Ivermectin

Based on their mechanism of action in humans, voriconazole, ritonavir, and cobicistat were classified as dual CYP/P-gp inhibitors [18 (link), 19 (link)], cyclosporine A and elacridar were classified as P-gp-specific inhibitors [37 (link)], and rifampicin was classified as a dual CYP/P-gp inducer [38 (link)]. The choice of inhibitors and inducers used in the present study was based on their ability to act as substrates for the CYP CYP3A4, which is the major enzyme involved in ivermectin metabolism in humans [39 (link)].
ivermectin, voriconazole, ritonavir, cobicistat, cyclosporine A, elacridar, and rifampicin were obtained from Sigma Aldrich (Spain). The active pharmacological ingredients were dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions for all compounds. Aliquots of the prepared solutions were frozen at −20 °C. For each experiment, the stock solutions were diluted in phosphate-buffered saline (PBS) to achieve the desired concentration.

Mice were infected with 50 L3 of the ML-susceptible MO 2005 FR3 isolate of D. immitis and then treated with ivermectin (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) diluted in corn oil/DMSO 50:50 on days 1, 15 and 30-post infection. Each mouse was weighed on the day of treatment for individual calculation of ivermectin doses by oral gavage, ranging from 0.005 to 3.0 mg/kg. After 6 weeks, mice were sacrificed and analyzed for parasite survival as described above. NSG mice were also infected with either the ML-susceptible MO 2005 isolate or the ML-resistant JYD-34 isolate and then treated with: (1) 0.01 mg/kg of ivermectin or (2) 0.01 mg/kg moxidectin (Activate Scientific, GmbH, Prien, Germany) on days 1, 15 and 30-post infection and were sacrificed at 6 weeks post-infection and parasites recovered.

N2A, SHEP, and HEK293T were described previously [13 (link)]. WT HCT116 and p53-KO HCT116 were kindly provided by B. Vogelstein (Ludwig Center at Johns Hopkins, Baltimore, MD) [24 (link)]. SHEP, LAN6, and CLB-Ga were kindly provided by V. Combaret (CRCL, Lyon)[12 (link)]. N2A cells were grown in DMEM/F-12, GlutaMAX (Life Technologies), supplemented with 10% FBS (Lonza); SHEP, LAN6, and CLB-Ga cells were grown in RPMI1640, GlutaMAX (Life Technologies), supplemented with 10% FBS (Lonza); and HEK293T, WT HCT116, and p53-KO HCT116 cells were grown in DMEM (Life Technologies), supplemented with 10% FBS (Lonza). The plasmid constructs and siRNA were transfected using JetPrime (PolyPlus) for cell death assays and Lipofectamine RNAiMAX (Life Technologies) for RT-QPCR assays following manufacturer’s instructions. Caspase activity was inhibited in SHEP cells by treatment with 20 μM of z-VAD-fmk (Merck-Millipore), a general caspase inhibitor. The pan-importins inhibitor Ivermectin (Sigma I8898) was added to the cells at a final concentration of 10 μM 2 h before cell collection. Pifithrin-α (Sigma P4359), p53 inhibitor was used at a concentration of 20 μM for 30 h.

A biological assay was conducted to evaluate nematicidal activity of pure compounds against Caenorhabditis elegans and H. filipjevi according to Helaly et al. [80 ]. Surface-sterilised cysts of H. filipjevi propagated in the greenhouse were incubated in sterile tap water under aseptic conditions for nematode hatching. The freshly hatched second stage juveniles (J2) were collected and used for the experiment. Caenorhabditis elegans was cultivated as described by Helaly et al. [80 ]. The number of nematodes was adjusted to 600/mL of J2 of H. filipjevi and 600/mL of adults and juveniles of C. elegans in sterile tap water. The assays were carried out in 24-well microtiter plates. Each well received 1 mL of nematode suspension. The compounds were tested against nematodes at the concentrations of 100, 50, 20 and 10 μg/mL in MeOH. Each treatment included three replications. Ivermectin (Sigma-Aldrich) was used as positive and MeOH in DMSO (v/v) as negative control. Nematodes were monitored for 30 min after inoculation and afterwards plates were incubated at 24°C for 18 h. Cytotoxicity (IC₅₀) of compounds was tested against different cell lines as described by Richter et al. [81 (link)].