1 School of Medicine, University of Manchester, Hope Hospital, Salford, Manchester M6 8HD; 2 Department of Microbiology, Salford Royal Hospital NHS Trust, Salford, Manchester M6 8HD; 3 Wythenshawe Hospital, Southmoor Road, Manchester M23 9LT, UK
Received 27 June 2001; returned 4 March 2002; revised 22 April 2002; accepted 12 May 2002
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Thus new agents are being studied in invasive aspergillosis. Some compounds are related to the existing compounds, such as posaconazole. Resistance within classes may become problematic.7a A different approach to the treatment of refractory infections is the use of combinations of drugs already in use, even if one of them lacks activity against Aspergillus. A good result would be synergy or an additive effect of both drugs in combination.
In this work we studied the in vitro interaction of the allylamine terbinafine with itraconazole, fluconazole, amphotericin B and flucytosine against four species of Aspergillus. Thus we have extended the existing data on interactions already studied8,9 by including new drugs and Aspergillus species combinations, as well as cidality tests. Terbinafine is a synthetic naphthalenemethanamine that inhibits squalene epoxidase, a key enzyme in ergosterol biosynthesis of fungi. Its mode of action is highly selective, i.e. it is much more inhibitory to fungal than to mammalian sterol biosynthesis. Terbinafine can be administered orally and extensive use in humans indicates that it is well tolerated. It is highly potent against dermatophytes in vitro10 and is also active in vitro against Aspergillus spp.11,12 It has also been reported to be as effective as amphotericin B and itraconazole in the treatment of mild bronchopulmonary aspergillosis in non-immunocompromised patients.13
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Nine selected Aspergillus clinical isolates from Hope Hospital, Salford, UK were studied: three A. fumigatus, two A. flavus, two Aspergillus niger and two A. terreus. They were chosen from a large collection that has been susceptibility tested because each isolate had either a high or low MIC of terbinafine.11 One of the A. fumigatus isolates, AF72, was selected because it is resistant to itraconazole.7
Antifungal agents
Terbinafine (Novartis Pharma, Basel, Switzerland) was provided as the standard hydrochloride salt. Itraconazole (Janssen Research Foundation, Beerse, Belgium), fluconazole (Pfizer Ltd, Kent, UK) and flucytosine (Roche Products Limited, Hertfordshire, UK) were obtained as powders. A commercially available preparation of amphotericin B (Fungizone; E.R. Squibb, Hounslow, UK) was used. Stock solutions (3200 mg/L) of all drugs were prepared using appropriate solventsterbinafine (dimethylsulphoxide containing 5% Tween 80), itraconazole (acetone and hydrochloric acid in a water bath at 60°C), fluconazole, amphotericin B and flucytosine (sterile distilled water)adjusting for potency where necessary. Each stock was then dispensed into aliquots and stored, protected from the light, at 20°C.
Antifungal susceptibility testing
Drug interactions were assessed using a chequerboard titration broth microdilution-based method, derived from published work from our laboratory, validated in vivo and capable of detecting itraconazole resistance in A. fumigatus but that failed to do so for amphotericin B.14,15 Testing of all drugs was performed in a single batch of casitone (pH 7.0; Difco, Detroit, MI, USA) supplemented with 2% glucose, or, for itraconazole only, also in RPMI-1640 medium (with L-glutamine, without bicarbonate) (Sigma) supplemented with 2% glucose, buffered to pH 7.0 with 0.165 M MOPS. Previous work has shown casitone and RPMI-1640 to be essentially equivalent for itraconazole, terbinafine and amphotericin B, with slightly lower MICs in casitone. Doubling dilutions of each drug, at a concentration four times the targeted final concentration, were prepared in 50 µL volumes of the medium in the wells of a flat-bottomed microtitre plate. Then 50 µL of the other drug tested, at a concentration of four times the targeted final concentration, was dispensed. Final drug concentration ranges employed were chosen according to the MIC previously obtained for each strain, in order to be able to demonstrate synergy or antagonism. They varied from 8 to 0.0001 mg/L of terbinafine, 80.007 mg/L of itraconazole, 5124 mg/L of fluconazole, 160.25 mg/L of amphotericin B and 10241 mg/L of flucytosine. Fungal inocula densities were counted with an improved Neubauer haemocytometer and then adjusted in medium to obtain a 2x concentration. One hundred microlitres were added to each well of the microdilution trays, giving a final inoculum in the wells of 5 x 104 cfu/mL. Microtitre trays were incubated at 37°C for 48 h. The MICs were read visually and were defined as the lowest drug concentration with no growth. For descriptive purposes an MIC of >1024 mg/L is considered as equal to 2048 mg/L, >512 mg/L as 1024 mg/L, >16 as 32 mg/L and >8 as 16 mg/L.
Drug interactions were classified on the basis of the fractional inhibitory concentration (FIC) index. The FIC index is the sum of the FICs for each drug; the FIC is defined as the MIC of each drug when used in combination divided by the MIC of the drug when used alone. The interaction was defined as synergic if the FIC index was 0.50, additive if the FIC index was >0.501.0, indifferent if the FIC index was >1.02.0 and antagonistic if the FIC index was >2.0.8 When only antagonism appeared, the higher FIC values were taken. The lower one was taken in the rest of cases. FIC reproducibility was studied for the drugs that gave synergy in combination with terbinafine (itraconazole and fluconazole).
To determine the minimal fungicidal concentrations (MFCs), 100 µL samples were withdrawn from the wells containing each drug alone and in combination and for wells containing all concentrations above the MIC. The samples were inoculated on to Sabouraud dextrose agar plates, and the liquid was allowed to dry. The plates were then streaked with a sterile loop and incubated at 37°C for 48 h. The MFC was defined as the lowest concentration of the drug alone and in combination at which no colonies grew (>99.99% kill). Fractional fungicidal concentrations (FFCs) were computed as FICs, with the same interpretation for interactions of fungicidality.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Itraconazole was synergic in its interaction with terbinafine (MICs) with two A. fumigatus, both A. niger and both A. terreus isolates, in both testing media (Tables 1 and 2). Remarkable synergy (FIC = 0.281) was seen with the itraconazole-resistant isolate AF72. For these isolates synergy was also demonstrated in MFCs, except for the three A. fumigatus isolates, for which an additive effect was seen with casitone and indifference for two of them in RPMI. Itraconazole MICs for susceptible isolates fell from 0.1250.5 mg/L in RPMI to 0.0070.125 mg/L in combination with terbinafine. The A. flavus isolates both showed an additive or synergic effect for MIC, as well as for MFC in RPMI, but indifference or antagonism was seen in casitone. When synergy was documented, the reductions in MIC were 2- to 32-fold for both terbinafine and itraconazole. The reductions in MFC were 2- to 16-fold for terbinafine and 4- to 64-fold for itraconazole.
|
|
The A. fumigatus and A. terreus and one of the A. flavus isolates showed synergy (for MIC and MFC) in their interaction with terbinafine. MICs of fluconazole fell from 256512 mg/L to 16128 mg/L in these isolates in combination with terbinafine. For the other isolates the interaction was additive or indifferent. With the A. niger isolates fluconazole MICs were higher than the maximum obtainable concentration of fluconazole (this antifungal is insoluble at higher concentrations). Thus a synergic effect could not be detected. When synergy was documented, the reductions in MIC were 4-fold for terbinafine and 4- to 16-fold for fluconazole. The reductions in MFCs were 4- to 8-fold for terbinafine and 4- to 16-fold for fluconazole.
Amphotericin B
The combination of terbinafine with amphotericin B was indifferent or antagonistic with MICs for all isolates, except for one A. fumigatus isolate in which it was additive. Synergy or an additive effect with MFCs was shown for both A. fumigatus isolates and one A. niger isolate but antagonism of killing was demonstrated for the other species.
Flucytosine
Regarding MICs, the interaction of flucytosine with terbinafine was mainly indifferent or antagonistic. No synergy was demonstrated. One isolate of A. niger showed an additive effect for MIC but antagonism of killing. For MFCs all the interactions were antagonistic.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In published work from our laboratory11 we reported that terbinafine has potent in vitro activity against A. flavus [geometric mean (GM) MIC = 0.10 mg/L], A. niger (GM MIC 0.19 mg/L) and A. terreus (GM 0.16 mg/L), but limited activity against A. fumigatus (GM 19.03 mg/L). Peak plasma concentrations in humans of 1.7 mg/L are typically seen within 1.5 h of an oral 250 mg dose.16 No intravenous preparation is available. Thus the pharmacodynamics in patients are favourable for non-fumigatus infections but not for those caused by A. fumigatus. However, in combination with an azole, terbinafine MICs fall 4-fold for A. fumigatus, providing a more favourable pharmacodynamic relationship for response. Nevertheless, the in vivo correlation for these in vitro interactions is difficult to establish due to the difficulties of ensuring bioavailability of terbinafine in animal models.
We believe that these observations of synergy may be significant for several reasons: (i) the in vitro method employed derives from an in vivo validated one, capable of detecting itraconazole resistance in A. fumigatus; (ii) we have used a stringent criterion for the definition of synergy; (iii) FIC reproducibility is good when synergy is observed; and (iv) both terbinafine and itraconazole ultimately block ergosterol synthesisterbinafine earlier in the pathway (squalene to squalene epoxide) than itraconazole (lanosterol to ergosterol). There are several examples of a proven synergic interaction between two antibacterial compounds acting at different steps of the same pathway. Possibly the best known example is the case of the combination of trimethoprim and the sulphonamides inhibiting folate metabolism.
With respect to amphotericin B and flucytosine, combination with terbinafine resulted in an indifferent or, often, even antagonistic effect on MICs and MFCs. A clinical trial of combined terbinafine (750 mg daily) and amphotericin B has been concluded and unpublished results indicate a worse outcome in the terbinafine combination arm compared with amphotericin B alone. Based on our in vitro results, a worse outcome in the combination group would have been anticipated.
The in vitro interaction of terbinafine with amphotericin B, fluconazole, itraconazole and voriconazole has already been studied against a number of difficult-to-treat fungal pathogens in vitro, showing potent synergy with these azoles, and in some cases with amphotericin B.8,17 Regarding Aspergillus, the activity of terbinafine in combination with itraconazole, fluconazole, voriconazole or amphotericin B has been shown to be synergic, or sometimes additive, against several A. fumigatus isolates and one of A. niger.8 Another study showed synergic activity of terbinafine with itraconazole against A. flavus, A. niger, A. terreus, Aspergillus glaucus, Aspergillus clavatus, Aspergillus candidus and particularly against A. fumigatus.9 Our results with amphotericin B differ from those obtained in these previous studies. Since no in vitro method has been validated to be capable of detecting in vivo amphotericin B resistance,15 any in vitro susceptibility testing result involving this drug should be interpreted with caution.
In conclusion, these findings suggest that addition of terbinafine to triazole regimens may be useful in the treatment of infections caused by Aspergillus. In contrast, the combination of amphotericin B and terbinafine should be avoided. Clinical studies are warranted to further elucidate the potential utility of this combination therapy.
![]() |
Acknowledgements |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Denning, D. W. (1998). Invasive aspergillosis. Clinical Infectious Diseases 26, 781805.[ISI][Medline]
3
.
Verweij, P. E., Oakley, K. L., Morrissey, J., Morrissey, G. & Denning, D. W. (1998). Efficacy of LY303366 against amphotericin B-susceptible and -resistant Aspergillus fumigatus in a murine model of invasive aspergillosis. Antimicrobial Agents and Chemotherapy 42, 8738.
4
.
Odds, F. C., Gerven, F. V., Espinel-Ingroff, A., Bartlett, M. S., Ghannoum, M. A., Lancaster, M. V. et al. (1998). Evaluation of possible correlations between antifungal susceptibilities of filamentous fungi in vitro and antifungal treatment outcomes in animal infection models. Antimicrobial Agents and Chemotherapy 42, 2828.
5
.
Dannaoui, E., Borel, E., Persat, F., Piens, M. A. & Picot, S. (2000). Amphotericin B resistance of Aspergillus terreus in a murine model of disseminated aspergillosis. Journal of Medical Microbiology 49, 6016.
6 . Dannaoui, E., Borel, E., Persat, F., Monier, M. F. & Piens, M. A. (1999). In-vivo itraconazole resistance of Aspergillus fumigatus in systemic murine aspergillosis. Journal of Medical Microbiology 48, 108793.[Abstract]
7 . Denning, D. W., Venkateswarlu, K., Oakley, K. L., Anderson, M. J., Manning, N. J., Stevens, D. A. et al. (1997). Itraconazole resistance in Aspergillus fumigatus. Antimicrobial Agents and Chemotherapy 41, 13648.[Abstract]
7
.
Mosquera, J. & Denning, D. W. (2002). Azole cross-resistance in Aspergillus fumigatus. Antimicrobial Agents and Chemotherapy 46, 5567.
8 . Ryder, N. S. (1999). Activity of terbinafine against serious fungal pathogens. Mycoses 42, 1159.[ISI][Medline]
9
.
Moore, C. B., Walls, C. M. & Denning, D. W. (2001). In vitro activities of terbinafine against Aspergillus species in comparison with those of itraconazole and amphotericin B. Antimicrobial Agents and Chemotherapy 45, 18825.
10 . Schmitt, H. J., Bernard, E. M., Andrade, J., Edwards, F., Schmitt, B. & Armstrong, D. (1988). MIC and fungicidal activity of terbinafine against clinical isolates of Aspergillus spp. Antimicrobial Agents and Chemotherapy 32, 7801.[ISI][Medline]
11 . Schiraldi, G. F., Colombo, M. D., Harari, S., Lo-Cicero, S., Ziglio, G., Ferrarese, M. et al. (1996). Terbinafine in the treatment of non-immunocompromised compassionate cases of bronchopulmonary aspergillosis. Mycoses 39, 512.[ISI][Medline]
12 . Denning, D. W., Radford, S. A., Oakley, K. L., Hall, L., Johnson, E. M. & Warnock, D. W. (1997). Correlation between in-vitro susceptibility testing to itraconazole and in-vivo outcome of Aspergillus fumigatus infection. Journal of Antimicrobial Chemotherapy 40, 40114.[Abstract]
13
.
Johnson, E. M., Oakley, K. L., Radford, S. A., Moore, C., Warn, P., Warnock, D. W. et al. (2000). Lack of correlation of in vitro amphotericin B susceptibility testing with outcome in a murine model of Aspergillus infection. Journal of Antimicrobial Chemotherapy 45, 8593.
14 . Ryder, N. S. & Leitner, I. (2001). Synergistic interaction of terbinafine with triazoles or amphotericin B against Aspergillus species. Medical Mycology 39, 915.[ISI][Medline]
15 . Kovarik, J. M., Mueller, E. A., Zehender, H., Denouel, J., Caplain, H. & Millerioux, L. (1995). Multiple-dose pharmacokinetics and distribution in tissue of terbinafine and metabolites. Antimicrobial Agents and Chemotherapy 39, 273841.[Abstract]
16
.
Meletiadis, J., Mouton, J. W., Rodriguez-Tudela, J. L., Meis, J. F. G. M. & Verweij, P. E. (2000). In vitro interaction of terbinafine with itraconazole against clinical isolates of Scedosporium prolificans. Antimicrobial Agents and Chemotherapy 44, 4702.
17 . Rodero, L., Vitale, R., Cordoba, S., Reinoso, E. H. & Davel, G. (1998). In vitro activity of terbinafine and the combination with itraconazole against Aspergillus species. In Programs and Abstracts of the Thirty-eighth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, 1998. Abstract E-63b, p. 187. American Society for Microbiology, Washington, DC, USA.