W. Lauwers

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 .

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.

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.

Comparative metabolism of flunarizine in rats, dogs and man: an in vitro study with subcellular liver fractions and isolated hepatocytes

1. The biotransformation of 3H-flunarizine ((E)-1-[bis(4-fluorophenyl)methyl]-4-(3-phenyl-2-propenyl)piperazine dihydrochloride, FLUN) was studied in subcellular liver fractions (microsomes and 12,000 g fraction) and in suspensions or primary cell cultures of isolated hepatocytes of rats, dogs and man. The major in vitro metabolites were characterized by h.p.l.c. co-chromatography and/or by mass spectrometric analysis. 2. The kinetics of FLUN metabolism was studied in microsomes of dog and man. The metabolism followed linear Michaelis-Menten kinetics over the concentration range 0.1-20 microM FLUN. 3. A striking sex difference was observed for the in vitro metabolism of FLUN in rat. In male rats, oxidative N-dealkylation at one of the piperazine nitrogens, resulting in bis(4-fluorophenyl) methanol, was a major metabolic pathway, whereas aromatic hydroxylation at the phenyl of the cinnamyl moiety, resulting in hydroxy-FLUN, was a major metabolic pathway in female rats. In incubates with hepatocytes, these two metabolites were converted to the corresponding glucuronides. 4. In human subcellular fractions, aromatic hydroxylation to hydroxy-FLUN was the major metabolic pathway. In primary cell cultures of human hepatocytes, oxidative N-dealkylation at the 1- and 4-piperazine nitrogen and glucuronidation of bis(4-fluorophenyl)methanol were observed. The in vitro metabolism of FLUN in humans, resembled more than in female rats and in dogs than that in male rats. 5. The present in vitro results are compared with data of previous in vivo studies in rats and dogs. The use of subcellular fractions and/or isolated hepatocytes for the study of species differences in the biotransformation of xenobiotics is discussed.

Mode of Action of Antifungals of Use in Immunocompromised Patients. Focus on Candida Glabrata and Histoplasma Capsulatum

During recent years considerable advances have been made in the identification of potential targets for antifungal agents. The most important antifungals for use in immunocompromised patients interfere with targets in the plasma membrane (polyenes), nucleus (5-fluorocytosine) or smooth endoplasmic reticulum (allylamines, morpholines, azole derivatives).

Plasma levels, biotransformation and excretion of oxatomide (R 35 443) in rats, dogs and man

Plasma levels, biotransformation and excretion of oxatomide were studied after single oral doses of 14C-oxatomide in male rats, dogs and humans. Oxatomide was very well absorbed, and almost completely metabolized in the three species. Excretion of the metabolites was very rapid and complete within a few days; the 14C label was excreted more in the faeces (54-62%) than in the urine (27-40%). Major metabolic pathways of oxatomide were oxidative N-dealkylations at the piperazine nitrogens and at the benzimidazolone nitrogen in rats and man, and also aromatic hydroxylation at the benzimidazolone moiety in man. The main urinary metabolite in the three species was 2,3-dihydro-2-oxo-1H-benzimidazole-1-propanoic acid, resulting from the oxidative N-dealkylation at the 1-piperazine nitrogen.

Laser microprobe mass spectrometry: Possibilities and limitations

Laser microprobe mass spectrometry (LMMS) offers a great potential for inorganic analysis and speciation as well as for organic structural characterization. This remarkable flexibility makes the results critically dependent on the experimental conditions, however, so an exact description is hard to achieve, since inaccessible local parameters are involved. The paper deals in detail with this aspect, which is often neglected but remains essential for making LMMS acceptable as analytical tool. Our methods of procedure are presented in the context of instrumental aspects and experimental evidence. Tentative hypotheses for desorption and ionization in LMMS provide an adequate framework for interpreting results consistently. Selected examples illustrate the potential gained from our experimental procedure as well as the limitations imposed. For organic compounds, analysis of pure products and simple mixtures, and microprobe applications are highlighted. Improved differentiation of inorganic compounds is also shown.