Comparison of the efficacies of amphotericin B, fluconazole, and ...

AND CHEMOTHERAPY, Feb. 1989, 0066-4804/89/020147-05$02.00/0 Copyright © 1989, American Society for Microbiology

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Comparison of the Efficacies of Amphotericin B, Fluconazole, and Itraconazole against a Systemic Candida albicans Infection in Normal and Neutropenic Mice JAN W. VAN 'T WOUT, HERMAN MATTIE, AND RALPH VAN FURTH* Department of Infectious Diseases, University Hospital, 2300 RC Leiden, The Netherlands Received 20 June 1988/Accepted 1 November 1988

We compared the efficacies of the new triazole antifungal drugs fluconazole and itraconazole with that of amphotericin B in vitro and in an animal model of systemic candidiasis in normal and neutropenic mice. Antifungal treatment with fluconazole (2.5 to 20 mg/kg orally twice daily), itraconazole (10 to 40 mg/kg orally twice daily), or amphotericin B (0.1 to 4 mg/kg intraperitoneally once daily) was started 1 day after intravenous injection of 104 Candida albicans into normal mice or 103 C. albicans into neutropenic mice; the drugs were administered for 2 days. In normal mice the efficacy of treatment, which was assessed on the basis of the number of C. albicans cultured from the kidney, was greater for amphotericin B than for the triazoles. Fluconazole was more potent than itraconazole on the basis of equivalent doses, although itraconazole was more potent on the basis of the amount of free drug that was available. In neutropenic mice amphotericin B was less effective than it was in normal mice, whereas the triazoles were equally effective in normal and neutropenic mice. This was not expected, since in vitro data showed that amphotericin B was highly fungicidal, whereas both fluconazole and itraconazole had only a minimal effect on the growth of C. albicans in vitro.

yeasts was counted in a hemacytometer, the suspension was diluted to the appropriate concentration and plated onto Sabouraud dextrose agar. Viability counts were made after overnight incubation at 37°C. Antifungal drugs. Amphotericin B (Fungizone) was purchased from E. R. Squibb & Sons (Rijswijk, The Netherlands). A standard solution of the drug was prepared by dissolving 50 mg of amphotericin B with 41 mg of sodium desoxycholate in 10 ml of distilled water. Further dilutions were made with distilled water. A fresh standard solution was prepared weekly. Standard solutions of 5 mg of fluconazole (kindly donated by Pfizer Central Research, Sandwich, United Kingdom) per ml and 5 mg of itraconazole (kindly donated by Janssen Pharmaceutica B. V., Beerse, Belgium) per ml were prepared daily. Fluconazole was dissolved and diluted in distilled water for all experiments. For in vitro experiments itraconazole was dissolved in dimethyl sulfoxide and immediately diluted with distilled water. For in vivo experiments the drug was dissolved in and further diluted with polyethylene glycol 200. Effect of the antifungal drugs on the growth of C. albicans in vitro. C. albicans (final concentration, 5 x 104/ml) and the antifungal drug were added to 20 ml of modified Eagle medium supplemented with 10% newborn calf serum (GIBCO) and buffered to pH 7.3 with sodium hydrogen carbonate. The controls contained only the solvent of the drug in appropriate dilutions. The suspension was incubated under 5% CO2 at 37°C for 24 h. At various times samples were taken and 10-fold dilutions were plated onto Sabouraud dextrose agar. Dimethyl sulfoxide had no effect on the growth of C. albicans at the concentrations that were used (up to 0.25%). The MICs of the various antifungal drugs were determined in a twofold dilution series by using the same medium, culture conditions, and inoculum as were used for the growth curves. The MIC was defined as the lowest concentration that prevented visible growth after 24 h of incubation. Experimental infection in mice. Neutropenia was estab-

The treatment of systemic candidal infections in neutropenic patients continues to be a major problem. According to a recent review, only 20% of patients survive despite treatment with amphotericin B (7). Although experimental and clinical data suggest that the combination of amphotericin B and flucytosine may be synergistic in candidal infections (7, 16), this combination may also be hazardous, since nephrotoxicity induced by amphotericin B can lead to impaired renal clearance of flucytosine and, subsequently, to toxic levels of this drug in plasma, unless the dose is adjusted. Moreover, fear of toxicity may prevent physicians from instituting empiric treatment with these drugs, which is often necessary because of problems in diagnosing systemic candidiasis. Therefore, there is a need for more effective and less toxic drugs to treat patients with this infection. The most important antifungal agents recently developed are the triazole drugs fluconazole and itraconazole (3, 8, 14, 15, 17-19, 22, 23). The efficacies of these drugs against disseminated candidiasis in neutropenic patients still must be established. In order to find support for the clinical application of fluconazole and itraconazole in neutropenic patients, we compared the efficacies of these drugs with that of amphotericin B in a model of systemic candidiasis in normal and neutropenic mice.

MATERIALS AND METHODS Animals. Specific-pathogen-free male Swiss mice (weight, 25 to 35 g; Broekman Institute, Someren, The Netherlands) were used. Culturing of C. albicans. C. albicans UC820 was maintained on agar slants at 4°C, inoculated into 100 ml of Sabouraud dextrose broth (Difco Laboratories, Detroit, Mich.), and cultured for 24 h at 37°C. The suspension was centrifuged, washed twice, and suspended in phosphatebuffered saline or modified Eagle medium (GIBCO Laboratories, Paisley, Scotland), as indicated. After the number of *

Corresponding author.

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lished by subcutaneous administration of 150 mg of cyclophosphamide per kg 4 days before and 100 mg/kg 1 day before the injection of C. albicans. Normal mice received 104 C. albicans and neutropenic mice received 103 C. albicans in 0.2 ml of phosphate-buffered saline injected into the lateral tail vein. This produced a nonlethal infection with the kidney as the main target organ (J. W. van 't Wout, I. Linde, P. C. J. Leijh, and R. van Furth, submitted for publication). Treatment with antifungal drugs was started 24 h after injection of C. albicans. Amphotericin B was injected intraperitoneally once daily; fluconazole or itraconazole was given orally twice daily via a mouse feeding tube. On day 3 of infection, after 2 days of treatment, the animals were sacrificed; and the left kidney was removed aseptically, weighed, and homogenized. The number of viable C. albicans cells in the kidney was determined by plating serial 10-fold dilutions of the suspension in duplicate onto Sabouraud dextrose agar. After overnight incubation at 37°C the colonies were counted. Preliminary experiments indicated that there was no difference in the number of C. albicans cultured from the left or the right kidney (data not shown). Pharmacokinetics. Normal mice were given 20 mg of fluconazole or itraconazole per kg orally; blood was drawn by cardiac puncture at 30 min and 1, 2, 4, 6, 8, 10, and 12 h later and collected in an heparinized tube. The blood samples of two to three mice were combined. After centrifugation for 10 min at 1,500 x g, the antifungal concentration in plasma was determined by bioassay. Antifungal assay. Concentrations of fluconazole and itraconazole were determined by an agar diffusion method (13) on a medium consisting of amino acids and yeast carbon base in buffered agar with Candida pseudotropicalis Carlshalton as the test organism. The medium was made according to the reference manual for investigators (Pfizer Central Research, 1986; a complete medium is now available from P. F. Troke, Pfizer Central Research, Sandwich, Kent CT13 9NJ, United Kingdom). Standards were prepared in pooled murine plasma in concentrations ranging from 0.2 to 6.3 ,ig/ml. Statistical analysis. The effects of the various antifungal drugs in normal and neutropenic mice were analyzed by multiple regression analysis and by the standard parallel line bioassay procedure (1). RESULTS In vitro studies. The effect of various concentrations of the antifungal drugs on the growth of C. albicans in vitro is

represents the mean of three experiments. (a)

shown in Fig. 1. Amphotericin B was fungicidal at a concentration of 1 p,g/ml (Fig. la); fluconazole (Fig. lb) and itraconazole (Fig. lc) had only a minimal effect on the growth of C. albicans in vitro even at concentrations of up to 100 ,ugIml. The same pattern was seen when other media (Sabouraud dextrose broth, yeast nitrogen base) were used (data not shown). The MICs in modified Eagle medium were 0.05 ,g/ml for amphotericin B, 0.8 ,ug/ml for fluconazole, and 0.01 ,ug/ml for itraconazole. In vivo efficacy in normal and neutropenic mice. After intravenous injection of 1 x 104 C. albicans into normal mice on day 0, the number of CFU per kidny, expressed as the geometric mean, rose from 1.2 x 103 on day 1 to 3.3 x 104 on day 3 in untreated controls (Fig. 2a to c). For neutropenic

mice a lower infecting dose was chosen because about 20% of the animals died within 3 days of an injection of 104 C. albicans. After intravenous injection of 1 x 103 C. albicans on day 0 no deaths occurred, and the mean number of CFU per kidney rose from 1.2 x 104 on day 1 to 7.4 x 105 on day 3 of infection in untreated controls (Fig. 2d to f). In normal mice, a dose of 1 mg of amphotericin B per kg decreased the mean number of CFU per kidney from days 1 to 3 (Fig. 2a). The efficacy of fluconazole (Fig. 2b) and itraconazole (Fig. 2c) in normal mice was less than that of amphotericin B. Although both triazoles had a clear dosedependent effect, there was no decrease in the number of CFU per kidney from days 1 to 3 of infection, despite the high doses (up to 20 mg of fluconazole per kg twice daily and up to 40 mg of itraconazole per kg twice daily) that were administered. In neutropenic mice neither amphotericin B (Fig. 2d) nor fluconazole (Fig. 2e) or itraconazole (Fig. 2f) caused a decrease in the number of CFU per kidney from days 1 to 3 of infection. The maximum effect that could be achieved seemed greater for the triazoles than for amphotericin B, although the differences were not statistically significant. The efficacy of amphotericin B could not be improved by increasing the daily dose to 4 mg/kg (data not shown). There was no significant difference between the slopes of the regression lines of the dose-response relationship for fluconazole and itraconazole for either normal or neutropenic mice (Fig. 3). Fluconazole was significantly more effective than itraconazole in both normal and neutropenic mice (P < 0.01). The horizontal distance between the lines represents the relative potency of these drugs, which was calculated by a parallel bioassay procedure. This revealed that fluconazole was 5.9 times more potent than itraconazole. Because the number of CFU per kidney before anti-

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FIG. 2. Effect of amphotericin B (O, *), fluconazole (0, 0), and itraconazole (A,*) on the number of C. albicans in the kidneys of normal mice (open symbols) (a to c) and neutropenic mice (closed symbols) (d to f). Treatment with antifungal drugs was initiated 24 h after intravenous injection of 104 C. albicans into normal mice and 103 C. albicans into neutropenic mice and lasted for 2 days. Amphotericin B was given intraperitoneally once daily; fluconazole and itraconazole were given orally twice daily. Since all drugs were compared in parallel experiments, the controls depicted in panels a to c and those depicted in panels d to f are the same. Each symbol represents the mean value for six mice.

fungal treatment was 10 times higher in neutrolpenic than in normal mice, we used the difference between th e numbers of CFU per kidney on days 1 and 3 of infection to calculate the relative potencies of the antifungal drugs in normal and neutropenic mice. This revealed no statistical ly significant

difference for the triazoles between normal and neutropenic mice (P > 0.60). For amphotericin B the relative potency in normal and neutropenic mice could not be calculated be-

cause there was a difference in the slopes of the doseresponse curves (0.05 < P < 0.1) (Fig. 3). However, a dose of 1 mg of amphotericin B per kg was significantly more effective in normal than in neutropenic mice (P < 0.01). In another series of experiments, the duration of treatment was 106 prolonged to 7 days in normal mice in order to obtain a microbiological cure. Eradication of the infection from the 105 was achieved in about half of the mice that were 1 - 4kidney C' with amphotericin B, 3 of the 18 mice that were treated a 104 treated with fluconazole, and none of the 18 mice that were treated with itraconazole (Table 1). LL. 103. Pharmacokinetic studies. Fluconazole was rapidly abL sorbed after oral administration, and the calculated apparent 02. elimination half-life was 3.3 h (Fig. 4a). The increase in itraconazole concentrations was much slower, since the maximum level in serum was only reached after 4 h (Fig. 4b). 10 0.1 1 2.5 10 20 40 0.5 Because absorption and elimination occur simultaneously and since the drug cannot be administered intravenously, it Dose (mg/kg) was impossible to calculate the true elimination half-life for FIG. 3. Effect of amphotericin B (l, U), fluconaz(ole (0, 0), and itraconazole. The area under the concentration-time curve itraconazole (A, A) on the number of C. albicans in the kidneys of (from 0 to 12 h) was 87.7 mg. h/liter for fluconazole and 22.8 normal (open symbols) and neutropenic (closed s iymbols) mice. mg h/liter for itraconazole. Since protein binding in serum Amphotericin B was given intraperitoneally once daiily; fluconazole has been shown to be 11% for fluconazole and 99% for and itraconazole were given orally twice daily. Each symbol represents the mean and + standard error of the mean fobr six mice. itraconazole (6, 10), the area under the concentration-time

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TABLE 1. Effect of 7 days of treatment of a systemic candidal infection in normal mice with amphotericin B, fluconazole, or itraconazolea No. of animals

No. of mice with infection

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Treatment (dose [mg/kg])

Fluconazole 10 20 40

Itraconazole 10 20 40

a Treatment was started 24 h after intravenous injection of 104 C. albicans. Amphotericin B was given intraperitoneally once daily; fluconazole and itraconazole were given orally twice daily.

curve for the free drug amounted to 78.1 mg h/liter for fluconazole and 0.23 mg h/liter for itraconazole.

DISCUSSION A comparison of the efficacies of amphotericin B, fluconazole, and itraconazole in an animal model of systemic candidiasis revealed that in normal mice the maximum effect that can be achieved with amphotericin B is greater than that which can be obtained with the triazoles. However, in neutropenic mice the efficacy of amphotericin B was greatly diminished, despite the use of very high doses (up to 4 mg/kg). Because of its toxicity, higher doses of amphotericin B could not be administered. The relative inefficacy of amphotericin B during neutropenia in both experimental animals and patients (11, 12, 21) is disappointing, in view of the highly fungicidal activity of the drug in vitro. This suggests that at least part of the antifungal effect of amphotericin B in vivo must be explained by a synergism between the drug and cellular host defense mechanisms. In the present study this hypothesis was supported by the divergence of the regression lines of the dose-response curves for amphotericin B in normal and neutropenic mice. One would expect these regression lines to be parallel if the effects of amphotericin B and cellular host defense mechanisms on

systemic candidiasis are additive. Bistoni et al. (2) have provided evidence that amphotericin B stimulates the activity of macrophages against not only C. albicans but also Staphylococcus aureus, a microorganism that is not susceptible to amphotericin B. Stimulation of macrophages by amphotericin B may have played a role in our experimental model, although we have already shown in an earlier study that mononuclear phagocytes do not contribute to host defenses against disseminated candidiasis in mice in the acute phase of infection (van 't Wout et al., submitted). The slopes of the dose-response curves for normal and neutropenic mice treated with fluconazole or itraconazole were not significantly different. After correction for the higher number of C. albicans in the kidneys of neutropenic mice before treatment, both fluconazole and itraconazole were equally effective in normal and neutropenic mice. Thus, we found no evidence for synergistic activity between the triazoles and cellular host defense mechanisms against systemic candidiasis. Fluconazole proved to be more potent than itraconazole. However, since the kidney was the main target organ in our model, the renal excretion of fluconazole may have favored the efficacy of this drug compared with those of amphotericin B and itraconazole. Our data on the in vivo efficacies of fluconazole and itraconazole are in agreement with the limited data available on the efficacy of the older azole drug ketoconazole in neutropenic animals with C. albicans infections (9, 25). Because of the discrepancy between the efficacy of the triazoles in vivo and their limited activity in vitro found in our studies, the possibility of an antagonizing effect of the culture medium must be considered. However, this seems unlikely since the same medium proved to be particularly useful for comparison of the relative activities of various azoles in vitro (14). Moreover, when we performed MIC determinations in the same medium, we found values comparable to those found by others in other media (3, 13, 18). Lastly, if there were antagonizing substances in the medium, one should expect that the effects of these substances could be overcome by raising the concentration of the drug. This was not the case. Because of this lack of correlation between in vitro activity and in vivo efficacy for the azoles (20), studies must be carried out in animals to determine which drugs should undergo clinical evaluation and to predict the concentrations at which these drugs are effective. In our study higher doses of fluconazole and itraconazole than have been used in animal experiments performed by others (17, 18, 22, 23) were needed to obtain an antifungal effect in vivo. This can probably be attributed to differences in the cellular host defense of the animals, the time elapsed between infection and treatment, and the method by which the antifungal effect was measured. To address these three issues we carried out

study as follows. First, to simulate more closely the clinical situation in which systemic candidal infections occur in humans, we used not only normal mice but also mice that were severely neutropenic throughout the course of the infection. Second, in our study the interval between the injection of C. albicans and treatment was long enough for the infection to become established. In other studies antifungal treatment was started simultaneously with or shortly after the administration of C. albicans (17, 23). Third, our assessment of the antifungal effect was based on quantification of the microbiological response rather than survival, as in other studies (18, 22). Clinical studies of the effect of new antifungal drugs in neutropenic patients are very difficult to perform because of

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the complicated conditions under which these infections occur and because most clinicians are reluctant to use antifungal drugs other than amphotericin B in such cases. However, in a recent comparative clinical trial, ketoconazole and amphotericin B were found to be equally effective in neutropenic patients with suspected or proven fungal infections, although the overall efficacy of both drugs against proven infections was low (4). The new triazoles seem to have important advantages over ketoconazole; itraconazole has an advantage because it is active against both Candida spp. and Aspergillus spp., and fluconazole has an advantage because it can be administered both intravenously and orally. The efficacies of itraconazole and fluconazole against deep-seated fungal infections in nonneutropenic patients have already been established (5, 19, 24). The results of the present study justify clinical trials to evaluate the efficacies of these two drugs against systemic candidal infections in neutropenic patients.

10. Humphrey, M. J., S. Jevons, and M. H. Tarbit. 1985. Pharmacokinetic evaluation of UK-49,858, a metabolically stable triazole antifungal drug, in animals and humans. Antimicrob.

ACKNOWLEDGMENTS This study was supported by a grant from Pfizer Central Research, Sandwich, Kent, United Kingdom. The technical assistance of Ineke Linde is greatly appreciated.

15. Perfect, J. R., D. V. Savani, and D. T. Durack. 1986. Comparison of itraconazole and fluconazole in treatment of cryptococcal meningitis and candida pyelonephritis in rabbits. Antimicrob. Agents Chemother. 29:579-583. 16. Rabinovitch, S., B. D. Shaw, T. Bryant, and S. T. Donta. 1974. Effect of 5-fluorocytosine and amphotericin B on Candida albicans infection in mice. J. Infect. Dis. 130:28-31. 17. Richardson, K., K. W. Brammer, M. S. Marriot, and P. F. Troke. 1985. Activity of UK-49,858, a bis-triazole derivative against experimental infections with Candida albicans and Trichophyton mentagrophytes. Antimicrob. Agents Chemother. 27:832-835. 18. Rogers, T. E., and J. N. Galgiani. 1986. Activity of fluconazole (UK 49,858) and ketoconazole against Candida albicans in vitro and in vivo. Antimicrob. Agents Chemother. 30:418-422. 19. Saag, M. S., and W. E. Dismukes. 1988. Azole antifungal agents: emphasis on new triazoles. Antimicrob. Agents Chemother. 32:1-8. 20. Speller, D. C. E., and D. W. Warnock. 1986. Sensitivity and resistance to antifungals. J. Antimicrob. Chemother. 17:514516. 21. Tremblay, C., M. Barza, C. Fiore, and F. Szoka. 1984. Efficacy of liposome-intercalated amphotericin B in the treatment of systemic candidiasis in mice. Antimicrob. Agents Chemother.

Agents Chemother. 28:648-653. 11. Lopez-Berestein, G., V. Fainstein, R. Hopfer, K. Metha, M. P. Sullivan, M. Keating, M. G. Rosenblum, R. Metha, M. Luna, E. M. Hersh, J. Reuben, R. L. Juliano, and G. P. Bodey. 1985. Liposomal amphotericin B for the treatment of systemic fungal infections in patients with cancer: a preliminary study. J. Infect. Dis. 151:704-710. 12. Lopez-Berestein, G., R. L. Hopfer, R. Metha, K. Metha, E. M. Hersh, and R. L. Juliano. 1984. Liposome-encapsulated amphotericin B for treatment of disseminated candidiasis in neutropenic mice. J. Infect. Dis. 150:278-283. 13. Mattie, H. 1978. Antimicrobial drugs, p. 107-115. In F. A. De Wolff, H. Mattie, and D. D. Breimer (ed.), Therapeutic relevance of drug assays. Leiden University Press, Leiden, The

Netherlands. 14. Odds, F. C., S. L. Cheesman, and A. B. Abbott. 1986. Antifungal effects of fluconazole (UK 49858), a new triazole antifungal, in

vitro. J. Antimicrob. Chemother. 18:473-478.

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crob. Agents Chemother. 27:625-631. 3. Espinel-Ingroff, A., S. Shadomy, and R. J. Gebhart. 1984. In vitro studies with R 51,211 (itraconazole). Antimicrob. Agents Chemother. 26:5-9. 4. Fainstein, V., G. P. Bodey, L. Elting, A. Maksymiuk, M. Keating, and K. B. McCredie. 1987. Amphotericin B or ketoconazole therapy of fungal infections in neutropenic cancer patients. Antimicrob. Agents Chemother. 31:11-15. 5. Ganer, A., E. Arathoon, and D. A. Stevens. 1987. Initial experience in therapy for progressive mycoses with itraconazole, the first clinically studied triazole. Rev. Infect. Dis. 9:S77-S86. 6. Heykants, J., M. Michiels, W. Meuldermans, J. Monbaliu, K. Lavrjsen, A. vah Peer, J. C. Levron, R. Woestenborghs, and G. Cauwenbergh. 1987. The pharmacokinetics of itraconazole in animals and man: an overview, p. 223-249. In R. A. Fromtling (ed.), Evaluation of antifungal agents. JR Prous Science Publishers, Barcelona, Spain. 7. Horn, R., B. Wong, T. E. Kiehn, and D. Armstrong. 1985. Fungemia in a cancer hospital: changing frequency, earlier onset, and results of therapy. Rev. Infect. Dis. 7:646-655. 8. Hughes, C. E., and W. H. Beggs. 1987. Action of fluconazole (UK-49,858) in relation to other systemic antifungal azoles. J. Antimicrob. Chemother. 19:171-174. 9. Hughes, C. E., L. R. Petersop, W. H. Beggs, and D. N. Gerding. 1986. Ketoconazole and flucytosine alone and in combination against Candida spp. in a neutropenic site in rabbits. J. Antimicrob. Chemother. 18:65-72.

26:170-173. 22. Troke, P. F., R. J. Andrews, K. W. Brammer, M. S. Marriot, and K. Richardson. 1985. Efficacy of UK-49,858 (fluconazole)

against Candida albicans experimental infections in mice. Antimicrob. Agents Chemother. 28:815-818. 23. Van Cutsem, J., J. Fransen, and P. A. J. Janssen. 1987. Therapeutic efficacy of itraconazole in systemic candidosis in guinea pigs. Chemother. 33:52-60. 24. Van It Wout, J. W., H. Mattie, and R. van Furth. 1988. A prospective study of the efficacy of fluconazole (UK-49,858) against deep-seated fungal infections. J. Antimicrob. Chemother. 21:665-672. 25. Weber, M. J., M. Keppen, K. E. Gawith, and R. B. Epstein. 1985. Treatment of systemic candidiasis in neutropenic dogs with ketoconazole. Exp. Hematol. 13:791-795.