In vitro interactions of anidulafungin with azole antifungals, amphotericin B and 5-fluorocytosine against Candida species

International Journal of Antimicrobial Agents 27 (2006) 174–177

In vitro interactions of anidulafungin with azole antifungals, amphotericin B and 5-fluorocytosine against Candida species James A. Karlowsky a,∗ , Daryl J. Hoban a , George G. Zhanel a , Beth P. Goldstein b a

Department of Medical Microbiology, Faculty of Medicine, University of Manitoba and Department of Clinical Microbiology, Health Sciences Centre, MS673–820 Sherbrook Street, Winnipeg, Man., Canada R3A 1R9 b Vicuron Pharmaceuticals, King of Prussia, PA 19406, USA Received 1 August 2005; accepted 30 October 2005

Abstract Anidulafungin, an echinocandin, is in late stage development for the treatment of fungal infections. We investigated the activity of anidulafungin in combination with other antifungal agents (fluconazole, itraconazole, ketoconazole, amphotericin B and 5-fluorocytosine) against four isolates each of Candida albicans, Candida glabrata, Candida parapsilosis and Candida tropicalis, and two isolates of Candida krusei using a macrobroth chequerboard method with interactions evaluated by fractional inhibitory concentration indices (FICIs). Additive activity (FICI > 0.5 to 1) or indifference (FICI > 1 to <4) was observed in 85 of 90 interactions of anidulafungin with another antifungal agent. Synergy with itraconazole (FICI ≤ 0.5) was observed for one strain of C. glabrata, and antagonism with ketoconazole (FICI ≥ 4), a drug rarely used systemically, was noted for four strains of C. tropicalis. The combination of anidulafungin and amphotericin B demonstrated additive activity for each of the 18 isolates of Candida tested. These results suggest additional studies are warranted, for example in animal models, to evaluate further the potential of combination antifungal therapy with anidulafungin for Candida infections. © 2005 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Anidulafungin; Candida; Antifungal combination; Azoles; Amphotericin B

1. Introduction Fungal infections have increased in recent decades. Those caused by Candida species are the most common, and these organisms are the fourth most frequent cause of nosocomial bloodstream infections in the USA [1] and are associated with significant morbidity and mortality [2]. Although Candida albicans is most frequently isolated, candidaemia due to nonalbicans species has increased in recent years [3] and these species are more frequently resistant to azole and polyene antifungal agents [4]. Echinocandins are a recently introduced class of antifungal agents. Anidulafungin is a semisynthetic echinocandin under development for the treatment of fungal infections including invasive candidiasis. Anidulafungin, like other ∗

Corresponding author. Tel.: +1 204 787 1275; fax: +1 204 787 4699. E-mail address: [email protected] (J.A. Karlowsky).

echinocandins, targets the fungal cell wall [5]. Anidulafungin has potent activity against all species of Candida, is fungicidal and is active against strains resistant to amphotericin B and azoles [4,6–16]. The efficacy of anidulafungin has been demonstrated in stringent animal models of Candida infections using susceptible strains as well as organisms resistant to other antifungal agents [17–19]. Recently concluded clinical trials have demonstrated the safety and efficacy of intravenously administered anidulafungin against oesophageal candidiasis and candidaemia [20,21]. Patients with fungal infections are often immunocompromised and/or seriously ill. Fungicidal drugs may offer an advantage in some of these patients and combination therapy may be beneficial in situations in which the mortality and morbidity of these infections is high. Using the chequerboard method, we evaluated the effect of antifungal combinations of anidulafungin with three azoles (fluconazole, itraconazole, ketoconazole), amphotericin B and 5-fluorocytosine against

0924-8579/$ – see front matter © 2005 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2005.10.014

J.A. Karlowsky et al. / International Journal of Antimicrobial Agents 27 (2006) 174–177

isolates of C. albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis and Candida krusei.

175

The results were interpreted as follows: synergy (FICI ≤ 0.5); additive activity (FICI > 0.5–1); indifference (FICI > 1 to <4); and antagonism (FICI ≥ 4). All FICs were read in test tubes demonstrating 100% growth inhibition.

2. Methods 2.1. Candida isolates

3. Results

Eighteen candidaemia isolates, obtained from the Department of Clinical Microbiology, Health Sciences Centre (Winnipeg, Manitoba, Canada), were used in this investigation. The isolates comprised: four C. albicans (S313, S381, S297, M1509), four C. glabrata (M391, E282, M735, L1575), four C. tropicalis (D1407, H212, H1114, L1193), four C. parapsilosis (L950, L1447, L1834, M1130) and two C. krusei (E175, K1865). Several fluconazole non-susceptible isolates were included. Isolates were subcultured twice on Sabouraud agar prior to initial susceptibility testing and subsequent synergy testing.

Table 1 lists the MICs of individual agents for the 18 Candida isolates and Table 2 lists the FICIs and interpretations for the various combinations. Additive activity or indifference was observed in 85 of the 90 interactions, including with fluconazole against fluconazole-resistant isolates. Anidulafungin and amphotericin B showed additive activity against all 18 isolates. Against C. glabrata, additive activity with fluconazole was seen against all four strains, including two strains that were non-susceptible (MIC = 32 mg/L) to fluconazole. Synergy was observed with anidulafungin and itraconazole for a single isolate of C. glabrata. Antagonism was noted for the combination with ketoconazole against all four strains of C. tropicalis.

2.2. Antifungal agents Stock solutions of anidulafungin (Eli Lilly, Indianapolis, IN), amphotericin B (Bristol-Myers Squibb, St Laurent, Canada), fluconazole (Pfizer Canada, Kirkland, Canada), itraconazole (Janssen/Ortho, North York, Canada) and ketoconazole (Janssen/Ortho) were prepared in dimethyl sulfoxide. 5-Fluorocytosine (Hoffman-La Roche, Mississauga, Canada) was prepared in water. 2.3. Chequerboard tests Minimum inhibitory concentrations (MICs) were determined in RPMI 1640 buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS) following 48 h incubation at 35 ◦ C. MICs were defined as 100% growth inhibition for anidulafungin and amphotericin B and as the lowest concentration that resulted in an 80% reduction of visual growth compared with growth of the control for fluconazole, ketoconazole, itraconazole and 5-fluorocytosine. Following initial MIC determinations, eight doubling dilutions of each pair of antifungal agents to be tested for synergy were prepared in RPMI 1640. The concentration ranges tested for synergy were strain specific and based on previous MIC determinations for each of the individual agents. Individual dilutions (0.5 mL) of each agent were placed in test tubes to provide 64 combinations for each pair of compounds. The MIC of each individual antifungal agent was confirmed along with each chequerboard test. Controls included fungal growth in drug-free medium and sterility testing of uninoculated medium. The final inoculum tested was 5 × 102 to 2.5 × 103 colony-forming units/mL. Viable counts of initial inocula were determined by 10-fold serial dilution in normal saline and spreading on Sabouraud agar. Fractional inhibitory concentrations (FICs), determined for each single agent in a combination, were summed to give FIC indices (FICIs).

4. Discussion Treatment of many fungal infections continues to be a challenge. Combination antifungal therapy may be of potential benefit for the treatment of candidiasis if it augments efficacy, permits the use of lower doses of toxic agents (such as amphotericin B) or shortens the duration of therapy. Other potential advantages may include expanding the spectrum of activity of antifungal agents, complementary pharmacokinetic profiles or reducing the emergence of resistance. It is also important to exclude antagonistic interactions, as switching patients from treatment with one single agent to another involves a period of overlap in which two agents may be present simultaneously for several hours. In vitro data regarding antifungal combinations against Candida species are not definitive; however, certain trends are apparent. The combination of amphotericin B and azoles has resulted in mostly indifferent or antagonistic interactions [22,23]. Antagonism has been attributed to the fact that both of these classes of antifungal agents affect sterol synthesis; reduction in membrane ergosterol content by azoles could reduce the targets available for polyene interaction. In contrast, 5-fluorocytosine, which has an unrelated target, has exhibited mostly additive or synergistic interactions with amphotericin B or azoles [22]. Anidulafungin, like other echinocandin antifungal agents, is a non-competitive inhibitor of (1,3)-␤-d-glucan synthase, an enzyme required for synthesis of an essential cell wall component of Candida, and is fungicidal for Candida species [5]. The FICIs obtained with the combination of anidulafungin and amphotericin B in this study against different species of Candida (0.56–1, additive activity) were similar to previous observations with another echinocandin,

J.A. Karlowsky et al. / International Journal of Antimicrobial Agents 27 (2006) 174–177

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Table 1 Minimum inhibitory concentrations (MICs) of individual antifungal agents against isolates of Candida species Candida species and isolate

MIC (mg/L) Anidulafungin

Fluconazole

Itraconazole

Ketoconazole

Amphotericin B

5-Fluorocytosine

4 16 2 0.5

1 1 0.015 0.015

0.5 0.5 0.06 0.5

4 0.25 0.12 0.12

1 2 1 1

1 0.015 0.015 0.015

0.5 0.25 1 0.5

0.12 0.06 0.06 0.06

C. albicans S313 S381 S297 M1509

0.04 0.08 0.02 0.08

C. glabrata M391 E282 M735 L1575

0.08 0.08 0.16 0.32

C. tropicalis D1407 H212 H1114 L1193

0.16 0.08 0.16 0.16

128 256 0.25 0.12

2 2 1 0.25

0.06 4 0.015 0.008

1 1 0.25 1

0.12 8 0.25 0.5

C. parapsilosis L950 L1447 L1834 M1130

1.28 2.56 2.56 1.28

0.25 2 4 0.5

0.06 0.06 0.12 0.12

0.06 0.03 0.06 0.03

0.5 0.5 0.25 1

0.12 0.12 0.06 0.12

C. krusei E175 K1865

0.25 1.28

2 2

0.06 0.5

2 2

128 256 1 0.125 32 32 2 2

16 32

32 16

Table 2 Fractional inhibitory concentration indices (FICIs) of antifungal combinations against isolates of Candida species and their interpretations Candida species and isolate

Anidulafungin + Fluconazole

Ketoconazole

Itraconazole

Amphotericin B

5-Fluorocytosine

FICI

Interpretation

FICI

Interpretation

FICI

Interpretation

FICI

Interpretation

FICI

Interpretation

C. albicans S313 S381 S297 M1509

2 1.5 1 0.75

Indifference Indifference Additive Additive

2 2 2 2

Indifference Indifference Indifference Indifference

0.75 3 1 0.75

Additive Indifference Additive Additive

0.75 0.75 1 0.75

Additive Additive Additive Additive

2 2 1 2

Indifference Indifference Additive Indifference

C. glabrata M391 E282 M735 L1575

0.63 0.75 0.63 0.75

Additive Additive Additive Additive

0.75 2 1 1

Additive Indifference Additive Additive

0.75 0.25 0.75 0.75

Additive Synergy Additive Additive

0.75 0.75 0.75 0.75

Additive Additive Additive Additive

2 2 3 3

Indifference Indifference Indifference Indifference

C. tropicalis D1407 H212 H1114 L1193

2 1.5 1.5 2

Indifference Indifference Indifference Indifference

6 4 5 4

Antagonism Antagonism Antagonism Antagonism

3 2.25 1 2.25

Indifference Indifference Additive Indifference

0.56 0.56 0.56 0.63

Additive Additive Additive Additive

0.75 1 2 0.75

Additive Additive Indifference Additive

C. parapsilosis L950 L1447 L1834 M1130

2 2 2 2

Indifference Indifference Indifference Indifference

2 2 2 2

Indifference Indifference Indifference Indifference

2 2 2 2

Indifference Indifference Indifference Indifference

0.75 0.75 0.75 0.75

Additive Additive Additive Additive

2 2 1 0.75

Indifference Indifference Additive Additive

C. krusei E175 K1865

2 3

Indifference Indifference

2 2

Indifference Indifference

0.63 2.5

Additive Indifference

0.63 0.56

Additive Additive

0.75 1

Additive Additive

J.A. Karlowsky et al. / International Journal of Antimicrobial Agents 27 (2006) 174–177

caspofungin, against C. albicans (FICIs, 0.74–0.90) [24,25]. Most azole–anidulafungin combinations showed indifference, although additive activity was uniformly seen with fluconazole against C. glabrata (including fluconazole-nonsusceptible strains) and against two of four strains of C. albicans. In a previous study using the time–kill method, fluconazole demonstrated an indifferent interaction with either anidulafungin or caspofungin against C. albicans, C. glabrata, C. krusei and C. tropicalis [26]. In the present study, antagonism occurred only with ketoconazole, a drug not currently used systemically, against one species (C. tropicalis). Antagonism was not observed for combinations of anidulafungin and commonly prescribed systemic antifungal agents. The chequerboard method is a convenient preliminary in vitro test of the interaction between different classes of antimicrobial agents. The observation that anidulafungin showed additive activity or indifference with other antifungal compounds against Candida spp. suggests that experiments in animal models might provide useful additional information about the potential for combination therapy for serious Candida infections in humans.

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