Control de las tricostrongilidosis ovinasdiseño, síntesis y ensayos clínicos de eficacia de nuevas moléculas de acción antihelmíntica

Resumo

Infections caused by helminth parasites, including nematodes, trematodes and cestodes, represent an important cause of disease and economic loss for the small ruminant livestock industry. Within helminths, gastrointestinal nematodes are the most relevant in terms of health and economic impact in both developing and industrialised countries. Haemonchus contortus, Teladorsagia circumcincta and Trichostrongylus spp. are the three species responsible for the highest economic losses worldwide. Under field conditions, most infections are usually caused by more than one species of nematodes located in the abomasum and intestine. These are generally endemic processes, with chronic course and low mortality. However, the impact of this parasitism depends on the infection intessity, as well on the physiological and immunological state of the host. Most common clinical signs include anorexia, diarrhoea, emaciation and anaemia. However, in cases of high levels of infection, especially in young animals, death can take place. Administration of anthelmintic drugs has been the treatment of choice for the control of these parasites for many years due to the availability of highly effective and safe drugs. However, the increasing prevalence of anthelmintic resistance worldwide has led to control programmes that are costly and unsustainable in the long term. In addition, climate change is increasing the incidence and intensity of these infections, which exarcebates their economic impact on livestock farming. Therefore, searching for new drugs with anthelmintic activity and new mechanisms of action is vital. To address this problem, the general objective of the present Thesis is focused on the search for new compounds with potential anthelmintic activity against trichostrongylid infections in sheep which could alleviate the problems arising from the the emergence of resistance. In this context, three out of five chapters described in the present Thesis are focused on the evaluation of the possible anthelmintic activity of several families of compounds synthesized by the Department of Pharmaceutical Chemistry of the University of Salamanca. First, the compounds were subjected to a primary screening at a dose of 50 μM to determine their in vitro efficacy against different parasitic stages of the nematode T. circumcincta. Those compounds with an efficacy higher than 90% were selected to carry out studies with a triple resistant isolate (to benzimidazoles, ivermectin and levamisole), as well as to calculate their effective dose 50 (ED50). At the same time, in vitro cytotoxicity studies were carried out to discard the most toxic compounds. Therefore, the three compounds with the highest anthelmintic activity and lowest in vitro toxicity were selected in order to study their in vivo toxicity and efficacy in gerbils and sheep. Two diamine and one benzimidazole derivatives were selected as candidate compounds, all of them with an ED50 below 3 μM in one of the tests carried out. Once the potential adverse effects of oral administration of the candidates in mice were discarded, their anthelmintic efficacy was evaluated using Mongolian gerbils (Meriones unguiculatus) infected with H. contortus. After observing a 95% reduction in the number of pre-adults in the stomach of the gerbils in the group treated with the benzimidazole derivative at a dose of 200 mg/kg, we decided to evaluate the efficacy of the compound in the definitive host, the sheep. In the last experiment, the group of sheep treated with the benzimidazole derivative at a dose of 120 mg/kg produced a 99% reduction in the number of eggs in faeces 7 days after administration, and a 95% reduction in the number of adults present in the sheep abomasum. Under this context, the BZ derivative demonstrated to be an interesting candidate for the development of an anthelmintic drug. For this reason, we believe it is necessary to carry out further studies to establish a minimum effective dose, as well as to determine its efficacy in different GIN species and isolates, and to carry out detailed pharmacokinetic and pharmacodynamic studies. In this context, during the international stay at the “Swiss Tropical and Public Health Institute”, in Basel (Switzerland), part of the compounds previously tested in the nematode T. circumcincta, were evaluated against another model of GIN infecting mice: Trichuris muris. Three compounds were selected for their potent activity against the first larvae stage in an initial screening at a dose of 100 μM, and subsequently their efficacy against the adult forms of the parasite was tested. The three candidates consisted of two benzimidazole and one aminoalcohol derivatives, being different from those selected in the previous experiment. Additionally, these candidates were tested against adult forms of Heligmosomoides polygyrus. As a final result, two of them showed efficacy, at least, against one of the two parasites; one derivative showed an ED50 of 8.1 μM against T. muris and BZ12 showed an ED50 of 5.3 μM against H. polygyrus. Therefore, both compounds could also be potential candidates for future in vivo efficacy studies using infected animals. Another approach that we have considered in the present Thesis, in order to combat the problem of the lack of new anthelmintic drugs, is related to the search for improved screening techniques that allow the evaluation of large number of compounds in a short period of time. Thus, during the second international stay at the "Laboratory of Growth Regulators" of the University of Palacký, in Olomouc (Czech Republic), a high-throughput compound screening protocol was set up using the wMicrotracker ONE device on T. circumcincta and H. contortus. This device is able to automatically measure the mobility of the different parasite stages after their incubation with the compounds by means of infrared light microbeams. Complementarily, we have also carried out studies to determine the level of benzimidazole resistance of gastrointestinal nematodes present in sheep flocks in Castilla y León by means of in vivo, in vitro and molecular techniques. Our study confirmed an increase in the occurrence of anthelmintic resistance compared to the last data analysed in previous years, being T. circumcincta the most resistant species. In this study, 35% of the sampled farms were resistant to netobimin by the in vivo faecal egg reduction test and 26% by the in vitro egg hatching test, with a very high correlation between both techniques (r= -0.770; p < 0.0001). At molecular level, we also observed that a novel polymorphism at codon 198 of the isotype-1 β-tubulin gene can confer BZ resistance on its own in T. circumcincta. A very high and similar correlation was demonstrated between the presence of the new polymorphism and resistance measured by in vivo and in vitro assays (r > 0.720; p < 0.0001).