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Antimicrobial Agents and Chemotherapy, November 2005, p. 4751-4753, Vol. 49, No. 11
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.11.4751-4753.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Combination Therapy with Terbinafine and Amphotericin B in a Rabbit Model of Experimental Invasive Aspergillosis

William R. Kirkpatrick,1* Ana C. Vallor,1 Robert K. McAtee,1 Neil S. Ryder,4 Annette W. Fothergill,2 Michael G. Rinaldi,2,3 and Thomas F. Patterson1,3

Departments of Medicine,1 Pathology, The University of Texas Health Science Center at San Antonio, Texas 78229-3900,2 Audie L. Murphy Division, South Texas Veterans Health Care System, San Antonio, Texas 78284,3 Novartis Institutes for Biomedical Research, Inc., Cambridge, Massachusetts 017424

Received 29 December 2004/ Returned for modification 2 February 2005/ Accepted 22 August 2005


   ABSTRACT
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Antagonistic effects of combination therapy using amphotericin B (AmB) with agents which block ergosterol synthesis are a concern. Terbinafine was evaluated with AmB to assess antagonism or synergy in a rabbit model of invasive aspergillosis. Terbinafine had relatively little activity but did not demonstrate antagonism against AmB in our model.


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One approach to improving antifungal therapies has been the use of combination therapy, which may offer the benefits of using reduced doses of toxic drugs through synergy (1, 2, 10, 11).

We employed a rabbit model of invasive aspergillosis to assess the efficacy of combination antifungal therapy (3, 8, 9, 12). In this lethal model, rabbits are made leukopenic and are further immunosuppressed with steroids. Extensive infection develops in liver, kidney, lung, and brain, similar to clinical disseminated invasive aspergillosis (6, 7).

New Zealand White rabbits (female, 2.5 kg) were immunosuppressed as previously described (3, 6, 7). Twenty-four hours after induction of immunosuppression, groups of 8 to 10 rabbits were challenged with a lethal bolus of 106 Aspergillus fumigatus conidia administered intravenously (i.v.) through a lateral ear vein. Each group contained at least one untreated control rabbit. Ceftazidime (200 mg) (Abbott Laboratories, North Chicago, IL) was administered intramuscularly daily from the day of challenge to prevent bacterial infection.

Antifungal therapy with oral terbinafine (Novartis Research Institute, Vienna, Austria) at 100 mg/kg of body weight/day (TRB 100), i.v. AmB (Fungizone; Bristol-Myers Squibb Co., Princeton, NJ) at either 0.4 (AmB 0.4) or 1.0 (AmB 1) mg/kg/day, and a combination of oral terbinafine at 100 mg/kg/day with AmB at 0.4 mg/kg/day was begun 24 h after i.v. challenge with A. fumigatus in immunocompromised rabbits and was continued for 5 days. AmB was diluted with 5% dextrose in sterile water at a ratio of 1 mg of drug to 10 ml of diluent and was given i.v. over 15 min through a lateral ear vein. Terbinafine was dissolved in polyethylene glycol 200 (Sigma Chemical, St. Louis, MO) and administered via a gastric gavage tube (Monoject, St. Louis, Mo.). All animal research procedures were approved by the Institutional Animal Care and Use Committee at the University of Texas Health Science Center at San Antonio, Texas.

Organ cultures were performed postmortem (after the death of the animal during treatment [n = 23] or 72 h after completion of therapy in the remaining treated rabbits [n = 17], thus examining one trial of 40 rabbits in 5 experimental groups). The brains, kidneys, livers, and lungs of each rabbit were aseptically removed and cultured for A. fumigatus (7).

Aspergillus fumigatus isolate P160, a clinical isolate which has been previously used in our animal studies, was grown on Sabouraud dextrose agar (Remel, Lenexa, Kan.) slants at 37°C for 24 h, and 106 conidia per milliliter suspension for injection was prepared as previously described (6, 7).

Synergy testing with terbinafine (0.008 to 2.0 µg/ml) and amphotericin B (0.015 to 2.0 µg/ml) was conducted (utilizing parameters as outlined in NCCLS document M27-A, modified for testing of A. fumigatus [4, 5]). Modifications included an inoculum of 1 x 104 CFU/ml and expansion of the test to a checkerboard broth macrodilution method. The MIC, defined as the lowest concentration without visible growth, was read at 24 and 48 h. Synergy was defined as a minimum fourfold decrease in the MIC of both agents compared to that of each drug alone, whereas antagonism occurs when one of the drugs has at least a fourfold increase in the MIC (2). The fractional inhibitory concentration index (FICI) was calculated as previously described and was defined as synergy with an FICI of ≤0.5; >0.5 but ≤4 was defined as indifference; and >4 was defined as antagonism (2). Testing was performed by The Fungus Testing Laboratory, San Antonio, Texas.

The Fisher exact test and the Wilcoxon rank sum test were used where appropriate. Statistical significance was defined as P < 0.05; adjustments were made for multiple-dose comparisons for each organ evaluated so that the level of significance was P < 0.005.

Antifungal therapy in this rabbit model of invasive aspergillosis was started 24 h after inoculation with conidia. Untreated control animals succumbed to the lethal challenge within 4 days of challenge, with a mean survival of 3.6 ± 0.2 days (range, 3 to 4 days). Rabbits receiving treatment with AmB 0.4, AmB 1, or TRB 100 alone or in combination with AmB 0.4 had reduced mortality compared to untreated, infected control rabbits, as shown in Fig. 1. Treatment with TRB 100 reduced mortality by 25% and also increased the mean days of survival to 5.1 ± 0.7 days compared to that of untreated controls. The three groups of rabbits treated with either dose of AmB or with the combination of AmB plus TRB all had 38% mortality, but while all three groups had increases in mean days of survival, only the rabbits treated with AmB 1 showed significant improvement compared to controls. The two groups treated with AmB 0.4 or AmB 0.4 plus TRB 100 had nearly identical increases in days of survival, and they survived nearly 2.5 days longer than controls and almost 1 day longer than those treated with TRB 100 alone.



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  		FIG. 1. Cumulative mortality of rabbits treated with terbinafine, amphotericin B, and a combination of terbinafine and amphotericin B. Rabbits (n = 8 per group) were challenged on the second day. Controls received no antifungal therapy. Treated rabbits had TRB 100 (TRB), AmB 0.4, AmB 1, or a combination of TRB 100 and AmB 0.4 initiated 24 h after challenge and were treated daily for 5 days. Mean days of survival (x.x ± standard errors) from the day of challenge with Aspergillus fumigatus are noted by group. *, P < 0.0007 versus controls.

 
Semiquantitative cultures of selected tissues are shown in Table 1. Each of these tissues was extensively infected in the untreated control rabbits. Rabbits treated with TRB 100 were indistinguishable from the control rabbits with respect to tissue burden of Aspergillus. The low dose of amphotericin B (AmB 0.4) and combination therapy regimen reduced the kidney (P < 0.005) and brain (P < 0.005 for combination therapy) tissue burden of Aspergillus nearly 10- to 100-fold versus that of controls, with a slight increase in efficacy (nonstatistical [NS]) seen with the combination of AmB 0.4 plus TRB 100. Small doses of AmB 0.4 and the combination of AmB 0.4 plus TRB 100 did reduce counts in the kidney and brain (NS). Reductions in colony counts were seen in all four tissues from the rabbits treated with AmB 1, with reductions of nearly 100-fold for both brain and lung (P < 0.0001) and nearly 1,000-fold for liver (P < 0.0001) and kidney (P < 0.0001).


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  		TABLE 1. Semiquantitative organ cultures of rabbits treated with antifungal therapy begun 24 h after challenge and sacrificed 96 h after completion of therapy

 
Positive organ culture results from rabbits treated with terbinafine and AmB are also shown in Table 1. Positive cultures were obtained from all samples of kidney, liver, and lung tissues examined from each of the untreated control animals and from seven of eight (88%) samples of brain tissue from the controls. In these experiments, none of the doses was significantly effective at sterilizing brain, kidney, liver, and lung tissues compared to controls, with the exception of kidney (P < 0.001) tissues for rabbits treated with AmB 1. Overall, combination therapy of TRB 100 plus AmB 0.4 as well as AmB 1 (P < 0.05) alone were more effective than TRB 100 alone at sterilizing brain, kidney, and liver (AmB 1) tissues compared to controls and showed improvement over AmB 0.4 (NS). Thus, these in vivo data suggest that while the combination of AmB 0.4 plus TRB 100 in this model had results similar to those of low-dose AmB 0.4 alone, these studies did not support the presence of antagonism with this combination.

In vitro studies with the A. fumigatus isolate used in our animal model showed the isolate to be susceptible to AmB (MIC at 48 h, 1 µg/ml), TRB (MIC at 48 h, 0.5 µg/ml), and the combination of AmB plus TRB (MIC at 48 h, 0.5 + 0.008 µg/ml). The combination of these two drugs in vitro shows that TRB has an indifferent effect in combination with AmB, inducing a 1-dilution reduction in MIC of AmB, as shown in Table 2. The absence of apparent antagonism is supported in our in vitro examination of the isolate we used in these in vivo studies.


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  		TABLE 2. MICs of TRB and AmB alone and in combination against Aspergillus fumigatus isolate P160

 
In our rabbit model using our single Aspergillus isolate, TRB had minimal activity alone but did not demonstrate antagonism against AmB. Therapy with antifungal combinations may yield therapeutic advantages against serious fungal disease through synergy or, to a lesser extent, through indifferent interactions.


   ACKNOWLEDGMENTS
 
This study was supported by a grant from the Novartis Research Institute, Vienna, Austria.


   FOOTNOTES
 
* Corresponding author. Mailing address: The University of Texas Health Science Center at San Antonio, Department of Medicine, Division of Infectious Diseases, 7703 Floyd Curl Drive, Mail Code 7881, San Antonio, TX 78229-3900. Phone: (210) 567-4823. Fax: (210) 567-3303. E-mail: kirkpatrick{at}uthscsa.edu. Back


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Antimicrobial Agents and Chemotherapy, November 2005, p. 4751-4753, Vol. 49, No. 11
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.11.4751-4753.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.




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