Synergism between ivermectin and the tyrosine kinase/P-glycoprotein inhibitor crizotinib against Haemonchus contortus larvae in vitro

Highlights

• We examined interaction of P-gp inhibitor crizotinib with ivermectin in Haemonchus contortus larvae in vitro.

• Crizotinib increased the toxicity of ivermectin towards a resistant isolate in migration assays.

• Less synergism observed in larval development assays.

• Assay differences suggest life-stage specific patterns of ivermectin/P-gp interaction.

• Study highlights potential of P-gp inhibitors to reverse ivermectin resistance.

Abstract

Anthelmintic resistance is a major problem in parasitic nematodes of livestock worldwide. One means to counter resistance is to use synergists that specifically inhibit resistance mechanisms in order to restore the toxicity, and hence preserve the usefulness, of currently available anthelmintics. P-glycoproteins (P-gps) eliminate a wide variety of structurally unrelated xenobiotics from cells, and have been implicated in anthelmintic resistance. Crizotinib is a tyrosine kinase inhibitor under development as a cancer therapeutic. The compound also inhibits P-gps, and has been shown to reverse multidrug resistance in cancer cells. We were therefore interested in determining if the compound was able to increase the sensitivity of Haemonchus contortus larvae to ivermectin, as measured by in vitro larval development and migration assays with a drug-resistant and a –susceptible isolate. In migration assays, co-administration of crizotinib increased the toxicity of ivermectin to resistant larvae (up to 5.7-fold decrease in ivermectin IC₅₀), and rendered the resistant larvae equally or more sensitive to ivermectin than the susceptible isolate. On the other hand, co-administration of crizotinib had no effect on ivermectin sensitivity in the susceptible isolate. In development assays, significant increases in the sensitivity of both the resistant (up to 1.9-fold) and susceptible (up to 1.6-fold) larvae to ivermectin were observed, although the magnitude of the observed synergism was less than seen in migration assays, and the resistant larvae retained significant levels of ivermectin resistance. By highlighting the ability of the P-gp inhibitor crizotinib to increase the sensitivity of H. contortus larvae to ivermectin, this study provides further evidence that P-gp inhibitors are potential tools for modulating the efficacy of anthelmintics. In addition, the differences in the outcomes of the two assays, with ‘resistance-breaking’ effects being much more marked in migration assays, suggest that some life-stage-specific aspects may exist in the interaction of ivermectin with P-gps in the two worm isolates.

Introduction

The development of resistance to almost all available anthelmintics threatens our ability to control parasitic nematodes in livestock enterprises worldwide. Given the time and cost of developing new drugs (Woods and Williams, 2007), there is a need to manage the use of the existing drugs to preserve their usefulness for as long as possible. Means to achieve this include the elucidation of resistance mechanisms in order to develop resistance diagnostics (Kotze et al., 2014), as well as the use of compounds that can act to inhibit resistance mechanisms, and hence restore anthelmintic susceptibility to resistant worms (for example Lespine et al., 2012).

ATP binding cassette (ABC) transporters are a superfamily of transmembrane proteins which mediate the ATP-dependent efflux of a wide range of structurally and mechanistically unrelated compounds including various anticancer and anthelmintic drugs (Gottesman and Pastan, 1993, Lespine et al., 2012). Multiple ABC transporter genes have been reported in free-living and parasitic nematodes (Sheps et al., 2004, Ardelli et al., 2010, Laing et al., 2013). The overexpression of some of these transporters has been observed in drug-resistant isolates of different nematode species compared to susceptible reference isolates (Dicker et al., 2011, Williamson et al., 2011, Sarai et al., 2013, Raza et al., 2016), suggesting a potential role for ABC transporters in anthelmintic resistance. Numerous in vitro and in vivo studies have shown that an anthelmintic/multi-drug-resistance inhibitor (MDRI) combination therapy increases the toxicity of the anthelmintic to both drug-susceptible and –resistant isolates of different nematode species (Bartley et al., 2009, Pérez et al., 2010, Heckler et al., 2014). Recently, Raza et al. (2015) reported that zosuquidar and tariquidar, members of the so-called third generation of MDRIs (Falasca and Linton, 2012), significantly increased ivermectin (IVM) toxicity to Haemonchus contortus larvae in vitro.

Tyrosine kinase inhibitors are an important new class of targeted chemotherapeutic agents that represent a promising group of anticancer drugs in current clinical trials and clinical use (Shawver et al., 2002). Crizotinib is a tyrosine kinase inhibitor that has been examined as a cancer therapeutic for the treatment of patients with anaplastic lymphoma kinase (ALK)-positive advanced non-small cell lung cancer (NSCLC). It works by inhibiting c-Met (a gene that encodes hepatocyte growth factor receptor) and ALK. In NSCLC, it also inhibits echinoderm microtubule-associated protein-like 4 anaplastic lymphoma kinase (EML-ALK 4) translocation (reviewed by Sahu et al., 2013). As well as being an inhibitor of tyrosine kinase, crizotinib also acts as a competitive inhibitor of P-gps (Zhou et al., 2012). The compound significantly increased the sensitivity of ABCB1 over-expressing cells to doxorubicin and paclitaxel, and the combination of crizotinib with paclitaxel markedly increased anti-tumour activity of paclitaxel in the KBv200 tumour xenograft model.

Given that, i) crizotinib increases toxicity of anti-cancer drugs in mammalian cells and xenograft experimental models by acting as an MDRI in inhibiting the activity of P-gps, ii) P-gps have been implicated in some anthelmintic resistances, and iii) other MDRIs have been shown to partially reverse anthelmintic resistances, we hypothesized that crizotinib might be able to inhibit nematode P-gps, and hence restore the sensitivity of resistant worms to anthelmintics. The present study therefore aimed to investigate whether crizotinib was able to synergise the toxicity of IVM against drug-resistant and – susceptible isolates of H. contortus using larval development and migration assays.