H. Van den Bossche

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.

Chemotherapy for larval echinococcosis in animals and humans: Report of a workshop

Mebendazole, its fluorine analogue flubendazole, and other benzimidazole derivatives are active against many gastrointestinal and tissue-stage helminths. This article reviews the published literature and proceedings of a workshop on the use of benzimidazoles against larval echinococcosis (hydatid disease). Orally administered high doses (30–50 mg/kg body weight) of mebendazole given daily for 20–90 days to rodents or sheep infected with larvalEchinococcus granulosus cause damage or destruction of the cyst wall, loss of cyst fluid, and death of protoscolices. Similar treatment of rodents infected withE. multilocularis with mebendazole, flubendazole, fenbendazole, and albendazole for 60–300 days leads to reduction of weight, inhibition of growth and of metastases formation ofE. multilocularis tissue, and to prolonged host survival time although the metacestodes are not killed. Mebendazole or flubendazole treatment of human patients infected withE. granulosus is followed by subjective improvement in most, and evidence of regression of cysts in some; in other patients, cysts continue to grow or have been proven viable even after several months of high-dose mebendazole therapy. In patients infected withE. multilocularis, the progressive course of the disease appeared to be arrested, but treatment apparently did not kill the parasite. Side effects in some patients have included allergic reactions, alopecia, and reversible neutropenia. Some possible reasons for differnet responses to treatment include inadequate plasma drug absorption from the gut and age, condition, and location of cysts. Many remaining questions concerning the risk versus benefits of mebendazole therapy can be answered only through controlled clinical trials.

Promotion of pseudomycelium formation of Candida albicans in culture: a morphological study of the effects of miconazole and ketoconazole.

The effects of miconazole and its new derivative ketoconazole on Candida albicans have been evaluated by light and electron microscopy. The growth characteristics and morphology of C. albicans in culture for various periods of time in a solution consisting of Eagles minimum essential medium supplemented with amino acids and fetal calf serum are emphasized. This medium, normally used for culturing mammalian cells, promotes a rather fast growth of C. albicans and favours the development of pseudomycelium. The obvious interest in using such culture conditions for drug evaluation is the prevalence of pseudomycelium, which in vivo is the predominant pathological form of C. albicans. Suppression of pseudomycelium formation is found in the 10-9 to 10-7 M concentration range of the imidazoles. Growth retardation and the destruction of both yeast and pseudomycelial forms brought about by incubating the cells with 10-9 to 10-4 M of the drugs are reported. At low doses these changes include the alteration of cell division, an increase in cell volume and a progressive deterioration of subcellular organelles at the cell periphery. At higher doses the involvement of all other organelles is observed finally leading to complete cell necrosis.

Mode of insertion of miconazole, ketoconazole and deacylated ketoconazole in lipid layers: A conformational analysis

The conformation of three imidazole derivatives, miconazole, ketoconazole and deacylated ketoconazole (R 39 519) inserted in a lipid layer was calculated using a procedure of conformational analysis. For each imidazole derivative all probable conformers were inserted into a dipalmitoyl phosphatidylcholine (DPPC) monolayer. Miconazole maintains its two dichlorophenyl groups in the hydrophobic phase whereas the imidazole moiety is orientated in the hydrophilic phase. Ketoconazole orientates its dichlorophenyl group in the hydrophobic phase whereas its acylated piperazine moiety is orientated towards the hydrophobic region. Deacylation inverses completely the orientation of the compound. The most probable conformer of R 39 519 is inserted in the lipid layer with its piperazine moiety orientated towards the aqueous phase. The inversion increases the area occupied per drug molecule from 30 A2 for ketoconazole to 90 A2 for R 39 519 equal to the mean area occupied per miconazole molecule and higher than that occupied per DPPC molecule (60 A2). Such a conformation should result in a destabilizing effect of miconazole and R 39 519; this was proved using differential scanning calorimetry.

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.

The activity of ketoconazole in mixed cultures of leukocytes and Candida albicans

A system is described which allows the semi-quantitative investigation of the interaction between Candida albicans and leukocytes in culture with and without the addition of chemotherapeutic agents. Both polymorphonuclear leukocytes and macrophages avidly engulfed added yeast cells. However, they did not succeed in eradicating the fungus even when only 450 yeast cells were added to 3 X 10(6) leukocytes. This is probably due to several factors, including the decline in the functiontional capacity of the leukocytes with time in culture. The major way for the fungus to escape intracellular killing, however, seems to be the switch to the mycellial form in the presence of leukocytes. Engulfed yeasts produce germ tubes, grow out of the leukocytes and form hyphae which are much more resistant to the lytic action of the leukocytes. The leukocytes become necrotic through their interaction with the mycelia. Ketoconazole, a potent, orally active systemic antifungal agent inhibited the growth of C. albicans and completely suppressed the formation of mycelia in culture at very low concentrations (0.01 microgram ml-1). It was toxic to the leukocytes themselves only at 100 microgram ml-1. Addition of ketoconazole (10 (10-1.01 microgram ml-1) to mixed cultures of leukocytes and C. albicans allowed complete elimination of the fungus, probably because the leukocytes could easily remove the remaining yeast cells. The data show the usefulness of the system in the search for systemic antifungals and provide a possible explanation for the efficacy of ketoconazole in vivo.

The action of miconazole on the growth of Candida albicans

The growth of Candida albicans was studied in control cultures and in the presence of miconazole or clotrimazole. Each drug prolonged the lag phase and reduced the total final population. Although miconazole, at the low concentrations used, was a less potent inhibitor than clotrimazole in the main logarithmic phase, it was more fungicidal. The antifungal activity of miconazole on C. albicans was inversely proportional to the number of cells inoculated in the media. The effects of miconazole on growth depended on the nutrients in the medium and were most pronouncedwhen it was added to cultures of C. albicans in the lag and main logarithmic phase of growth. The growth inhibitory effects of sub-fungicidal doses of micronazole (smaller than or equal to 10-6 M) on C. albicans seemed to be reversible.

Subcellular distribution of digestive enzymes in Ascaris suum intestine

Abstract Van den Bossche H. and Borgers M. 1973. Subcellular distribution of digestive enzymes in Ascaris intestine. International Journal for Parasitology3: 59–65. The microvilli of the intestinal cells of Ascaris suum resemble the microvilli of the mammalian intestine in respect to their morphologic structure; their behaviour to homogenization in the presence of a chelating agent; the presence of the disaccharide hydrolases, maltase, sucrase and trehalase and the presence of an enzyme which hydrolyses 5′-AMP at neutral pH. The microvilli of the Ascaris intestinal cells differ completely from those present in mammalian intestine in respect to the presence of non-specific phosphatases. The brush border fraction contains the bulk of acid phosphatase present in the intestinal cells. Although some pinocytotic vesicles have been observed only low endocytotic activity was found. We therefore suggest that the acid hydrolases found on the brush border membrane may be functionally related to extracellular digestion of macromolecules.

Studies on the carbohydrate metabolism of third-stage Haemonchus contortus larvae.

Abstract Third-stage Haemonchus contortus larvae can derive additional energy from an aerobic metabolism. This is concluded from the following observations: 1 - the larvae exhibit a Pasteur effect, 2 - an electron transport system from NADH 2 and succinate to oxygen, which is rotenone, antimycin A and KCN sensitive, is demonstrable, 3 - cytochrome oxidase is present. Under anaerobic conditions these larvae may meet some of their energy requirements by a NADH 2 -coupled fumarate-succinate system which is tetramisole-sensitive.

Synergism of the Antimicrobial Agents Miconazole, Bacitracin and Polymyxin B

The antibacterial activity of a combination of miconazole with bacitracin against Staphylococcus epidermidis or Staphylococcus aureus, was greater than the sum of the effects observed with the two drugs independently. Since no uptake changes were observed, this synergism may originate from a combined effect of miconazole and bacitracin on the biosynthesis of the carrier lipid, undecaprenyl-phosphate, resulting in an impairment of cell wall integrity. The synergism between these drugs and polymyxin B may be due to an increased accessibility of the cell membrane to polymyxin B. Miconazole and bacitracin, alone or in combination, are completely inactive against Escherichia coli. Since polymyxin B disturbs the permeability barrier and stimulates the uptake of miconazole, the synergism of polymyxin B, miconazole and bacitracin may originate from the ability of miconazole and bacitracin to penetrate the bacterial membranes in the presence of polymyxin B.