Effectiveness of the Experimental Fosfatriclaben in Comparison with Two Commercial Fasciolicides in Cattle

The aim of the present study was to compare the fasciolicidal efficacy in cattle of an experimental water-soluble compound, named fosfatriclaben, a prodrug of triclabendazole, with two commercial fasciolicides. For this, twenty-four cross mixed milking cows, aged between 1 to 3 years, naturally infected with Fasciola hepatica, were used. To apply the treatment, animals were divided into 4 groups of 6 animals each being ranked according to their fecal flukeegg count from the highest to the lowest number of eggs. The compounds were administered in the groups with a single dose. Group 1 (G1) was treated with 6 mg/kg/IM of fosfatriclaben, G2 was treated with triclabendazole at 12 mg/kg/PO; G3 was treated with 5% closantel at 10 mg/kg/SC and G4 was administered with 5 mL/IM of deionized water and served as untreated control. Fecal samples were analyzed on days—8, 0 (treatment day), 7, 14, 21 and 28 to count the number of fasciola eggs before and after the treatment using the sedimentation test. The efficacy was assessed as the percentage of fluke-egg reduction on treated groups relative to the untreated control. The resulting data were analyzed by the Kruskal-Wallis non-parametric test, using the statistical IBM SPSS 24.0 (2016) program. Results indicated a fluke-egg reduction of 100% for fosfatriclaben along the study; triclabendazole showed an efficacy of 99.7% 100% from day 7 to day 28. In the case of closantel, values from 98.8% to 99.6% were recorded from day 7 to day 28. No statistical differences between treatments were observed (P < 0.05). It was concluded that the experimental fosfatriclaben administered at 6 mg/kg/IM to naturally infected cattle exerted a fasciolicidal efficacy similar to the drugs of choice for the treatment of fasciolosis (triclabendazole) when administered at 12 mg/kg/ PO, and closantel injected at 10 mg/kg/SC. How to cite this paper: Rojas-Campos, T., Vera-Montenegro, Y., Flores-Ramos, M., Castillo, R., Hernández-Campos, A. and IbarraVelarde, F. (2019) Effectiveness of the Experimental Fosfatriclaben in Comparison with Two Commercial Fasciolicides in Cattle. Pharmacology & Pharmacy, 10, 498-506. https://doi.org/10.4236/pp.2019.1011041 Received: September 5, 2019 Accepted: November 8, 2019 Published: November 11, 2019 Copyright © 2019 by author(s) and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access T. Rojas-Campos et al. DOI: 10.4236/pp.2019.1011041 499 Pharmacology & Pharmacy


Introduction
Fasciolosis is a zoonotic parasitic disease of worldwide distribution [1] caused by the liver fluke Fasciola hepatica which affects different kind of animals [2]. Its relevance relies in huge economic losses in cattle production [3] [4]. 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 [5] [6]. Closantel is also a fasciolicide of choice; in addition, it also kills nematodes such as Haemonchus contortus, and the fly Oestrus ovis [7] [8].
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 [9] ( Figure 1).
In this work, we present the fasciolicidal efficacy of the fosfatriclaben compared with two commercials well known fasciolicides in naturally infested cattle.

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.

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   [10]. 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 [10].
The animals were identified by ear tag.

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.

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 [11].

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 [13]. Table 1 shows the coprological analyses in which the total and mean number of fluke-eggs, observed in the experimental groups.

Results and Discussion
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.    It is well known that an experimental compound should have high solubility, suitable chemical stability and neutral pH to be adequate for intramuscular application [14]. 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 oraldosing 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 [15] [16].
Another important point is that the injectable drugs require less active ingredient to provoke a clinical effect and therefore less quantity of compound [15].
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, [14] described the synthesis of a benzimidazole derivative named alfa-pro the compound being highly soluble in water. This compound was in- pecting 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. [11] 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 [6] [18] [19] [20].
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 [21]. 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 [22] who treated 41 cows with closantel at 5 mg/kg/SC; then, they found fluke-eggs 3 weeks after the treatment.

Conclusion
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.

Availability of Data and Material
All datasets are included in this manuscript.

Funding
The present study was carried out thanks to the project PAPIIT-DEGPA-UNAM No. RT201018.

Animal Research
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.

Conflicts of Interest
The authors declare no conflicts of interest regarding the publication of this paper.