In Vivo Activity of Posaconazole against Mucor spp. in an Immunosuppressed-Mouse Model

Abstract

The in vivo activities of posaconazole, itraconazole, and amphotericin B in neutropenic mice with zygomycosis were compared. The in vitro MICs of posaconazole and itraconazole for the strains of Mucor spp. used in this study ranged from 0.125 to 8 μg/ml and 0.25 to 8 μg/ml, respectively. The in vitro MIC range for amphotericin B is 0.125 to 0.25 μg/ml. At twice-daily doses of ≥15 mg/kg of body weight, posaconazole prolonged the survival of the mice and reduced tissue burden.

Zygomycosis is a relatively uncommon but highly aggressive fungal infection that affects diabetics, neutropenic patients, and subjects with burns or iron overload but that only rarely affects healthy people (15, 19). Illness may rapidly progress with angioinvasion and tissue infarction. The existing methods for treatment are often ineffective (4, 9, 17). Presently, therapy utilizes aggressive surgical measures and high doses of amphotericin B. There are scattered reports of lipid-associated amphotericin B in salvage, but there is no evidence that these forms are more efficacious than amphotericin B (2, 3, 5). Even with aggressive therapy, mortality is often above 50% (6). Of the alternative antifungals, itraconazole has been effective in vitro, particularly against Absidia spp. One mouse study has also shown some activity against Absidia, but the clinical evidence of efficacy is less clear (2, 6, 10, 16; E. Dannaoui, J. Meletiadis, J. Meis, J. W. Moulton, and P. E. Verweij, Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 939, 2000). Posaconazole is a new broad-spectrum triazole with activity against many filamentous fungal pathogens (1, 8, 12, 18). This study describes the in vivo activity of posaconazole against three Mucor spp. isolates in a neutropenic-mouse model.

Three clinical Mucor isolates were tested in vitro by the NCCLS microdilution method for triazoles adapted to filamentous fungi (11). The endpoint was a visual MIC at which azoles were seen to reduce growth by 80% compared to that in the drug-free control tube. The MIC of amphotericin B was taken as the least drug producing a visually clear tube. MICs of posaconazole and itraconazole at 48 h were 0.125 and 0.25 μg/ml for Mucor ramosissimus strain 98-1763, 0.25 and 0.25 μg/ml for Mucor ramosissimus 95-2650, and 8 and 8 μg/ml for Mucor circinelloides 00-1194, respectively. The 48-h MIC of amphotericin B was 0.25 μg/ml for all three isolates. The isolates were grown on potato flake agar plates at room temperature for 1 week. They were harvested by scraping the plates with sterile isotonic saline and filtering the suspension through glass wool. The inoculum was calculated by determining hemacytometer counts.

For the mouse model, we used 18-week-old BALB/c males. One day before infection, mice were rendered neutropenic with single doses of 5-fluorouracil administered intravenously at 150 mg/kg of body weight and with cyclophosphamide administered intraperitoneally at 200 mg/kg. In groups of five uninfected mice, this treatment reduced the neutrophil count from a median pretreatment value of 1,100/μl to less than 50/μl 7 days after treatment with 5-fluorouracil and to less than 200/μl 11 days later. Mice were infected intravenously using a 0.2-ml volume of the inoculum as shown below.

The antifungal agents used included posaconazole oral suspension (Schering-Plough Research Institute), itraconazole oral solution (Janssen), and amphotericin B powder (Bristol-Myers Squibb). Posaconazole and itraconazole were diluted as appropriate with sterile water. Amphotericin B powder was suspended in water as stock and diluted as needed in 5% dextrose. Treatment began 1 day after infection and continued for seven consecutive days. Mice received posaconazole orally by gavage at 5, 15, or 30 mg/kg twice per day, once in the early morning and once in the late afternoon. Itraconazole was administered orally by gavage at 30 mg/kg three times a day within an 8-h period. This is the maximum dose we could deliver without causing the mice to develop diarrhea from the cyclodextrin vehicle. Amphotericin B was administered intraperitoneally at 1 mg/kg once daily. Control mice were treated orally by gavage with sterile water. Mortality was recorded for 15 days. For the tissue burden studies, the treatment regimens were similar to those of the survival studies. One day after the last dose of the antifungal drugs, the mice were sacrificed. Kidneys and spleens were removed aseptically. The organs were weighed and homogenized in 2 ml of sterile saline. We took care to ensure that each tissue specimen received modest and uniform trauma. Semiquantitative culture data, obtained by serial dilution methods, were expressed as CFU per gram. Survival times were compared by using the log-rank test of life tables. The Mann-Whitney test was used for comparison of the results of tissue burden studies. A P value of ≤0.05 was required for significance.

Table 1 gives the results of the survival studies. For all three isolates, most controls succumbed within 6 days of infection. Itraconazole recipients did not fare better. The survival of posaconazole recipients increased significantly in a dose-dependent manner over that of the controls, with 60 to 83% survival at the 30-mg/kg twice-daily dose. Amphotericin B gave 84% survival in one study (P = 0.0004). Table 2 shows the tissue burdens of kidneys in two studies done with isolate 00-1194. Control mice infected with 2.8 × 10⁵ CFU (study 5) had 3.7 × 10⁴ to 22 × 10⁴ CFU/g of kidney, while mice infected in study 6 with 2.6 × 10⁴ CFU had kidney counts in the range of 0.05 × 10⁴ to 64 × 10⁴ CFU/g. Posaconazole doses of 15 and 30 mg/kg significantly lowered counts in tissue, while itraconazole did not. Amphotericin B and posaconazole at a dose of 30 mg/kg reduced many counts to undetectable levels (<18 CFU/g).

TABLE 1.

Survival of mice treated with posaconazole, itraconazole, or amphotericin B

Isolate	Inoculum (CFU/mouse)	Drug	Dose (mg/kg)	Survival (median no. of days)	Survival range (no. of days)	P valuea
98-1763	3.2 × 105	None	NA	4	3->15	NA
		Posaconazole	5	5	3->15	0.2535
		Posaconazole	15	5	3->15	0.0458*
		Posaconazole	30	10	5->15	0.0019*
		Itraconazole	30	4	3-6	0.0810
00-1194	2.7 × 105	None	NAb	5	4-10	NA
		Posaconazole	5	5	4-6	0.3460
		Posaconazole	15	5	4->15	0.0015*
		Posaconazole	30	7	5->15	0.0001*
		Itraconazole	30	4	3-6	0.2320
95-2650	5.3 × 104	None	NA	5	4-6	NA
		Posaconazole	30	9	5->15	0.0001*
00-1194	5.8 × 105	None	NA	4	3-6	NA
		Posaconazole	15	6	4->15	0.0002*
		Posaconazole	30	8	4->15	0.0001*
		Itraconazole	30	4	3-5	0.5086
		Amphotericin B	1	>15	2->15	0.0004*

^aValues marked with an asterisk show significance in prolonging survival over that of the controls. A P value of ≤0.05 is needed for significance.

^bNA, not applicable.

TABLE 2.

Tissue burdens in the kidneys of mice infected with isolate 00-1194

Inoculum (CFU/mouse)	Drug	Dose (mg/kg)	Median no. of CFU/g (104)	Range of CFU/g (104)	P valuea
2.8 × 105	None	NAb	6.4	3.7-22	NA
	Posaconazole	15	3	0.5-7	0.0148*
	Posaconazole	30	4	0.2-2.8	0.0002*
	Itraconazole	30	6.8	2.6-2	1.000
2.6 × 104	None	NA	16	0.05-64
	Posaconazole	15	3.7	0.0001-28	0.0281*
	Posaconazole	30	0.02	0.0001-2.6	0.0011*
	Itraconazole	30	9	0.5-20	0.2345
	Amphotericin B	1	0.0001	0.0001-0.0053	0.0002*

^aValues marked with an asterisk show significance in reducing kidney counts to levels lower than those found in the controls. A P value of ≤0.05 is needed for significance.

^bNA, not applicable.

The record of triazoles against zygomycetes is mixed at best. SCH 42427, the active stereoisomer of genaconazole, has also been used successfully in the treatment of murine zygomycosis (7). Unfortunately, because of animal carcinogenicity, this drug could not be developed for clinical use. Limited clinical data with triazoles have suggested that fluconazole or itraconazole may be useful on occasion (13, 14).

Posaconazole may be a novel triazole for treatment of zygomycosis. Dannaoui et al. previously reported that posaconazole is active in vitro against Mucor spp. (12; Dannaoui et al., 40th ICAAC). In this study, we have shown that posaconazole is also active in vivo against three isolates of Mucor spp. in a neutropenic-mouse model of disseminated infection. The in vitro MICs for two isolates were low and the MIC for another isolate was high (8 μg/ml). This disparity may relate to differences in species (M. ramosissimus versus M. circinelloides) which may have different in vitro growth rates; these in turn may affect MICs. Alternatively, the results may vary because the methods for in vitro testing used different media or conidial versus hyphal forms. In any case, in vivo activity was consistently reflected both in the prolonged survival and in the reduction of tissue burden. Mucor forms a fragile mycelium which can readily be disrupted and may be nonviable in vigorously disrupted tissues. However, our findings of high counts for controls and clear dose-associated reductions in counts indicate that our methods are useful at least for semiquantitation. Further, our findings agree with those of the survival studies, in which posaconazole given twice daily at 30 mg/kg was as potent as our amphotericin B regimen. A maximum dose has not been established in our model, and it is possible that even higher doses may show more benefit. Posaconazole has potential for the therapy of systemic infection caused by Mucor spp. in humans.