W. Cools

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.

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.

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.

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.

Liarozole fumarate inhibits the metabolism of 4-keto-all-trans-retinoic acid

The metabolism of 4-keto-all-trans-retinoic-acid (4-keto-RA), a biologically active oxygenated metabolite of all-trans-retinoic (RA), has been examined. In vitro, incubation of [14C]4-keto-RA with hamster liver microsomes in the presence of NADPH produced two major radioactive metabolites which were more polar than the parent compound. Following isolation, appropriate derivatization and analysis by GC-MS, these compounds were tentatively identified as 2-hydroxy- and 3-hydroxy-4-ketoretinoic acid. Formation of both hydroxy-keto derivatives was suppressed by the imidazole-containing P450 inhibitor liarozole fumarate (IC50, 1.3 microM). In vitro, an i.v. injection of 4-keto-RA (20 micrograms) into rats was followed by rapid disappearance of the retinoid from plasma with a half-life of 7 min. Pretreatment with liarozole fumarate (40 mg/kg, -60 min) reduced the elimination rate of 4-keto-RA: it prolonged the plasma half-life of the retinoid to 12 min, without affecting its distribution volume. These results indicate the important role of the P450 enzyme system in the metabolism of 4-keto-RA both in vitro and in vivo. The inhibitory effect of liarozole fumarate on this metabolic process may contribute to the reported retinoid-mimetic activity of this drug.

Induction of the oxidative catabolism of retinoid acid in MCF-7 cells.

Cytochrome P450-dependent oxidation is a pathway for all-trans-retinoic acid (all-trans-RA) catabolism. Induction of this catabolic pathway was studied in MCF-7 breast cancer cells. MCF-7 cells showed low constitutive all-trans-RA catabolism. Concentration-dependent induction was obtained by preincubation of the cells with all-trans-RA (10(-9) to 10(-6) M). Onset of induction was fast, being detectable within 60 min, with maximal induction (45-fold) obtained after 16 h. Enzymatic characterization of induced all-trans-RA catabolism showed an estimated Km value (Michaelis-Menten constant) of 0.33 microM and a Vmax value (maximal velocity of an enzyme-catalysed reaction) of 54.5 fmol polar all-trans-RA metabolites 10(6) cells(-1) h(-1). These kinetic parameters represent the overall formation of polar metabolites from all-trans-RA. Induction of all-trans-RA catabolism was also obtained with other retinoids, CH55 >> 13-cis-RA = all-trans-RA > 9-cis-RA > 4-keto-all-trans-RA > 4-keto-13-cis-RA > retinol. The potency of the retinoids to induce all-trans-RA catabolism was correlated to their retinoic acid receptor affinity (Crettaz et al, 1990; Repa et al, 1990; Sani et al, 1990). Induction of all-trans-RA catabolism was inhibited by actinomycin D. Furthermore, all-trans-RA did not increase cytosolic retinoic acid-binding protein (CRABP) mRNA levels. These data suggest that induction of all-trans-RA catabolism in MCF-7 cells is a retinoic acid receptor-mediated gene transcriptional event. Induced all-trans-RA catabolism was inhibited by various retinoids with decreasing potency in the order: all-trans-RA > 4-keto-all-trans-RA > 13-cis-RA > 9-cis-RA > 4-keto-13-cis-RA > retinol > CH55. The antitumoral compound liarozole-fumarate inhibited all-trans-RA catabolism with a potency similar to that of all-trans-RA.

COMPARATIVE EFFECTS OF KETOCONAZOLE ON RAT, DOG AND HUMAN TESTICULAR STEROIDOGENESIS

Ketoconazole is an antifungal azole derivative which also inhibits the cytochrome P-450(17)alpha, catalyzing the conversion of progestins into androgens. The effects of ketoconazole on human, dog and rat testosterone biosynthesis were compared using short term incubations of dispersed testicular cells. The results showed that ketoconazole inhibited androgen biosynthesis at lower concentrations in dispersed human testicular cells (IC50: 0.08 mumol/l) than in canine (IC50: 0.1 mumol/l) and rat cells (IC50 greater than or equal to 0.2 mumol/l). Furthermore, they demonstrated that ketoconazole first inhibited the 17,20-lyase activity and then the 17-hydroxylation in rat and dog cells whereas only the 17-hydroxylation was affected in human cells.

Analysis of the oxidative catabolism of retinoic acid in rat Dunning R3327G prostate tumors

We studied the enzymatic characteristics of the oxidative catabolism of retinoic acid (RA) and its inhibition by liarozole‐fumarate in homogenates of rat Dunning R3327G prostate tumors. Homogenates of rat liver were used as reference material. Both tumor and liver homogenates were able to catabolize retinoic acid. HPLC analysis revealed only very polar metabolites in tumors, while in the liver both metabolites with intermediate polarity and more polar metabolites were found. Kinetic analysis of retinoic acid catabolism showed a Km of 1.7 ± 0.7 μM and a Vmax of 4.2 ± 4.4 pmol polar RA metabolites/mg protein/hr for Dunning G tumor homogenates. In liver homogenates a Km value of 4.3 ± 0.5 μM and a Vmax value of 290 ± 120 pmol polar RA metabolites/mg protein/hr were obtained. Liarozole‐fumarate inhibited retinoic acid catabolism in Dunning tumors and liver with IC50 values of 0.26 ± 0.16 μM and 0.14 ± 0.05, respectively. The results suggest that rat Dunning R3327G tumors are able to metabolize retinoic acid in a manner similar to that found in rat liver but with a lower metabolizing capacity.