In vitro evaluation of ivermectin, moxidectin, albendazole and pyrantel against cyathostomins of horses / Avaliação in vitro da ivermectina, moxidectina, albendazole e pirantel contra ciatostomíneos de equinos

Abstract Cyathostomins are the most prevalent nematodes of horses, and multidrug resistance has been reported worldwide. There is a need to implement alternative drug monitoring analytical tests. The objective of this study was to determine the consistency (5 repetitions) of the larval migration on agar test (LMAT) using ivermectin, moxidectin, pyrantel or albendazole against cyathostomin infective-stage larvae in eight different concentrations. LMAT showed a strong coefficient of determination (R2 > 0.91), between the test repetitions (n=5). The average 50% effective concentration (EC50) for ivermectin, moxidectin, pyrantel and albendazole were 0.0404, 0.0558, 0.0864 and 0.0988 nMol, respectively. The results of the EC50 for albendazole showed the greatest range of concentration. Ivermectin and moxidectin had the lowest in between-test variation. In the future, internationally certified susceptible isolates could be used for screening new drug candidates, or to follow up the pattern of drug efficacy from field populations.

Resumo Ciatostomíneos são os nematodas mais prevalentes em equinos e a resistência múltipla foi relatada em todo o mundo. Existe a necessidade de implementar o monitoramento dos produtos com testes analíticos alternativos. O objetivo deste estudo foi determinar a consistência (5 repetições) do teste de migração larval em ágar (TMLA) usando ivermectina, moxidectina, pirantel e albendazole contra larvas infectantes de ciatostomíneos em oito concentrações diferentes. O TMLA demonstrou um coeficiente de determinação (R2) acima de 0,91 entre as repetições do teste. A concentração efetiva para 50% (CE50) para ivermectina, moxidectina, pirantel e albendazole foi de 0,0404; 0,0558; 0,0864 e 0,0988 nMol, respectivamente. A CE50 do albendazole demonstrou a maior amplitude entre os testes. A ivermectina e a moxidectina tiveram as menores variações das doses entre as repetições. No futuro, isolados certificados susceptíveis poderão ser testados com o TMLA para indicação de novos produtos e mesmo para acompanhar o perfil de eficácia de populações do campo.

In vitro evaluation of ivermectin, moxidectin, albendazole and pyrantel against cyathostomins of horses.

Cyathostomins are the most prevalent nematodes of horses, and multidrug resistance has been reported worldwide. There is a need to implement alternative drug monitoring analytical tests. The objective of this study was to determine the consistency (5 repetitions) of the larval migration on agar test (LMAT) using ivermectin, moxidectin, pyrantel or albendazole against cyathostomin infective-stage larvae in eight different concentrations. LMAT showed a strong coefficient of determination (R2 > 0.91), between the test repetitions (n=5). The average 50% effective concentration (EC50) for ivermectin, moxidectin, pyrantel and albendazole were 0.0404, 0.0558, 0.0864 and 0.0988 nMol, respectively. The results of the EC50 for albendazole showed the greatest range of concentration. Ivermectin and moxidectin had the lowest in between-test variation. In the future, internationally certified susceptible isolates could be used for screening new drug candidates, or to follow up the pattern of drug efficacy from field populations.

In vitro evaluation of ivermectin, moxidectin, albendazole and pyrantel against cyathostomins of horses / Avaliação in vitro da ivermectina, moxidectina, albendazole e pirantel contra ciatostomíneos de equinos

Cyathostomins are the most prevalent nematodes of horses, and multidrug resistance has been reported worldwide. There is a need to implement alternative drug monitoring analytical tests. The objective of this study was to determine the consistency (5 repetitions) of the larval migration on agar test (LMAT) using ivermectin, moxidectin, pyrantel or albendazole against cyathostomin infective-stage larvae in eight different concentrations. LMAT showed a strong coefficient of determination (R2 > 0.91), between the test repetitions (n=5). The average 50% effective concentration (EC50) for ivermectin, moxidectin, pyrantel and albendazole were 0.0404, 0.0558, 0.0864 and 0.0988 nMol, respectively. The results of the EC50 for albendazole showed the greatest range of concentration. Ivermectin and moxidectin had the lowest in between-test variation. In the future, internationally certified susceptible isolates could be used for screening new drug candidates, or to follow up the pattern of drug efficacy from field populations.(AU)

Ciatostomíneos são os nematodas mais prevalentes em equinos e a resistência múltipla foi relatada em todo o mundo. Existe a necessidade de implementar o monitoramento dos produtos com testes analíticos alternativos. O objetivo deste estudo foi determinar a consistência (5 repetições) do teste de migração larval em ágar (TMLA) usando ivermectina, moxidectina, pirantel e albendazole contra larvas infectantes de ciatostomíneos em oito concentrações diferentes. O TMLA demonstrou um coeficiente de determinação (R2) acima de 0,91 entre as repetições do teste. A concentração efetiva para 50% (CE50) para ivermectina, moxidectina, pirantel e albendazole foi de 0,0404; 0,0558; 0,0864 e 0,0988 nMol, respectivamente. A CE50 do albendazole demonstrou a maior amplitude entre os testes. A ivermectina e a moxidectina tiveram as menores variações das doses entre as repetições. No futuro, isolados certificados susceptíveis poderão ser testados com o TMLA para indicação de novos produtos e mesmo para acompanhar o perfil de eficácia de populações do campo.(AU)

Lack of Cyathostomin sp. reduction after anthelmintic treatment in horses in Brazil.

The increase of anthelmintic resistance in the last years in the nematode population of veterinary importance has become a major concern. The objective of the present study was to evaluate the efficacy of the main anthelmintic drugs available in the market against small strongyles of horses in Brazil. A total of 498 horses from 11 horse farms, located in the states of Paraná, São Paulo, Rio de Janeiro and Minas Gerais, in Brazil, were treated with ivermectin, moxidectin, pyrantel and fenbendazole, orally at their recommended doses. The fecal egg count reduction test (FECRT) was used to determine the product's efficacy and fecal culture was used to determine the parasite genus. Reduction on anthelmintic efficacy was found for fenbendazole in all horse farms (11/11), pyrantel in five yards (5/11) and ivermectin had low efficacy in one of the yards studied (1/11). Multidrug resistance of up to 3 drugs classes was found in one of the tested farms (1/11). Cyathostomin were the most prevalent parasite. The results showed that resistance to fenbendazole is widespread; the efficacy of pyrantel is in a critical situation. Although the macrocyclic lactones compounds still showed high efficacy on most farms, suspected resistance to macrocyclic lactones is of great concern.

Glutamine 57 at the complementary binding site face is a key determinant of morantel selectivity for {alpha}7 nicotinic receptors.

Nicotinic receptors (AChRs) play key roles in synaptic transmission. We explored activation of neuronal alpha7 and mammalian muscle AChRs by morantel and oxantel. Our results revealed a novel action of morantel as a high efficacy and more potent agonist than ACh of alpha7 receptors. The EC(50) for activation by morantel of both alpha7 and alpha7-5HT(3A) receptors is 7-fold lower than that determined for ACh. The minimum morantel concentration required to activate alpha7-5HT(3A) channels is 6-fold lower than that of ACh, and activation episodes are more prolonged than in the presence of ACh. By contrast, oxantel is a weak agonist of alpha7 and alpha7-5HT(3A), and both drugs are very low efficacy agonists of muscle AChRs. The replacement of Gln(57) in alpha7 by glycine, which is found in the equivalent position of the muscle AChR, decreases the efficacy for activation and turns morantel into a partial agonist. The reverse mutation in the muscle AChR (epsilonG57Q) increases 7-fold the efficacy of morantel. The mutations do not affect activation by ACh or oxantel, indicating that this position is selective for morantel. In silico studies show that the tetrahydropyrimidinyl group, common to both drugs, is close to Trp(149) of the principal face of the binding site, whereas the other cyclic group is proximal to Gln(57) of the complementary face in morantel but not in oxantel. Thus, position 57 at the complementary face is a key determinant of the high selectivity of morantel for alpha7. These results provide new information for further progress in drug design.

Activation of single nicotinic receptor channels from Caenorhabditis elegans muscle.

Nicotinic acetylcholine receptors (nAChRs) are pentameric neurotransmitter-gated ion channels that mediate synaptic transmission throughout the nervous system in vertebrates and invertebrates. Caenorhabditis elegans is a nonmammalian model for the study of the nervous system and a model of parasitic nematodes. Nematode muscle nAChRs are of considerable interest because they are targets for anthelmintic drugs. We show single-channel activity of C. elegans muscle nAChRs for the first time. Our results reveal that in the L1 larval stage acetylcholine (ACh) activates mainly a levamisole-sensitive nAChR (L-AChR). A single population of 39 pS channels, which are 5-fold more sensitive to levamisole than ACh, is detected. In contrast to mammalian nAChRs, open durations are longer for levamisole than for ACh. Studies in mutant strains reveal that UNC-38, UNC-63, and UNC-29 subunits are assembled into a single L-AChR in the L1 stage and that these subunits are irreplaceable, suggesting that they are vital for receptor function throughout development. Recordings from a strain mutated in the LEV-1 subunit show a main population of channels with lower conductance (26 pS), prolonged open durations, and reduced sensitivity to levamisole. Thus, although LEV-1 is preferentially incorporated into native L-AChRs, receptors lacking this subunit can still function. No single-channel activity from levamisole-insensitive nAChRs is detected. Thus, during neuromuscular transmission in C. elegans, the majority of ACh-activated current flows through L-AChRs. This study contributes to the understanding of the molecular mechanisms underlying functional diversity of the nAChR family and offers an excellent strategy to test novel antiparasitic drugs.

Molecular determinants of pyrantel selectivity in nicotinic receptors.

Nicotinic receptors (acetylcholine receptors, AChRs) play key roles in synaptic transmission throughout the nervous system. AChRs mediate neuromuscular transmission in nematodes, and they are targets for antiparasitic drugs. The anthelmintic agents levamisole and pyrantel, which are potent agonists of nematode muscle AChRs, are partial agonists of mammalian muscle AChRs. To further explore the structural basis of the differential activation of AChR subtypes by anthelmintics, we studied the activation of alpha7 AChRs using the high-conductance form of the alpha7-5-hydroxytryptamine-3A receptor, which is a good model for pharmacological studies involving the extracellular region of alpha7. Macroscopic and single-channel current recordings show that levamisole is a weak agonist of alpha7. It is interesting that pyrantel is a more potent agonist of alpha7 than acetylcholine (ACh). To identify determinants of this differential activation, we replaced residues of the complementary face of the binding site by the homologous residues in the muscle epsilon subunit and evaluated changes in activation. The mutation Q57G does not affect the activation by either ACh or levamisole. However, it increases EC50 values and decreases the maximal response to pyrantel. Kinetic analysis shows that gating of the mutant channel activated by pyrantel is profoundly impaired. The decreased sensitivity of alpha7-Q57G to pyrantel agrees with its weak action at muscle AChRs, indicating that when glycine occupies position 57, as in the mammalian muscle AChR, pyrantel behaves as a partial agonist. Thus, position 57 located at the complementary face of the binding site plays a key role in the selective activation of AChRs by pyrantel.

The anthelmintic pyrantel acts as a low efficacious agonist and an open-channel blocker of mammalian acetylcholine receptors.

Pyrantel is an anthelmintic which acts as an agonist of nicotinic receptors (AChRs) of nematodes and exerts its therapeutic effects by depolarizing their muscle membranes. Here we explore at the single-channel level the action of pyrantel at mammalian muscle AChR. AChR currents are elicited by pyrantel. However, openings do not appear in clearly identifiable clusters over a range of pyrantel concentrations (1-300 microM). The mean open time decreases as a function of concentration, indicating an additional open-channel block. Single-channel recordings in the presence of high ACh concentrations and pyrantel demonstrate that the anthelmintic acts as a high-affinity open-channel blocker. When analyzed in terms of a sequential blocking scheme, the calculated forward rate constant for the blocking process is 8x10(7) M(-1) x s(-1), the apparent dissociation constant is 8 microM at a membrane potential of -70 mV and the process is voltage dependent. Pyrantel displaces alpha-bungarotoxin binding but the concentration dependence of equilibrium binding is shifted towards higher concentrations with respect to that of ACh binding. Thus, by acting at the binding site pyrantel activates mammalian AChRs with low efficacy, and by sterical blockade of the pore, the activated channels are then rapidly inhibited.

Atualizaçäo em anti-helmínticos para cavalos / Bringing up to date anthelmintics for horses

Neste trabalho säo discutidos os fatores positivos e negativos dos modernos anti-helmínticos. Muitas säo as escolhas com que os veterinários se defrontam em relaçäo aos anti-helmínticos e programas de vermifugaçäo, e uma vez feita a escolha, näo há garantias de que a droga ou programa de controle vá permanecer indefinidamente eficiente. O autor faz consideraçöes sobre os benzimidazóis, o ivermectin e o pirantel, tecendo alguns comentários sobre a rotaçäo rápida e lenta de vermífugos para eqüinos

Estudio comparativo de flubendazol, oxantel-pirantel, albendazol y mebendazol en el tratamiento de helmintos transmitidos por el suelo / Comparative study of flubendazol, oxantel-pirantel, albendazol and mebendazol in treatment of helminths transmited by the soil

Se trataron 141 personas que tenian infeccion por Ascaris lumbricoides, Trichuris trichiura o Necator americanus. Las drogas empleadas fueron: Flubendazol, con tres dosificaciones: 500 mg dosis unica; 300 mg/dia por 2 dias; y 200 mg/dia por 3 dias. Oxantel-Pirantel, 400 mg de cada una en dosis unica o 600 mg de cada una, tambien en dosis unica. Mebendazol con tres dosificaciones: 500 mg dosis unica; 300 mg/dia por 2 dias; y 200 mg/dia por tres dias. Los resultados indican que todas las drogas empleadas tuvieron una efectividad para el tratamiento de la ascariasis con una curacion entre el 84.6 y el 100% y un porcentaje de reduccion del 88.5 al 100%. El flubendazol a la dosis de 300 mg/dia por 2 dias fue la droga mas efectiva para la tricocefalosis y la uncinariasis, curacion y reduccion de huevos del 100% para ambas. Todas las drogas fueron bien toleradas y casi no se observaron efectos secundarios. Si se requeire una droga para tratamientos en masa con dosis unica, se debe seleccionar el flubendazol, a la dosis de 500 mg; con ella se cubren los tres helmintos con una alta efectividad.

Dosis única antihelmíntica: estudio comparativo con dosis única, entre oxantel/pirantel y albendazol en ascariasis, tricocefalosis y uncinarias / Single antihelminthic dosage: comparative study with single doses between oxantel/pirantel and albendazole in ascariasis, trichocephaliasis and uncinarias

Se presentan los resultados de un estudio comparativo entre el oxantel-pirantel y el albendazol, en un grupo de 60 años parasitados por alguno de los siguientes helmintos transmitidos por el suelo: áscaris, tricocéfalos y uncinarias. Los medicamentos fueron administrados en dosis única: el oxantel-pirantel a razón de 20 miligramos-/kilogramo de peso, y el albendazol en dosis total de 400 milígramos. Se observaron excelentes resultados con ambos medicamentos en el tratamiento de la ascariasis. En la curación parasitológica de la uncinariasis el oxantel-pirantel exhibió superioridad (85.7%) de los casos tratados) sobre el albendazol (66.7% de los casos tratados); y, en lo que respecta a la reducción en el número de huevecillos, ambas drogas demostraron ser de utilidad. En las tricocefalosis de grados, I, II y III la curación obtenida con oxantel-pirantel fué superior. En relación a la disminución de huevecillos en las heces, existió una ligera superioridad por parte del oxantel-pirantel. En el grado IV de tricocefalosis, el efecto terapéutico de ambos medicamentos mostró muy poca utilidad cuando son suministrados en dosis única. Se propone una escala de los grados de parasitosis para ser utilizada en la evaluación de drogas antihelmínticas

Estudio comparativo con dosis única entre Oxantel/Pirantel y Albenzol en Ascariasis, tricocefalosis y uncinarias en niños

Se presenta los resultados de un estudio comparativo entre el oxante/pirantel y el albendazol, en un grupo de 60 niños parasitados por alguno de los siguientes helmintos transmitidos por el suelo: áscaris, tricocéfalos y uncinarias. Los medicamentos fueron administrados en dosis única: el oxantel/pirantel a razón de 20 miligramos/Kilogramo de peso, y el albendazol en dosis total de 400 miligramos. Se observaron excelentes resultados con ambos medicamentos en el tratamiento de las ascariasis. En la curación parasitológica de la uncinariasis el oxantel/pirantel exhibió superioridad (85.7% de los casos tratados) sobre el albendazol (66.7% de los casos tratados); y en lo que respecta a la reducción en el número de huevecillos, ambas drogas demostraron ser de utilidad. En las tricocefalosis de grados I, II y III la curación obtenida con oxante/pirantel fue superior. En relación a la disminución de huevecillos en las heces, existió una ligera superioridad por parte del oxante/pirantel. En el grado IV de tricocefalosis, el efecto terapéutico de ambos medicamentos mostró muy poca utilidad cuando son suministrados en dosis única. Se propone una escala de los grados de parasitosis para ser utilizada en la evaluación de drogas antihelmínticas