Interaction between Posaconazole and Caspofungin in Concomitant Treatment of Mice with Systemic Aspergillus Infection

Abstract

The interaction of posaconazole and caspofungin was evaluated in concomitant treatment of Aspergillus fumigatus (two strains) or A. flavus (one strain) systemic infections in immunocompetent mice. Survival curves for mice treated with the combinations were compared statistically with those for mice treated with the component monotherapies. No antagonism was observed.

Invasive aspergillosis continues to be a serious threat to immunocompromised patients, resulting in very significant mortality (2, 8) despite antifungal therapy (15). Outcomes could potentially be improved through the use of combination antifungal therapy (13, 14).

Combination treatment using antifungal agents with different mechanisms of action could potentially be synergistic against serious fungal infections. Posaconazole (POS) is a broad-spectrum antifungal triazole which recently completed phase III clinical trials (6). POS, like other triazoles, inhibits 14-demethylase activity, resulting in inhibition of the synthesis of ergosterol, a key component of fungal cell membranes. Caspofungin (CSP), an echinocandin, inhibits the synthesis of 1,3-beta-d-glucan, thereby interfering with cell wall synthesis (1). The different mechanisms of action of POS and CSP suggest that they may be complementary when used in combination. In this report, we examine the interaction between POS and CSP in combination therapy against systemic Aspergillus infections in mice.

Aspergillus fumigatus ND158 and ND231 and A. flavus ND83 were obtained from the Schering-Plough Research Institute culture collection. The minimum effective concentration (MEC) end points for CSP were described by Arikan et al. (4). MICs for POS and CSP were determined and end points read according to CLSI methodology (M38-A) (10). Drug interactions between POS and CSP were determined by a checkerboard microdilution MIC method. Preparation of the inoculum, drug dilutions, and reading of end points for drug interaction tests were performed as described in document M38-A. The drug interaction studies were scored by using the azole end point (100% inhibition of growth) and, consequently, were primarily a measure of the impact of CSP on the POS MIC. The fractional inhibitory concentration (FIC) index (5) was defined as synergistic if the FIC was ≤0.5, indifferent if the FIC was >0.5 but ≤4, and antagonistic if the FIC was >4.

For infection studies, A. fumigatus ND158 and ND231 were grown on Sabouraud dextrose agar slants and A. flavus ND83 was grown on malt extract agar slants for 7 days at 28°C. The conidia were washed off the slants and used to inoculate tissue culture flasks containing malt extract agar, which were incubated for 7 to 8 days at 28°C. Conidia from four flasks were suspended and diluted 1:5 in saline, and this suspension was used for infection of immunocompetent mice on day 0 by intravenous injection of 0.1 ml into the tail vein. Inocula (CFU/mouse), determined by plate counts on Sabouraud dextrose agar, were 1.5 × 10⁶ and 9.5 × 10⁶ for ND158, 1.9 × 10⁶, 4.4 × 10⁶, and 4.9 × 10⁶ for ND231, and 6.7 × 10⁵ and 1.7 × 10⁶ for ND83 in two, three, and two experiments, respectively.

A POS clinical oral suspension was diluted in sterile water for injection. CSP (Cancidas) was obtained from Merck & Co. Inc., Whitehouse Station, NJ, and was prepared according to the manufacturer's directions. Dilutions of CSP were made in sterile 0.9% NaCl for injection. Drug therapy for groups of eight mice began at 4 h postinfection on day 0 and continued once daily through day 7 for A. fumigatus ND158 and ND231 and day 3 for A. flavus ND83. The shorter treatment period for A. flavus ND83 was based on earlier studies which showed that POS was more active in vivo against A. flavus than A. fumigatus (6). POS and CSP were each tested at three dose levels as monotherapies and in all possible combinations in a checkerboard fashion, comprising nine drug combination groups per strain (a total of 27 combinations for all three strains; see Table 2). The doses were selected from preliminary dose-response experiments (data not shown) to ensure that POS and CSP would be tested in combinations involving high, intermediate, and low doses of each drug. Concomitant combination therapy was achieved by administering POS orally, followed immediately by CSP administered intraperitoneally. Control animals were administered sterile water by oral injection and given a 0.9% NaCl injection intraperitoneally. Survival was monitored for 14 or 15 days. Ninety to 100% of control mice were dead by days 4, 7, and 6 postinfection with strains ND158, ND231, and ND83, respectively. The results from two experiments each with A. fumigatus ND158 and A. flavus ND83 and from three experiments with A. fumigatus ND231 were pooled for statistical analysis. Log-rank tests were performed to compare the survival curve (Kaplan-Meier analysis, day 0 to end of experiment) for each combination to that for the most active monotherapy. A P value of <0.05 indicated that the survival curves were statistically different. No multiplicity adjustments were made. Fisher's exact test was applied when log-rank tests were invalid. CF1 mice (white, male, 18 to 20 g at time of infection) from Charles River Laboratories (Wilmington, MA) were used in these studies, which were carried out in accordance with the Guide to the Care and Use of Laboratory Animals of the National Institutes of Health (11) and the Animal Welfare Act in an Association for Assessment Accreditation of Laboratory Animal Care-accredited program.

TABLE 2.

Effect of POS and CSP combinations on mouse survival against Aspergillus strains

Strain	Combination				% Survival with POS alonea,c		% Survival with CSP alonea,c
	Dose levels (POS + CSP) (mg/kg)b	% Survivalc		P valued	Dld	End	Dld	End
		Dld	End
A. fumigatus ND158	25 + 10	100	100	1.00	94	94	25	25
	25 + 5	100	100	1.00	94	94	19	19
	25 + 1	100	100	1.00	94	94	7	7
	5 + 10	69	69	0.53	63	63	25	25
	5 + 5	50	44	0.51	63	63	19	19
	5 + 1	63	57	0.92	63	63	7	7
	1 + 10	32	32	0.97	7	7	25	25
	1 + 5	19	19	0.64	7	7	19	19
	1 + 1	0	0	0.89	7	7	7	7
A. fumigatus ND231	5 + 5	92	92	0.99	92	92	50	50
	5 + 1	88	88	0.66	92	92	54	54
	5 + 0.2	92	92	0.97	92	92	13	4
	1 + 5	79	79	0.04	50	38	50	50
	1 + 1	75	75	0.13	50	38	54	54
	1 + 0.2	79	63	0.05	50	38	13	4
	0.2 + 5	71	71	0.16	8	2	50	50
	0.2 + 1	79	79	0.08	8	2	54	54
	0.2 + 0.2	38	34	0.04	8	2	13	4
A. flavus ND83	5 + 1	100	82	0.12	100	19	88	57
	5 + 0.2	100	25	0.49	100	19	88	7
	5 + 0.05	100	19	0.94	100	19	82	0
	0.5 + 1	100	38	0.74	100	0	88	57
	0.5 + 0.2	94	0	<0.0001	100	0	88	7
	0.5 + 0.05	100	0	0.0072	100	0	82	0
	0.1 + 1	100	69	0.41	100	0	88	57
	0.1 + 0.2	100	0	<0.0001	100	0	88	7
	0.1 + 0.05	82	0	0.14	100	0	82	0

^aPOS or CSP alone at the dose levels used in the combination.

^bTreatment occurred once daily starting at 4 h postinfection through day 7 for A. fumigatus ND158 and ND231 and day 3 for A. flavus ND83.

^cSurvival of mice on the day after the last dose (dld; day 8 for ND158 and ND231 and day 4 for ND83) or the end of the experiment (day 14 for ND158 and day 15 for ND231 and ND83). Data are averages of two experiments for ND158 and ND83 and of three experiments for ND231 (eight mice per group per experiment).

^dP values from log-rank or Fisher's exact tests comparing survival curves (from day 0 to end of experiment) of combinations versus the most active monotherapy at the dose level used in the combination. A significant difference between survival curves was observed if the P value is <0.05.

Data from several independent in vitro assays are shown in Table 1. The POS MICs ranged from 0.03 to 0.125 μg/ml against each of the strains. The CSP MICs ranged from 32 to 128 μg/ml, and the MECs ranged from 0.03 to 0.06 μg/ml. The POS-CSP interactions with A. fumigatus ND158 and ND231 were synergistic and indifferent in different experiments, while those with A. flavus ND83 were indifferent. No antagonism was observed.

TABLE 1.

In vitro interaction between POS and CSP against Aspergillus strains

Straina	MIC (μg/ml)b			POS-CSP FIC	Interpretation	CSP MEC (μg/ml)c
	POS	CSP	POS, CSP (in combination)
A. fumigatus ND158	0.03	32	0.016, 1	0.53	Indifferent	0.03
	0.03	32	0.008, 0.5	0.27	Synergy
	0.03	128	0.016, 32	0.75	Indifferent
A. fumigatus ND231	0.125	64	0.016, 4	0.19	Synergy	0.03
	0.125	64	0.008, 32	0.56	Indifferent
A. flavus ND83	0.06	32	0.03, 2	0.56	Indifferent	0.06
	0.06	32	0.03, 1	0.53	Indifferent

^aCheckerboard assays were repeated at least twice (three times for ND158) on different days, and the results for each replicate are shown.

^bMICs were determined using CLSI methodology M38-A (10).

^cMECs were determined as described by Arikan et al. (4).

Table 2, shows the combinations used for each strain, the average percentages of survival on the day after the last dose and the day the experiments ended for the combinations and monotherapies, and the P values for the survival curve comparisons. Survival curves are shown as examples of the data in Fig. 1. As shown in Fig. 1A, mice infected with A. fumigatus ND231 and treated with a combination of POS (1 mg/kg) and CSP (5 mg/kg) survived longer than those treated with POS (1 mg/kg) or CSP (5 mg/kg) monotherapy (P = 0.04 compared to CSP monotherapy). As shown in Fig. 1B, mice infected with the same strain and treated with another combination of POS (1 mg/kg) and CSP (1 mg/kg) also survived longer than those treated with POS (1 mg/kg) or CSP (1 mg/kg) monotherapy (statistically similar to CSP monotherapy [P = 0.13]).

FIG. 1.

Concomitant combination treatments with POS and CSP of systemic A. fumigatus ND231 infections in mice. Survival results for two combinations of POS and CSP are shown (A and B), along with those for POS alone, CSP alone, and controls. Dose levels (mg/kg) are indicated in parentheses. Drugs were administered once daily starting at 4 h postinfection through day 7. Controls were administered sterile water and 0.9% NaCl. The P values were determined by comparing the survival curve for each combination to that for the most active monotherapy (in these graphs, CSP alone was slightly more active than POS alone and was used for the survival curve comparisons).

Table 3 summarizes the interactions between POS and CSP from Table 2. Against all three Aspergillus strains, 5 (18.5%) of the 27 POS and CSP combinations tested were more effective than the most active monotherapy (P < 0.05), while 22 (81.5%) were statistically similar to the most active monotherapy (P > 0.05). No combinations were antagonistic, defined as less effective than the most active monotherapy. Similar results were seen when kidney burdens were determined by culture methods (data not shown).

TABLE 3.

Summary of interactions based on mouse survival results with POS and CSP combinations against Aspergillus strains^a

Strain	No. of combinations (%)
	Total tested	More effective than most active monotherapyb	Similar to most active monotherapyb	Less effective than most active monotherapyb
A. fumigatus ND158	9	0	9	0
A. fumigatus ND231	9	2	7	0
A. flavus ND83	9	3	6	0
Total	27	5 (18.5)	22 (81.5)	0

^aTreatment occurred once daily starting at 4 h postinfection through day 7 for A. fumigatus ND158 and ND231 and day 3 for A. flavus ND83. The interactions are based on statistical analysis of survival curves through day 14 or 15 postinfection, using three dose levels of each drug alone and in all possible combinations (data from two experiments were pooled for strains ND158 and ND83 and from three experiments were pooled for ND231).

^b“More effective” or “less effective” means the survival curves for the combinations were significantly different from those for the most active monotherapy at the dose level used in the combination (P < 0.05). “Similar” means the survival curves for the combinations and the most active monotherapy were not significantly different (P > 0.05).

Overall, our studies indicate that both POS and CSP are efficacious as monotherapies or in combination and are not antagonistic against Aspergillus in mice, consistent with in vitro results. In another study, POS and CSP used in combination in vitro against 59 strains of Aspergillus, including A. fumigatus and A. flavus, were mainly indifferent or synergistic, with no antagonism (F. Sabatelli, C. Mendrick, J. Halpern, C. Norris, R. Patel, A. Cacciapuoti, and D. Loebenberg, Abstr. 43rd Intersci. Conf. Antimicrob. Agents Chemother., abstr. M-990, p. 453, 2003). Similarly, Manavathu et al. demonstrated in vitro synergy, but no antagonism, between POS and CSP against A. fumigatus strains (9).

Although pharmacokinetic (PK) data and pharmacokinetic-pharmacodynamic (PK-PD) analyses were not obtained or performed in our studies, the PK and/or PK-PD of POS or CSP have been reported previously. Wiederhold et al. (16) reported that the PK-PD index predictive of efficacy for CSP was the peak plasma level/MEC ratio. The effect of CSP on the reduction of pulmonary Aspergillus burdens in immunosuppressed mice was most prominent at a ratio of 10 to 20. In addition, following oral administration of POS to mice, Nomeir et al. (12) observed a dose-related increase in the maximum concentration in serum (up to 80 mg/kg) and the area under the curve (AUC; up to 120 mg/kg). Andes et al. (3) studied the PK-PD of POS against Candida albicans in neutropenic mice and reported that the 24-h AUC/MIC ratio was the PK-PD index associated with POS efficacy in this model. The mean free-drug AUC/MIC ratio of 16.9 for POS was comparable to the ratio of 25 observed with other triazoles. In addition, Andes et al. also indicated that POS exhibited a prolonged (20 to 30 h) postantifungal effect of free drug, potentially due to sub-MIC effects. The prolonged postantifungal effect of POS may have contributed to the antifungal efficacy of monotherapy with POS or combined treatment with POS and CSP in our studies.

There is a need for properly designed clinical trials to test the efficacy of combination antifungal therapy (7). Although the correlation between the efficacies of antifungal combination therapy in infection models and clinical fungal infections is uncertain, the lack of antagonism observed with POS and CSP in experimental combination therapy in this report suggests that this combination should be evaluated in clinical trials. The potential exists, with the different mechanisms of action of the two drugs, for beneficial outcomes of combination therapy with POS and CSP against clinical Aspergillus infections.