Primary site of action of ketoconazole on Candida albicans

Abstract

Ketoconazole, an antifungal drug, completely inhibited the growth of Candida albicans 7N at concentrations of . 50 ,ug/ml (94 ,uM). However, ketoconazole incompletely inhibited the growth of this opportunistic yeast at concentrations of 25 to 0.2 ,ug/ml (47 to 0.4 ,uM). At these lower concentrations, 2,3,5-triphenyl tetrazolium chloride, an electron acceptor, was reduced by several strains of C. albicans. This effect resulted in red coloration of colonies. Concomitantly, this phenomenon was not antagonized in the presence of ergosterol. Furthermore, neither ketoconazole nor antimycin A inhibited the growth of C. albicans under anaerobic conditions, as revealed by a paper disk method. Ketoconazole at the concentrations stated above inhibited endogenous and exogenous respiration immediately after it was added to a system containing log phase C. albicans cells, as determined polarographically. At the same time, ketoconazole inhibited the activity of NADH oxidase at the mitochondrial level. In contrast, higher concentrations of ketoconazole (>100 ,uM) were required to inhibit the activity of succinate oxidase from rat liver mitochondria. In addition, concentrations of ketoconazole greater than 100 ,uM were required to impair the uptake of labeled leucine and adenine and, subsequently, the incorporation of the former into protein and the latter into DNA and RNA in intact cells. On the other hand, ketoconazole at concentrations of 10, 1.0, and 0.4 ,uM had no effect on either membrane permeability or macromolecular synthesis.Ketoconazole (R41,400) is a new antifungal imidazole compound which has been found to be very active against both superficial and systemic fungal infections when it is administered orally, and its efficacy has been proved in clinical trials. It is known that ketoconazole is much more water soluble and readily absorbed from the gastrointestinal tract than any other chlorinated imidazole derivative synthesized to date. Nevertheless, the excellent in vivo activity of ketoconazole against Candida albicans cannot be explained by its fair blood level after oral administration alone, because the blood level attained is far below the minimal (complete) inhibitory concentration (MIC) in vitro (10).A number of studies have been undertaken to elucidate the mode(s) of action of antifungal imidazole derivatives. The fact that these compounds interact with cell membranes seems to have been well established by the results of Iwata et al. (4,5) and Yamaguchi and Iwata (21,22) with clotrimazole, of with miconazole, and of (3) and by Marriot with some imidazole-containing antifungal agents (9). These interesting findings may provide some clue concerning the primary site of action of these compounds. However, these results do not differentiate the biochemical actions of miconazole and ketoconazole, and the function of the cell membrane components substituted by the precursors of ergosterol in C. albicans is not yet known. Similarly, the majority of these results were obtained after long exposures to the drugs.In ...

In-vitro resistance to azoles associated with mitochondrial DNA deficiency in Candida glabrata

“…It is now well established that the synthesis of P-450DM is stimulated under anaerobic conditions or by the presence of a high concentration of glucose (Crabtree effect) [35]. Similarly, in C. albicans, anaerobic conditions inhibit the antifungal activity of ketoconazole [36]. Therefore, it can be speculated that the absence of respiration imposed by culture conditions (anaerobic conditions, catabolic repression, respiratory inhibitors) or by spontaneous or induced mutations, may stimulate the biosynthesis of P-450DM, leading to the resistance of the yeast to azole derivatives.…”

Defined Anaerobic Growth Medium for Studying Candida albicans Basic Biology and Resistance to Eight Antifungal Drugs

“…Two previous reports also examined the effectiveness of antifungals during the anaerobic growth of C. albicans. Using yeast morphology agar and the paper disk method in GasPak jars, Uno et al (36) showed that ketoconazole inhibited growth aerobically but not anaerobically. In contrast, Szawatkowski and Hamilton-Miller (35) incorporated various levels of amphotericin B and clotrimazole into Sabouraud agar.…”

“…Our literature search recovered only eight articles (10,16,17,33,(35)(36)(37)(38), two of which (16,33) describe survival under anaerobic conditions rather than actual anaerobic growth and three more of which (10,17,38) merely confirm that anaerobic growth is possible. However, Szawatkowski and Hamilton-Miller (35) reported that 20 strains of C. albicans could grow anaerobically on either Sabouraud agar or brucella agar supplemented with 10% whole horse blood, and Webster and Odds (37) showed that seven Candida species, including C. albicans, could grow anaerobically on four media: Eagle's minimal medium with 10% horse serum, a peptone-glucose broth, and two defined but complicated media (yeast nitrogen base-glucose broth and yeast nitrogen baseasparagine-glucose-phosphate [YAGP] broth).…”

“…Research has been done to test the sensitivity and resistance of C. albicans cells grown aerobically (21,32) and anaerobically (35,36,38) to several antifungal agents. We have tested five classes of antifungal agents on cells grown anaerobically: polyenes, allylamines, azoles, zaragozic acid B, and cerulenin.…”

Role of the naphthoquinone moiety in the biological activities of sakyomicin A.

“…The oxidation of NADH or NADPH by mitochondria was measured at 30°C by oxygen consumption as determined with a Clark type electrode (Yellow Spring Instrument Co., Yellow Spring, Ohio) 19) Respiration of Rat Liver Mitochondria Respiration of mitochondria on glutamate was measured by the same method as described in the measurement of the oxidation of NADH or NADPH. The activity of AMV reverse transcriptase was markedly inhibited by 1, 2, 3 or 4 at 40 tag/ml, while moderate inhibition was observed by sakyomicin A and no significant inhibition by either 5, 6 or 7 at the same concentration (Table 1).…”