Molecular mechanisms of drug resistance in fungi.

Failures of treatment in fungal infections have drawn attention recently to the problem of antifungal resistance and its underlying mechanisms. The number of fungal isolates that are resistant to the orally active azole antifungals, especially fluconazole, is growing. Amphotericin-B-resistant isolates have been recovered during treatment of patients with candidiasis, and resistance to flucytosine is so common that this antifungal is no longer recommended as a single-drug therapy.

The interaction of miconazole and ketoconazole with lipids.

Staphylococcus aureus can be protected by unsaturated unesterified fatty acids against the growth inhibitory effects of miconazole and ketoconazole observed at concentrations greater than 10(-6) M and greater than 10(-5) M, respectively. Miconazole's fungicidal activity is partly antagonized by oleic acid. However, the effect of ketoconazole on the viability of Candida albicans was not affected by this fatty acid. Cytochrome oxidase and ATPase activities are more sensitive to miconazole (10(-5) M) than to ketoconazole (greater than 10(-4) M) and also liposomes are more susceptible to lysis induced by miconazole. Using differential scanning calorimetry it is shown that high concentrations of miconazole shift the lipid transition temperature of multilamellar vesicles to lower values without affecting the enthalpy of melting. Ketoconazole induces a broadening of the main transition peak only. It is suggested that miconazole changes the lipid organization without binding to the lipids, whereas ketoconazole is localized in the multilayer without having an important direct effect on the lipid organization. The results indicate that miconazole, and to a lesser extent ketoconazole, at doses that can be reached by topical application only, interfere with a third target (the two others are ergosterol synthesis and fatty acid elongation plus desaturation). It is hypothesized that the induced change in lipid organization may play some role in miconazole's topical antibacterial and fungicidal activity, whereas it does not seem to play a significant role in ketoconazole's activities.

Mechanisms of resistance to azole antifungals.

Until the late eighties, clinical resistance to azole antifungals was a rare phenomenon. Only a few cases of resistance to ketoconazole were found in patients with chronic mucocutaneous candidiasis (CMC). The spread of AIDS and the widespread prophylactic and therapeutic use of the hydrophilic azole compound fluconazole resulted both in the selection and induction of resistant strains and in a shift in the nature of the infecting organisms. Most azole antifungals such as itraconazole, ketoconazole and fluconazole are active against a variety of fungal diseases. However, the concentration needed to inhibit growth is dependent on the nature of the infecting species. Mucor spp., e.g., are almost insensitive to present available azole compounds and can be regarded as intrinsically resistant to azole treatment. Physiochemical features, such as the hydrophobicity and pKa, of a given azole, define whether or not it will be active or cross-resistant against a given species. Fluconazole is almost inactive against Candida krusei and Aspergillus fumigatus, whereas the lipophilic itraconazole is active against these species. A third type of resistance is acquired or induced resistance. This is the most controversial type because, even within a given species, organisms may differ in their response to the same azole. For these strains, convincing evidence can only be obtained when there is a genotypically related strain, which does not show resistance. In a limited number of biochemical or molecular biological studies the mechanisms of resistance have been investigated at the molecular level. These studies show that resistance can occur when there is an insufficient intracellular content of the azole. This can be due to impermeability problems, inactivated uptake systems or, and more likely, the presence of active multidrug resistance gene products of the P-glycoprotein type. Alteration or overexpression of the target for azole antifungals, the cytochrome P450-dependent 14 alpha-demethylase, also induces resistance. The nature and amount of the accumulating sterols also are of great importance for azole-induced growth inhibition. This may explain why mutations in other enzymes of the ergosterol biosynthesis pathway, e.g. the delta 5-6 desaturase, can contribute to azole resistance.

Lipid-itraconazole interaction in lipid model membranes.

Itraconazole, a lipophilic, fungal sterol-biosynthesis inhibitor, does not disturb membrane organization parameters measured by differential scanning calorimetry and infrared spectroscopy. Conformational analysis studies suggest that the molecular volume and the position of itraconazole in the lipid membrane is similar to that of dipalmitoyl phosphatidylcholine. The mean energy of interaction between itraconazole and the phospholipid is -60.6 kJ mol-1 whereas this energy in the pure lipid matrix is -54.3 kJ mol-1. The mean molecular area of itraconazole calculated by projecting the molecule on the lipid-water interface is equal to that occupied by the pure lipid (60 A2/molecule).

A hemodynamic study of epidural versus intravenous anesthesia for aortofemoral bypass surgery.

The hemodynamic effects of two types of anesthesia on aortofemoral bypass surgery were studied in a randomised prospective trial. Epidural anesthesia supplemented with nitrous oxide (group I) and total intravenous anesthesia combining fentanyl and a continuous infusion of etomidate (group II) were compared. A high incidence of preoperative disease was found and all 18 patients were classified in ASA classes III-IV. It is concluded that epidural anesthesia provides excellent anesthetic and hemodynamic stability provided that an optimal filling pressure is maintained. Total intravenous anesthesia resulted in significant hypertensive reactions during surgery, which were not specifically related to crossclamping. Decreasing the high SVRI with vasodilatory treatment was necessary to treat hypertension in all those patients with preoperative hypertensive disease. No problems were seen in the intravenous group patients without preoperative hypertension. Cardiac work was higher in the intravenous group due to the high impedance of the cardiovascular system provoked by the absence of vasodilatory properties with this type of intravenous anesthesia. Monitoring of PWP and CI by Swan-Ganz catheter is shown to be very useful for optimalization of hemodynamics and fluid management especially during crossclamping, when normal Frank-Starling relationships might not be valid anymore. The effect of vasodilatory treatment, crossclamping and declamping could be carefully evaluated.

Mode of action of anti-Candida drugs: focus on terconazole and other ergosterol biosynthesis inhibitors.

A large proportion of the presently available antifungal agents are claimed to derive their activity from interaction with the biosynthesis of ergosterol, the key sterol in most pathogenic fungi. An important target for the allylamines, naftifine and terbinafine, is the squalene epoxidase. Interaction with the epoxidation step results in a decreased availability of ergosterol and an accumulation of squalene. Although the squalene epoxidase is clearly the primary target for this class of antifungals, it still remains an open question whether the fungistatic or fungicidal effects originate from a decrease in ergosterol or squalene accumulation. Indeed, preliminary evidence suggests that squalene does not change the physicochemical properties of membranes. Much more is known about the primary and secondary effects of the azole antifungals, such as miconazole, ketoconazole, terconazole, and itraconazole. Most of the imidazole and triazole derivatives are highly potent and selective inhibitors of the cytochrome P-450-dependent 14 alpha-demethylation of lanosterol (P-45014DM). Their potency and selectivity are determined by the nitrogen heterocycle and to a much greater extent by the hydrophobic N-1 substituent. The triazole antifungals, terconazole and itraconazole, combine a high affinity for Candida P-45014DM with an exceptionally low effect on mammalian cytochrome P-450.

Novel antifungals based on 4-substituted imidazole: solid-phase synthesis of substituted aryl sulfonamides towards optimization of in vitro activity.

The in vitro activity of novel 4-substituted imidazole antifungals was optimized by solid-phase chemistry and parallel synthesis. Potent yeast-selective as well as broad-spectrum antifungal compounds (32 and 20) were discovered.

Anti-Candida drugs--the biochemical basis for their activity.

The past years have seen a continuous effort toward the synthesis of new antifungal agents. Most of them belong to the N-substituted imidazoles and triazoles. Another interesting series of antifungals are the allylamines. Biochemically, both the azole derivatives and the allylamines belong to the class of ergosterol biosynthesis inhibitors and thus differ from the polyene macrolide antibiotics. Indeed, it is now believed that the antifungal action of the polyenes, nystatin and amphotericin B, is due to a direct interaction with ergosterol itself. A more detailed analysis of the ergosterol biosynthesis inhibitors revealed that ergosterol depletion is the consequence of the interaction of the azole derivatives, e.g., miconazole, ketoconazole, and itraconazole, with the cytochrome P-450 involved in the 14 alpha-demethylation of lanosterol. Both the accumulation of 14 alpha-methylsterols and the concomitant decreased ergosterol content affect the membranes and membrane-bound enzymes of yeast and fungi. The allylamines seem to act by inhibition of the squalene epoxidase resulting in ergosterol depletion and accumulation of squalene. The target for the fluorinated pyrimidine, flucytosine, is completely different. Its antifungal properties may result from its conversion to 5-fluorouracil. The latter is then phosphorylated and incorporated into RNA, thus disrupting the protein synthesis in the yeast cell. These different biochemical targets for the antifungals of use in candidosis are discussed in this paper.

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.

Characterization of an azole-resistant Candida glabrata isolate.

A Candida (Torulopsis) glabrata strain (B57149) became resistant to fluconazole after a patient carrying the organism was treated with the drug at 400 mg once daily for 9 days. Growth of the pretreatment isolate (B57148) was inhibited by 50% with 0.67 microM ketoconazole, 1.0 microM itraconazole, and 43 microM fluconazole, whereas growth of B57149 was inhibited slightly by 10 microM ketoconazole but was unaffected by 10 microM itraconazole or 100 microM fluconazole. This indicates cross-resistance to all three azole antifungal agents. The cellular fluconazole content of B57149 was from 1.5- to 3-fold lower than that of B57148, suggesting a difference in drug uptake between the strains. However, this difference was smaller than the measured difference in susceptibility and, therefore, cannot fully explain the fluconazole resistance of B57149. Moreover, the intracellular contents of ketoconazole and itraconazole differed by less than twofold between the strains, so that uptake differences did not account for the azole cross-resistance of B57149. The microsomal cytochrome P-450 content of B57149 was about twice that of B57148, a difference quantitatively similar to the increased subcellular ergosterol synthesis from mevalonate or lanosterol. These results indicate that the level of P-450-dependent 14 alpha-demethylation of lanosterol is higher in B57149. Increased ergosterol synthesis was also seen in intact B57149 cells, and this coincided with a decreased susceptibility of B57149 toward all three azoles and amphotericin B. B57149 also had higher squalene epoxidase activity, and thus, more terbinafine was needed to inhibit the synthesis of 2,3-oxidosqualene from squalene. P-450 content and ergosterol synthesis both decreased when isolate B57149 was subcultured repeatedly on drug-free medium. This repeated subculture also fully restored the strain's itraconazole susceptibility, but only partly increased its susceptibility to fluconazole. The results suggest that both lower fluconazole uptake and increased P-450-dependent ergosterol synthesis are involved in the mechanism of fluconazole resistance but that only the increased ergosterol synthesis contributes to itraconazole cross-resistance.

Hypothesis on the mechanism of resistance to fluconazole in Histoplasma capsulatum.

An AIDS patient with disseminated histoplasmosis who improved during treatment with fluconazole but remained fungemic and subsequently relapsed is described. Isolates obtained from blood during therapy showed a progressive increase in fluconazole MIC from 0.625 to 20 micrograms/ml. The pretreatment, or parent, isolate and the posttreatment, or relapse, isolate demonstrated identical genetic patterns by PCR fingerprinting with three different primers. Fluconazole was less potent inhibitor of the growth of the relapse isolate than of the pretreatment isolate (50% inhibitory concentration [IC50] = 11.7 microM), while itraconazole was more potent (relapse isolate IC50 = 0.0011 microM versus pretreatment isolate IC50 = 0.0064 microM). Neither the increased sensitivity to itraconazole nor the decreased activity of fluconazole on the growth of the relapse isolate results from changes in the intracellular content of these agents. To reach 50% inhibition of ergosterol synthesis in both the parent and relapse isolates, about 2 nM itraconazole was needed; with fluconazole, 50% inhibition was achieved at 20.9 microM and 55.5 microM, respectively. Resistance to fluconazole may develop during treatment and results from decreased sensitivity of ergosterol synthesis.

Rhodamine 6G efflux for the detection of CDR1-overexpressing azole-resistant Candida albicans strains.

We investigated the drug efflux mechanism in azole-resistant strains of Candida albicans using rhodamine 6G (R6G). No significant differences in R6G uptake were observed between azole-sensitive B2630 (9.02 +/- 0.02 nmol/10(8) cells) and azole-resistant B67081 (8.86 +/- 0.03 nmol/10(8) cells) strains incubated in glucose-free phosphate buffered saline. A significantly higher R6G efflux (2.0 +/- 0.21 nmol/10(8) cells) was noted in the azole-resistant strain (B67081) when glucose was added, compared with that in the sensitive strain B2630 (0.23 < or = 0.14 nmol/10(8) cells). A fluconazole-resistant strain C40 that expressed the benomyl resistance gene (CaMDR) also showed a low R6G efflux (0.16 +/- 0.06 nmol/10(8) cells) as did the sensitive strains. Accumulation of R6G in growing C. albicans cells was inversely correlated with the level of CDR1 mRNA expression. Our data also suggest that measurement of intracellular accumulation of R6G is a useful method for identification of azole-resistant strains due to CDR1-expressed drug efflux pump.

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.

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 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.

Synergic effects of tactolimus and azole antifungal agents against azole-resistant Candida albican strains.

We investigated the effects of combining tacrolimus and azole antifungal agents in azole-resistant strains of Candida albicans by comparing the accumulation of [3H]itraconazole. The CDR1-expressing resistant strain C26 accumulated less itraconazole than the CaMDR-expressing resistant strain C40 or the azole-sensitive strain B2630. A CDR1-expressing Saccharomyces cerevisiae mutant, DSY415, showed a marked reduction in the accumulation of both fluconazole and itraconazole. A CaMDR-expressing S. cerevisiae mutant, DSY416, also showed lower accumulation of fluconazole, but not of itraconazole. The addition of sodium azide, an electron-transport chain inhibitor, increased the intracellular accumulation of itraconazole only in the C26 strain, and not in the C40 or B2630 strains. Addition of tacrolimus, an inhibitor of multidrug resistance proteins, resulted in the highest increase in itraconazole accumulation in the C26 strain. The combination of itraconazole and tacrolimus was synergic in azole-resistant C. albicans strains. In the C26 strain, the MIC of itraconazole decreased from >8 to 0.5 mg/L when combined with tacrolimus. Our results showed that two multidrug resistance phenotypes (encoded by the CDR1 and CaMDR genes) in C. albicans have different substrate specificity for azole antifungal agents and that a combination of tacrolimus and azole antifungal agents is effective against azole-resistant strains of C. albicans.

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.

Novel antifungals based on 4-substituted imidazole: a combinatorial chemistry approach to lead discovery and optimization.

A series of 4-substituted imidazole sulfonamides has been prepared by solid-phase chemistry. These compounds were found to have good in vitro antifungal activity and constitute the first examples of C-linked azoles with such activity. The most potent inhibitor (30) demonstrated inhibition of key Candida strains at an in vitro concentration of < 100nM and compared favorably with in vitro potency of itraconazole.

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.