Research Article: Diethylcarbamazine Increases Activation of Voltage-Activated Potassium (SLO-1) Currents in Ascaris suum and Potentiates Effects of Emodepside

Date Published: November 20, 2014

Publisher: Public Library of Science

Author(s): Samuel K. Buxton, Alan P. Robertson, Richard J. Martin, Timothy G. Geary. http://doi.org/10.1371/journal.pntd.0003276

Abstract: Diethylcarbamazine is a drug that is used for the treatment of filariasis in humans and animals; it also has effects on intestinal nematodes, but its mechanism of action remains unclear. Emodepside is a resistance-busting anthelmintic approved for treating intestinal parasitic nematodes in animals. The novel mode of action and resistance-breaking properties of emodepside has led to its use against intestinal nematodes of animals, and as a candidate drug for treating filarial parasites. We have previously demonstrated effects of emodepside on SLO-1 K+-like currents in Ascaris suum. Here, we demonstrate that diethylcarbamazine, which has been proposed to work through host mediated effects, has direct effects on a nematode parasite, Ascaris suum. It increases activation of SLO-1 K+ currents and potentiates effects of emodepside. Our results suggest consideration of the combination of emodepside and diethylcarbamazine for therapy, which is predicted to be synergistic. The mode of action of diethylcarbamazine may involve effects on parasite signaling pathways (including nitric oxide) as well as effects mediated by host inflammatory mediators.

Partial Text: Infections with parasitic nematodes are a global concern for human and animal health. These infections come in the form of gastrointestinal nematodes, like ascariasis infections, hookworm infections and trichuriasis infections, as well as infections transmitted by biting insects, like filariasis. Over 1 billion people are infected with parasitic nematodes [1], especially in the tropical regions where the combination of poor sanitation, warm and moist conditions creates the conducive environment for survival and spread of these parasites. Infections of parasitic nematodes of farm animals cause massive loss of food production and also lead to animal welfare issues. Parasitic nematode infections cause cognitive impairment of humans and, in humans and animals, stunted growth, anemia, sometimes swollen limbs and sometimes death. In the absence of effective vaccines or sanitation, anthelmintic drugs are required for both treatment and prophylaxis. There are a limited number of drug classes available and their frequent use has produced resistance in animals [2] and concerns about development of resistance in humans [3], [4]. One way to slow the speed of development of resistance is to use the drugs that are available in a more targeted manner [5] and to use synergistic combinations of drugs [6]. In this paper we explore effects of diethylcarbamazine and describe the interactive effects of the combination of emodepside and diethylcarbamazine.

Adult Ascaris suum were collected weekly from JBS Swift and Co. pork processing plant, Marshalltown, IA and maintained for up to 4 days in Locke’s solution (NaCl 155 mM, KCl 5 mM, CaCl2 2 mM, NaHCO3 1.5 mM, glucose 5 mM) at 32°C. About 1 cm of the anterior part of the worm, 4 cm from the head, was cut-out and the cylindrical worm piece cut open along a lateral line to form a muscle flap. After removing the gut to expose muscle cells, the muscle flap was pinned to a 35×10 mm Sylgard-lined Petri-dish containing low-potassium, high-calcium Ascaris perienteric fluid (APF) (mM: NaCl 23, Na acetate 110, KCl 3, CaCl2 6, MgCl2 5, glucose 11, HEPES 5, pH adjusted to 7.6 with NaOH). A 20-gauge perfusion needle, placed directly over the muscle bag being recorded from, delivered the drugs in APF at a rate of 4 mL min−1. We employed the two-micropipette current-clamp and voltage-clamp techniques to investigate the effects of diethylcarbamazine and emodepside on A. suum muscle bag. Micropipettes were pulled on a Flaming Brown Micropipette Puller (Sutter Instrument Co., Novato, CA, USA) and filled with 3 M potassium acetate. Resistance of the voltage-sensing micropipettes was between 20–30 MΩ but the tip of the current-injecting micropipette was broken to have a resistance of 3–6 MΩ. A 1320A Digidata, an Axoclamp 2B amplifier and pClamp 8.2 software (Molecular Devices, Sunnyvale, CA, USA) were used for the recordings. The resting membrane potential of cells selected for recording were stable and between −25 mV and −35 mV and had input conductances less than 4.0 µS. The current-clamp protocol consisted of injection of 40 nA hyperpolarizing pulses for 500 ms and recording the change in membrane potential with the voltage-sensing micropipette. In the voltage-clamp protocol, the muscle bag was held at −35 mV and then stepped to 0, 5, 10, 15, 20, 25 and 30 mV to activate the K+ currents. We used a leak subtraction protocol [11] that averaged four 5 mV hyperpolarizing pre-pulses to obtain the leak subtraction current before each depolarizing step and which was scaled by the amplitude of the depolarizing step for leak subtraction. The leak subtraction was under the control of pClamp software. The leak subtraction procedure was not modified otherwise by voltage, emodepside or emodepside and diethylcarbamazine. Recordings were rejected if the conductance of the muscle cells increased abruptly, indicating cell membrane damage, or if the conductance increased above 4 µS. Acquired data were displayed on a Pentium IV desktop computer and the currents were leak-subtracted. We analyzed the leak subtracted K+ current at the 0 mV step potential because the emodepside effect was biggest at this potential [11]. All chemicals and drugs were purchased from Sigma Aldrich (St Louis, MO, USA) except emodepside, which was generously supplied by Achim Harder (Bayer HealthCare AG, Leverkusen, Germany). Emodepside stocks of 2 mM in 100% DMSO were prepared every two weeks. The working emodepside concentration was prepared so that the final DMSO concentration did not exceed 0.1%. To avoid problems with emodepside coming out of solution, we did not keep it longer than the two weeks and in some cases, we prepared fresh emodepside for every experiment. Effects of drug applications were measured after 10 min and post-drug measurements made after a 20 min wash in drug-free solutions. Graph Pad Prism Software (version 5.0, San Diego, CA, USA) and Clampfit 9.2 (Molecular Devices) were used for data analysis. The activation curve was fitted by the Boltzmann equation G  =  Gmax/[1 + exp {(V50 – V)/Kslope}], where G =  conductance, Gmax  =  maximal conductance change, V50  =  half-maximal voltage and Kslope  =  slope factor.

Diethylcarbamazine is used mostly for treatment of filariasis in humans but has been used for treatment of intestinal nematode parasites [20]. Details of its mode of action remain to be defined however; it has been suggested that the effects of diethylcarbamazine are mediated via the host innate immune system [15] rather than by a direct effect on the parasite. Our observations show that 100 µM (but not 10 µM) diethylcarbamazine has a direct effect on the parasite (independent of the host) raising the possibility that its therapeutic mode of action also involves a direct effect. The antifilarial action of diethylcarbamazine appears to involve host arachidonic acid metabolism via cyclooxygenase & 5-lipoxygenase and, in addition nitric oxide metabolic pathways via inducible nitric oxide synthase [16]. A role for nitric oxide and the inducible nitric oxide pathway is suggested by the experiments involving microfliarial infected iNOS−/− mice which showed no clearance response following treatment with diethylcarbamazine [16].

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http://doi.org/10.1371/journal.pntd.0003276

 

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