This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Patera, A. C.
Right arrow Articles by Loebenberg, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Patera, A. C.
Right arrow Articles by Loebenberg, D.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, August 2004, p. 3154-3158, Vol. 48, No. 8
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.8.3154-3158.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Effect of Granulocyte Colony-Stimulating Factor Combination Therapy on Efficacy of Posaconazole (SCH56592) in an Inhalation Model of Murine Pulmonary Aspergillosis

Andriani C. Patera,* Frederick Menzel, Craig Jackson, Joan K. Brieland,{dagger} Judy Halpern, Roberta Hare, Anthony Cacciapuoti, and David Loebenberg

Schering Plough-Research Institute, Kenilworth, New Jersey 07033

Received 29 July 2003/ Returned for modification 25 November 2003/ Accepted 25 April 2004


arrow
ABSTRACT
 
Using an inhalation model of pulmonary aspergillosis, we observed modest differences in the survival rates of mice treated with granulocyte colony-stimulating factor (G-CSF) and posaconazole (POS) and those treated with POS alone. This finding is in contrast to a previous report that suggested that G-CSF had a significant antagonistic effect on the antifungal activity of POS.


arrow
TEXT
 
Invasive aspergillosis is most prevalent, and often fatal, in patients with defective macrophage and neutrophil function, such as leukopenic cancer patients and bone marrow transplant recipients (1, 15). Consequently, these patients receive therapy with immunomodulators such as granulocyte colony-stimulating factor (G-CSF) to boost host defense against inhaled Aspergillus spp. (10, 19). It has been suggested that G-CSF not only augments neutrophil activity (14, 21, 22) but may also improve the effects of antifungal drugs (6, 24, 28). However, a recent report demonstrated that posaconazole (POS)-G-CSF cotreatment of corticosteroid-immunocompromised mice infected intranasally with Aspergillus fumigatus resulted in significantly increased mortality compared with that resulting from POS monotherapy (9).

By use of an inhalation model of murine aspergillosis, male CF-1 mice (17 to 18 g; Charles River Laboratories) were infected with A. fumigatus clinical isolate ND158 or ND208 (isolate 94-2766 used by Graybill et al. [9]) or Aspergillus flavus isolate ND83 by inhalation as previously described (17, 20). Mice were immunocompromised with cortisone acetate (100 mg/kg of body weight/day administered subcutaneously on days –1, 0, 1, and 6). Recombinant human G-CSF (Amgen Inc., Thousand Oaks, Calif.) was administered intraperitoneally from day –3 to day 5 at 125 or 600 µg/kg/day. Sterile water for injection (control) or POS (Schering-Plough Research Institute, Kenilworth, N.J.) was given orally on days 1 to 9 at 5, 25, or 100 mg/kg/day for A. fumigatus infection or at 0.2, 1, or 10 mg/kg/day for A. flavus infection.

The infective dose was determined 2 h postinfection by plating serial dilutions of lung homogenates from individual mice on Sabouraud dextrose agar plates. Up to three individual experiments were performed per strain. A 30-s exposure of the mice in the inhalation chamber resulted in a mean infectious dose of 4.68 x 106± 2.09 x 106 CFU (A. fumigatus ND158), 9.33 x 107± 0.8 x 107 CFU (A. fumigatus ND208), or 1.26 x 106 CFU (A. flavus ND83) for the lungs of each mouse. Intranasal infection with A. fumigatus ND208 yielded 1.6 x 107 CFU for the lungs of each mouse.

The survival of the mice was monitored throughout the treatment. Mean survival data for all treatment groups are summarized in Table 1. Representative survival curves from individual experiments are shown in Fig. 1. Similar trends were observed with the intranasal and inhalation routes of infection and with different strains and species of Aspergillus. G-CSF monotherapy at 125 or 600 µg/kg/day was not protective against Aspergillus infection since all mice died by day 10. However, POS monotherapy was protective, enhancing survival from 0 to 41.7% (inhaled A. fumigatus), 83.3% (inhaled A. flavus), or 30% (intranasal A. fumigatus). Similarly, POS increased the survival of mice treated with G-CSF from 0% without POS to 22.2 to 66.6% (125 µg of G-CSF/kg/day) and to 19.4 to 66.7% (600 µg of G-CSF/kg/day) with POS (Table 1). Thus, in contrast to a previous study by Graybill et al. (9), we observed a dose-dependent increase in survival irrespective of whether POS was used alone or with G-CSF (Table 1).


View this table:
[in this window]
[in a new window]
  		TABLE 1. Mean percentage and range of survival of mice 10 days after infection with Aspergillus and combination therapy with G-CSF and POS or POS monotherapy



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 1. Kaplan-Meier survival curves showing percentages of CF-1 mice infected with Aspergillus either by inhalation in a chamber (A. fumigatus ND158 [a], A. fumigatus ND208 [b], and A. flavus ND83 [c]) or by intranasal administration (A. fumigatus ND208 [d]). Each graph represents a single study (with 10 to 12 mice). Treatment consisted of sterile water for injection as a control ({circ}), 600 µg of G-CSF/kg/day (x), or POS monotherapy at 100 mg/kg/day (A. fumigatus) or 10 mg/kg/day (A. flavus) (•). Combination therapy consisted of treatment with POS at 100 mg/kg/day for A. fumigatus or 10 mg/kg/day for A. flavus ND83 and G-CSF at either 125 µg/kg/day ({blacksquare}) or 600 µg/kg/day ({blacktriangleup}). P values for all combinations were >0.05 by Wilcoxon and log rank test analyses.

A comparison of survival of Aspergillus-infected mice treated with POS alone with survival of those treated with POS and G-CSF suggested a trend towards a decrease in survival of as much as 10 to 15%. However, an equivalent increase in survival was observed in some cases for individual experiments (e.g., for groups to which POS was given at 5 mg/kg/day with or without G-CSF [data not shown]). Moreover, at higher doses of POS, an increase in G-CSF from 125 to 600 µg/kg/day appeared to have either no effect or a modestly beneficial effect (Table 1). Overall, the differences between groups treated with POS alone and those treated with POS in combination with G-CSF were modest (~10%; P > 0.05). These findings are in contrast to an earlier report that suggested that POS monotherapy was more efficacious than the combination of POS and G-CSF, with a 20 to 50% difference in mortality between the monotherapy and combination therapy groups (9).

To determine the effect of POS-G-CSF combination therapy on the fungal burden in the lungs, the surviving mice from the high-dose POS groups were sacrificed on day 10 postinfection. The results for individual and combined drug experiments are shown in Table 2 and Fig. 2. It is interesting that in contrast to the results of the earlier study by Graybill et al. (9), mice surviving intranasal infection with A. fumigatus ND208 following POS monotherapy or POS-G-CSF combination therapy completely cleared infection (Fig. 2d). The mice infected via the inhalation route did not clear infection. A modest trend toward an increased mean number of CFU per lung was observed with POS-G-CSF combination therapy in mice infected with A. fumigatus when the data from three individual studies were combined (n = 5 to 10) (Fig. 2a and b). The number of data collected varied per individual study (n = 2 to 5), and in some cases there was either no change or a decrease in the mean lung burden (Table 2). However, it should be noted that the number of animals tested in these groups was very small; therefore, no clear conclusions can be drawn from the lung burden data.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Mean lung burden by group of surviving Aspergillus-infected mice following combination therapy with G-CSF and POS



View larger version (11K):
[in this window]
[in a new window]
  		FIG. 2. Lung fungal burden of mice surviving inhalation (A. fumigatus ND158 [a], A. fumigatus ND208 [b], and A. flavus ND83 [c]) or intranasal infection (A. fumigatus ND208 [d]). Each point represents the fungal burden in the lungs of an individual mouse on day 10 postinfection, and horizontal bars indicate the mean per group. The combined results for the survivors of three (a) or two (b) independent experiments and for a single experiment (c and d) are shown. P values were <0.05 by parametric (Student's t test and Tukey-Kramer test) and nonparametric (Wilcoxon test and Kruskal-Wallis rank sum) methods for A. fumigatus ND208 infection by inhalation for panel b. The surviving mice cleared intranasal infection with A. fumigatus ND208. P values were >0.05 for all other groups.

Interestingly, A. flavus ND83 infection, which resulted in the lowest mortality (>70% survival) (Fig. 1c), also resulted in the least difference in mean numbers of CFU per lung between treatment groups (P > 0.05) (Fig. 2c), suggesting that G-CSF has no significant effect on the antifungal activity of POS under these conditions. While lower mortality appears to present a clearer picture of similarities between monotherapy and combination therapy, this possibility could not be further substantiated by using a lower infecting dose of A. fumigatus, due to practical constraints of the inhalation model. Furthermore, in our hands, intranasal administration of A. fumigatus also resulted in >70% mortality at a dose for which >70% survival was observed by Graybill et al. (9), highlighting some limitations of in vivo analyses.

Several factors may contribute to the differences in the survival rates and pathogen burdens between the present study and the previous study by Graybill et al. (9). The mouse strain, age, and weight and the route of infection have all been shown to affect pathogen clearance and survival in pulmonary disease models of cryptococcosis (16) and coccidioidomycosis (4).

Both the mouse weight (18 versus 30 g) and the strain used (CF-1 versus CD-1 [ICR]; both with the H-2q haplotype) differed between this and the previous study by Graybill et al. (9). However, no difference in susceptibility to Aspergillus infection or response to G-CSF or triazoles has been documented for these strains. Differences due to the Aspergillus strain used can be ruled out, since we observed similar trends with one strain of A. flavus and two strains of A. fumigatus, including the same clinical isolate, ND208 (94-2766), used by Graybill et al. (9).

To address the possibility that the route of infection may play a role in susceptibility and response to therapy, we repeated the study using an intranasal route of infection with A. fumigatus ND208. An interesting difference was found in the clearance of A. fumigatus ND208 infection after intranasal or inhalation administration (Fig. 2d; Table 2). This finding may suggest possible differences in infectivity or mechanisms and efficiency of pathogen clearance following different routes of infection. It is conceivable that the infectivity and dissemination of Aspergillus may differ in the lungs depending on whether the infective dose is administered in solution or by aerosol, and as a result, the host response may also be affected. Generally, aerosolized infections result in diffuse bronchopneumonia with even distribution and replication primarily in lung tissue, whereas intranasal administration often results in upper respiratory tract infection (2). Thus, diffuse Aspergillus infection resulting from aerosol administration may contribute to the increased mortality and lung burden observed in the present study.

Triazoles, including POS, inhibit lanosterol 14{alpha}-demethylase. The net result is a depletion of ergosterol and the accumulation of methylated precursors in the fungal membrane (25). These changes in membrane composition may result in increased sensitivity to oxygen-dependent immune mechanisms such as neutrophil attack (5, 24). For example, ex vivo studies have shown that POS had a synergistic effect on the fungicidal activity of neutrophils against Scedosporium species (6) similar to properties of another triazole, voriconazole, against A. fumigatus (26, 27). Similarly, fluconazole in combination with granulocyte macrophage-CSF increased monocyte killing of Candida albicans (3). Thus, the combination of POS and G-CSF offers the possibility of direct inhibition of fungal growth, triazole-induced susceptibility to neutrophil-mediated killing, and enhancement of innate immunity by G-CSF (11, 18, 19, 23).

In vivo studies using G-CSF-triazole combination therapy have reported either a beneficial response (7, 8, 13, 28) or no significant difference (12, 13) in the survival of infected mice. For example, fluconazole-G-CSF combination therapy had no effect on survival (~10% difference) (12). In another study, the same combination prolonged the survival of neutropenic mice at a low infecting dose of Candida (7). However, at high infecting doses, the differences in survival were marginal to none (10 to 30% decrease). In the present study, we report that combining G-CSF with POS does not substantially affect the antifungal efficacy of POS in a murine model of invasive aspergillosis, with differences observed in ranges similar to those of previous studies that reported a lack of effect of G-CSF on triazole efficacy.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Schering-Plough Research Institute, K15-4-4800, 2015 Galloping Hill Rd., Kenilworth, NJ 07033. Phone: (908) 740-2651. Fax: (908) 740-3918. E-mail: andriani.patera{at}spcorp.com. Back

{dagger} Present address: Pfizer Global R&D, Ann Arbor, Mich. Back


arrow
REFERENCES
 
    1
  1. Baddley, J. W., T. P. Stroud, D. Salzman, and P. G. Pappas. 2001. Invasive mold infections in allogeneic bone marrow transplant recipients. Clin. Infect. Dis. 32:1319-1324.[CrossRef][Medline]
    2. 2
  2. Bakker-Woudenberg, I. A. J. M. 2003. Experimental models of pulmonary infection. J. Microbiol. Methods. 54:295-313.[CrossRef][Medline]
    3. 3
  3. Baltch, A. L., R. P. Smith, M. A. Franke, W. J. Ritz, P. B. Michelsen, and L. H. Bopp. 2001. Effects of cytokines and fluconazole on the activity of human monocytes against Candida albicans. Antimicrob. Agents Chemother. 45:96-104.[Abstract/Free Full Text]
    4. 4
  4. Cox, R. A., W. Kennell, L. Boncyk, and J. W. Murphy. 1988. Induction and expression of cell-mediated immune responses in inbred mice infected with Coccidioides immitis. Infect. Immun. 56:13-17.[Abstract/Free Full Text]
    5. 5
  5. Ghannoum, M. A., and L. B. Rice. 1999. Antifungal agents: mode of action, mechanisms of resistance, and correlation of these mechanisms with bacterial resistance. Clin. Microbiol. Rev. 12:501-517.[Abstract/Free Full Text]
    6. 6
  6. Gil-Lamaignere, C., E. Roilides, J. Mosquera, A. Maloukou, and T. J. Walsh. 2002. Antifungal triazoles and polymorphonuclear leukocytes synergize to cause increased hyphal damage to Scedosporium prolificans and Scedosporium apiospermum. Antimicrob. Agents Chemother. 46:2234-2237.[Abstract/Free Full Text]
    7. 7
  7. Graybill, J. R., R. Bocanegra, and M. Luther. 1995. Antifungal combination therapy with granulocyte colony-stimulating factor and fluconazole in experimental disseminated candidiasis. Eur. J. Clin. Microbiol. Infect. Dis. 14:700-703.[CrossRef][Medline]
    8. 8
  8. Graybill, J. R., R. Bocanegra, C. Lambros, and M. F. Luther. 1997. Granulocyte colony stimulating factor therapy of experimental cryptococcal meningitis. J. Med. Vet. Mycol. 35:243-247.[Medline]
    9. 9
  9. Graybill, J. R., R. Bocanegra, L. K. Najvar, D. Loebenberg, and M. F. Luther. 1998. Granulocyte colony-stimulating factor and azole antifungal therapy in murine aspergillosis: role of immune suppression. Antimicrob. Agents Chemother. 42:2467-2473.[Abstract/Free Full Text]
    10. 10
  10. Guiot, H. F. L., W. E. Fibbe, and J. W. van't Wout. 1994. Risk factors for fungal infection in patients with malignant hematologic disorders: implications for empirical therapy and prophylaxis. Clin. Infect. Dis. 18:525-532.[Medline]
    11. 11
  11. Hamood, M., P. F. Bluche, C. De Vroey, F. Corazza, W. Bujan, and P. Fondu. 1994. Effects of recombinant human granulocyte colony-stimulating factor on neutropenic mice infected with Candida albicans: acceleration of recovery from neutropenia and potentiation of anti-C. albicans resistance. Mycoses 37:93-99.[Medline]
    12. 12
  12. Kullberg, B. J., M. G. Netea, J. H. A. J. Curfs, M. Keuter, J. F. G. M. Meis, and J. W. M. van der Meer. 1998. Recombinant murine granulocyte colony-stimulating factor protects against acute disseminated Candida albicans infection in non-neutropenic mice. J. Infect. Dis. 177:175-181.[Medline]
    13. 13
  13. Kullberg, B. J., M. G. Netea, A. G. Vonk, and J. W. M. van der Meer. 1999. Modulation of neutrophil function in host defense against disseminated Candida albicans infection in mice. FEMS Immunol. Med. Microbiol. 26:299-307.[CrossRef][Medline]
    14. 14
  14. Liles, W. C., J. E. Huang, J. A. Van Burik, and R. A. Bowden. 1997. Granulocyte colony-stimulating factor administered in vivo augments neutrophil-mediated activity against opportunistic fungal pathogens. J. Infect. Dis. 175:1012-1015.[Medline]
    15. 15
  15. 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]
    16. 16
  16. Lipscomb, M. F., C. R. Lyons, A. A. Izzo, J. Lovchik, and J. A. Wilder. 1999. Experimental pulmonary cryptococcal infection in mice, p 681-696. In O. Zak and M. A. Sande (ed.), Handbook of animal models of infection. Academic Press, London, England.
    17. 17
  17. Loebenberg, D., A. Cacciapuoti, R. Parmegiani, E. L. Moss, Jr., F. Menzel, Jr., B. Antonacci, C. Norris, T. Yarosh-Tomaine, R. S. Hare, and G. H. Miller. 1992. In vitro and in vivo activities of SCH 42427, the active enantiomer of the antifungal agent SCH 39304. Antimicrob. Agents Chemother. 36:498-501.[Abstract/Free Full Text]
    18. 18
  18. Matsumoto, M., S. Matsubara, and T. Yokota. 1991. Effect of combination therapy with recombinant human granulocyte colony-stimulating factor (rG-CSF) and antibiotics in neutropenic mice unresponsive to antibiotics alone. J. Antimicrob. Chemother. 28:447-453.[Abstract/Free Full Text]
    19. 19
  19. Ozsahin, H., M. von Planta, I. Müller, H. C. Steinert, D. Nadal, R. Lauener, P. Tuchschmidt, U. V. Willi, M. Ozsahin, N. E. A. Crompton, and R. A. Seger. 1998. Successful treatment of invasive aspergillosis in chronic granulomatous disease by bone marrow transplantation, granulocyte colony-stimulating factor-mobilized granulocytes, and liposomal amphotericin-B. Blood 92:2719-2724.[Abstract/Free Full Text]
    20. 20
  20. Piggot, W. R., and C. W. Emmons. 1960. Device for inhalation exposure of animals to spores. Proc. Soc. Exp. Biol. Med. 103:805-806.
    21. 21
  21. Polak-Wyss, A. 1991. Protective effect of human granulocyte colony stimulating factor (hG-CSF) on Candida infections in normal and immunosuppressed mice. Mycoses 34:109-118.[Medline]
    22. 22
  22. Polak-Wyss, A. 1991. Protective effect of human granulocyte colony-stimulating factor (hG-CSF) on Cryptococcus and Aspergillus infections in normal and immunosuppressed mice. Mycoses 34:205-215.[Medline]
    23. 23
  23. Rex, J. H., S. C. Bhalla, D. M. Cohen, J. P. Hester, S. E. Vartivarian, and E. J. Anaissie. 1995. Protection of human polymorphonuclear leukocyte function from the deleterious effects of isolation, irradiation, and storage by interferon-gamma and granulocyte colony-stimulating factor. Transfusion 35:605-611.[CrossRef][Medline]
    24. 24
  24. Shimokawa, O., and H. Nakayama. 1992. Increased sensitivity of Candida albicans cells accumulating 14{alpha}-methylated sterols to active oxygen: possible relevance to in vivo efficacies of azole antifungal agents. Antimicrob. Agents Chemother. 36:1626-1629.[Abstract/Free Full Text]
    25. 25
  25. Vanden Bossche, H., G. Willemsens, and P. Marichal. 1987. Anti-Candida drugs—the biochemical basis for their activity. Crit. Rev. Microbiol. 15:57-72.[Medline]
    26. 26
  26. Vora, S., S. Chauhan, E. Brummer, and D. A. Stevens. 1998. Activity of voriconazole combined with neutrophils or monocytes against Aspergillus fumigatus: effects of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor. Antimicrob. Agents Chemother. 42:2299-2303.[Abstract/Free Full Text]
    27. 27
  27. Walsh, T. J., I. Lutsar, T. Driscoll, B. DuPont, M. Roden, P. Gharamani, M. Hodges, A. H. Groll, and J. R. Perfect. 2002. Voriconazole in the treatment of aspergillosis, scedosporiosis, and other invasive fungal infections in children. Pediatr. Infect. Dis. J. 21:240-248.[CrossRef][Medline]
    28. 28
  28. Yamamoto, Y., K. Uchida, T. W. Klein, H. Friedman, and H. Yamaguchi. 1992. Immunomodulators and fungal infections: use of antifungal drugs in combination with GCSF, p. 231-241. In H. Friedman (ed.), Microbial infections. Plenum Press, New York, N.Y.

Antimicrobial Agents and Chemotherapy, August 2004, p. 3154-3158, Vol. 48, No. 8
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.8.3154-3158.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Ullmann, A. J., Krammes, E., Sommer, S., Buschmann, I., Jahn-Muehl, B., Cacciapuoti, A., Schmitt, H.-J. (2007). Efficacy of posaconazole and amphotericin B in experimental invasive pulmonary aspergillosis in dexamethasone immunosuppressed rats. J Antimicrob Chemother 60: 1080-1084 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Patera, A. C.
Right arrow Articles by Loebenberg, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Patera, A. C.
Right arrow Articles by Loebenberg, D.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»