Accumulation of 3-ketosteroids induced by itraconazole in azole-resistant clinical Candida albicans isolates.

The effects of itraconazole on ergosterol biosynthesis were investigated in a series of 16 matched clinical Candida albicans isolates which had been previously analyzed for mechanisms of resistance to azoles (D. Sanglard, K. Kuchler, F. Ischer, J. L. Pagani, M. Monod, and J. Bille, Antimicrob. Agents Chemother., 39:2378-2386, 1995). Under control conditions, all isolates contained ergosterol as the predominant sterol, except two strains (C48 and C56). In isolates C48 and C56, both less susceptible to azoles than their parent, C43, substantial concentrations (20 to 30%) of 14alpha-methyl-ergosta-8,24(28)-diene-3beta,6alpha-dio l (3, 6-diol) were found. Itraconazole treatment of C43 resulted in a dose-dependent inhibition of ergosterol biosynthesis (50% inhibitory concentration, 2 nM) and accumulation of 3,6-diol (up to 60% of the total sterols) together with eburicol, lanosterol, obtusifoliol, 14alpha-methyl-ergosta-5,7,22,24(28)-tetraene-3betaol, and 14alpha-methyl-fecosterol. In strains C48 and C56, no further increase of 3,6-diol was observed after exposure to itraconazole. Ergosterol synthesis was less sensitive to itraconazole inhibition, as was expected for these azole-resistant isolates which overexpress ATP-binding cassette transporter genes CDR1 and CDR2. In addition to 3,6-diol, substantial amounts of obtusifolione were found after exposure to itraconazole. This toxic 3-ketosteroid was demonstrated previously to accumulate after itraconazole treatment in Cryptococcus neoformans and Histoplasma capsulatum but has not been reported in Candida isolates. Accumulation of obtusifolione correlated with nearly complete growth inhibition in these azole-resistant strains compared to that found in the susceptible parent strain, although the onset of growth inhibition only occurred at higher concentrations of itraconazole. ERG25 and ERG26 are the only genes assigned to the 4-demethylation process, of which the 3-ketoreductase is part. To verify whether mutations in these ERG25 genes contributed to obtusifolione accumulation, their nucleotide sequences were determined in all three related isolates. No mutations in ERG25 alleles of isolates C48 and C56 were found, suggesting that this gene is not involved in obtusifolione accumulation. The molecular basis for the accumulation of this sterol in these two strains remains to be established.

Origin of differences in susceptibility of Candida krusei to azole antifungal agents.

Two Candida krusei isolates were used to compare the effects of fluconazole, ketoconazole and itraconazole on growth and ergosterol synthesis, and to measure intracellular drug contents. Fifty per cent inhibition (IC50) of growth was achieved at 0.05-0.08 microM itraconazole and 0.56-1.2 microM ketoconazole, whereas 91-->100 microM fluconazole was needed to reach the IC50 value. Similar differences in sensitivity to these azole antifungal agents were seen when their effects on ergosterol synthesis from [14C]acetate were measured after 4 h and 24 h of growth. However, when the effects of the azoles on ergosterol synthesis from [14C]mevalonate by subcellular fractions were measured, fluconazole was only 2.3-6.1 times less active than itraconazole, and the IC50 values for ketoconazole were almost similar to those obtained with itraconazole. These results indicate that differences in susceptibility to itraconazole and ketoconazole are unrelated to differences in affinity for the C. krusei cytochrome P450. The much lower growth-inhibitory effects of fluconazole can also be explained partly only by a lower affinity for the P450-dependent 14 alpha-demethylase. The differences in sensitivity of both C. krusei isolates appeared to arise from differences in the intracellular itraconazole, ketoconazole and fluconazole contents. Depending on the experimental conditions, these isolates accumulated 6-41 times more itraconazole than ketoconazole and the intracellular ketoconazole content was 3.0-19.0 times higher than that of fluconazole.

Effects of itraconazole on cytochrome P-450-dependent sterol 14 alpha-demethylation and reduction of 3-ketosteroids in Cryptococcus neoformans.

As in other pathogenic fungi, the major sterol synthesized by Cryptococcus neoformans var. neoformans is ergosterol. This yeast also shares with most pathogenic fungi a susceptibility of its cytochrome P-450-dependent ergosterol synthesis to nanomolar concentrations of itraconazole. Fifty percent inhibition of ergosterol synthesis was reached after 16 h of growth in the presence of 6.0 +/- 4.7 nM itraconazole, and complete inhibition was reached at approximately 100 nM itraconazole. This inhibition coincided with the accumulation of mainly eburicol and the 3-ketosteroid obtusifolione. The radioactivity incorporated from [14C]acetate in both compounds represents 64.2% +/- 12.9% of the radioactivity incorporated into the sterols plus squalene extracted from cells incubated in the presence of 10 nM itraconazole. The accumulation of obtusifolione as well as eburicol indicates that itraconazole inhibits not only the 14 alpha-demethylase but also (directly or indirectly) the NADPH-dependent 3-ketosteroid reductase, i.e., the enzyme catalyzing the last step in the demethylation at C-4. This latter inhibition obviates the synthesis of 4,4-demethylated 14 alpha-methylsterols that may function at least partly as surrogates of ergosterol. Eburicol and obtusifolione are unable to support cell growth, and the 3-ketosteroid has been shown to disturb membranes. The complete inhibition of ergosterol synthesis and the accumulation of the 4,4,14-trimethylsterol and of the 3-ketosteroid together with the absence of sterols, such as 14 alpha-methylfecosterol and lanosterol, which can partly fulfill some functions of ergosterol, are at the origin of the high activity of itraconazole against C. neoformans. Fifty percent inhibition of growth achieved after 16 h of incubation in the presence of 3.2 +/- 2.6 nM itraconazole.

Biochemical basis for the activity and selectivity of oral antifungal drugs.

The ergosterol biosynthesis-inhibiting (EBI) antifungals constitute the most important group of compounds developed for the control of fungal diseases in man. Currently, representatives of two classes of EBI antifungals are available: the squalene epoxidase inhibitors and those that interfere with cytochrome P450-dependent ergosterol synthesis. The allylamines (eg, terbinafine) inhibit squalene epoxidase in sensitive fungi, Trichophyton mentagrophytes being the most sensitive species. The most important developments have come from the introduction of the N-substituted imidazoles and triazoles, the so-called azole antifungals. Most of the currently available imidazoles (eg, miconazole, clotrimazole, econazole) and the triazole derivative terconazole are mainly for topical treatment. Ketoconazole was the first azole derivative orally active against yeasts, dermatophytes and dimorphic fungi. The new triazole, itraconazole, appears to be among the most promising orally active systemic agents. All the azole antifungals inhibit the cytochrome P450-dependent, 14 alpha-demethylase, a key enzyme in the synthesis of ergosterol, the main sterol in most fungal cells. Of all the azoles tested, itraconazole shows the highest affinity for the cytochrome P450 involved. It is about three and ten times more active in vitro than miconazole and the bis-triazole, fluconazole, respectively. Itraconazole's high affinity for the fungal P450 originates from its triazole group as well as from the nonligating lipophilic tail.

Saperconazole: a selective inhibitor of the cytochrome P-450-dependent ergosterol synthesis in Candida albicans, Aspergillus fumigatus and Trichophyton mentagrophytes.

The N-1-substituted triazole antifungal, saperconazole, is a potent inhibitor of ergosterol synthesis in Candida albicans, Aspergillus fumigatus and Trichophyton mentagrophytes. Fifty % inhibition is already achieved at nanomolar concentrations. The saperconazole-induced inhibition of ergosterol synthesis coincides with an accumulation of 14-methylated sterols, such as 24-methylenedihydrolanosterol, lanosterol, obtusifoliol, 14 alpha-methylfecosterol, 14 alpha-methylergosta-8,24(28)-dien-3 beta-6 alpha-diol and 14 alpha-methylergosta-5,7,22,24(28)-tetraenol. This indicates that saperconazole interferes with the cytochrome P-450 (P-450)-dependent 14 alpha-demethylation of lanosterol and/or 24-methylenedihydrolanosterol. Saperconazole forms stable drug-P-450-complexes by binding via its free triazole nitrogen to the heme iron and via its N-1 substituent to the apoprotein moiety. The triazole derivative is a highly selective inhibitor of the 14 alpha-demethylase in fungal cells. It is a poor inhibitor of the 14 alpha-demethylation of lanosterol in rat and human liver cells. Saperconazole is, at concentrations as high as 10 microM, devoid of effects on the P-450-dependent cholesterol side-chain cleavage and 11 beta-hydroxylase, 17,20-lyase,21-hydroxylase and aromatase. Saperconazole does not interfere with the 2 alpha, 6 alpha-, 6 beta- and 7 alpha-hydroxylations of testosterone in microsomes from male rat liver. At high concentrations (greater than 5 microM) an inhibition of the 16 beta-hydroxylations is seen.

Biochemical approaches to selective antifungal activity. Focus on azole antifungals.

Azole antifungals (e.g. the imidazoles: miconazole, clotrimazole, bifonazole, imazalil, ketoconazole, and the triazoles: diniconazole, triadimenol, propiconazole, fluconazole and itraconazole) inhibit in fungal cells the 14 alpha-demethylation of lanosterol or 24-methylenedihydrolanosterol. The consequent inhibition of ergosterol synthesis originates from binding of the unsubstituted nitrogen (N-3 or N-4) of their imidazole or triazole moiety to the heme iron and from binding of their N-1 substituent to the apoprotein of a cytochrome P-450 (P-450(14)DM) of the endoplasmic reticulum. Great differences in both potency and selectivity are found between the different azole antifungals. For example, after 16h of growth of Candida albicans in medium supplemented with [14C]-acetate and increasing concentrations of itraconazole, 100% inhibition of ergosterol synthesis is achieved at 3 x 10(-8) M. Complete inhibition of this synthesis by fluconazole is obtained at 10(-5) M only. The agrochemical imidazole derivative, imazalil, shows high selectivity, it has almost 80 and 98 times more affinity for the Candida P-450(s) than for those of the piglet testes microsomes and bovine adrenal mitochondria, respectively. However, the topically active imidazole antifungal, bifonazole, has the highest affinity for P-450(s) of the testicular microsomes. The triazole antifungal itraconazole inhibits at 10(-5) M the P-450-dependent aromatase by 17.9, whereas 50% inhibition of this enzyme is obtained at about 7.5 x 10(-6)M of the bistriazole derivative fluconazole. The overall results show that both the affinity for the fungal P-450(14)DM and the selectivity are determined by the nitrogen heterocycle and the hydrophobic N-1 substituent of the azole antifungals. The latter has certainly a greater impact. The presence of a triazole and a long hypdrophobic nonligating portion form the basis for itraconazole's potency and selectivity.

Effects of ketoconazole and itraconazole on growth and sterol synthesis in Pityrosporum ovale.

The effects of ketoconazole and itraconazole on growth and sterolsynthesis in Pityrosporum ovale was studied. Itraconazole was at least 10 times more active than ketoconazole. Sterol synthesis was inhibited more rapidly than growth, suggesting that the antifungal activity of both azoles originates from an effect on the 14 alpha demethylase system, as seen in other species.

The action of itraconazole and ketoconazole on growth and sterol synthesis in Aspergillus fumigatus and Aspergillus niger.

Growth and sterol synthesis of Aspergillus fumigatus and A. niger were studied in control cultures and in the presence of ketoconazole or itraconazole, the latter compound being 100 times more growth inhibitory than the former. Sterol synthesis is inhibited more rapidly than any visible fungal outgrowth. This inhibition results in an accumulation of 4,14 dimethyl- and 4,4',14 trimethylsterols. The presence of these membrane-disturbing sterols may result in a pertubation of membrane-bound enzyme systems such as chitin synthase.