Weak phenotypic reversion of ivermectin resistance in a field resistant isolate of Haemonchus contortus by verapamil

Pesquisa Veterinária Brasileira

Buzzulini, Carolina¹  Velludo, Patrícia P.¹  Borges, Fernando A.¹  Molento, Marcelo B.²  Rossini, Julhiano B.²  Costa, Gustavo H.¹  Costa, Alvimar J.² 

[1] Universidade Federal de Mato Grosso do Sul;Universidade Estadual Paulista

关键词: Haemonchus contortus;    ivermectin;    P-glycoprotein;    resistance;    reversion;    verapamil. RESUMO;   

DOI  :  10.1590/S0100-736X2011000900002

来源: Colegio Brasileiro de Patologia Animal-CBPA

【 摘 要 】

Recent advances in anthelmintic resistant phenotype reversion by Pgp modulating drugs in ruminant nematodes indicate that this can be a useful tool to helminth control. The aim of the present study was to evaluate the efficacy of ivermectin (IVM) in combination with verapamil (VRP), in oil or water-based vehicle, against an IVM-resistant field isolate of Haemonchus contortus through a larval migration assay and experimental infection trial. In the in vitro assay was observed a phenotypic reversion of H. contortus resistance to ivermectin at a high concentration of VRP, increasing IVM efficacy from 53.1% to 94.3. In the in vivo trial, IVM + VRP demonstrated 36.02% efficacy compared to the 7.75% of IVM alone. The vehicle formulation showed no influence in efficacy. These are the first results demonstrating the effect of VRP as a partial IVM-resistance phenotype reverser in a field isolate of IVM-resistant H. contortus experimentally inoculated in sheep. Index terms: Haemonchus contortus, ivermectin, P-glycoprotein, resistance, reversion, verapamil. RESUMO Avanços recentes na reversão fenotípica da resistência anti-helmíntica por drogas moduladoras de Pgp em nematódeos de ruminantes indicam que esta pode ser uma ferramenta útil no controle de helmintos. O objetivo do presente estudo foi avaliar a eficácia da ivermectina (IVM), em combinação com o verapamil (VRP), em veículo oleoso ou à base de água, contra um isolado de campo de H. contortus resistente por meio de teste de migração de larvas e infecção experimental em ovinos. No teste in vitro, observou-se reversão fenotípica da resistência de Haemonchus contortus à ivermectina com alta concentração de VRP, aumentando a eficácia da IVM de 53,1% para 94,3. No teste in vivo, IVM + VRP demonstrou 36,02% de eficácia em relação a 7,75% de IVM sozinha. O veículo da formulação não apresentou influência na eficácia. Estes são os primeiros resultados que demonstram o efeito da VRP como reversor parcial do fenótipo da resistência de IVM-fenótipo em um isolado de campo de H. contortus resistente, inoculado experimentalmente em ovinos. Termos de indexação: Haemonchus contortus, ivermectina, Pglycoprotein, resistance, reversion, verapamil    INTRODUCTIONMacrocyclic lactones (ML) are a third-generation broad-spectrum anthelmintic agent for ruminants, but the exact resistance mechanisms are still far from being totally understood. Such mechanisms seem to be polygenic, involving several or many genes (Köhler 2001). Mutations in glutamate-gated chloride (GluCl) receptor genes are associated with ML-resistance (Blackhall et al. 1998), along with mutations in the dye-filling defective (Dyf) genes that encode proteins related to IVM uptake (Dent et al. 2000). The first gene associated to ML resistance in Haemonchus contortus was Pgp-A, which encodes a P-glycoprotein, a member of the ATP-binding cassette transporter family (Xu et al. 1998). Pgp homologues are expressed in many organisms, including vertebrates, and are involved in the absorption, distribution, metabolism and excretion processes of xenobiotic compounds (Mealey 2004).Pgp has been largely studied, as its overexpression can cause drug resistance in human tumor cells due to drug transport from the target cell, resulting in the phenomena known as multidrug resistance (MDR) when several chemical groups are involved (Juliano & Ling 1976, Gottesman & Pastan 1993). Pgp is also responsible for MDR in Trypanosoma cruzi and T. brucei brucei, which is a very important protozoan in human and animal health (Kerboeuf et al. 2003).MDR reversion by modulating drugs is a recent treatment protocol employed in chemotherapy targeting tumor cells. These drugs directly act as competitive inhibitors of Pgp-mediated drug efflux and/or through indirect mechanisms that induce an increase in cellular ATP consumption and block the Pgp function (Watanabe et al. 1995, Garrigos et al. 1997). Verapamil (VRP) is a calcium channel blocker (Tsuruo et al. 1981) that has been used with this aim. There are a number of in vitro studies related to Pgp-activity modulation by VRP demonstrating partial reversion to benzimidazole and IVM resistance in nematodes (Beugnet et al. 1997, Molento & Prichard 1999, 2001, Bartley et al. 2009).Along with the direct action of resistance reversion, VRP can significantly increase some pharmacokinetic parameters (AUC and Cmax) of co-administrated IVM in sheep, probably due to liver and intestinal Pgp inhibition, thereby reducing biliary excretion and increasing the intestinal absorption of IVM (Molento et al. 2004).Lespine et al. (2008) pointed that the study of Pgp modulators should follow the following steps: selection of the drug, evaluation of the ability of this drug to modulate Pgp action in cells, pharmacokinetic studies in the host, in vitro tests with resistant parasites and in vivo coadministration of anthelmintic and Pgp modulator on experimentally infected hosts. These authors alerted to some possible problems such as side effects, different pharmacokinetic profile of the anthelmintic and the Pgp modulator, costs and the need of posterior studies on the withdraw period.Recent studies showed the increase of ivermectin efficacy against resistant nematode in sheep (Lifschitz et al. 2010a) and cattle (Lifschitz et al. 2010b) experimentally infected and also the enhance of ivermectin systemic concentration by the coadministration of loperamide, a Pgp modulator.All the studies on Pgp modulators in ruminants (Molento et al. 2004, Lifschitz et al. 2010a,b) used the drug diluted in saline solution. The use of a formulation containing the association of a Pgp modulator and ivermectin in an oil-based vehicle could reduce the rate of absorption from the injection site, resulting in prolonged residence time and longer effect of the drug on Pgp modulation.Considering the absence of studies on Pgp-modulation by verapamil, a classical Pgp inhibitor, in sheep experimentally infected with an IVM-resistant field strain of Haemonchus contortus, the aim of the present study was to evaluate the in vitro and in vivo efficacy of ivermectin co-administered with verapamil and the possible effect of the vehicle formulation.   MATERIALS AND METHODSThe Ivermectin-resistant field strain of Haemonchus contortus was isolated from the Ovine Sector of the School of Agrarian and Vete rinary Sciences, São Paulo State University, Jaboticabal, São Paulo, Brazil. A previous controlled anthelmintic efficacy test following the methodology recommended by Wood et al. (1995) confirmed the extremely high phenotypic resistance status of this field strain of H. contortus to IVM, that was ineffective (-33.97% efficacy).For the isolation of the monospecific strain, H. contortus females were obtained from abomasums and immediately transferred to a saline solution in Petri dishes at 36 o C for two hours. The eggs produced were transferred to recipients containing vermiculite and incubated at 27 o C for 10 days, when the third-stage infective larvae were obtained. Two worm-free sheep were inoculated with 1000 viable L3 larvae and maintained in individual pens at the Animal Health Research Center (CPPAR/Unesp) in order to minimize accidental re-infection.Infective larvae from an IVM-resistant field strain of H. contortus were used in the in vitro agar migration assay test (D'Assonville et al. 1996, modified by Molento & Prichard, 2001). The larvae were submitted to the following treatments: Group I: 1, 2, 4, 8, 16, 32, 64, 128 and 256μM of IVM (ivermectin, Ivomec ® Solução Oral, Merial Saúde Animal); Group II: 1, 2, 4, 8, 16, 32, 64, 128 and 256 μM of IVM co-administrated with 2mM of VRP (verapamil hydrochloride, Sigma, St Luis, Missouri, USA) - this concentration of VRP showed to be able to increase the efficacy of IVM in other work (Molento & Prichard 2001); Group III: 1, 2, 5, 10 and 100mM of VRP; Group IV: IVM EC50 co-administrated with 0, 1, 2, 5, 10 and 100mM of VRP.The drugs were diluted in distilled water, instead of oil to avoid interference in the larvae migration in agar, and vortexed immediately prior to use. In each experimental group, there was a triplicate that only received distilled water, used as the control group. The larva number used in each of the four groups was variable, depending on the availability. Thus, efficacy percentages between treatments were statistically compared rather than mean larva survival rate.The in vivo evaluation of reversion of ivermectin resistance design and procedures were performed based on the guidelines of the WAAVP (Wood et al. 1995) and VICH (Vercruysse et al. 2001). The management of the experimental sheep was carried out in compliance with the Ethics Committee of the institution and "Good Clinical Practice" guidelines (VICH GL9, 2000, http://vich.eudra.org/pdf/2000/GL09_st7.pdf).As it was not possible to produce a large number of naive animals, the decision was made to use lambs that were treated with an anthelmintic agent until the total elimination of any pre-experimental nematode infection. All animals were housed in stalls designed to avoid parasitic infections and provided with daily food and water ad libitum.After an acclimation period, each worm-free animal was orally inoculated with 8460 IVM-resistant H. contortus L3 larvae. Thirty of the 35 inoculated sheep were selected based on eggs per gram (EPG) counts on Days 32, 33 and 34 post-inoculation. The animals were ranked based on the EPG count and allocated into five groups (n=6) in a randomized block design: I- Control; II- 1% IVM (200 μg/kg); III- 15% VRP (3mg/kg), water-based vehicle, administrated three times at 12-hour intervals; IV- 1% IVM (200 μg/kg), single dose, co-administered with 15% VRP (3mg/kg), water-based vehicle, administrated three times at 12-hour intervals (separate formulations); V-1% IVM (200μg /kg) plus 15% VRP (3mg/kg), oil-based vehicle (sterile pure corn oil), single dose (combined formulation).All treatments were performed subcutaneously on Day 35 postinoculation and the animals were observed for possible adverse reactions. The established VRP dose of 3mg/kg is the safest for sheep when diluted in saline solution and administered subcutaneously (Molento et al. 2004). The aim of using an oil-based vehicle is to allow slow absorption of the drug from the injection site, as VRP is absorbed and eliminated very quickly, whereas macrocyclic lactones have a longer absorption half-life and mean residence time.All sheep were sacrificed seven days post-treatment and subjected to a parasitological necropsy in order to determine the worm burden. Fecal samples were collected and eggs per gram (EPG) were determined for each animal (Gordon & Whitlock 1939) in order to evaluate the effect of the treatments on nematode reproduction.The half maximal effective concentration (EC)50 of IVM against the resistant field strain was calculated using the GraphPad Prism software program (GraphPad Prism, version 5.01 for Windows, San Diego, California, USA, www.graphpad.com). The total number of surviving larvae in each treatment was log-transformed and normalized to fit a dose-response sigmoid curve. The curves were statistically compared (fitted values log EC50 and Hill slope) by the Tu

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