Anticandidal Activities of Terconazole,

Transcription

1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 1986, p /86/ $02.00/0 Copyright 1986, American Society for Microbiology Vol. 29, No. 6 Anticandidal Activities of Terconazole, a Broad-Spectrum Antimycotic EDWARD L. TOLMAN,'* DAVID M. ISAACSON,' MARVIN E. ROSENTHALE,1 JOHN L. McGUIRE,1 JAN VAN CUTSEM,2 MARCEL BORGERS,3 AND HUGO VAN DEN BOSSCHE3 Research Laboratories, Ortho Pharmaceutical Corporation, Raritan, New Jersey 08869,1 and Department of Bacteriology and Mycology2 and Department of Life Sciences,3 Research Laboratories, Janssen Pharmaceutica, B 2340 Beerse, Belgium Received 25 November 1985/Accepted 20 March 1986 Terconazole is a new triazole ketal derivative with broad-spectrum in vitro and in vivo antifungal activities. This study further characterizes the effects of terconazole in vitro on yeast cell growth, viability, and morphology. Terconazole inhibited the growth of Candida albicans ATCC in a concentration-related manner, but with modest. effects noted at levels from 10-8 to 10-5 M when the yeast was grown on media favoring the cell form. The inhibitory potency of terconazole on yeast cell viability varied with the strain and species of Candida tested. The susceptibility of C. albicans ATCC to terconazole was markedly enhanced when the yeast was grown on Eagle minimum essential medium, which favors mycelium formation. The effects of terconazole on the morphology of yeast cells (grown on Eagle minimum essential medium) were shown by phase-contrast and electron microscopy. There is a progression of changes, from loss of mycelia formation at 10-8 M terconazole through complete necrosis at 10-4 M. Van Cutsem et al. (5) have reported the broad spectrum of in vitro and in vivo antifungal activities of terconazole, a new triazole ketal derivative. The chemical structure of terconazole is presented in Fig. 1. In vitro terconazole inhibits the growth of a wide variety of yeasts and myceliumforming fungi, though the potency of its inhibitory activities is dependent on both yeast strain and growth medium. It is well documented that the in vitro antifungal potencies of the azole class of antimycotics depend on several growth condition factors, including inoculum size, ph, medium nutrient constituents, and temperature (4, 5). These factors contribute to the morphological state of the yeast, i.e., whether it grows in the yeast cell form or mycelial form stage. Van Cutsem et al. (5) demonstrated that terconazole is most potent in medium favoring mycelial formation, as it inhibits the transformation of the yeast cell into its mycelial form. The experiments presented here were designed to characterize further and define the in vitro antifungal effects of terconazole. We report on the effects of terconazole on the growth and viability of several strains of Candida albicans and various Candida spp., including C. tropicalis, C. pseudotropicalis, C. krusei, C. parapsilosis, and C. stellatoidea. The yeast were grown on media favoring either the yeast cell form (casein-yeast extract-glucose; CYG) or the mycelial form (Eagle minimum essential medium; EMEM), and the effects of terconazole on yeast cell growth and yeast cell viability were monitored. In addition, a study of the effects of terconazole on the morphology of C. albicans was carried out by electron microscopy. MATERIALS AND METHODS Media. CYG contained, per liter, 5 g of casein hydrolysate (Merck Sharp & Dohme, West Point, Pa.) or 5 g of Casamino Acids (Difco Laboratories, Detroit, Mich.), 5 g of yeast extract (Difco), and 5 g of glucose. Sabouraud broth contained, per liter, 10 g of neopeptone (Difco) and 20 g of glucose. Sabouraud agar contained 25 g of agar per liter added * Corresponding author. to Sabouraud broth. The preparation of EMEM was done by the method of Aerts et al. (1). Yeast strains and inocula preparation. All strains of C. albicans were obtained from the American Type Culture Collection (Rockville, Md.) and are identified where appropriate. For testing, cells were first grown at 37 C on Sabouraud agar for 24 h, and then a loopful was transferred into 4.5 ml of Sabouraud broth and incubated at 37 C for 24 h. A 0.5-ml fraction of this culture was added to 100 ml of CYG and again incubated with shaking at 37 C for 24 h. This was followed by transfer of 0.2 ml of this culture into 100 ml of CYG and incubation by shaking at 37 C for 24 h. Fractions of 0.2 ml (about 3 x 107 CFUs) of this last cell preparation were added as the inoculum to each 100 ml of prewarmed (37 C) CYG medium or EMEM. Growth studies. For studies in CYG medium (H. Van den Bossche and F. Cornelisson, data on file, Janssen Pharmaceutica, Beerse, Belgium), cells were grown at 37 C aerobically by shaking in a reciprocating shaker, (model R7 or G76; New Brunswick Scientific Co., Inc., Edison, N.J.). Terconazole was dissolved initially in dimethyl sulfoxide and then further diluted into 2.5% dimethyl sulfoxide and added to the CYG medium before the cells were added. Controls were set up similarly with equivalent quantities of solvent (final concentration, 0.05%). For studies in EMEM, 10 ml of the EMEM test system-containing cells, terconazole and dimethyl sulfoxide were placed into tissue culture dishes and incubated by shaking at 37 C in a 5% CO2 atmosphere. Transmission and scanning electron microscopy. After centrifugation, the pelleted yeast cells were fixed for 2 h at room temperature in 3% glutaraldehyde buffered to ph 7.4 with 0.1 M sodium cacodylate. The cells were rinsed overnight in the same solution to which 0.22 M sucrose was added. The cells were postfixed in 2% osmium tetroxide buffered to ph 7.4 with 0.05 M Veronal acetate (Winthrop Laboratories, 986 Div. Sterling Drug Co., New York, N.Y.) and then were impregnated for 40 min in 0.5% uranyl acetate brought to ph 5.2 with Veronal acetate. After dehydration in a graded series of increasing alcohol concentrations, the cells were

2 VOL. 29, 1986 ANTICANDIDAL EFFECTS OF TERCONAZOLE 987 N CH 2 c L.LCH 2-0 N N-CH(CH3 )2 FIG. 1. Chemical structure of terconazole (molecular weight, ). embedded in epon for transmission electron microscopy (3). Semithin sections were prepared for light microscopic examination, and ultrathin sections were prepared for electron microscopic examination by counterstaining with uranium acetate and lead citrate. The cells were examined in a Philips EM 300 microscope. The cells were fixed for scanning electron microscopy in a manner similar to that for transmission electron microscopy. After postfixation in 2% osmium tetroxide, the cells were passed through a series of ethanol-acetone mixtures with a gradient of increasing acetone concentrations up to 100%. Thereafter, they were dried to the critical point, sputtered with a thin layer of gold, and examined in a Philips PSEM 500 microscope. RESULTS Effects on growth. The effects of terconazole on the growth of C. albicans ATCC were determined by total cell count (Fig. 2). The results demonstrate the effects of a wide range of terconazole concentrations (10-8 to 10-4 M) on the growth of C. albicans ATCC through a 70-h growth period in CYG medium. After a lag of 4 h there was an exponential increase in the total number of cells from hours 4 through 8. Terconazole had a slight depressant effect on the increase in cell number when added at concentrations of 10-8 to 10-5 M, and there was marked inhibition at 10-4 M terconazole. It should be noted that when specifically viable cell counts were performed, marked effects of terconazole on ATCC and ATCC C. albicans strains were observed at concentrations of 10-6 M (Table 1). As noted, terconazole reduced viability by 97.1% when tested against strain ATCC at a concentration of 10-6 M, while a similar effect (i.e., 98.3% loss of viability) was noted when strain ATCC was grown in the presence of 10-4 M terconazole. Both strains were grown under the same experimental conditions. When C. albicans was grown on EMEM, the yeast were rapidly transformed into their mycelial phase (2). The results (Fig. 3) depict the effects of terconazole on the growth of the mycelial phase of C. albicans ATCC grown in EMEM. IJ w 0 L. 0 w7 z IU 0 6 I- 0 5 J TIME (h) FIG. 2. Effect of terconazole on the growth of C. albicans. C. albicans ATCC was exposed to various concentrations of terconazole through a 70-h growth period. Data are the total number of cells counted. Terconazole was added at concentrations of 10-8, 10-5, or 10-4 M. By using dry weight or cell mass as the measure of growth, terconazole inhibited growth of the yeast at all concentrations tested, with complete inhibition occurring at CA) 1-0) E X- cc az TABLE 1. Reduction of C. albicans viability by terconazole Viable cells as % of control in the Terconazole concn (M) following C. albicans strains a: ATCC ATCC a Mean of two experiments: the number of CFUs in control cultures of strain ATCC were 3.58 x 108 (n = 5) and of strain ATCC were 3.74 x 108 (n = 2) TIME (h) FIG. 3. Effect of terconazole on the growth of Candida albicans ATCC Cells were grown at 37 C in a humidified 5% CO2 atmosphere in tissue culture dishes containing 10 ml of EMEM without serum, supplemented with dimethyl sulfoxide (0) or 10-8 (A), l0-5 (0), or 10-4 (M) M terconazole. Solvent and drug were added just before inoculation. Dry weight was determined after drying at 70 C under reduced pressure.

3 988 TOLMAN ET AL. ANTIMICROB. AGENTS CHEMOTHER. FIG. 4. Phase-contrast microscopic evaluation of the anticandidal activity of terconazole. C. albicans ATCC was grown for 8 h in EMEM with fetal bovine serum, which was added in the presence of vehicle or terconazole at the stated concentration. Cells were washed, diluted, and recultured in EMEM-serum, but without terconazole, for an additional 24 h M. Relative to the effects of terconazole on C. albicans ATCC grown on the CYG medium, terconazole was more potent at inhibiting the growth of this strain of yeast when incubated in medium favoring its mycelial form. It was also observed that the addition of fetal bovine serum to EMEM had no effect on the potency of terconazole as an inhibitor of the growth of C. albicans ATCC (data not shown). Effects on viability. The effects of terconazole on yeast cell viability were tested by using a broad spectrum of additional C. albicans strains and candidal species. The yeast grown in CYG medium were exposed to terconazole at 10-6 to 10-4 M or to its vehicle for up to 48 h. The yeast were then transferred after washing in Sabouraud broth and further incubated for 48 h, and the CFUs per ml of inoculum were quantified (Table 2). The results depicted demonstrate the different sensitivities exhibited by various strains of C. albicans and other species of Candida to terconazole. In all cases, yeast cell viability was decreased with exposure to an increased concentration of terconazole. Note that certain of the C. albicans strains, in particular ATCC 38247, ATCC 32470, and ATCC 11651, were more sensitive to the lower concentration (10-6 M) of terconazole than were the remaining C. albicans strains. Similar results were obtained with the other Candidal spp. Terconazole exhibited greater potency against C. krusei and C. tropicalis ATCC than against C. tropicalis ATCC 750, C. pseudotropicalis, or C. parapsilosis after exposure for 24 h. The efficacy, i.e., the total effect, of terconazole differed with the various Candida spp. Note the greater than 99% loss of viability after exposure for 24 h to 10-4 M terconazole by C. albicans ATCC 38247, C. krusei, C. parapsilosis, and C. pseudotropicalis. These results emphasize that the potency of terconazole as an inhibitor of yeast cell growth and viability is dependent on yeast strain and species. Morphological effects of terconazole. C. albicans ATCC was grown for 8 h in EMEM without the addition of serum in the absence or presence of various concentrations of terconazole. The yeast were then transferred to EMEM medium, without terconazole, and incubated for an additional 24 h. The results of these experiments are shown in Fig. 4 in which the effects of terconazole yeast cell growth are observed through phase-contrast microscopy. In the control cultures, colonies of mainly long-radiating hyphae were observed. With a first incubation in medium containing 5 x 10-6 M terconazole, shortened germ tubes were present. When the concentration of terconazole was raised to 5 x FIG. 5. Scanning electron micrographs of C. albicans ATCC (A) exposed to various concentrations of terconazole for 24 h. Control culture; note predominance of mycelial form. (B) Terconazole (10-8 M); arrows point to swollen yeast with few mycelial elements remaining. (C) Terconazole (1l-7 M); arrows point to abnormal germ tube growth; no mycelial elements remains. (D) Terconazole (10-4 M); yeast cells exhibit irregular surface with few germ tubes remaining. Magnification, x2,500.

4 VOL. 29, 1986 ANTICANDIDAL EFFECTS OF TERCONAZOLE 989

5 990 TOLMAN ET AL. TABLE 2. Species Effect of terconazole on the viability of candidal species Terconazole Viable yeast as % concn (M) of control at 24 h C. albicans ATCC C. albicans ATCC C. albicans ATCC C. albicans ATCC C. albicans ATCC C. albicans ATCC C. albicans ATCC C. tiropicalis ATCC C. tropicalis ATCC C. krusei ATCC C. parapsilosis ATCC C. pseudotropicalis ATCC C. stellatoidea ATCC M, only small clusters of yeast form cells were seen, and with 10-4 M terconazole, only scarce clusters of budding cells were present. In an additional study (data not shown), when yeast were preincubated for 24 h in EMEM containing 10-4 M terconazole and then recultured in the same medium without terconazole for an additional 120 h, only a single intact yeast cell was noted. Somewhat less sensitivity was observed when the yeasts were grown in EMEM, to which fetal bovine serum was added, though it can be speculated that the serum affected the growth of the yeast and not the activity of terconazole per se. Scanning electron micrographs depicting the effects of terconazole on the morphology of C. albicans ATCC cultured on EMEM with added fetal bovine serum are shown in Fig. 5. Note the predominance of the mycelial form in the control culture (Fig. 5A). As terconazole was added to the medium at increasing concentrations, the number of mycelial elements was diminished and yeast cells began to swell (10-8 M terconazole; Fig. 5B); the formation of mycelia was inhibited completely and yeast cell and germ tube shape became abberant (10'- M terconazole; Fig. 5C); there was a further swelling of the yeast cells at i0-5 M terconazole; and at 10-4 M terconazole (Fig. 5D), no germ tubes were visible and yeast cell surfaces became irregular. When viewed through the transmission electron microscope (data not shown), terconazole at 10-8 M was seen to cause the appearance of osmophilic granules just with the cell wall. Necrotic changes, including loss of clearly defined cytoplasmic organelles, were seen following exposure to 106 M terconazole; and almost total necrosis, including a vacuolated appearance and highly irregular cell shapes, were observed with 10-4 M terconazole. DISCUSSION Terconazole is a new triazole ketal derivative with potent and broad-spectrum antifungal activities. Data presented in ANTIMICROB. AGENTS CHEMOTHER. this report and previously by Van Cutsem et al. (5) document that terconazole can inhibit the growth of a variety of yeasts and filamentous fungi that are pathogenic to humans. The broad spectrum of in vitro antifungal activity translates into in vivo effects in a number of animal models of fungal diseases, including rat vaginal candidosis and guinea pig dermatophytoses (5). Interpretation of in vitro results is made difficult for the azole class of antimycotic agents because of the dependency of their potencies on growth conditions and fungal species. We have demonstrated that though terconazole has a broad spectrum of antifungal activity, its precise in vitro potencies vary among different strains of C. albicans and among various Candidal spp. Furthermore, the potency of the anticandidal activity of terconazole is enhanced when the yeast cells are grown in media favoring mycelia formation. These results were documented by microscopic examination of the morphology of yeast grown in EMEM either with or without added serum, a medium which favors mycelial formation. At concentrations as low as 10-8 or 10-7 M, terconazole produced abnormal candidal cell growth, with the disappearance of the predominating (under control conditions) mycelial form and the appearance of yeast cells with abnormal external appearance. As the concentration of terconazole was increased, yeast cell necrosis occurred. These gross changes coincided with changes (e.g., swelling or lipid inclusions) in the morphology of intracellular organelles and the appearance of the cell wall. It has been demonstrated (6-10; Bossche and Cornelisson, data on file, Janssen Pharmaceutica) that antifungal agents of the terconazole type act by inhibiting the cytochrome P-450- dependent 14a-demethylase, resulting in an accumulation of membrane-disturbing 14a-demethylsterols and depletion of ergosterol. This selective inhibition was the result of the interaction of these azole derivatives with cytochrome P-450 in the yeast and fungal membranes. Alterations in the sterol composition results in membrane permeabilty changes, affecting membrane-bound enzymes and growth. A study of the effects of terconazole on the biochemistry of yeast cells is in progress, and the results will be reported in the near future. These data indicate the potential of terconazole as a therapeutically useful antimycotic agent. The successful clinical use of terconazole in both cream and suppository formulation, in the treatment of vulvovaginal candidosis, has been documented (data on file, Ortho Pharmaceutical Corp.). ACKNOWLEDGMENTS We express our appreciation to Barbara Foleno and Jamese Hilliard for assistance. We also thank Margaret Westlake for aid in the preparation of this manuscript. LITERATURE CITED 1. Aerts, F., M. De Brabander, H. Van den Bossche, J. Van Cutsem, and M. Borgers The activity of ketoconazole in mixed cultures of fungi and human fibroblasts. Mykosen 23: , Borgers, M., M. DeBrabender, H. Van den Bossche, and J. Van Cutsem Promotion of pseudomycelium formation of Candida albicans in culture: a morphological study of the effects of miconazole and ketoconazole. Postgrad. Med. J. 55: Borgers, M., and S. DeNollin The preservation of the subcellular organelles of C. albicans using conventional fixatives. J. Cell. Biol. 62:

6 VOL. 29, 1986 ANTICANDIDAL EFFECTS OF TERCONAZOLE Odds, F. C Laboratory evaluation of antifungal agents: a comparative study of five imidazole derivatives of clinical importance. J. Antimicrob. Chemother. 6: Van Cutsem, J., F. Van Gerven, R. Zaman, and P. A. J. Janssen Terconazole, a new broad spectrum antifungal. Chemother. 29: Van den Bossche, H Biochemical effects of miconazole on fungi. I. Effects on uptake and/or utilization of purines, pyrimidines, nucleosides, amino acids and glucose by Candida albicans. Biochem. Pharmacol. 23: Van den Bossche, H., J. M. Ruysschaert, F. DeFrise-Quertain, G. Willemsens, F. Cornelissen, P. Morchal, W. Cools, and J. Van Cutsem The interaction of miconazole and ketoconazole with lipids. Biochem. Pharmacol. 31: Van den Bossche, H., G. Willemsens, W. Cools, and F. Cornelissen Inhibition of ergosterol synthesis in Candida albicans by ketoconazole. Arch. Int. Physiol. Biochim. 87: Van den Bossche, H., G. Willemsens, W. Cools, W. F. J. Lauwers, and L. Lejuene Biochemical effects of miconazole on fungi; inhibition of ergosterol biosynthesis in Candida albicans. Chem. Biol. Interact. 21: Van den Bossche, H., G. Willemsens, and J. M. Van Cutsem The action of miconazole on the growth of Candida albicans. Sabouraudia 13:63-73.