatriclaben uses only half of the dose (6 mg/kg/ IM) to achieve 100% egg reduction, in comparison with its precursor triclabendazole (12 mg/kg/PO). This can be considered relevant since the environment could be less contaminated.
In 2014,  described the synthesis of a benzimidazole derivative named alfa-pro the compound being highly soluble in water. This compound was initially tested under in vitro conditions rendering 100% efficacy against newly excysted flukes. Afterwards, the authors tested the compound in artificially infected sheep at a dose of 4 mg/kg/IM removing 87.8% of flukes. Recently,  determined the effective dose of the alfa-pro against immature and adult Fasciola hepatica in experimentally infected sheep. This benzimidazole soluble drug cleared 97.6%, 98.5% and 98.8% of adult flukes when administered at 6, 8 and 10 mg/kg/IM, respectively, and 81.2% at 6 mg/kg/IM against 5-week-old flukes.
Considering that nowadays the bioavailability of benzimidazoles have increased tremendously, and been aware of the great potential of triclabendazole as fasciolicide, it was decided to create the new fosfatriclaben for parenteral use expecting to obtain similar efficacy to its oral precursor (triclabendazol) with the advantage of using half of the dose required to remove adult and juvenile flukes.
Wood et al.  mentioned that studies like this in which the fluke-egg reduction is evaluated, give valuable information in cattle, even when the number of adult flukes removed is unknown. Unfortunately, critical tests in cattle involving sacrifice are very expensive to run, therefore the alternative to assess egg-reduction in cattle is widely accepted and considered of a great value.
On the other hand, no significant differences were observed between treatments (P < 0.05), indicating that possibly fosfatriclaben, applied once by the intramuscular route to cattle is as effective as its precursor triclabendazole (PO), recognized to date as the most effective and safer fasciolicidal agent. However, is important to note that not in all cases triclabendazole has removed 100% of flukes, perhaps this is in part because of the appearance of resistance to this drug in different parts of the world    .
It is important to emphasize that none of the other commercial fasciolicides tested were able to remove fluke eggs at 100% along period of the experiment.
Different studies have demonstrated the high fasciolicidal efficacy exerted by closantel . However, this drug only exerts high efficacy to flukes older than 6 weeks of age. Certainly, this confer the probability to the remaining flukes after treatment could be continue maturing to produce more eggs.
In coincidence with data obtained by  who treated 41 cows with closantel at 5 mg/kg/SC; then, they found fluke-eggs 3 weeks after the treatment.
In addition,  observed fluke-eggs after treatment with closantel, obtaining 72% and 92% of efficacy. Nevertheless, closantel is still being well recognized as a good fasciolicide and it also shows good efficacy against other parasites such as Haemonchus contortus and Oestrus ovis.
In contrast to this situation, fosfatriclaben at present reaches up to 100% efficacy since as a new experimental drug is used in a very limited manner.
Results obtained in this study suggest that the experimental fosfatriclaben is a good candidate to be consider for the treatment of fasciolosis in cattle. Further studies will determine the real potential of this experimental drug indicated for parenteral use in sheep and cattle.
At present, this drug is under different biological tests aimed to know the mode of action, drug stability as well as the withholding period. This compound is patented by the Mexican Institute for Industrial Protection (Instituto Mexicano de la Protección Industrial) (MX2014014417A) and it also obtained the patent of USA (US10239842 B2).
The experimental fosfatriclaben, a highly water-soluble prodrug, administered at 6 mg/kg/IM to naturally infected cattle exerted similar fasciolicidal efficacy to its precursor triclabendazole when it is dosed at 12 mg/kg/PO, and closantel injected at 10 mg/kg/SC.
This work was supported by UNAM-PAPIIT-IT201018. M. Flores-Ramos is very grateful to Dirección General de Asuntos del Personal Académico (DGAPA, UNAM) for the Postdoctoral fellowship granted. The authors are indebted to Laboratorios Loeffler for kind donation of triclabendazole. The authors are grateful to the owners of the farm “Mi Alma”, for permission to use their cattle.
Availability of Data and Material
All datasets are included in this manuscript.
The present study was carried out thanks to the project PAPIIT-DEGPA-UNAM No. RT201018.
MFR, RC, AHC contributed with the synthesis of the fosfatriclaben, manuscript revision and discussion. All other authors contributed to the study design, carried out the field study, performed laboratory analysis, collaborated with results interpretation, data analysis, manuscript revision and discussion: Performed statistical data analysis and suggested the experimental design: FIV, YVM, and Wrote the paper: FIV with input from YVM Grant funding: FIV. All authors supervised the experimental procedures, read and approved the final version of the manuscript.
The work was carried out adhering to the guidelines of the Institutional Committee for Use and Care of Experimental Animals of the institution, according to the Mexican Official Regulation NOM-062-ZOO-1999 and Animal Research: Reporting of In Vivo Experiments guidelines was followed.
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