In vitro Data

The good in vitro activity of polyenes, particularly amphotericin B is known for many years (Otcenasek & Buchta, 1994) and has been regularly confirmed in several studies using standardized methodologies (Sun et al., 2002a; Minassian et al., 2003;

Gomez-Lopez et al., 2001; Dannaoui et al., 2002b, 2003b). Some differences could be noted between species (Table 7.4), Apophysomyces elegans and Cunninghamella bertholletiae being less susceptible to amphotericin B than other species (Dannaoui et al., 2002b, 2003b). The relative resistance of C. bertholletiae has been confirmed in an animal model of pulmonary zygomycosis in severely immunosuppressed mice (Honda et al., 1998) and is in accordance with the poor clinical response in patients (Ribes et al., 2000). For A. elegans an animal model of infection in non-neutropenic mice has also been setup and showed that this fungus is considerably more pathogenic than other zygomycetes (Dannaoui et al., 2002c, 2003c). Nevertheless, in this model, despite high MICs of amphototericin B, the drug was fully active in vivo (Dannaoui et al., 2002c).

Azoles are a widely used antifungal group of compounds that comprise marketed drugs with activity against filamentous fungi, such as itraconazole, vori-conazole, and posaconazole. Other azoles are still in development. All azole drugs mainly act as inhibitors of ergosterol biosynthesis by inhibition of 14-alpha-demethylase (Cyp51). It was generally assumed that azole antifungal agents had no activity against zygomycetes (Sheehan et al., 1999; Kwon-Chung & Bennett, 1992). The recent in vitro data have shown that voriconazole had indeed no activity against all tested species of zygomycetes (Dannaoui et al., 2003b; Espinel-Ingroff, 1998b; Gomez-Lopez et al., 2001; McGinnis et al., 1998; Sabatelli et al., 2006; Sun et al., 2002a). In contrast, itraconazole exhibited varied in vitro activity depending on the species. Low MICs has been observed for Absidia spp. and Rhizomucor spp. while higher MICs, with some variability between isolates, were noted for Rhizopus spp. and Mucor spp. (Sabatelli et al., 2006; Sun et al., 2002a; Pfaller et al., 1998; Johnson et al., 1998; Dannaoui et al., 2003c). More interestingly, posaconazole, a new triazole with broad-spectrum activity, showed good in vitro activity against various species of zygomycetes (Table 7.4). Again, some differences were noted between species, with significantly lower MICs for Absidia spp. and Rhizomucor spp. compared to Rhizopus spp. and Mucor spp. (Dannaoui et al., 2003b; Sun et al., 2002a; Sabatelli et al., 2006). Very recently, the comparative activity of posaconazole, itraconazole, voriconazole, and fluconazole has been evaluated in vitro against Rhizopus oryzae and Absidia corymbifera (Chau et al., 2006). It has been shown that

Table 7.4 In vitro antifungal susceptibility of different species to amphotericin B (AMB), voriconazole (VRZ), and posaconazole (PSZ)

Species (no. of strains)

MIC* (|lg/mL) of

AMB

VRZ

PSZ

Rhizopus spp. (20)

0.06-1

4-64

\-1

Mucor spp. (10)

0.03-0.25

16-64

0.5-2

Rhizomucor spp. (10)

0.06

2-4

0.06-0.5

Absidia corymbifera (15)

0.06-0.5

2-16

0.06-0.25

Cunninghamella bertholletiae (2)

2-8

8-32

0.06-0.5

Apophysomyces elegans (1)

2

16

0.5

* MIC: Minimum Inhibitory Concentration.

* MIC: Minimum Inhibitory Concentration.

posaconazole and itraconazole were more active than fluconazole and voricona-zole for inhibition of ergosterol synthesis in cell extract and that posaconazole was the most active azole for inhibition of ergosterol synthesis in whole cells. Moreover, expression of Cyp51 from R. oryzae in an azole-susceptible Saccharomyces cerevisiae decreased susceptibility to voriconazole by >32-fold, but only by eight- and twofold for itraconazole and posaconazole, respectively (Chau et al., 2006).

Among other azole drugs that are still in development, some have shown potential activity against zygomycetes. For example, ravuconazole, which was considered initially as poorly active against zygomycetes (Fung-Tomc et al., 1998), has been recently reevaluated in vitro. Modal MICs of 0.5-2 |g/mL were found for most zygomycetes tested except for Mucor spp. that were less susceptible (Minassian et al., 2003).

Echinocandins such as caspofungin that are glucan-synthase inhibitors, are not active in vitro against zygomycetes with MIC or MEC (Minimum Effective Concentration) values usually >8 |g/mL (Espinel-Ingroff, 1998a; Pfaller et al., 1998; Singh et al., 2005; Gil-Lamaignere et al., 2005). Nevertheless, a recent study showed the presence in R. oryzae of both a FKS gene and a membrane-associated glucan-synthase activity, demonstrating that the drug target is present in this fungus (Ibrahim et al., 2005a). Although this glucan-synthase activity was inhibited by caspofungin, a 1,000-fold higher concentration was needed to achieve similar inhibition than those observed against other fungi such as Candida albicans or Aspergillus fumigatus.

Among other antifungal drugs, terbinafine has shown in vitro activity against some zygomycetes species (Jessup et al., 2000; Dannaoui et al., 2003b). Interestingly, a sharp difference was noted between R. oryzae (geometric mean MIC of terbinafine of 64 |g/mL) and R. microsporus (geometric mean MIC of ter-binafine of 0.15 |g/mL) (Dannaoui et al., 2003b) and this could be used as one of the identification criteria, along with others, to distinguish these two morphologically close species.

Other compounds such as statins, which are not used as antifungal drugs, have been shown to have inhibitory activity against a variety of fungi. Recently, the in vitro activity of lovasatin has been evaluated against some species of zygomycetes (Chamilos et al., 2006). It has been shown that lovastatin exhibited fungicidal activity by a microdilution broth technique with MIC50s and MFC50s of 48 and 56 |g/mL, respectively. The fungicidal effect against hyphae was further confirmed by fluorescence microscopy after staining with DiBAC, a dye that specifically stain damaged hyphae. The effect of lovastatin has also been recently evaluated against two Rhizomucor species (Lukacs et al., 2004). There was a dramatic difference of susceptibility between the species, R. pusillus being inhibited by 1-2 |g/mL of lovastatin while concentration of >8 |g/mL was necessary for growth inhibition of the R. meihei isolates. This difference of lovastatin susceptibility has been proposed as a simple test for differentiating these two closely related species (Lukacs et al., 2004).

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