Interaction of ivermectin with multidrug resistance proteins (MRP1, 2 and 3)

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Abstract

Ivermectin is a potent antiparasitic drug from macrocyclic lactone (ML) family, which interacts with the ABC multidrug transporter P-glycoprotein (Pgp). We studied the interactions of ivermectin with the multidrug resistance proteins (MRPs) by combining cellular and subcellular approaches. The inhibition by ivermectin of substrate transport was measured in A549 cells (calcein or 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, BCECF) and in HL60-MRP1 (calcein). Ivermectin induced calcein and BCECF retention in A549 cells (IC50 at 1 and 2.5 μM, respectively) and inhibited calcein efflux in HL60-MRP1 (IC50 = 3.8 μM). The action of ivermectin on the transporters ATPase activity was followed on membranes from Sf9 cells overexpressing human Pgp, MRP1, 2 or 3. Ivermectin inhibited the Pgp, MRP1, 2 and 3 ATPase activities after stimulation by their respective activators. Ivermectin showed a rather good affinity for MRPs, mainly MRP1, in the micromolar range, although it was lower than that for Pgp. The transport of BODIPY-ivermectin was followed in cells overexpressing selectively Pgp or MRP1. In both cell lines, inhibition of the transporter activity induced intracellular retention of BODIPY-ivermectin. Our data revealed the specific interaction of ivermectin with MRP proteins, and its transport by MRP1. Although Pgp has been considered until now as the sole active transporter for this drug, the MRPs should be taken into account for the transport of ivermectin across cell membrane, modulating its disposition in addition to Pgp. This could be of importance for optimizing clinical efficacy of ML-based antiparasitic treatments. This offers fair perspectives for the use of ivermectin or non-toxic derivatives as multidrug resistance-reversing agents.

Introduction

Among antiparasitic drugs, macrocyclic lactones (MLs) are the most powerful agents used worldwide in livestock to fight against a broad spectrum of ecto- and endoparasites. Several millions of humans are also treated with ivermectin, the first commercialized compound belonging to ML family, for the control of onchocerciasis and lymphatic filiarisis [1]. Ivermectin and other MLs are large highly hydrophobic molecules characterized by the macrocyclic lactone ring as a common structural feature (Fig. 1) [2]. The antiparasitic activities of ivermectin and other MLs are related to the presence of effective concentration for a suitable length of time in the systemic circulation and in target tissues.

The MDR1 gene product P-glycoprotein (Pgp), is a membrane protein whose main function is the ATP-dependent transport of various structurally unrelated exogenous compounds [3]. It belongs to the ATP-binding cassette (ABC) protein family, among which MDR transporters are involved in the multidrug resistance of cancers by severely restricting the effectiveness of chemotherapy. Pgp, like the other MDR transporters, functions as a permeability barrier for the passage of xenobiotics across the cell membrane by expelling them out of the cells, the tissues and ultimately out of systemic circulation, exerting a protecting action against their toxicity. Due to its broad substrate specificity, Pgp also plays a clinically relevant role in drug–drug interactions.

Pgp plays a pivotal role in the bioavailability and tissue distribution of ivermectin in host organisms as well as in target parasites. Located on the blood–brain barrier, this transporter protects mammals against brain penetration of ivermectin and its subsequent neurotoxicity [4], [5], [6]. Also present on intestinal epithelium and on bile canalicules, it contributes to the massive faecal elimination of MLs [7], [8] and modulates ivermectin and the structurally related moxidectin bioavailabilities in the cells and the whole organism [4], [9], [10], [11]. Moreover, a Pgp homologue is expressed in parasites where it expels ivermectin, contributing to reduction of drug activity and development of ML resistance [12], [13]. In a wider range of therapeutic applications, recent data have revealed the ability of MLs to reverse multidrug resistance in tumor cells [14].

Besides Pgp, the multidrug resistance proteins MRP1, 2 and 3 (ABCC1, 2 and 3) are also involved in multidrug resistance [15], [16] providing complementary and overlapping functionality as drug efflux pumps. MRP1 shows relatively ubiquitous expression whereas MRP2 and 3 expressions are more restricted to renal, intestinal and hepatic epithelia [17]. MRP2 is localized to apical membranes [18] and may assist Pgp for apical drug efflux, while MRP1 and 3 are localized to basolateral membranes [17]. The key role of MDR transporters in drug availability, metabolism and toxicity is now well established, but their individual specific contribution remains to be defined more precisely. The complexity of the system lies both in the presence of numerous transporters and the large overlapping of substrate specificities. Pgp extrudes large hydrophobic molecules that are uncharged or positively charged, while the members of the MRP family can extrude both hydrophobic uncharged molecules and water soluble anionic compounds.

In the light of these informations and because MLs are large uncharged hydrophobic molecules, we asked the question whether besides Pgp, other MDR proteins may interact with ivermectin, modulating thereby their transport function. Because the efficacy of MLs is directly related to the drug concentration in target tissues, one of the strategies for therapeutic optimization lies in the identification of compounds able to inhibit MLs transport, improving their bioavailability in host and parasite. A better knowledge of the transporters with which MLs interact will allow specific inhibitors to be selected and targeted more efficiently. Also, on the basis of the capacity of ivermectin to suppress multidrug resistance in tumor cells, the potential use of ivermectin or derivatives in cancer therapy deserves to be considered [14], [19].

In the present work, we studied the interactions of ivermectin with MDR transporters of the Pgp and MRP family. In a first approach, we studied the influence of ivermectin on the transmembrane transport of fluorescent substrates by using cell lines overexpressing either Pgp or MRPs. Then, we characterized the interaction of ivermectin with the ATPase activity of several isolated human MDR transporters: Pgp, MRP1, 2 and 3, by using membrane vesicles derived from infected Sf9 insect cells overexpressing the respective transporters. We also studied the retention of the fluorescent derivative BODIPY-ivermectin in cells overexpressing Pgp or MRP1.

Section snippets

Chemicals

Ivermectin, indomethacin, probenecid, rhodamine 123 (Rho123), dimethyl sulfoxide (DMSO), HEPES, ascorbic acid, sodium dodecyl sulfate and collagen were purchased from Sigma Chimie (Saint-Quentin Fallavier, France). Valspodar was a generous gift from Novartis (Basel, Switzerland). BODIPY-ivermectin and 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) was purchased from Molecular Probes (Invitrogen, Cergy Pontoise, France). Calcein acetoxymethyl ester (calcein-AM)

Influence of ivermectin on substrate transport in cells overexpressing Pgp or MRP

LLCPK1 pig kidney epithelial cells are characterized by a low drug transport activity attributable to Pgp or MRPs, making them suitable for the over-expression of an efflux pump of interest [27]. Transduction with mdr1a gene guarantees thus high level of Pgp expression as previously reported [28]. To study Pgp functionality, we followed Rho123, a widely reported fluorescent dye substrate [29], in LLCPK1-mdr1 cells. In this model, 1 μM of valspodar led to a marked increase of intracellular Rho123

Discussion

The major physiological role of multidrug resistance transporters is the protection of the cells and tissues against xenobiotics by controlling drug exposure and toxicity. Due to its pivotal role in the biodisposition of ivermectin, Pgp is considered as a main target in the antiparasitic chemotherapy strategy. Beside Pgp, proteins of the MRP family are also largely represented on the apical and basolateral poles of hepatocytes and other cells, playing a major role in the efflux into bile or

Conclusion

For the first time, we showed that ivermectin is able to inhibit MRP1 transport function and to specifically interact with MRP1, MRP2 and MRP3. Moreover, we provide evidence of the transport by MRP1 of the fluorescent derivate BODIPY-ivermectin. Other ABC transporters such as MRP4, 5, 6 or the half-multidrug transporter MXR (ABCG2) may also be concerned by ivermectin inhibitory effect and transport, and additional data need to be generated.

The identification of transporters interacting with and

Acknowledgements

We are grateful to Christine Comera for her helpful discussions. This work was partly supported by OTKA T043141 grant to P.K.

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