Interaction between Posaconazole and Amphotericin B in Concomitant Treatment against Candida albicans In Vivo

The interaction of posaconazole and amphotericin B was evaluatedin concomitant treatment of Candida albicans systemic infectionsin immunocompetent mice by using four strains of C. albicanswith different susceptibilities to fluconazole. Posaconazoleand amphotericin B were each tested at four dose levels aloneand in all possible combinations against each C. albicans strain.Survival curves of mice treated with combinations of posaconazoleand amphotericin B were statistically compared with those ofmice treated with the component monotherapies. Of the 64 totalcombinations evaluated against the C. albicans strains (16 combinationsper strain), 20.3% were more effective in prolonging mouse survivalthan both of the monotherapies, 45.3% were more effective thanone of the monotherapies, and 32.8% were similar to both monotherapies.No evidence of antagonism was observed between posaconazoleand amphotericin B in this mouse model, consistent with in vitroresults against the same strains.

INTRODUCTION

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Introduction

The clinical use of azoles in combination with amphotericinB (AMB) is still controversial because of the potential forantagonism between the two drugs (9, 12, 18, 22). This potentialcomes from their mechanisms of action; azoles block ergosterolbiosynthesis, while AMB causes membrane damage by binding toergosterol. Various experimental fungal infection models havebeen used to address the issue of combinational dosing, butthe results have been mixed. In systemic candidiasis modelsin mice with Candida albicans, Louie et al. (11) observed antagonismbetween the triazole fluconazole (FLC) and AMB, while Sugarand Liu (23) reported antagonism between another triazole, itraconazole,and AMB. Louie et al. (10, 12) also found that FLC was antagonisticto AMB therapy against experimental C. albicans endocarditis,endophathalmitis, and pyelonephritis in rabbits. However, Sanatiet al. (17) did not observe antagonism when FLC and AMB wereused in combination against C. albicans in invasive candidiasisin neutropenic mice or in endocarditis in rabbits. Sugar etal. (21) reported no antagonism between FLC and AMB in invasivecandidiasis with C. albicans in immunocompetent or immunocompromisedmice.

Posaconazole (POS) is a broad-spectrum antifungal triazole whichrecently completed phase III clinical trials (7). The experimentsdescribed in this report were performed to determine the interactionbetween POS and AMB in concomitant combination therapy againstsystemic C. albicans infection in mice.

(A preliminary report of this research was presented at the42nd Interscience Conference on Antimicrobial Agents and Chemotherapy,San Diego, Calif., 27 to 30 September 2002 [A. F. Cacciapuoti,M. Gurnani, J. Halpern, F. Gheyas, R. Hare, and D. Loebenberg,Abstr. 42nd Intersci. Conf. Antimicrob. Agents Chemother., abstr.M-1814, p. 415, 2002].)

MATERIALS AND METHODS

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Materials and Methods

Antifungal agents. POS clinical oral suspension was used in these experiments,and dilutions were made in sterile water for injection. AMB(Fungizone) was obtained from Apothecon, Bristol-Myers Squibb,Princeton, N.J., and prepared according to the manufacturer'sdirections.

C. albicans strains and in vitro activity testing. All C. albicans strains were from the Schering-Plough ResearchInstitute fungal culture collection and included one FLC-susceptible(FLC-S) strain C43, one FLC-susceptible, dose-dependent (FLCS-DD) strain C210, and two FLC-resistant (FLC-R) strains C284and C335. MICs were determined by the standard NCCLS methodM27-A (14). MICs of FLC for strains C43, C210, C284, and C335were 0.125, 16, 64, and 64 µg/ml, respectively. Drug interactionsbetween POS and AMB were determined by a checkerboard microdilutionmethod. The endpoints for POS alone and for the POS-AMB combinationswere read at 80% inhibition, while that for AMB alone was readat 100% inhibition. The fractional inhibitory concentration(FIC) index (5) was defined as synergistic if the FIC was $≤$ 0.5,indifferent if it was >0.5 but $≤$ 4, and antagonistic if itwas >4.

Mice. Charles River Laboratories (Wilmington, Mass.) CF1 mice (white,male) were used in these studies. At the time of infection,the mice weighed 18 to 20 g. These studies were carried outin accordance with the Guide to the Care and Use of LaboratoryAnimals of the National Institutes of Health (15) and the AnimalWelfare Act in an Association for Assessment Accreditation ofLaboratory Animal Care-accredited program.

Systemic infection model and drug therapy. C. albicans strains were grown for 48 h on Sabouraud dextroseagar, and inocula were prepared as saline suspensions as describedpreviously (6). Initiation of systemic infection occurred onday 0 by intravenous injection (tail vein). Inocula ranged fromca. 5 x 10⁶ (strains C43 and C210) to ca. 1 x 10⁷ (strains C284and C335) CFU/mouse. Drug therapy to groups of 10 mice beganat 4 h postinfection on day 0 and continued once daily throughday 3. POS and AMB were each tested at 4 dose levels alone andin all possible combinations in a checkerboard fashion againsteach C. albicans strain (see Table 2). The 4 dose levels foreach drug were selected from preliminary dose-response experiments(data not shown) to include the full range of survival efficacy,from maximum to intermediate to minimal, in the survival curves.This was done to ensure that POS and AMB would be tested incombinations involving high-, intermediate-, and low-dose levelsof each drug (similar to doing a checkerboard in vitro MIC test).For each strain, there were 16 drug combination groups and 8monotherapy groups of mice. Concomitant combination therapywas achieved by administering POS orally followed immediatelyby AMB intraperitoneally. Control animals were administeredsterile water for injection. Survival was monitored for 10 days.Mice were not cultured for organ fungal burdens.

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TABLE 2. Effect of POS-AMB combinations on mouse survival compared to POS or AMB alone against C. albicans

Statistical analysis. Each C. albicans strain was tested twice, and the results werecombined for statistical analysis. Wilcoxon tests were performedto compare the survival curves (Kaplan Meier). A P value of<0.05 indicated that the overall survival curves were statisticallysignificantly different. No multiplicity adjustments were made.

RESULTS

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Results

In vitro interaction between POS and AMB. POS and AMB were tested in vitro alone and in combination againstthe four C. albicans strains which exhibited a range of susceptibilitiesto FLC (Table 1). The MICs of POS ranged from 0.03 to 1, whilethe MICs of AMB ranged from 1 to 4. The drug interaction resultswere mixed but were primarily indifferent. The POS-AMB interactionswith strains C43 and C284 were synergistic and indifferent indifferent experiments, while those with strains C284 and C335were indifferent. No antagonism was observed.

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TABLE 1. In vitro interaction between POS and AMB against four C. albicans strains with different susceptibilities to FLC

Effect of combination dosing against systemic candidiasis in mice. The four C. albicans strains were tested in a systemic infectionmodel with immunocompetent mice. Table 2 shows the 16 POS andAMB combinations used for each strain and the averaged percentsurvival data from two experiments per strain on day 4 (theday after the last dose) and day 10 (end of experiment) forthe combinations and their component monotherapies. Survivalcurves from day 0 to day 10 for each concomitant combinationof POS and AMB were statistically compared to those for thePOS and AMB monotherapies, and the resulting P values are alsoshown in Table 2. Survival curves are shown as examples of thedata in Fig. 1A (strain C43) and 1B (strain C284). Both graphsshow the combination of the highest dose level of POS and thesecond-highest dose level of AMB used against that strain, comparedto the component monotherapies and controls. The emphasis inthe graphs on the second-highest dose level of AMB is to showthe drug interaction with a lower, but still efficacious, levelof AMB, where potential antagonism could still be observed,as opposed to the highest level of AMB, where antagonism maybe masked by the maximum efficacy of AMB. In Fig. 1A, for theFLC-S strain C43, mice treated with the POS and AMB combinationsurvived significantly longer than those treated with the componentmonotherapies (P < 0.05) or the controls. In Fig. 1B, forthe FLC-R strain C284, mice treated with the POS and AMB combinationsurvived similarly to those treated with the component monotherapies(P > 0.05) but longer than the controls.

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FIG. 1. Concomitant combination treatment with POS and AMB of systemic infection in mice with FLC-S C. albicans C43 (A) or FLC-R C. albicans C284 (B). The combination of POS and AMB, POS alone, or AMB alone was administered once daily for 4 days starting 4 h postinfection on day 0. Dose levels (in milligrams/kilogram of body weight) are indicated in parentheses. Controls were administered sterile water for injection. The survival data were averaged from two experiments (10 mice per group in each experiment). The P values were determined by comparing the survival curve for the combination to each of those for the drugs alone.

Table 3 shows a summary of all of the interactions between POSand AMB listed in Table 2. In these studies, an antagonisticcombination was defined as providing less survival efficacythan one or both of the component drugs alone. Against all fourC. albicans strains, 20.3% of the POS and AMB combinations testedwere more effective than either POS or AMB alone (P < 0.05)in prolonging the survival of mice. In addition, 45.3% of thecombinations were more effective than one of the drugs alone(P < 0.05) and similar to the other drug alone (P > 0.05),while 32.8% of the combinations were similar to both drugs alone(P > 0.05). Only one combination of POS and AMB was lesseffective than one of the drugs alone (POS in this case), andno combinations were less effective than both POS and AMB alone.Overall, 98.4% of the 64 combinations tested against the fourC. albicans strains showed no evidence of antagonism betweenPOS and AMB in this model of systemic candidiasis in immunocompetentmice.

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TABLE 3. Summary of interactions of POS-AMB combinations compared to one or both of POS or AMB alone against the four C. albicans strains^a

DISCUSSION

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Discussion

Our studies demonstrate that concomitant combination treatmentof systemic candidiasis in immunocompetent mice with POS andAMB was not antagonistic, as determined by using survival asthe efficacy endpoint. Instead, 20.3, 45.3, and 32.8% of thetotal combinations tested against four C. albicans strains resultedin survival of mice, which was more effective than both monotherapies,more effective than one of the monotherapies, and similar toboth monotherapies, respectively. In addition, the combinationof POS and AMB was effective and not antagonistic even againststrains with reduced susceptibility to FLC, where much higherdoses of POS were used in the combinations. The finding of noantagonism in the in vivo experiments was consistent with thein vitro results against the same strains.

In these studies we did not investigate the effect of POS andAMB on fungal burdens in organs or the effect of sequentialdosing of POS relative to AMB. However, Najvar et al. (13) reportedthat concomitant POS and AMB or sequential dosing (initial dosingwith POS followed a day later by AMB) in a pulmonary Aspergillusflavus infection model in immunocompromised mice were not antagonistic,as determined by both survival and lung burden results.

Although pharmacokinetic (PK) data and PK-pharmacodynamic (PD)analyses were not obtained or performed in our combination dosingstudies, the PK data for POS and the PK-PD data for POS monotherapyand AMB monotherapy were reported previously. Following oraladministration of POS to mice, Nomeir et al. (16) observed adose-related increase in the maximum concentration in serum(up to 80 mg/kg) and area under the concentration-time curve(AUC, up to 120 mg/kg). Andes et al. (4) studied PK-PD datafor POS against C. albicans in neutropenic mice and reportedthat the 24-h AUC/MIC ratio was the PK-PD parameter associatedwith POS efficacy in this model. The mean free drug AUC/MICratio of 16.9 for POS was similar to the ratio of 25 observedwith other triazoles. In addition, these authors also indicatedthat POS exhibited prolonged (20 to 30 h) postantifungal effect(PAFE) for free drug, potentially due to sub-MIC effects. Andeset al. (3) also reported that the PK-PD parameter predictiveof efficacy for AMB was the peak serum level/MIC ratio and thatAMB also had a prolonged (23 to 30 h) PAFE. The prolonged PAFEsof POS and AMB may have contributed to the antifungal efficacyof combination treatment with these drugs observed in our studies.

Antagonism between azoles and AMB has been observed in some,but not other, literature reports involving animal models offungal infections. Louie et al. (10, 11) showed antagonism betweenFLC and AMB, and Sugar and Liu (23) showed the same result withitraconazole and AMB with C. albicans infection models. Lewiset al. (9) also reported that preexposure to itraconazole reducedthe efficacy of subsequent treatment with AMB in murine pulmonaryaspergillosis. The absence of FLC-AMB antagonism was observedin murine candidiasis studies (17, 21) and by George et al.(8) in an immunosuppressed rabbit model of aspergillosis, byAnaissie et al. (2) against Trichosporon beigelii infectionin mice, and by Barchiesi et al. (5) against murine systemiccryptococcosis. Other triazole-AMB combinations were also notantagonistic, including saperconazole against murine systemiccandidiasis (20) and SCH 39304 against murine systemic candidiasis(19) and cryptococcal meningitis (1).

We anticipate that POS will be used in combination with otherantifungals, potentially including AMB, for treatment of seriousfungal infections in patients. The efficacy observed in micewith the combination of POS and AMB suggests this combinationcould be effective in clinical fungal infections. However, thelack of antagonism in our studies indicates that the combinationcould potentially be tried clinically with less concern forantagonism.

ACKNOWLEDGMENTS

We thank Ferdous Gheyas for statistical analysis and Paul McNicholasfor critical review of the manuscript.

FOOTNOTES

* Corresponding author. Mailing address: Schering-Plough Research Institute, 2015 Galloping Hill Rd., Kenilworth, NJ 07033. Phone: (908) 740-3139. Fax: (908) 740-3918. E-mail: anthony.cacciapuoti{at}spcorp.com.

REFERENCES

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