Fungicidal Synergism of Fluconazole and Cyclosporine in Candida albicans Is Not Dependent on Multidrug Efflux Transporters Encoded by the CDR1 , CDR2 , CaMDR1 , and FLU1 Genes

Abstract

Progress in modern medicine has led to a worldwide increase in the incidence of Candida infections (2, 25). Amphotericin B, a fungicidal agent, has been the standard treatment for these infections for decades, but the toxicity of its conventional form and the costs of its lipid forms limit its use. Other antifungal agents, such as azoles, have excellent efficacy-toxicity profiles and play an important role in the treatment of candidal infections in nonneutropenic patients, although they have mostly fungistatic activities (13,18,19). However, the treatment of candidiasis during neutropenia and the emergence of azole-resistant Candida species continue to represent major challenges (1,17,19,(25)(26)(27). Thus, new therapeutic strategies need to be developed. In Candida albicans, efflux pumps play a major role in azole susceptibility and may represent a new therapeutic target (22). Promising results were reported in studies in which efflux of cytotoxic agents was inhibited in multiresistant human cancer cells (10, 24). As mammalian and fungal multidrug efflux transporters (METs) have strong structural homologies, human efflux pump inhibitors were screened in vitro to determine whether they have synergistic activities with fluconazole (FLC) against C. albicans. We found that the combination of FLC and cyclosporine (CY) is fungicidal against FLC-susceptible C. albicans strains (13). This powerful synergism was confirmed in experimental endocarditis. The association of FLC and CY was fungicidal against infection in aortic valve vegetations, a model of localized neutropenia, as well as in the kidney, an organ with a neutrophil-phagocytic host response (11). Although this phenomenon was discovered during the screening of inhibitors of efflux pumps in cancer cells, the mechanism of the synergism of FLC and CY in C. albicans is unknown. CY has several cellular targets including the cell membrane, METs, and the cyclophilin-calmodulin-calcineurin pathway. Thus, its interaction with FLC might intervene at different sites (9). The objective of the present work was to investigate the involvement of METs encoded by the CDR1, CDR2, CaMDR1, and FLU1 genes, which have been found to mediate FLC efflux in C. albicans. We postulated that their deletion would result in the loss of the fungicidal synergism of FLC-CY. For this purpose, the in vitro and in vivo activities of the combination of FLC and CY were compared against FLC-susceptible parent strain C. albicans CAF2-1 and FLC-hypersusceptible mutant C. albicans DSY1024, obtained by targeted deletion of the MET genes CDR1, CDR2, CaMDR1, and FLU1 (3,21).

Combination Treatment of Invasive Fungal Infections

“…The following criteria are commonly followed: synergy, a Ն2-log 10 decrease in CFU/ ml compared to the most active constituent; antagonism, a Ն2-log 10 increase in CFU/ml compared to the least active agent; additivity, a Ͻ2-but Ͼ1-log 10 decrease in CFU/ml compared to the most active agent; and indifference, a Ͻ2-but Ͼ1-log 10 increase in CFU/ml compared to the least active agent. (97,98,130,154,156,211), rats (140), or rabbits (172). Factors that need to be considered while performing in vivo studies include variable drug absorption, distribution, and metabolism among animal species.…”

Regulatory Circuitry Governing Fungal Development, Drug Resistance, and Disease

“…It is important to note that FK506 and cyclosporine have other targets in the cell besides calcineurin, including multidrug transporters (483,513). However, synergy between cyclosporine and fluconazole was confirmed in vitro and in vivo for strains lacking drug transporters implicated in azole resistance (364). In addition, the deletion of the regulatory subunit of calcineurin, CNB1, renders cells hypersensitive to the azoles, providing compelling evidence for calcineurin as a key regulator of azole resistance (126).…”

Stress, Drugs, and Evolution: the Role of Cellular Signaling in Fungal Drug Resistance

“…While cyclosporine and FK506 can also inhibit multidrug transporters (50,79,179,196), the specificity of their effects on calcineurin in mediating crucial responses to triazoles has been validated in C. albicans. The FK506 binding protein FKBP12, which FK506 must bind to in order to inhibit calcineurin, is required for FK506 synergy with triazoles (39), and the synergy persists even in strains lacking the multidrug transporters associated with azole resistance (119). Furthermore, abrogating calcineurin function genetically by deleting the regulatory subunit encoded by Cnb1 or the catalytic subunit encoded by Cna1 renders cells hypersensitive to azoles (39,191).…”