Effect of Ketoconazole and Amphotericin B on Encapsulated and Non-Encapsulated Strains of Cryptococcus neoformans

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1 ANTIMICROBIAL AGNTS AND CHMOTHRAPY, Dec. 983, p. 8-8 Vol. 24, No. "484/83/28-$2./ Copyright C 983, American Society for Microbiology ffect of Ketoconazole and Amphotericin B on ncapsulated and Non-ncapsulated Strains of Cryptococcus neoformans DIANA SMITH, HARRY W. McFADDN, JR., AND NORMAN G. MILLR* Department of Medical Microbiology, University of Nebraska College of Medicine, Omaha, Nebraska 8 Received May 983/Accepted 23 September 983 Growth inhibition studies were done on an encapsulated and non-encapsulated strain of Cryptococcus neoformans at the minimal inhibitory concentration and one-half the minimal inhibitory concentration of ketoconazole and amphotericin B alone and in combination. Growth of both strains was significantly inhibited by ketoconazole, amphotericin B, and the combined drugs at the minimal inhibitory concentration of each drug over a -day period. Calculation of the expected inhibition of growth for both strains with both drugs showed antagonism at 24 h followed by an additive effect and synergy for the remaining 4 days of the assay. Although similar results were obtained for both strains with one-half the minimal inhibitory concentration, an additive effect was observed with the drug combination at 24 h for the encapsulated strain, and an antagonistic effect was observed with the non-encapsulated strain. Although amphotericin B has reduced the mortality from cryptococcal meningitis (), the necessity of its intravenous or intrathecal administration and its nephrotoxicity have prompted the search for an effective orally administered antifungal drug of low toxicity. Ketoconazole was recently introduced as an orally administered, broad-spectrum antifungal drug with relatively low toxicity (8, 4). High serum levels of ketoconazole are usually obtained, but concentrations in the cerebrospinal fluid are low (4, 8). In this study a comparison was made of the effects of ketoconazole and amphotericin B alone and combined on Cryptococcus neoformans over a -day period. All studies were done with the minimal inhibitory concentration (MIC) and one-half the MIC of ketoconazole and amphotericin B corresponding to attainable serum and cerebrospinal fluid concentrations of these drugs. An encapsulated and a non-encapsulated strain of C. neoformans were used to evaluate the effect of encapsulation on the susceptibility of C. neoformans to these drugs. MATRIALS AND MTHODS Test organisms. An encapsulated strain and a nonencapsulated strain of C. neoformans were used throughout this study. The encapsulated C. neoformans NU-2 strain was obtained from the cerebrospinal fluid of a patient with cryptococcal meningitis at the University of Nebraska Medical Center Hospital. The non-encapsulated C. neoformans M- strain was obtained from G. S. Bulmer, University of Oklahoma Health Sciences Center, Oklahoma City. Culture media. Sabouraud dextrose agar (Difco Laboratories, Detroit, Mich.) slants supplemented with %o thiamine hydrochloride were used to maintain cultures of C. neoformans NU-2 and M- strains. Freshly prepared yeast-nitrogen broth (Difco) supplemented with % dextrose and.% asparagine at ph was used for growth inhibition assays. The inoculum of C. neoformans for each assay was prepared in saline with.% Tween 8 to prevent aggregation, particularly of the M- strain. Samples taken for colony counts were also diluted in saline with.% Tween 8. Preliminary studies indicated that.% Tween 8 had no deleterious effect on the cell and did not interfere in the activity of the drugs either individually or in combination. Appropriate dilutions were plated on 3 ml of Sabouraud dextrose agar in -mm petri dishes. 8 Drugs used in growth inhibition assays. Ketoconazole obtained from Janssen Pharmaceutica Co., New Brunswick, N.J., was dissolved in.2 N HCI, diluted in sterile distilled water to Fjg/ml, and stored at 4 C. Additional dilutions were made in yeast-nitrogen broth containing.2 dimethyl sulfoxide on the days when growth inhibition assays were done. Amphotericin B (Fungizone;. R. Squibb and Sons, Princeton, N.J.) was obtained as a sterile lyophilized powder in vials containing mg of amphotericin B and 4 mg of sodium deoxycholate buffered with 2.2 mg of sodium phosphate. The powder was reconstituted and further diluted with sterile distilled water to a concentration of p.g/ml. Samples of this stock solution were stored at - C for no longer than weeks. On the day the assay was started, a sample of the stock solution was thawed and diluted appropriately in yeast-nitrogen broth. All solutions of amphotericin B were protected from the adverse effects of light. Growth inhibition assays. C. neoforman'v NU-2 or M- stock cultures were subcultured to 2 ml of yeastnitrogen broth in 2-ml rlenmeyer flasks and grown at 3 C for 24 h with constant shaking on a Junior Orbit Shaker (Labline Instruments, Inc., Melrose Park, Ill.). Downloaded from on September, 28 by guest

2 82 SMITH, McFADDN, AND MILLR After 24 h an inoculum was prepared in saline with.% Tween 8 by adjusting the culture to %,o Tween 8 at 2 nm with a Coleman Junior II model /2 spectrophotometer (Coleman Instruments, Maywood, Ill.). An inoculum of ml was added to 2. ml of broth with or without appropriate concentrations of amphotericin B or ketoconazole (or both). Three flasks were prepared for each drug or drug combination and the control. Preliminary studies showed that the concentration of sodium deoxycholate equivalent to that in amphotericin B and.2% dimethyl sulfoxide in yeast nitrogen broth used to maintain ketoconazole in solution had no inhibitory effect on either strain of C. neoformans. At the completion of the drug dilution (time zero) and at 24-h intervals over a -day period, ml of broth was removed from each flask and diluted : in saline with.% Tween 8 to enhance dispersal of the yeast cells. Samples (,ul) of this and further dilutions were plated on Sabouraud dextrose agar with a calibrated loop. Two plates were prepared from each dilution and incubated at 3 C for 2 h. Colony counts were averaged, and the CFU per milliliter for each flask were determined at each time interval. All flasks were incubated at 3 C in the dark with constant shaking at x g. All assays were repeated twice. Basic definitions. The MIC was defined as the lowest concentration that prevented an increase in the number of CFU per milliliter during the time the drug remained active. In this study, this was reflected in colony counts that did not statistically deviate from the original colony count at time zero. The activity of combined antibiotic action was interpreted as described by Jawetz (9). An additive effect was defined as a combined drug effect that equalled the arthimetic sum of the effects produced by each drug alone. In cases where one drug was active and the other drug was not active, the effect was referred to as indifference. Synergy was defined as a combined drug effect that was greater than the arithmetic sum of the effects produced by each drug alone. The combined drug effect that was less than the sum of the effect of each individual drug was considered to be antagonism. Statistical analysis. The mean CFU of C. neoformans NU-2 and M- strains per milliliter was calculated for each of the various drug solutions in growth inhibition assays and plotted against time of incubation in days. The statistical significance of growth inhibition when compared with the drug-free control was determined by Student's t test. This was calculated for each day of the assay. A P value of less than. was considered to be significant. A variation of Student's t test as described by Armitage () was used to determine whether the various drug combinations were additive, indifferent, synergistic, or antagonistic on days when combinations varied significantly from drug-free controls. The expected growth inhibition of the two combined drugs was calculated by subtracting the mean CFU per milliliter of the control from the sum of the two individual drugs. This expected value was compared with the mean CFU per milliliter of the experimental drug combination (drug and 2) plus or minus t times the standard error (S) of this combination, where t is the tabulated value of significance for P =., as follows: (drug ) + (drug 2) - control = (drug and 2) + (t x S). When the expected value (left side of ANTIMICROB. AGNTS CHMOTHR. equation) equalled or fell within the combined experimental range (right side of equation), the combination was considered additive or indifferent. An expected value greater than the experimental range indicated synergy, whereas an expected value less than this range indicated antagonism. RSULTS The MIC of ketoconazole was,ug/ml for both C. neoformans NU-2 and M- strains. It was reached in 24 h and was maintained throughout the -day test period (Fig. and 2). The MIC of amphotericin B was. p.g/ml for the NU-2 strain and. pug/ml for the nonencapsulated M- strain of C. neoformans. Significant growth inhibition was observed at 24 h, but not at 48 h, in both strains (Fig. and 2). All drug combination studies were done with either the MIC or one-half the MIC of ketoconazole or amphotericin B. Ketoconazole in combination with amophotericin B at the MIC of each of these drugs significantly (P <.) inhibited the growth of C. neoformans NU-2 over all days of the assay - z c -l U- U I _-. yg/ml ASS *-. jig/ml ASS &. pg/ml KTZ --. ag/mi KTZ -- control Time (days) FIG.. Growth inhibition of C. neoformans NU-2 at the MIC ( pg/ml) of ketoconazole (KTZ) in combination with the MIC (.,ug/ml) of amphotericin B (AMB) over a -day period. Values marked with an asterisk are statistically significant when compared with the control (P <.). The combined drug effect, calculated as described in the text, was as follows: day, antagonistic; day 2, additive; days 3 through, synergistic. Downloaded from on September, 28 by guest

3 VOL. 24, N U l S I - II U J Time KTOCONAZOL, AMPHOTRICIN B ON C. NOFORMANS 83 (days) FIG. 2. Growth inhibition of C. neoformans M- at the MIC (,ug/ml) of ketoconazole (KTZ) in combination with the MIC (.,ug/ml) of amphotericin B (AMB) over a -day period. Values marked with an asterisk are statistically significant when compared with the control (P <.). The combined drug effect, calculated as described in the text, was as follows: day, antagonistic; days 2 through, synergistic. (Fig. ). Although the inhibitory effect of the combined drugs appeared greater than that obtained with each drug alone, when the expected inhibition of growth was calculated as described above, the combined drug effect was antagonistic on day, additive on day 2, and synergistic on days 3 through. Studies with the M- non-encapsulated strain at the MIC of both ketoconazole and amphotericin B produced results similar to those obtained with the NU-2 strain (Fig. 2). The inhibition of growth with the combination of drugs was also greater than that with each drug alone, but was more pronounced on days 3, 4, and than in the case of the encapsulated NU-2 strain. As with the encapsulated strain, the calculated combined drug effect was antagonistic on day, but synergistic on days 2 through. At one-half the MIC, amphotericin B showed no significant growth inhibition of the encapsulated NU-2 strain, whereas ketoconazole showed a minimal, but significant (P <.), growth inhibition of this strain (Fig. 3). The combination at one-half the MIC of each drug had the greatest effect on the encapsulated NU-2 strain (Fig. 3). This was determined as being additive at all 4 days when there was a significant difference (P <., Fig. 3) in growth inhibition. ssentially the same pattern of growth inhibition was seen with the non-encapsulated M- strain as with encapsulated NU-2 strain when one-half the MIC of ketoconazole and amphotericin B was used alone and in combination. However, growth inhibition of the non-encapsulated M- strain was greater and lasted longer than in the case of the encapsulated NU-2 strain (Fig. 3 and 4). Another difference between the two strains was the antagonism with the combined drugs at 24 h followed by synergy with the non-encapsulated M- strain throughout day (Fig. 4). DISCUSSION In general, the patterns of growth inhibition of the encapsulated and non-encapsulated strain of o I C c A IL %O. pg/mi AMB ---. pg/ml KTZ -*-. Mg/ml AMS & -v.- control. pg/ml KTZ TIm* (days) FIG. 3. Growth inhibition of C. neoformans NU-2 at one-half the MIC (.,ug/ml) of ketoconazole (KTZ) in combination with one-half the MIC (. plg/ml) of amphotericin B (AMB) over a -day period. Values marked with an asterisk are statistically significant when compared with the control (P <.). The combined drug effect, calculated as described in the text, was as follows: days through 4, additive; not done on day. Downloaded from on September, 28 by guest

4 84 SMITH, McFADDN, AND MILLR a z P.f i c o J A --*-.2 pg/n -.-. pg/ml pg/n...o- 2 3 control A'NTIMICROB. AGNTS CHMOTHR. compared with the untreated control over the -... _ day assay period, determination of the expected ;sa3 value of combined drug activity showed antagonism at 24 h that was followed by an additive effect or synergy (Fig. and 2). A similar effect was recently reported by Brajtburg et al. (3), who showed antagonism with amphotericin B and miconazole or ketoconazole against Candida albicans after a short incubation period followed by "potentiation" (a term used by these * authors to include either an additive effect or synergy) at 3 to days. Antagonism was also reported by Cosgrove et al. (), who exposed various yeasts to combinations of amphotericin B and clotrimazole or miconazole for min. ni AMS Beggs et al. (2) also found that clotrimazole KTZ antagonized the fungicidal activity of amphoterinl AMS & cin B with respect to several yeasts within the,g/ml KTZ first 24 h. This was not seen when amphotericin B was used alone. Regardless of the assay system used, there is general agreement that antagonism occurs between amphotericin B and,the imidazoles in their early interaction with 4 yeasts, which then proceeds to an additive or even synergistic effect after 24 h. The mecha- Time (days) nism of antagonism between amphotericin B and FIG. 4. Growth inhibition of C. neoformtans M- at ketoconazole may be similar to that advocated one-half the MIC (.,ug/ml) of ketoconazoole (KTZ) in by Cosgrove et al. () based on adsorption combination with one-half the MIC (.2',ug/ml) of studies with amphotericin B and clotrimazole in amphotericin B (AMB) over a -day periiod. Values combination on cell membranes of Saccharomysignificant ces cerevisiae. Their studies showed that there marked with an asterisk are statistically when compared with the control (P <.). The is competition for the same binding sites on the combined drug effect, calculated as described in the cell membrane by the two drugs. text, was as follows: day, antagonist ic; days 2 Although not as pronounced, the combined through, synergistic. effect of amphotericin B and ketoconazole at one-half the MIC of each drug was the same for the non-encapsulated M- strain as when one C. neoformans to the MIC of amphotericin B MIC was used in combination (Fig. 2 and 4). The and ketoconazole over the -day period were additive effect seen with the encapsulated NU-2 similar in our assay system. A noticeaible differ- strain at 24 h (Fig. 3) is difficult to explain, but ence in the reaction of both strains to almphoteri- may reflect a variation in the rate of penetration cin B and ketoconazole was the exten( ded activi- of the capsule by the two drugs not detectable ty of ketoconazole over the entire -clay period when the MIC of each drug was used in combiof the assay, whereas the activity of almphoteri- nation. Graybill et al. () also reported an addicin B on the cells was limited to 24 h. This most tive effect in 8 h by amphotericin B and ketolikely reflected the differences betwee.n the two conazole in combination on a strain of C. drugs with respect to their mode of acttion on the neoformans pathogenic for mice at concentra- binding tions approximating one-half the MIC used in fungal cell. Amphotericin B acts boy directly to ergosterol within the cell nnembrane, our study. resulting in leakage of vital protoplasmlic constit- Of particular interest was the synergistic or uents of the cell (). Since the action oif ketocon- additive effect seen after 24 h with the MIC or azole on the cell depends upon the in] hibition of one-half the MIC of ketoconazole and amphoter- icin B with either the encapsulated NU-2 strain ergosterol synthesis to effect cell permeability (), it is possible that the detrimental effects on or the non-encapsulated M- strain. Since ampared with photericin B was no longer active after 24 h, its the cell would be delayed when com; the effects of amphotericin B. Althouigh inhibi- most likely role in inhibiting growth after 24 h tion of growth of both the encapsulate( d and non- would be to enhance penetration of ketocona- by keto- zole into the cell by increasing cell membrane encapsulated strains of C. neoformanis conazole plus amphotericin B at the M[IC of each permeability as described for antifungal action was statistically significant (P < O.) when by combinations of amphotericin B and the Downloaded from on September, 28 by guest

5 VOL. 24, 983 tetracyclines or rifampin (, 2). This would permit ketoconazole to enter the cell more quickly and in larger quantities than if ketoconazole were used alone. Once inside the cell, ketoconazole at the low concentrations used in this study may have interfered with the demethylation of lanosterol to ergosterol, which is necessary to maintain the integrity of the cell membrane (). It is encouraging that in vivo studies with ketoconazole plus amphotericin B for the treatment of disseminated cryptococcosis in mice and cryptococcal meningitis in rabbits for 4 days have shown that this combination is slightly more effective than amphotericin B alone in the treatment of mice, and at least as effective as amphotericin B and -fluorocytosine in the treatment of rabbits (, 3). Our in vitro studies suggest that the treatment of experimental cryptococcosis may be more effective with these two drugs if amphotericin B were given initially followed by the continuous treatment of ketoconazole and amphotericin B only at regular weekly intervals for a prolonged (longer than 4 days) period of time. ACKNOWLDGMNTS We thank Kashinath D. Patil for the statistical consultation and Lisa Karnish for the preparation of the manuscript. LITRATU CITD. Armultage, P. 9. Statistical inference, p. 4-. In Statistical methods in medical research. Blackwell Scientific, Oxford. 2. Beggs, W. H., G. A. Swos, and N. M. Steele. 9. Inhibition of potentially pathogenic yeast-like fungi by clotrimazole in combination with -fluorocytosine or amphotericin B. Antimicrob. Agents Chemother. 9: Brajtburg, J., D. Kobayshl, G. Medo", ad G. S. Kobayasl Antifungal action of amphotericin B in KTOCONAZOL, AMPHOTRICIN B ON C. NOFORMANS 8 combination with other polyene or imidazole antibiotics. J. Infect. Dis. 4: Bras, C., J. N. Gagigani, T. F. Blashe, R. Defelice, R. A. O'Rellly, and D. A. Stevens Disposition of ketoconazole, an oral antifungal in humans. Antimicrob. Agents Chemother. 2: Cogove, R. F., A.. Beezer, And R. J. Miles. 98. In vitro studies of amphotericin B in combination with the imidazole antifungal compounds clotrimazole and miconazole. J. Infect. Dis. 38:8-8.. Graybi, J. R., D. M. WUIams,. Va Cutsem, and D. J. Drutz. 98. Combination therapy of experimental histoplasmosis and cryptococcosis with amphotericin B and ketoconazole. Rev. Infect. Dis. 2:-8.. Hamilton-Miler, J. M. T. 93. Chemistry and biology of the polyene macrolide antibiotics. Bacteriol. Rev. 3: Heel, R. C Toxicology and safety studies, p. 4-. In H. B. Levine (ed.), Ketoconazole in the management of fungal disease ADIS Press, New York. 9. Jawetz,. 98. Combined antibiotic action: some definitions and correlations between laboratory and clinical results, p Antimicrob. Agents Chemother. 9.. Kwan, C. N., G. Medoff, G. S. Kobayasb, D. Scleser, and H. J. Raska. 92. Potentiation of the antifungal effects of antibiotics by amphotericin B. Antimicrob. Agents Chemother. 2:4.. Llttman, M. L., and J.. Wakter. 98. Cryptococcosis: current issues. Am. J. Med. 4: Medoff, G., G. S. Kobabyah, C. N. Kwan, D. Schlessinger, and P. Venkov. 92. Potentiation of rifampicin and -fluorocytosine as antifungal antibiotics by amphotericin B. Proc. NatI. Acad. Sci. U.S.A. W Perfect, J. R., and D. T. Durack Treatment of experimental meningitis with amphotericin B, -fluorocytosine and ketoconazole. J. Infect. Dis. 4: Thlepont, D., J. Van Ctem, F. Van Gerven, J. Here, and P. A. J. Ja_nae. 99. Ketoconazole, a broad spectrum orally active antimycotic. xperientia 3:. S. Va den Bossche, H. G. We W. Coo, and F. Cornellssen. 99. Inhibition of ergosterol synthesis in Candida albicans by ketoconazole. Arch. Int. Physiol. Biochem. 8: Van den BDouhe, H. G. Wileues, W. Cools, F. Cornelisse-, W. F. J. Lauwers, and J. M. Van Cutsem. 98. In vitro and in vivo effects of the antimycotic drug ketoconazole on sterol synthesis. Antimicrob. Agents Chemother. : Downloaded from on September, 28 by guest