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Antimicrobial Agents and Chemotherapy, December 2006, p. 4202-4205, Vol. 50, No. 12
0066-4804/06/$08.00+0     doi:10.1128/AAC.00485-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Efficacy of Caspofungin against Aspergillus flavus, Aspergillus terreus, and Aspergillus nidulans{triangledown}

J. C. Bowman,1* G. K. Abruzzo,1 A. M. Flattery,1 C. J. Gill,1 E. J. Hickey,1 M. J. Hsu,1 J. Nielsen Kahn,1 P. A. Liberator,1 A. S. Misura,1 B. A. Pelak,1 T. C. Wang,2 and C. M. Douglas1

Departments of Infectious Disease,1 Biometrics Research, Merck Research Laboratories, Rahway, NJ 07065-09002

Received 19 April 2006/ Returned for modification 2 July 2006/ Accepted 14 September 2006


   ABSTRACT
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The echinocandin caspofungin is a potent inhibitor of the activity of 1,3-ß-D-glucan synthase from Aspergillus flavus, Aspergillus terreus, and Aspergillus nidulans. In murine models of disseminated infection, caspofungin prolonged survival and reduced the kidney fungal burden. Caspofungin was at least as effective as amphotericin B against these filamentous fungi in vivo.


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The incidence of life-threatening Aspergillus infections has risen in recent years (21, 23, 26). Although Aspergillus fumigatus accounts for the majority of cases of human aspergillosis, the number of infections caused by other Aspergillus species has increased (4, 28). The emerging threat posed by these species is especially important to understand because of their inherent reduced susceptibilities to many antifungal agents (8, 13, 30).

Caspofungin (CAS) was first approved for the treatment of invasive aspergillosis in patients refractory to or intolerant of other therapies. CAS derives its antifungal activity by inhibiting the synthesis of 1,3-ß-D-glucan, an essential cell wall polymer. The FKS gene encodes an integral membrane protein that is part of the 1,3-ß-D-glucan synthase (GS) complex (10), and the FKS gene family is highly conserved among a number of clinically relevant fungal pathogens (9). CAS inhibits the growth of A. fumigatus in vitro and has significant efficacy against A. fumigatus in animal models of pulmonary (27), disseminated (5), and central nervous system (17) disease, as well as clinical efficacy (19, 22).

CAS is active in vitro against several Aspergillus species (3, 29). CAS has been demonstrated to have activity against A. terreus in animal models (4, 12), and case reports have described the efficacy of CAS in patients infected with A. flavus (15) or A. terreus (7). Here we describe efforts to better characterize the activity of CAS against A. flavus, A. terreus, and A. nidulans.

(This work was presented in part at the 43rd Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 15 September 2003.)

Clinical isolates of A. flavus (CLF9089), A. terreus (CLF9062), and A. nidulans (CLF14) were used in these studies. Spores were harvested from cultures incubated for 5 to 7 days at 35°C on Sabouraud dextrose agar (SDA). Prior to infection, the viability of the spore suspensions was determined by spreading dilutions onto SDA plates and enumerating the CFU. CAS (Merck & Co., Inc. Rahway, NJ) was solubilized and serially diluted in sterile distilled water. Amphotericin B (AMB; Fungizone; Bristol-Myers Squibb, Princeton, NJ) was reconstituted according to the manufacturer's instructions and serially diluted in sterile distilled water.

Purified GS was prepared from each isolate by using product entrapment (18, 20), and the 50% inhibitory concentration of CAS was determined. Whole-cell susceptibility was measured by performing broth microdilution assays according to method M38-A of the Clinical and Laboratory Standards Institute (6).

Female DBA/2 mice (weight, 18 to 22 g; Taconic or Jackson Laboratories) were used for in vivo studies with A. flavus and A. terreus; female CD-1 mice (weight, 23 to 27 g; Charles River) were used for studies with A. nidulans. For survival studies, disseminated Aspergillus infections were induced by injecting 0.2 ml of a spore suspension containing 1.8 x 105 A. flavus, 3.7 x 106 A. terreus, or 8.6 x 104 A. nidulans CFU into the lateral tail vein. The compounds were administered intraperitoneally (i.p.) once daily for 7 days, beginning 15 to 30 min after infection (10 mice per group). Survival was monitored daily for 28 days. For the studies with A. nidulans, CD-1 mice were rendered chronically pancytopenic as described previously (1).

To assess the effect of therapy on the fungal burden, mice were infected by intravenous injection of 0.2 ml of a spore suspension containing 2.8 x 105 A. flavus, 4.1 x 106 A. terreus, or 9.0 x 104 A. nidulans CFU. A. nidulans-infected CD-1 mice were rendered pancytopenic as described above for the survival studies. CAS, AMB, or vehicle was administered i.p. once daily for 7 days, beginning 15 to 30 min after challenge (n = 10). Animals were euthanized 24 h after the last dose, and the kidney fungal burden was determined by quantitative PCR (qPCR) (5). Sense and antisense primers and a dual-labeled hybridization probe designed for the 18S rRNA gene of A. fumigatus (5) were used for A. flavus and A. terreus. For A. nidulans, the same primers but a different hybridization probe (5'-FAM-AGCCAGCGGCCCGCGGACG-TAMRA-3', where FAM is 6-carboxyfluorescein and TAMRA is 6-carboxytetramethylrhodamine) were used. qPCR values were normalized for genomic DNA recovery and are expressed as conidial equivalents (CE) per gram kidney (14, 16). Statistical significance relative to vehicle-treated animals (P ≤ 0.05) was determined by the log rank test (survival) or Student's t test (kidney burden).

The predicted protein sequence of the full-length A. fumigatus Fks was aligned with Fksp orthologues from A. flavus, A. terreus, and A. nidulans. The alignment suggests strong Fksp sequence conservation (Table 1). Specific amino acids that play a role in the echinocandin susceptibilities of Candida albicans and Saccharomyces cerevisiae (25) are conserved in these Fks proteins (data not shown). The high degree of homology is consistent with the susceptibility of the partially purified GS enzyme activity to inhibition by CAS (Table 1). CAS also demonstrates whole-cell activity against these isolates, with MIC80 values that are comparable to those seen for CAS-susceptible A. fumigatus isolates. The susceptibility to AMB was commensurate with the values reported previously for these species (11).


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  		TABLE 1. Whole-cell and GS enzyme susceptibilities to CAS and conservation of the FKS target gene for A. flavus, A. terreus, and A. nidulans

 
CAS prolonged survival in mice infected with A. flavus, A. terreus, or A. nidulans in a dose-dependent manner (Fig. 1). CAS was effective (P ≤ 0.05) in the A. flavus mouse model at doses of 0.25, 0.5, and 1 mg per kilogram of body weight, while AMB provided no protection. In A. terreus-infected mice, CAS was efficacious (P ≤ 0.05) at doses greater than 0.06 mg/kg (Fig. 1B). AMB was ineffective at 1 mg/kg but did provide a modest survival benefit (≥60%) at doses from 0.125 to 0.5 mg/kg (data not shown). In A. nidulans-infected mice, CAS at 1.0 and 0.5 mg/kg provided a moderate to significant improvement in survival (P = 0.11 and 0.03, respectively) (Fig. 1C), while AMB was inactive.


Figure 1
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  		FIG. 1. Survival of mice infected with A. flavus, A. terreus, or A. nidulans. Therapy (1, 0.5, 0.25, 0.125, or 0.06 mg/kg CAS or 1 mg/kg AMB administered i.p. once daily) was initiated immediately after infection and was continued for 7 days. Survival was monitored for 28 days, and Kaplan-Meier plots were generated. A. nidulans-infected mice remained immunosuppressed throughout the study. There were 10 mice in each group. *, P ≤ 0.05; mpk, milligram per kilogram.

 
The kidney fungal burden was significantly reduced by treatment with CAS at several dose levels (Fig. 2). CAS reduced the tissue burden in A. flavus-infected mice compared to that in vehicle-treated mice, with a peak reduction of ca. 3.5 log10 CE/g kidney (Fig. 2A). A reduction of the A. flavus burden was also seen in animals given AMB, with a peak reduction of ca. 3 log10 CE/g kidney when AMB was administered at 1 mg/kg (Fig. 2A). The A. terreus kidney burden was reduced by CAS, with a dose-dependent trend (Fig. 2B), but the peak burden reduction was only ca. 1 log10. As in the survival study, some AMB doses were efficacious in A. terreus-infected mice (Fig. 2B); however, significant mortality and large standard errors were observed in these AMB-treated animals. In A. nidulans-infected mice, all doses of CAS that we tested reduced the burden ca. 0.8 to 1 log10 CE/g kidney, while there was no apparent titration with AMB (Fig. 2C).


Figure 2
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  		FIG. 2. Reduction of kidney burden in mice infected with A. flavus, A. terreus, or A. nidulans and treated with CAS or AMB. Infected mice were treated with vehicle or drug (CAS or AMB; 1, 0.5, 0.25, 0.125, 0.06, or 0.03 mg/kg per day administered i.p. once daily), and the kidney fungal burden at 7 days postinfection was determined by qPCR. The mean burdens (log10 conidial equivalents/g kidney ± standard error) for vehicle-treated animals were as follows: A. flavus, 6.70 ± 0.15; A. terreus, 6.72 ± 0.17; A. nidulans, 5.68 ± 0.19. For each drug-treated group, the reduction in the mean burden (± standard error) relative to that in mice that received vehicle treatment is given. Values were derived from 10 mice per therapy group except when specified by numbers within or just above or below the bars. The y-axis scale for the A. flavus plot is different from those for A. terreus and A. nidulans. *, P ≤ 0.05; mpk, milligram per kilogram.

 
Recently, Graybill et al. (12) reported that CAS prolonged survival in A. terreus-infected neutropenic mice at doses of 0.5, 5, and 10 mg/kg and reduced the spleen CFU at 10 and 15 mg/kg. Barchiesi et al. (4) observed that CAS therapy provided protection in neutropenic mice infected with either of two A. terreus strains, with a significant fungal burden reduction in the kidneys achieved with 1 and 5 mg/kg CAS and a significant fungal burden reduction in the brain achieved with 5 mg/kg CAS. Of note, the peak kidney CFU reduction was comparable to the kidney CE reduction (~1 log10) shown here. The higher signal in vehicle-treated mice from our study is consistent with the increased sensitivity of the qPCR assay (5, 24, 31).

Here we report the results of both in vitro and in vivo studies which support the potential utility of CAS as treatment for infections with A. flavus, A. terreus, or A. nidulans. FKS, the likely molecular target of the echinocandins, is conserved in these organisms. The activity of the GS enzyme prepared from each isolate is sensitive to inhibition by CAS, consistent with growth inhibition in liquid MIC assays. Finally, CAS is at least as effective as AMB in murine models of disseminated infection, measured both by survival prolongation and by a reduction in fungal burden. Perhaps most importantly, reports of efficacy in patients (7, 22) suggest a clinical role for CAS against these Aspergillus species.


   ACKNOWLEDGMENTS
 
We thank S. Zachwieja, M. Smarsh, J. Widger, F. Patterson, Z. Zhong, and S. C. Power from Cell & Molecular Technologies, Inc. (Phillipsburg, NJ), for performing the qPCR assays.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Infectious Disease Research, Merck Research Laboratories, RY80Y-260, P.O. Box 2000, Rahway, NJ 07065-0900. Phone: (732) 594-1946. Fax: (732) 594-6708. E-mail: joel_bowman{at}merck.com. Back

{triangledown} Published ahead of print on 2 October 2006. Back


   REFERENCES
Top
 Abstract
 Text
 References
 

  1. Abruzzo, G. K., C. J. Gill, A. M. Flattery, L. Kong, C. Leighton, J. G. Smith, V. B. Pikounis, K. Bartizal, and H. Rosen. 2000. Efficacy of the echinocandin caspofungin against disseminated aspergillosis and candidiasis in cyclophosphamide-induced immunosuppressed mice. Antimicrob. Agents Chemother. 44:2310-2318.[Abstract/Free Full Text]
  2. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402.[Abstract/Free Full Text]
  3. Arikan, S., M. Lozano-Chiu, V. Paetznick, and J. H. Rex. 2001. In vitro susceptibility testing methods for caspofungin against Aspergillus and Fusarium isolates. Antimicrob. Agents Chemother. 45:327-330.[Abstract/Free Full Text]
  4. Barchiesi, F., E. Spreghini, A. Santinelli, A. W. Fothergill, S. Fallani, E. Manso, E. Pisa, D. Giannini, A. Novelli, M. I. Cassetta, T. Mazzei, M. G. Rinaldi, and G. Scalise. 2005. Efficacy of caspofungin against Aspergillus terreus. Antimicrob. Agents Chemother. 49:5133-5135.[Abstract/Free Full Text]
  5. Bowman, J. C., G. K. Abruzzo, J. W. Anderson, A. M. Flattery, C. J. Gill, V. B. Pikounis, D. M. Schmatz, P. A. Liberator, and C. M. Douglas. 2001. Quantitative PCR assay to measure Aspergillus fumigatus burden in a murine model of disseminated aspergillosis: demonstration of efficacy of caspofungin acetate. Antimicrob. Agents Chemother. 45:3474-3481.[Abstract/Free Full Text]
  6. Clinical and Laboratory Standards Institute. 2002. Reference method for broth dilution antifungal susceptibility testing of conidium-forming filamentous fungi. Approved standard M38-A. Clinical and Laboratory Standards Institute, Wayne, Pa.
  7. Cooke, F. J., E. Terpos, J. Boyle, A. Rahemtulla, and T. R. Rogers. 2003. Disseminated Aspergillus terreus infection arising from cutaneous inoculation treated with caspofungin. Clin. Microbiol. Infect. 9:1238-1241.[CrossRef][Medline]
  8. Dotis, J., and E. Roilides. 2004. Osteomyelitis due to Aspergillus spp. in patients with chronic granulomatous disease: comparison of Aspergillus nidulans and Aspergillus fumigatus. Int. J. Infect. Dis. 8:103-110.[CrossRef][Medline]
  9. Douglas, C. M. 2001. Fungal beta(1,3)-D-glucan synthesis. Med. Mycol. 39(Suppl. 1):55-66.
  10. Douglas, C. M., J. A. D'Ippolito, G. J. Shei, M. Meinz, J. Onishi, J. A. Marrinan, W. Li, G. K. Abruzzo, A. Flattery, K. Bartizal, A. Mitchell, and M. B. Kurtz. 1997. Identification of the FKS1 gene of Candida albicans as the essential target of 1,3-beta-D-glucan synthase inhibitors. Antimicrob. Agents Chemother. 41:2471-2479.[Abstract]
  11. Espinel-Ingroff, A. 2003. Evaluation of broth microdilution testing parameters and agar diffusion Etest procedure for testing susceptibilities of Aspergillus spp. to caspofungin acetate (MK-0991). J. Clin. Microbiol. 41:403-409.[Abstract/Free Full Text]
  12. Graybill, J. R., S. Hernandez, R. Bocanegra, and L. K. Najvar. 2004. Antifungal therapy of murine Aspergillus terreus infection. Antimicrob. Agents Chemother. 48:3715-3719.[Abstract/Free Full Text]
  13. Hachem, R. Y., D. P. Kontoyiannis, M. R. Boktour, C. Afif, C. Cooksley, G. P. Bodey, I. Chatzinikolaou, C. Perego, H. M. Kantarjian, and I. I. Raad. 2004. Aspergillus terreus: an emerging amphotericin B-resistant opportunistic mold in patients with hematologic malignancies. Cancer 101:1594-1600.[CrossRef][Medline]
  14. Hickey, E., G. Abruzzo, J. Bowman, C. Douglas, C. Gill, P. Liberator, A. Misura, B. Pikounis, and K. Bartizal. 2005. Caspofungin versus amphotericin B treatment of Aspergillus fumigatus in kidneys of chronically immunosuppressed infected mice. Therapy 2:615-621.[CrossRef]
  15. Howell, A., J. Midturi, M. Sierra-Hoffman, J. Carpenter, D. Hurley, and R. Winn. 2005. Aspergillus flavus scleritis: successful treatment with voriconazole and caspofungin. Med. Mycol. 43:651-655.[CrossRef][Medline]
  16. Ibrahim, A. S., J. C. Bowman, V. Avanessian, K. Brown, B. Spellberg, J. E. Edwards, Jr., and C. M. Douglas. 2005. Caspofungin inhibits Rhizopus oryzae 1,3-beta-D-glucan synthase, lowers burden in brain measured by quantitative PCR, and improves survival at a low but not a high dose during murine disseminated zygomycosis. Antimicrob. Agents Chemother. 49:721-727.[Abstract/Free Full Text]
  17. Imai, J., G. Singh, B. Fernandez, K. V. Clemons, and D. A. Stevens. 2005. Efficacy of Abelcet and caspofungin, alone or in combination, against CNS aspergillosis in a murine model. J. Antimicrob. Chemother. 56:166-171.[Abstract/Free Full Text]
  18. Kahn, J. N., M. J. Hsu, F. Racine, R. Giacobbe, and M. Motyl. 2006. Caspofungin susceptibility in Aspergillus and non-Aspergillus moulds: inhibition of glucan synthase and reduction of beta-D-1,3-glucan levels in culture. Antimicrob. Agents Chemother. 50:2214-2216.[Abstract/Free Full Text]
  19. Kartsonis, N. A., A. J. Saah, C. Joy Lipka, A. F. Taylor, and C. A. Sable. 2005. Salvage therapy with caspofungin for invasive aspergillosis: results from the caspofungin compassionate use study. J. Infect. 50:196-205.[CrossRef][Medline]
  20. Kelly, R., E. Register, M. J. Hsu, M. Kurtz, and J. Nielsen. 1996. Isolation of a gene involved in 1,3-beta-glucan synthesis in Aspergillus nidulans and purification of the corresponding protein. J. Bacteriol. 178:4381-4391.[Abstract/Free Full Text]
  21. Lin, S. J., J. Schranz, and S. M. Teutsch. 2001. Aspergillosis case-fatality rate: systematic review of the literature. Clin. Infect. Dis. 32:358-366.[CrossRef][Medline]
  22. Maertens, J., I. Raad, G. Petrikkos, M. Boogaerts, D. Selleslag, F. B. Petersen, C. A. Sable, N. A. Kartsonis, A. Ngai, A. Taylor, T. F. Patterson, D. W. Denning, and T. J. Walsh. 2004. Efficacy and safety of caspofungin for treatment of invasive aspergillosis in patients refractory to or intolerant of conventional antifungal therapy. Clin. Infect. Dis. 39:1563-1571.[CrossRef][Medline]
  23. Marr, K. A., R. A. Carter, F. Crippa, A. Wald, and L. Corey. 2002. Epidemiology and outcome of mould infections in hematopoietic stem cell transplant recipients. Clin. Infect. Dis. 34:909-917.[CrossRef][Medline]
  24. O'Sullivan, C. E., M. Kasai, A. Francesconi, V. Petraitis, R. Petraitiene, A. M. Kelaher, A. A. Sarafandi, and T. J. Walsh. 2003. Development and validation of a quantitative real-time PCR assay using fluorescence resonance energy transfer technology for detection of Aspergillus fumigatus in experimental invasive pulmonary aspergillosis. J. Clin. Microbiol. 41:5676-5682.[Abstract/Free Full Text]
  25. Park, S., R. Kelly, J. N. Kahn, J. Robles, M. J. Hsu, E. Register, W. Li, V. Vyas, H. Fan, G. Abruzzo, A. Flattery, C. Gill, G. Chrebet, S. A. Parent, M. Kurtz, H. Teppler, C. M. Douglas, and D. S. Perlin. 2005. Specific substitutions in the echinocandin target Fks1p account for reduced susceptibility of rare laboratory and clinical Candida sp. isolates. Antimicrob. Agents Chemother. 49:3264-3273.[Abstract/Free Full Text]
  26. Perea, S., and T. F. Patterson. 2002. Invasive Aspergillus infections in hematologic malignancy patients. Semin. Respir. Infect. 17:99-105.[CrossRef][Medline]
  27. Petraitiene, R., V. Petraitis, A. H. Groll, T. Sein, R. L. Schaufele, A. Francesconi, J. Bacher, N. A. Avila, and T. J. Walsh. 2002. Antifungal efficacy of caspofungin (MK-0991) in experimental pulmonary aspergillosis in persistently neutropenic rabbits: pharmacokinetics, drug disposition, and relationship to galactomannan antigenemia. Antimicrob. Agents Chemother. 46:12-23.[Abstract/Free Full Text]
  28. Pfaller, M. A., and D. J. Diekema. 2004. Rare and emerging opportunistic fungal pathogens: concern for resistance beyond Candida albicans and Aspergillus fumigatus. J. Clin. Microbiol. 42:4419-4431.[Free Full Text]
  29. Pfaller, M. A., F. Marco, S. A. Messer, and R. N. Jones. 1998. In vitro activity of two echinocandin derivatives, LY303366 and MK-0991 (L-743,792), against clinical isolates of Aspergillus, Fusarium, Rhizopus, and other filamentous fungi. Diagn. Microbiol. Infect. Dis. 30:251-255.[CrossRef][Medline]
  30. Segal, B. H., E. S. DeCarlo, K. J. Kwon-Chung, H. L. Malech, J. I. Gallin, and S. M. Holland. 1998. Aspergillus nidulans infection in chronic granulomatous disease. Medicine (Baltimore) 77:345-354.[CrossRef][Medline]
  31. Singh, G., J. Imai, K. V. Clemons, and D. A. Stevens. 2005. Efficacy of caspofungin against central nervous system Aspergillus fumigatus infection in mice determined by TaqMan PCR and CFU methods. Antimicrob. Agents Chemother. 49:1369-1376.[Abstract/Free Full Text]

Antimicrobial Agents and Chemotherapy, December 2006, p. 4202-4205, Vol. 50, No. 12
0066-4804/06/$08.00+0     doi:10.1128/AAC.00485-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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