ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Dec. 1999, p. 2841–2847 0066-4804/99/$04.00⫹0 Copyright © 1999, American Society for Microbiology. All Rights Reserved.
Vol. 43, No. 12
Interaction between Fluconazole and Amphotericin B in Mice with Systemic Infection Due to Fluconazole-Susceptible or -Resistant Strains of Candida albicans ARNOLD LOUIE,1,2* PARTHA BANERJEE,1,2 GEORGE L. DRUSANO,1,3 MEHDI SHAYEGANI,2 AND MICHAEL H. MILLER1,3 Division of Infectious Diseases1 and Clinical Pharmacology,3 Department of Medicine, Albany Medical College, Albany, New York 12208, and Wadsworth Center, New York State Department of Health, Albany, New York 122012 Received 10 July 1998/Returned for modification 13 December 1998/Accepted 8 September 1999
The interaction between fluconazole (Flu) and amphotericin B (AmB) was evaluated in a murine model of systemic candidiasis for one Flu-susceptible strain (MIC, 0.5 g/ml), two strains with intermediate Flu resistance (Flu mid-resistant strains) (MIC, 64 and 128 g/ml), and one highly Flu-resistant strain (MIC, 512 g/ml) of Candida albicans. Differences in fungal densities in kidneys of infected mice after 24 h of therapy and in survival rates at 62 days of mice treated with an antifungal drug or a combination of antifungal drugs for 4 days were compared. For the Flu-susceptible and Flu mid-resistant strains, the combination of Flu and AmB was antagonistic, as shown by both quantitative culture results and survival. The interaction was additive for the highly Flu-resistant strain. These results suggest that the combination of Flu and AmB should be used with caution in infections due to fungi that are usually susceptible to both antifungal agents and as empirical antifungal drug therapy. these drugs are being documented for these immunocompromised patients with greater frequency. Also, the use of combination therapy may decrease the development of Flu or AmB resistance by Candida isolates, a problem that may complicate the treatment of mucosal Candida infections in the human immunodeficiency virus-infected patient (19, 21). Furthermore, if the combination is synergistic, it is possible that the dose of AmB used in combination therapy may be less than the doses used with AmB monotherapy, thereby decreasing the AmB-related toxicity experienced by the patient without compromising treatment efficacy. In the present study we evaluated the interaction between AmB and Flu in mice infected with one Flu-susceptible strain (MIC, 0.5 g/ml), two strains with intermediate Flu resistance (Flu mid-resistant strains; MICs, 64 and 128 g/ml), and one highly Flu-resistant strain (MIC, 512 g/ml) of C. albicans. For the two Flu mid-resistant isolates and one highly Flu-resistant isolate, we also conducted dose range studies with Flu monotherapy to determine the highest dose that was associated with no efficacy and correlated these findings with the MIC breakpoint for Flu that was recently established by the National Committee for Clinical Laboratory Standards (17). The pharmacodynamic variable for Flu that best correlates with outcome is the ratio of the area under the concentration-time curve (AUC) to the MIC (12). Thus, we used doses of Flu that resulted in AUCs over 24 h that mimicked the 24-h AUCs measured in humans who were given 100, 200, 400, and 800 mg of Flu per day (10, 13). Since the pharmacodynamic parameter that predicts the efficacy of AmB is unknown, we used a dose in mice which resulted in serum peak and trough concentrations and 24-h AUC values similar to those seen in the serum of humans treated with 0.6 mg of AmB/kg of body weight per day (1). This dose of AmB is commonly used to treat systemic C. albicans infections in humans.
With advances in the treatment of oncologic malignancies with high-dose antineoplastic chemotherapy and bone marrow transplantation and improvements in medicine’s ability to support critically ill patients in intensive care units, the incidence of deep-seated fungal infections is rising (6). Recently, it was reported that 10% of all nosocomial bloodstream infections were due to fungi, particularly Candida albicans (6). In a matched, case-controlled study, Wey et al. (24) reported that the attributable mortality due to systemic fungal disease is 38%, despite antifungal drug therapy. Amphotericin B (AmB) has traditionally been considered the cornerstone of therapy for deep-seated fungal infections and fungemia. Recently two blinded, multicenter, randomized controlled trials suggested that fluconazole (Flu) is as efficacious as AmB in the treatment of C. albicans fungemia in the neutropenic and nonneutropenic host (3, 20). However, in these studies, treatment failure was seen in as many as 30% of patients (3). Because of this high failure rate, there is much interest in using Flu and AmB as combination therapy in an attempt to improve the outcome. However, the interaction between Flu and AmB remains poorly defined. In vitro studies using the checkerboard method and flow cytometry show that AmB and Flu are antagonistic for C. albicans (4, 16, 18). In contrast, in vivo models of C. albicans endocarditis in rabbits (22) and systemic candidiasis in mice (22, 23) reveal additivity. However, the in vivo models were not optimally designed to detect antagonism if it existed. Yet the characterization of the interaction between Flu and AmB is of utmost importance. If antagonism is not seen in vivo, this combination may have practical utility as empirical antifungal therapy in the febrile, neutropenic patient. Infections due to fungal species that are resistant to either one of * Corresponding author. Mailing address: Division of Infectious Diseases, Mail Code-49, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208. Phone: (518) 262-6548. Fax: (518) 262-6727. E-mail:
[email protected].
MATERIALS AND METHODS C. albicans isolates. C. albicans ATCC 36082 was purchased from the American Type Culture Collection (Manassas, Va.). C. albicans 208 and Y-12-99 were
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gifts from L. Steele Moore (Christiana Care Health System, Wilmington, Del.), and strain B59630 was a gift from F. Odds (Janssen Research Foundation, Beerse, Belgium). The MICs for Flu and AmB were determined on eight separate occasions by the broth macrodilution method described by the National Committee for Clinical Laboratory Standards (17). The median MIC of Flu after 48 h of incubation was 0.5 g/ml (range: 0.25 to 0.5 g/ml) for C. albicans ATCC 36082 (a Flu-susceptible strain). The median MICs were 64 g/ml (range: 64 to 128 g/ml) and 128 g/ml (range: 64 to 128 g/ml) for strains 208 and Y-12-99, respectively (both designated Flu mid-resistant strains). For strain B59630 the median MIC was 512 g/ml (range: 256 to 1,024 g/ml; designated a highly Flu-resistant strain). The median MICs for AmB ranged from 0.125 to 0.25 g/ml for the various fungal strains. The microorganisms were maintained on Sabouraud dextrose agar (BBL Microbiology Systems, Cockeysville, Md.) at 4°C until use. For each study, two or three colonies of a fungal isolate were subcultured onto fresh potato dextrose agar (BBL) and incubated at 35°C for 48 h. A fungal suspension was prepared by transferring three or four colonies of C. albicans to 5 ml of sterile, pyrogen-free normal saline (Baxter Inc., Chicago, Ill.) and quantified by hemocytometry. The suspension was diluted with normal saline to a concentration of 1.5 ⫻ 106 CFU/ml for C. albicans ATCC 36082 and 5 ⫻ 105 CFU/ml for the other fungal strains. Preliminary studies demonstrated that these inocula resulted in the death of mice between 2 and 8 days after fungal inoculation with each of these strains. For each of the fungal isolates the viability of the yeast was ⬎90% by trypan blue exclusion analysis. Antifungal agents. Flu powder was supplied by Pfizer Inc. (New York, N.Y.). AmB-desoxycholate power (Adria Laboratories, Columbus, Ohio) was purchased from the hospital pharmacy. Flu was dissolved in sterile, pyrogen-free saline to a stock solution of 4 mg/ml, aliquoted, and stored at ⫺70°C. For each study, the drug was thawed and further diluted to the desired concentration(s) with sterile, pyrogen-free normal saline. AmB was dissolved in sterile water to the desired concentration and was used immediately. Animals. Female NYLAR mice (weight 18 to 20 g) were raised at the Animal Research Facility of the Wadsworth Center for Laboratories and Research (Griffin Laboratories, Guilderland, N.Y.). These outbred Swiss mice were housed in plastic boxes at four or five animals per container. They received food and water ad libitum. All animal experimentation procedures were approved by and conducted in accordance with the guidelines of the Institutional Animal Care and Use Committees of the New York State Department of Health, Albany, N.Y., and Albany Medical College. Dose range pharmacokinetics of fluconazole in infected mice. Dose range studies were conducted to determine the pharmacokinetics of Flu and AmB when each drug was administered intraperitoneally (i.p.) as a single dose. The pharmacokinetic studies with Flu were conducted to determine the doses to give to mice that resulted in 24-h AUCs in serum that were similar to the 24-h AUCs that are measured in humans who are given 100, 200, 400, and 800 mg of Flu per day. Dose range AmB studies were conducted to determine the dose of AmB that would result in serum peak and trough concentrations and AUCs similar to those measured in humans who are given 0.6 mg of AmB/kg per day (1). NYLAR mice were intravenously infected with 3 ⫻ 105 CFU of blastoconidia of C. albicans ATCC 36082 via a lateral tail vein. The organism was administered in 0.2 ml of sterile saline. Five hours later, mice were injected i.p. with one of various doses of Flu or AmB in 0.2 ml of saline or water, respectively. The doses of Flu examined were 0, 1, 5, 25, 50, 75, 100, 150, 200, and 250 mg/kg. AmB doses of 0.5 and 1.0 mg/kg were evaluated. Three or four animals from each group were humanely sacrificed by CO2 asphyxiation 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12, 16, and 24 h after drug administration. Blood was collected by cardiac puncture and allowed to clot on ice. The serum was separated from the clot by centrifugation and stored at ⫺70°C. Comparison of AmB and Flu serum concentrations in animals treated with AmB, Flu, and AmB in combination with Flu. To define the effect of AmB on the concentration of Flu in serum, we compared levels of Flu and/or AmB in serum from infected animals that received once-daily doses of Flu, AmB, or Flu in combination with AmB on the second and fourth days of treatment. The doses of Flu and AmB examined were determined after analysis of the pharmacokinetic studies described above. Serum was collected from euthanized animals at 1, 4, and 23.45 h after the drug administration and stored at ⫺70°C until assayed. Antifungal drug assays. The concentration of Flu in each serum sample was determined by a well diffusion microbiological assay developed by Jorgensen et al. (11) with modifications described by Madu et al. (15). Candida pseudotropicalis (ATCC 46764) was used as the assay organism. Pour plates of the fungus were prepared with molten SAAMF (synthetic amino acid medium fungal) agar and allowed to solidify at room temperature. Four-millimeter-diameter wells were made in the agar. Twenty-microliter aliquots of serum collected from mice or standards were pipetted into wells and kept at 4°C for 1 h and then incubated overnight for 16 h at 30°C in an ambient air incubator. The diameters of inhibition for each serum sample and standards were measured with a vernier caliper to the nearest 0.1 mm. Antifungal drug concentrations in serum samples were calculated with the curves derived from Flu standards. The standard curve was linear for concentrations of Flu between 0.5 and 100 g/ml of serum. For serum samples that resulted in diameters of inhibition that were greater than those associated with the linear portion of the standard curve, the serum samples were diluted 1:4 with saline and retested. The calculation of the concentration of the
ANTIMICROB. AGENTS CHEMOTHER. drug in serum accounted for this. The intraday and interday coefficients of variation of the microbiological assay were 4.9 and 6.8%, respectively. Concentrations of AmB in serum were determined by a microbiological assay described by Bannatyne et al. (5) and Granich et al. (9) with modifications. Paecilomyces variotii (ATCC 22319) was used as the assay organism. The fungus was grown on Sabouraud dextrose agar slants for 5 to 7 days at 35°C. Mature spores were harvested with sterile cotton-tipped applicators and placed in normal saline. The total concentration of spores was determined by hemocytometry. Spores were added to molten SAAMF agar to a final concentration of 106 spores/ml. Pour plates of the fungal spores were made and allowed to solidify at room temperature. Ten-millimeter-diameter wells were made in the agar, and 100 l of serum or standards was pipetted into wells. After incubation for 24 h at 35°C, the diameters of zones of inhibited growth were measured to the nearest 0.1 mm with a vernier caliper. Antifungal drug concentrations in samples were calculated with the curves derived from AmB standards. The standard curve was linear from concentrations of 0.1 to 20 g/ml. The intraday and interday coefficients of variation of the microbiological assay were 4.3 and 6.2%, respectively. For animals that received AmB and Flu in combination, AmB concentrations in serum were measured with a Flu-resistant C. albicans strain (B59630; gift from F. Odds) in the biological assay. Otherwise the bioassay was conducted as described above. Initial studies demonstrated that serum that contained 0.1 to 4.0 g of AmB/ml together with 1 to 150 g of Flu/ml produced zones of growth inhibition whose diameters were the same as those of zones produced by serum containing only AmB. The lower limit of sensitivity of the assay was 0.125 g/ml. Flu concentrations in sera of animals that received AmB plus Flu were determined by a high-pressure liquid chromatography (HPLC) method described elsewhere (15). Previously, we demonstrated that Flu concentrations measured by the bioassay and HPLC were equivalent (15). The intraday and interday coefficients of variation of the HPLC at 1 g/ml were 4.5 and 5.6%, respectively. Pharmacokinetic analysis. Pharmacokinetic analysis of the serum samples for Flu and AmB concentration-time relationships were performed with the nonlinear least-squares regression program RSTRIP II (Micromath Scientific Software, Salt Lake City, Utah). The most appropriate pharmacokinetic models were determined by using model selection criteria based on a modified form of Akaike’s information criterion (2). Cmax and Cmin (i.e., trough) were defined as the highest and lowest concentrations, respectively, of drug measured in serum after the drug was administered. To determine the AUC in serum, the trapezoidal method was used for the data obtained from time zero to the last time point. Infection model for evaluating the interaction between Flu and AmB. NYLAR mice were infected intravenously with 3 ⫻ 105 CFU (C. albicans ATCC 36082) or 105 CFU (all other fungal strains) of yeast. With these inocula, mice succumbed of their infections within 2 to 8 days of fungal injection. The blastoconidia were administered via a lateral tail vein in 0.2 ml of pyrogen-free saline. For the studies that used C. albicans ATCC 36082, infected mice were randomly divided into four groups. Each group consisted of 29 to 31 animals. Group I received Flu once per day at a dose that resulted in a serum 24-h AUC that was equivalent to the 24-h AUC measured in humans that received 400 mg of Flu per day (10). The drug was given 5, 30, 54, and 78 h after fungal inoculation. Group II received AmB at 0.5 mg/kg every 24 h for four doses beginning 5 h after fungal inoculation. Group III was given Flu in combination with AmB at the doses and dosing schedules indicated for the individual drugs. For this group, AmB was given 1 h before Flu. Group IV received saline once every 24 h and served as untreated controls. The animals were observed for 62 days. Preliminary studies demonstrated that the administration of four daily doses of Flu and AmB, either alone or in combination, was not toxic to healthy mice for a 62-day observation period. Therefore, noninfected treatment controls were not studied in subsequent trials. Mortality was assessed at least twice each day for the duration of the study. Moribund animals (defined as mice that were unable to ambulate or to rise from a supine position) were humanely sacrificed by CO2 asphyxiation followed by induction of bilateral pneumothoraces. Euthanized animals were included in the mortality counts of the following day. Previously, we found that 90% of treated animals that demonstrated either of these conditions died within 24 h (mean: 12.6 h; range: 4 to 34 h) (unpublished results). This study was conducted twice. Quantitative cultures were conducted with kidneys of infected animals after 24 h of treatment. Briefly, seven or eight animals from each of the groups described above were randomly chosen to receive just one dose of Flu and/or one dose of AmB. The dose of Flu used was the dose that resulted in a 24-h AUC in mice that was equivalent to the 24-h AUC measured in the serum of humans who are given 400 mg of Flu per day. AmB at 0.5 mg/kg was used. Both drugs were given i.p. in 0.2 ml of solution 5 to 6 h after fungal injection. These animals were humanely sacrificed 24 h after fungal inoculation. The right kidney was removed aseptically. Each kidney was weighed, homogenized, and serially diluted with saline. Two hundred microliters of each dilution was plated onto potato dextrose agar that was supplemented with 100 IU of penicillin and 100 g of streptomycin per ml of agar. After 48 h of incubation at 35°C, the colonies were counted and the results for different groups were compared. The cultures reproducibly detected ⱖ50 organisms/g of tissue. The studies were conducted at least twice for each fungal strain. For the two Flu mid-resistant strains and the one highly Flu-resistant strain of C. albicans, the protocol described above for C. albicans ATCC 36082 was used with the following changes. First, the fungal inoculum used was 105 CFU/mouse
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since higher inocula resulted in the death of untreated animals between 12 and 24 h after the fungus was administered. Second, additional treatment groups were studied to evaluate the effect of increasing Flu doses on the interaction between this azole and AmB at 0.5 mg/kg per dose. The groups consisted of mice treated with Flu at doses that resulted in 24-h AUCs that were similar to those measured in humans given 100, 200, 400, and 800 mg of Flu per day (10, 13) and each of these Flu dosages in combination with AmB at 0.5 mg/kg/day. Additional groups included untreated controls and mice treated with AmB at 0.5 mg/kg/day as monotherapy. Animals that received both antifungal drugs were given AmB 1 h before Flu was administered. There were 21 to 30 infected mice per group. The survival rate in each group was evaluated at least twice daily for up to 62 days. Seven or eight mice from groups that received Flu at a dose resulting in a 24-h AUC equivalent to the 24-h AUC measured in humans who received 400 mg of Flu per day, this dose of Flu in combination with AmB, AmB monotherapy, or saline were sacrificed 24 h after fungal inoculation. The kidneys from these animals were assessed for fungal densities. To monitor for drug carryover, kidneys were collected from uninfected mice that were given Flu, AmB, or Flu plus AmB at the doses described previously. The AmB or Flu or both were administered i.p. approximately 18 h before the mice were euthanized. Kidneys collected from uninfected mice that received saline served as controls. The kidneys were weighed and homogenized. One hundred microliters of homogenate was added to 100 l of saline containing one of the C. albicans strains used in the in vivo studies. The entire volume of material was cultured onto drug-free potato dextrose agar plates. The plates were incubated for 48 h at 35°C, and the colonies were counted. The study was conducted in duplicate on two separate occasions for each fungal strain. No differences in counts between cultures containing homogenates of kidneys collected from saline- and antifungal drug-treated animals were seen, indicating that drug carryover did not occur for the doses of the drugs examined (data not shown). Statistical analysis. Comparisons of colony counts among the different treatment groups were performed by the Kruskal-Wallis test with multiple comparisons followed by Newman-Keuls analysis with the software program True Epistat, version 5.3 (Epistat Services, Richardson, Tex.). A difference was considered statistically significant at P ⬍ 0.05. Differences in survival after 62 days of observation were assessed by Kaplan-Meier analysis followed by the Wilcoxon test. A P ⬍ 0.05 was considered a statistically significant difference. Correction of P values for multiple comparisons was not done. The interaction between AmB and the various doses of Flu was defined as an enhanced effect if combination therapy resulted in a statistically significant improvement in survival compared with the most active monotherapeutic agent. The combination was antagonistic if it resulted in a statistically significant decrease in survival versus the most active monotherapy. If the combination did not meet either criterion it was deemed additive. For the quantitative culture results, the combination of Flu and AmB was defined as producing enhanced effect if it resulted in a statistically significant decrease in fungal density in kidneys versus the most active monotherapeutic agent. The combination was defined as antagonistic if it resulted in a significant increase in counts versus the most active monotherapy. It was additive if neither criterion was met.
RESULTS Dose range pharmacokinetic studies in infected mice. The pharmacokinetics of Flu were determined in mice that received a single i.p. injection of drug 5 h after they were inoculated intravenously with C. albicans. The Cmax was observed 1 h after the drug was administered. Both the Cmax and AUC increased in proportion to the dose of Flu administered. The Cmax was described by the linear equation Cmax ⫽ 1.2893 ⫻ dose ⫺ 2.3651, with r2 ⫽ 0.996. The AUC was described by the linear equation: AUC ⫽ 3.3271 ⫻ dose ⫹ 7.4691, with r2 ⫽ 0.998. The pharmacokinetics was best described by a twocompartment model with a terminal half-life of 3.4 h. The terminal half-life did not change with increasing Flu doses. For Flu, the AUC/MIC ratio is the pharmacodynamic parameter that best predicts outcome (12). Others observed that the 24-h AUCs in healthy human volunteers given 100, 200, and 400 mg of Flu were 90, 170, and 350 mg 䡠 h/liter, respectively (10). The 24-h AUC for a dose of 800 mg of Flu/day was 712 mg 䡠 h/liter (13). Using the equation described above for the AUC, we determined that the dosages of Flu that should be given to mice to result in the same 24-h AUCs as those measured in the serum of humans given 100, 200, 400, and 800 mg of Flu per day were 24.8, 48.9, 103.5, and 211.8 mg/kg each day. To simplify drug preparation, Flu dosages given to mice were 25, 50, 100, and 200 mg/kg per day, respectively. The
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FIG. 1. Pharmacokinetics of Flu at 25, 50, 100, and 200 mg/kg i.p. (A) and AmB at 0.5 and 1.0 mg/kg i.p. (B) in mice systemically infected with C. albicans.
serum concentration-versus-time relationships for these dosages of Flu are shown in Fig. 1A. The serum concentration-versus-time relationships for AmB doses of 0.5 and 1.0 mg/kg given i.p. are depicted in Fig. 1B. The Cmax values were seen between 1 and 1.5 h after drug administration. The Cmax and Cmin for 0.5-mg/kg i.p. doses of AmB were 1.60 ⫾ 0.04 and 0.22 ⫾ 0.01 g/ml, respectively. For 1.0-mg/kg doses of AmB these values were 3.21 ⫾ 0.03 and 0.43 ⫾ 0.02 g/ml, respectively. The 24-h AUCs for AmB doses of 0.5 and 1.0 mg/kg were 14.91 ⫾ 3.02 and 22.26 ⫾ 4.47 g 䡠 h/ml, respectively. In humans the Cmax, Cmin, and 24-h AUC for an AmB dosage of 0.6 mg/kg per day were 1.06 g/ml, 0.2 g/ml, and 17.06 g 䡠 h/ml, respectively (1). Since AmB at 0.5 mg/kg/day in mice resulted in Cmax, Cmin, and 24-h AUC values that were most similar to the corresponding parameters in humans, this dosage was used in our in vivo studies. Effect of AmB on serum Flu concentrations with combination therapy. Concentrations of Flu and AmB in serum were measured for infected mice that were treated for 2 or 4 days with Flu at 25, 50, or 100 mg/kg per day, AmB at 0.5 mg/kg per day, or each of these dosages of Flu in combination with AmB at 0.5 mg/kg/day. Animals were infected with one of the four C. albicans strains used in this project. The purpose of these studies was to determine whether AmB and/or Flu serum concentrations were altered when these drugs were used together. Table 1 shows the concentrations of drugs in mice infected
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TABLE 1. Comparison of serum Flu and AmB concentrations in mice given daily doses of Flu, AmB, or Flu plus AmB i.p. on days 2 and 4 of treatmenta Mean concn (g/ml) of indicated drug in serum ⫾ 1 SD after treatment on day: Drug
Time (h)
2 AmB
AmB Flu AmB ⫹ Flu
a
1 4 23.45 1 4 23.45 1 4 23.45
1.62 ⫾ 0.04 0.96 ⫾ 0.04 0.23 ⫾ 0.01
1.60 ⫾ 0.08 1.06 ⫾ 0.06 0.22 ⫾ 0.03
4 Flu
118.0 ⫾ 8.5 64.5 ⫾ 6.7 3.3 ⫾ 1.2 122.7 ⫾ 7.9 56.4 ⫾ 6.6 5.2 ⫾ 2.4
AmB
1.56 ⫾ 0.06 0.94 ⫾ 0.05 0.22 ⫾ 0.02
1.59 ⫾ 0.06 1.05 ⫾ 0.04 0.20 ⫾ 0.02
Flu
124.6 ⫾ 7.9 68.6 ⫾ 9.1 5.3 ⫾ 1.1 120.4 ⫾ 8.6 58.1 ⫾ 7.2 4.4 ⫾ 1.2
Dosages of 100 mg/kg/day for Flu and 0.5 mg/kg/day for AmB were examined. Data are from four mice per time point.
with C. albicans ATCC 36082 at 1 (peak level in serum), 4, and 23.45 h after the animals received their second and fourth daily doses of Flu (100 mg/kg) and/or AmB (0.5 mg/kg). On both days, the serum Flu concentrations for groups that received Flu alone and together with AmB were similar. Also, the serum AmB concentrations did not differ between groups treated with AmB alone and those treated with AmB in combination with Flu. Similar results were seen in mice that were treated with Flu at 25 or 50 mg/kg/day alone and in combination with AmB (data not shown). Also, these findings were independent of the strain of C. albicans used to infect the animals (data not shown). Survival of infected animals treated with Flu, AmB, or Flu in combination with AmB. For C. albicans ATCC 36082, a Flu-susceptible strain, the survivals of the various treatment groups are shown in Fig. 2A. The results were similar in two separate trials. Therefore, the results were combined. None of the animals were excluded from analysis. All treatment regimens were better than controls. AmB as monotherapy was the most active regimen. The combination of Flu and AmB was superior to Flu monotherapy (P ⫽ 0.000003). However, the combination of Flu and AmB was less active than AmB monotherapy (P ⬍ 0.000001). Therefore, this combination was antagonistic. For C. albicans strain 208, an isolate with intermediate resistance to Flu (Flu MIC, 64 g/ml), similar results were observed. AmB as monotherapy was the most active regimen (Fig. 2B), with 60% of the mice surviving the 62-day observation period. There were no survivors in the other groups. The survival rates associated with Flu at 25, 50, and 100 mg/kg per day were similar. However, all the Flu regimens were superior to no treatment (P ⬍ 0.000001 for any of these treatment group versus controls). Flu at 200 mg/kg per day was more effective than the lower dosages of Flu examined (P ⫽ 0.0001 versus Flu at 100 mg/kg per day). The combination of AmB and Flu at 25, 50, and 100 mg/kg per day demonstrated marked antagonism (P ⫽ 0.0002, 0.0002, and 0.004, respectively, versus AmB as monotherapy). The results of two separate trials using C. albicans Y-12-99 were similar. Therefore the results were combined and are shown in Fig. 2C. For C. albicans Y-12-99, a strain for which the Flu MIC is twofold higher (MIC of 128 g/ml) than that for strain 208, a dose-response relationship was seen with an increase of the Flu dosage from 12.5 to 50 mg/kg per day. The results for Flu at 50 and 100 mg/kg per day were similar. The combination of AmB and Flu at the 50- and 100-mg/kg doses was antagonistic relative to AmB monotherapy (P ⫽ 0.00002
and 0.00006, respectively, versus AmB). Of note, AmB plus Flu at 25 mg/kg per day resulted in a survival rate that was similar to that for AmB monotherapy. This finding was observed in both trials that were conducted with this fungal strain. For the highly Flu-resistant strain (Flu MIC of 512 g/ml), Flu therapy was no better than controls. All the doses of Flu in combination with AmB that were examined resulted in outcomes similar to that for AmB monotherapy (P ⱖ 0.6; Fig. 2D). This finding supports the results of the studies using C. albicans 208 and Y-12-99; the interaction between AmB and Flu is dependent on the doses of Flu used relative to the MIC of Flu for that organism. Of note, the results of two separate trials were similar. Therefore, the figure represents the combined outcomes of two trials. Fungal density in kidneys of mice treated with Flu, AmB, or Flu plus AmB. Flu monotherapy was superior to no therapy for the treatment of systemic fungal infection due to the Flususceptible strain (ATCC 36082). For all three Flu-resistant strains Flu therapy was no better than controls (Table 2). For the one Flu-susceptible strain and one of the Flu mid-resistant strains (isolate 208; Flu MIC of 64 g/ml), the combination of Flu and AmB demonstrated antagonism relative to AmB monotherapy (Table 2; P ⬍ 0.01). For the two C. albicans strains for which the Flu MICs were the highest (128 and 512 g/ml), Flu monotherapy had little effect in reducing the fungal densities in kidneys and the efficacies of the AmB-plus-Flu regimens were not different from that for AmB monotherapy. These results are consistent with the survival data for each of the strains of C. albicans examined. DISCUSSION In this study we showed that the interaction between Flu and AmB was antagonistic for one Flu-susceptible strain and two Flu mid-resistant (MIC, 64 to 128 g/ml) strains of C. albicans as determined by both survival and quantitative cultures of the kidneys. The efficacy of Flu plus AmB was similar to that of AmB monotherapy for the highly Flu-resistant isolate (MIC, 512 g/ml). The results of the present study confirmed our observation, in a rabbit model of aortic valve endocarditis and pyelonephritis, that the combination of Flu and AmB is antagonistic (14). Furthermore, the present study demonstrates that the degree of antagonism between Flu and AmB that is observed is dependent upon the dose of Flu used relative to the Flu MIC of the fungal isolate. AmB is the more active of the two antifungal agents studied. Based on the known mechanisms of action of Flu and AmB, it
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FIG. 2. Survival of mice infected with C. albicans ATCC 36082 (Flu MIC, 0.5 g/ml) (A), 208 (MIC, 64 g/ml) (B), Y-12-99 (MIC, 128 g/ml) (C), and B59630 (MIC, 512 g/ml) (D). The mice were treated for 4 days with various doses of Flu alone or in combination with AmB (0.5 mg/kg/day). The dose of Flu administered to each group is indicated. Two trials were conducted for each fungal strain. For each fungal strain, the results of the trials were combined since the results were similar. None of the animals from any trial were excluded. The numbers of animals in each group are in parentheses.
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TABLE 2. Fungal densities in kidneys of mice infected with various strains of C. albicansa Fungal strain
ATCC 36082 208 Y-12-99 B59630
Flu MIC (g/ml)
0.5 64 128 512
Fungal density (103 CFU/g) ⫾ 1 SD for indicated treatment groupb Control ⫹⫹
1,691 ⫾ 484 2,756 ⫾ 178⫹ 6,234 ⫾ 1,041⫹⫹⫹ 3,000 ⫾ 252⫹⫹⫹
Flu
Flu ⫹ AmB
AmB
453 ⫾ 129** 3,040 ⫾ 638⫹⫹ 6,986 ⫾ 758⫹⫹⫹ 3,299 ⫾ 294⫹⫹⫹
713 ⫾ 76** 2,091 ⫾ 154* 1,255 ⫾ 1,161*** 278 ⫾ 80***
81 ⫾ 13***,⫹⫹ 835 ⫾ 164***,⫹⫹⫹ 528 ⫾ 282*** 316 ⫾ 54***
a The mice were treated with a single i.p. dose of Flu (100 mg/kg) and/or AmB (0.5 mg/kg). The drug or drugs were given 5 h after fungal injection. Animals that received both drugs were given AmB 1 h before Flu was administered. The mice were humanely sacrificed 24 h after fungal inoculation. Untreated animals served as controls. There were 8 to 10 mice per group. b ⴱ, P ⬍ 0.05 versus control; ⴱⴱ, P ⬍ 0.01 versus control; ⴱⴱⴱ, P ⬍ 0.001 versus control; ⫹, P ⬍ 0.05 versus Flu plus AmB; ⫹⫹, P ⬍ 0.01 versus Flu plus AmB; ⫹⫹⫹, P ⬍ 0.001 versus Flu plus AmB.
is reasonable to expect that the interaction between these drugs would be antagonistic. AmB exerts its fungicidal effect by binding to ergosterol, a lipid constituent of the fungal membrane (7). Ergosterol-bound AmB aggregates to form channels across the fungal membrane through which essential nutrients and electrolytes exit from the fungus, resulting in the death of the organism (7). Flu, an alpha-14 demethylase inhibitor, incompletely inhibits the production of ergosterol by certain fungal species, including C. albicans, and is fungistatic (8, 10). Since this azole decreases ergosterol production, less targets are available in the fungal membrane for AmB to interact with, thus decreasing the activity of the more active of the two agents. Most in vitro studies report antagonism between Flu and AmB (4, 16, 18). In contrast, on the basis of in vivo studies with a non-neutropenic-mouse model of systemic candidiasis, Sugar et al. (23) reported additivity between these drugs. However, these in vivo studies were not optimally designed to identify an antagonistic interaction since the 100% survival associated with AmB monotherapy places the outcome at the top of the dose-response curve. This makes it extremely difficult to identify antagonism between Flu and AmB unless Flu completely abolishes the activity of AmB. On the basis of a neutropenic-mouse model of systemic candidiasis, Sugar et al. (23) reported a “positive effect” between Flu and AmB. In this model, Flu and Flu-plus-AmB recipients received Flu beginning 1 h after fungal inoculation. However, AmB was initiated 2 days after fungal injection in the AmB-plus-Flu group and AmB monotherapy group. If one excludes the deaths that occurred before AmB therapy was initiated in the latter group, the survival rates seen in the Flu-plus-AmB and AmB groups would be similar. Of note, in another trial (23) these investigators reported a 40% reduction in survival rates, from 62.5% for AmB alone to 37.5% for Flu plus AmB. The difference was not statistically significant. However, the number of animals in each group was small. Sanati et al. (22) evaluated the interaction between Flu and AmB in a neutropenic-mouse model of systemic candidiasis. Eight days after fungal inoculation the survival rates were approximately 15, 43, 60, and 72% for mice that received placebo, Flu, Flu plus AmB, and AmB, respectively. Although differences in survival between groups did not reach statistical significance, the investigators could not discount the possibility that their study lacked sufficient power to identify antagonism (22). In a rabbit model of C. albicans endocarditis, Sanati (22) reported that the interaction between Flu and AmB was indifferent. In contrast, using the same fungal strain, Louie et al. (14) noted the combination of Flu and AmB to be antagonistic. Both investigators found the fungal densities in cardiac tissues of Flu-plus-AmB recipients to lie between those of the two
monotherapies. The discrepancy in results may be explained by the fact that Louie et al. observed a larger difference between the fungal densities in the cardiac vegetations of the Flu and AmB monotherapy groups than that observed by Sanati et al. (a 5-log10 difference versus a 2-log10 difference). Thus, the model of Louie et al. was more sensitive than that of Sanati et al. for identifying statistically significant differences between Flu-plus-AmB and the other treatment groups. Of note, Louie et al. (14) also reported antagonism between Flu and AmB in the clearance of C. albicans from the kidneys of the same infected rabbits. With 5 and 14 days of therapy, the combination of Flu plus AmB was significantly less effective that AmB but more active than Flu. However, by day 21 of therapy Flu, AmB, and Flu plus AmB all sterilized this site. Thus, antagonism in the kidney was manifested by a delay in the sterilization of this organ. In summary, in the present study we found the interaction between Flu and AmB to be antagonistic in our murine model of systemic candidiasis. This was manifested by a decrease in the clearance of fungi from the kidneys and a worsening of survival with combination therapy relative to the most active regimen, AmB monotherapy. These findings are consistent with the antagonistic interaction that was observed in vitro (4, 16, 18) and in our rabbit model of endocarditis and pyelonephritis (14). However, our study and those of others show that the efficacy of Flu plus AmB is no worse than Flu monotherapy. Thus, for infections in which clinical studies have shown Flu and AmB monotherapies to have equivalent efficacies, such as catheter-related fungemia due to C. albicans (20), outcomes associated with Flu plus AmB, Flu, and AmB should be similar. Of note, in a nonfatal rabbit model of systemic candidiasis, we demonstrated that antagonism between Flu and AmB was manifested as a slower rate of clearance of the fungus from the kidney than that for AmB monotherapy. However, the kidneys of AmB, Flu-plus-AmB, and Flu recipients all were sterilized with 21 days of treatment. Flu resistance in human immunodeficiency virus-infected patients who are receiving long-term Flu for mucocutaneous candidiasis is well documented (19, 21). The results of our nonfatal rabbit model of Candida pyelonephritis (14) suggest that the combination of Flu and AmB should be evaluated in the treatment of nonlife-threatening C. albicans infections as a means of treating the infection while, perhaps, reducing the emergence of Flu or AmB resistance during therapy. However, the results of our murine model of fatal systemic candidiasis suggest that this antifungal drug combination should be used with caution in deep-seated fungal infections in which the relative activities of AmB and Flu for the fungus are not known or the activity of AmB for the fungus is greater than that of Flu.
VOL. 43, 1999
FLUCONAZOLE PLUS AMPHOTERICIN B FOR CANDIDIASIS ACKNOWLEDGMENT
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