G. Willemsens

Biochemical effects of miconazole on fungi. II. Inhibition of ergosterol biosynthesis in Candida albicans.

Abstract The effects of the antifungal agent miconazole nitrate on the ergosterol biosynthesis in Candida albicans were investigated after in vitro contact with the drug for 1, 4, 16 and 24 h. A time- and dose-(2.10 −10 –10 −4 M) dependent inhibition of [ 14 C]acetate incorporation into ergosterol was observed. Fifty percent inhibition of the acetate incorporation into ergosterol was found after 1 h incubation in the presence of 10 −9 M miconazole. Simultaneously 24-methylenedihydrolanosterol, lanosterol, obtusifoliol, 4,14-dimethylzymosterol and 14-methylfecosterol accumulated. The accumulation of 14 α-methyl sterols suggests that this antifungal agent is a potent inhibitor of one of the metabolic steps involved in the demethylation at C-14. The absence of 24-methyl sterols and of sterols with a C-22 [23] double bond in miconazole treated C. albicans indicates that miconazole also inteferes with the reduction of the 24(28)-double bond and with the introduction of the 22(23)-double bond. Miconazole also intervenes to a small extent in triglyceride synthesis. However, in all circumstances studied, ergosterol biosynthesis was affected at lower doses than those interfering with the acetate incorporation into triglycerides. 16 and 24 h of incubation in the presence of miconazole (≥ 10 −6 M) also resulted in an increased fatty acid synthesis. It is suggested that the miconazole-induced inhibition of the C-14 demethylation may be at the origin of the previously observed permeability changes in miconazole treated C. albicans .

In vitro and in vivo effects of the antimycotic drug ketoconazole on sterol synthesis.

Ketoconazole, an orally active antimycotic drug, is a potent inhibitor of ergosterol biosynthesis in Candida albicans when added to culture media which support yeast or mycelial growth or to cultures containing outgrown mycelium. This inhibition coincides with accumulation of sterols with a methyl group at C-14 and can thus be attributed to an interference with one of the reactions involved in the removal of the 14 alpha-methyl group of lanosterol. When administered to rats infected with C. albicans, ketocanazole also inhibits fungal synthesis of ergosterol. A six-times-higher dose is required to effect cholesterol synthesis by rat liver.

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.

Biochemical Approaches to Selective Antifungal Activity. Focus on Azole Antifungals

Summary: Azole antifungals (e.g. the imida‐zoles: miconazole, clotrimazole, bifona‐zole, imazalil, ketoconazole, and the tria‐zoles: diniconazole, triadimenol, propico‐nazole, fluconazole and itraconazole) inhibit in fungal cells the 14α‐demethylation of lanosterol or 24–methylenedihydro‐lanosterol. 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‐45014DM) of the endo‐plasmic reticulum.

Effects of etomidate on steroid biosynthesis in subcellular fractions of bovine adrenals

The imidazole derivative, etomidate, inhibits the 11 beta-hydroxylase in cell-free systems and mitochondria isolated from bovine adrenal cortex. Fifty per cent inhibition is achieved at 3.10(-7) M. The less active hypnotic L-enantiomer is also a less potent inhibitor of the 11-hydroxylation. At a 2 times higher concentration, etomidate affects the cholesterol side chain cleavage. The inhibition of both steroidogenic enzyme systems may be due to binding of the unhindered nitrogen of the imidazole ring of etomidate to the heme iron atom of the adrenal cortex mitochondrial cytochrome P-450 species.

R 76713 and enantiomers: Selective, nonsteroidal inhibitors of the cytochrome P450-dependent oestrogen synthesis

The triazole derivative, R 76713 and its enantiomers R 83839(-) and R 83842(+) are effective inhibitors of the aromatization of androstenedione. For human placental microsomes, the (+) enantiomer (R 83824) is about 1.9- and 32-times more active than the racemate (IC50 2.6 nM) and the (-) enantiomer, respectively. R 83842 is about 30- and 1029-times more active than 4-hydroxyandrostene-3,17-dione and aminoglutethimide. This potency might originate from its high affinity for the microsomal cytochrome P450 (P450). Indeed, R 83842, compared to R 76713 and R 83839, forms a more stable P450-drug complex. Difference spectral measurements indicate that the triazole nitrogen N-4 coordinates to the haem iron. The reversed type 1 spectral changes suggest that R 76713 is able to displace the substrate from its binding place and the stable complex formed in particular with the (+) enantiomer suggests that its N-1-substituent occupies a lipophilic region of the apoprotein moiety. Kinetic analysis implies that there is a competitive part in the inhibition of the human placental aromatase by R 76713. The Ki values for R 76713, R 83842 and R 83839 are 1.3 nM, 0.7 nM and 18 nM, respectively. These results are indicative of stereospecificity for binding. Up to 10 microM, R 76713 and its enantiomers have no statistically significant effect on the regio- and stereoselective oxidations of testosterone in male rat liver microsomes. All three compounds have no effect on the P450-dependent cholesterol synthesis, cholesterol side-chain cleavage and 7 alpha-hydroxylation and 21-hydroxylase. At 10 microM, R 76713 has a slight effect on the bovine adrenal 11 beta-hydroxylase. This effect originates mainly from R 83839, the less potent aromatase inhibitor. On the other hand, the inhibition of the 17,20-lyase of rat testis observed at concentrations greater than or equal to 0.5 microM, originates rather from R 83842. However, 50% inhibition is only achieved at 1.8 microM R 83842, i.e. at a concentration about 1300-times higher than that needed to reach 50% inhibition of the human placental aromatase.

Inhibition of all-TRANS-retinoic acid metabolism by R116010 induces antitumour activity

All-trans-retinoic acid is a potent inhibitor of cell proliferation and inducer of differentiation. However, the clinical use of all-trans-retinoic acid in the treatment of cancer is significantly hampered by its toxicity and the prompt emergence of resistance, believed to be caused by increased all-trans-retinoic acid metabolism. Inhibitors of all-trans-retinoic acid metabolism may therefore prove valuable in the treatment of cancer. In this study, we characterize R116010 as a new anticancer drug that is a potent inhibitor of all-trans-retinoic acid metabolism. In vitro, R116010 potently inhibits all-trans-retinoic acid metabolism in intact T47D cells with an IC50-value of 8.7 nM. In addition, R116010 is a selective inhibitor as indicated by its inhibition profile for several other cytochrome P450-mediated reactions. In T47D cell proliferation assays, R116010 by itself has no effect on cell proliferation. However, in combination with all-trans-retinoic acid, R116010 enhances the all-trans-retinoic acid-mediated antiproliferative activity in a concentration-dependent manner. In vivo, the growth of murine oestrogen-independent TA3-Ha mammary tumours is significantly inhibited by R116010 at doses as low as 0.16 mg kg−1. In conclusion, R116010 is a highly potent and selective inhibitor of all-trans-retinoic acid metabolism, which is able to enhance the biological activity of all-trans-retinoic acid, thereby exhibiting antitumour activity. R116010 represents a novel and promising anticancer drug with an unique mechanism of action.

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. Miconazoles 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 miconazoles topical antibacterial and fungicidal activity, whereas it does not seem to play a significant role in ketoconazoles activities.

Perturbation of sterol biosynthesis by itraconazole and ketoconazole in Leishmania mexicana mexicana infected macrophages

The azole antifungals ketoconazole and itraconazole possess in vitro antileishmanial activity against Leishmania mexicana mexicana amastigotes in macrophages (cell line J774G8). As in yeast and fungi, the activity is likely to be due to inhibition of the cytochrome P-450-dependent 14 alpha-demethylation of lanosterol and/or 24,25-dihydrolanosterol. Indeed, 50% inhibition of ergosterol synthesis was observed at 0.21 microM ketoconazole and 0.15 microM itraconazole. At 5 microM ketoconazole, traces of ergosterol could be found, whereas no ergosterol could be detected in cells treated with 5 microM itraconazole. The inhibition of ergosterol biosynthesis was concomitant with an accumulation of the 14 alpha-methylsterols lanosterol and 24,25-dihydrolanosterol. Fifty percent inhibition of cholesterol synthesis in uninfected macrophages was achieved at 0.95 microM and 1.5 microM itraconazole and ketoconazole, respectively. In infected macrophages all [14C]acetate was incorporated in ergosterol, suggesting an inhibition in cholesterol synthesis in the host cells. An inhibition of ergosterol synthesis coincided with increasing cholesterol synthesis. The latter synthesis was inhibited at concentrations greater than 1 microM. However, even at 5 microM cholesterol synthesis was higher than under control conditions.

Cytochrome-P-450-Dependent Metabolism of Retinoic Acid in Rat Skin Microsomes: Inhibition by Ketoconazole

Epidermal microsomes, prepared from neonatal Wistar or Sprague-Dawley rats, show low levels of retinoic acid (RA) metabolism. The specific activities (as fmol/min/mg protein) of epidermal microsomes, using [15-14C]-RA as substrate, are 232 (Wistar rats) and 222 (Sprague-Dawley rats). Topical application of RA (1 mg) on 4-day-old rats induces a 3.3- and 3.9-fold increase in epidermis microsomal RA metabolism. A 4.6- to 8.1-fold increase is observed 24 h after topical application of 3-methylcholanthrene (0.5 mg). By contrast, phenobarbital (1 mg topically) has a much smaller inducing effect. So far the chemical structure of the metabolites has not been identified. The Rf values of two major compounds correspond with those of 4-hydroxy- and 4-ketoretinoic acid, formed after incubation of hamster liver microsomes in the presence of [15-14C]-RA. The RA metabolism in rat epidermal microsomes shows the typical characteristics of a cytochrome-P-450 (P450)-dependent enzyme system, i.e. a requirement for NADPH and oxygen and inhibition by CO and SKF-525A. Ketoconazole and miconazole, imidazole antifungal agents and inhibitors of some fungal and mammalian P450-dependent enzymes, inhibit in vitro RA metabolism by rat epidermal microsomes. 50% inhibition is achieved at 6.5 X 10(-7) and greater than or equal to 10(-5) mol/l, respectively. The triazole antifungal agent, itraconazole, has no effect at concentrations up to 10(-5) mol/l. Topical treatment of 4-day-old Wistar rats with ketoconazole, at doses of 1,5 and 10 mg/kg, 1 h before the application of RA (1 mg/rat) results in a dose-dependent inhibition of RA metabolism by epidermal microsomes, prepared 24 h later. Our data show a P450-dependent RA metabolism in rat epidermal microsomes and suggest that ketoconazole may prove to be effective in maintaining biologically active levels of RA in epidermal cells.