Glibenclamide modulates glucantime activity and disposition in Leishmania major Maritza Padrón-Nieves, Emilia Díaz, Claudia Machuca, Amarilis Romero, Alicia Ponte Sucre * Laboratorio de Fisiología Molecular, Instituto de Medicina Experimental, Facultad de Medicina, Universidad Central de Venezuela, Caracas, Venezuela article info Article history: Received 18 September 2008 Received in revised form 1 December 2008 Accepted 15 December 2008 Available online 25 December 2008 Keywords: Leishmania major-glucantime Glibenclamide-ABC transporter Drug activity-combination therapy abstract A source of chemotherapeutic failure in anti-infective therapies is the active movement of drugs across membranes, through ATP-binding cassette (ABC) transporters. In fact, simultaneous administration of therapeutic drugs with ABC transporter blockers has been invoked to be the way to actively prevent the emergence of drug resistance. Herein, we demonstrate that glucantime’s efficacy in decreasing the infection rate of Leishmania-infected macrophages is strongly enhanced when used in combination with glibenclamide, a specific blocker of ABC transporters. Intracellular ABC transporters mediate glucantime sequestration in intracellular organelles. Their selective inhibition may effectively increase the cytoplas- mic concentration of glucantime and its leishmanicidal activity. Our results reveal for the first time that glibenclamide targets in Leishmania major a compartment associated with a multivesicular system that is simultaneously labeled by the acidic marker LysoTracker-red and may represent the organelle where antimonials are sequestered. These results constitute a proof of concept that conclusively demonstrates the potential value that combination therapy with an ABC transporter blocker may have for leishmaniasis therapy. Ó 2009 Elsevier Inc. All rights reserved. 1. Introduction Chemotherapy against leishmaniasis is mainly based on the pentavalent antimonial [Sb (V)] compounds sodium stibogluconate (Pentostam Ò ) and meglumine antimoniate (Glucantime Ò ). In order to be active against Leishmania infection, Sb (V) must be reduced to trivalent antimonials [Sb (III)], the active form of the drug against the parasite (Shaked-Mishan et al., 2001; Frezard et al., 2001). However, the underlying molecular mechanism of how antimoni- als act against Leishmania infections is not fully understood. Their toxicity causes serious side-effects that often result in patients terminating their treatment, thereby contributing to the world- wide escalating frequency of Leishmania resistance to antimonials (Davis et al., 2004; Ouellette et al., 2004; Croft et al., 2006; Loiseau and Bories, 2006). The drug amphotericin B was introduced more recently; however, its use is not widely spread due to the cost of the treatment. Finally, since 2001 the oral drug miltefosine is also in use (Escobar et al., 2002; Davis et al., 2004). Unfortunately, single point mutations in the Leishmania putative miltefosine transporter can lead to resistance against miltefosine (Pérez-Victo- ria et al., 2006) and a gene that encodes a putative polypeptide, without similarities to known proteins, has recently been isolated from Leishmania infantum (Choudhury et al., 2008). Upon over- expression, this protein confers resistance against miltefosine, but also against Sb (III), the active principle of anti-leishmanial antimonials (Choudhury et al., 2008). In leishmaniasis, ATP-binding cassette (ABC) transporters are responsible for much of the decreased sensitivity to antimonial drugs. The augmented expression of ABC transporters enhances drug extrusion and disposition (Shaked-Mishan et al., 2001) and constitutes one of the main impediments to successful chemother- apy against Leishmania (El Fadili et al., 2005). Hence, the specific inhibition of ABC transporter function could be a fundamental strategy in helping to maintain effective drug concentrations with- in the cell. Furthermore, the simultaneous administration of the therapeutic drug with an ABC transporter blocker has been invoked to be a way to actively prevent the emergence of drug resistance and stop its increasing frequency. By blocking the transporters’ activity, the efficacy of a selected drug would be either maintained or enhanced. Unfortunately, this approach has the potential draw- back that resistance to the inhibitor itself might emerge (Leonard et al., 2003), and hence the risk/benefit profile of the combined use of inhibitors and drugs should be evaluated on a case-by-case basis. Still, this approach is certainly a promising avenue for devel- oping new drugs and novel strategies against the disease that may slow down drug resistance development. Glibenclamide (GLIB) is a sulfonylurea that inhibits ABC proteins with dissimilar functions, such as the K + - ATP channel associated sul- fonylurea receptor (Inagaki et al., 1995), the cystic fibrosis transport regulator (Schultz et al., 1996), the ABC1 transporter of immune cells (Becq et al., 1997; Hamon et al., 1997), the P-glycoprotein (P-gp) (Golstein et al., 1999), the Arabidopsis multidrug resistance-related 0014-4894/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2008.12.008 * Corresponding author. Present address: Alicia Ponte CCS 81720, P.O. Box 025323, Miami, Fl 33102-5323, USA. Fax: +58 212 6934351. E-mail address: aiponte@gmail.com (A.P. Sucre). Experimental Parasitology 121 (2009) 331–337 Contents lists available at ScienceDirect Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr