Improving the mouse model for studying the efficacy of voriconazole

John R. Graybill1,*, Laura K. Najvar1, Gloria M. Gonzalez1,2, Steve Hernandez1 and Rosie Bocanegra1

1 The University of Texas Health Science Center at San Antonio, Department of Medicine, Division of Infectious Diseases (7881), 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA; 2 Departamento de Microbiología, Facultad de Medicina, Universidad Autónoma de Nuevo León, Monterrey, N.L., Mexico

Received 19 February 2003; accepted 12 March 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Outbred ICR mice were rendered neutropenic, infected intravenously with Fusarium solani and treated orally with voriconazole. When given alone, voriconazole was not protective up to 40 mg/kg/day. When grapefruit juice was administered before infection, mice were protected by voriconazole. The mechanism may be inhibition of gut mucosal cytochrome enzymes that rapidly degrade voriconazole in the mouse. These murine studies support expansion of voriconazole therapy in other highly resistant systemic mycoses.

Keywords: voriconazole, mice, grapefruit juice


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Voriconazole has been developed as a broad-spectrum antifungal triazole. Like itraconazole and posaconazole, voriconazole is degraded by hepatic cytochrome enzymes. However, whereas itraconazole is degraded largely by cyp 3A4, cyp 2B9 and 2B19 are mostly responsible for the degradation of voriconazole. Clearance of voriconazole is very rapid in the mouse (terminal half-life 1 h), and generally mice have not been used successfully in studies of this antifungal. Guinea pigs, which clear voriconazole more slowly, have been the major animal models for the study of systemic mycoses. Although guinea pigs are very useful for some studies, they are costly, occupy considerable laboratory space and are not suitable for some mycoses.1 In unpublished studies, we have infected severely neutropenic guinea pigs with Fusarium solani intravenously, but have been unable to produce disease, and usually unable to recover any Fusarium from tissues of guinea pigs.

In prior studies, Sugar & Liu2 demonstrated that mice treated orally with grapefruit juice substituted for drinking water have detectable voriconazole up to 1.6 mg/L 4 h after an oral dose of 20 mg/kg/day. Furthermore, Sugar & Liu3 found that mice infected with Blastomyces dermatitidis responded clinically to treatment with voriconazole and grapefruit juice. Grapefruit juice contains naringin and 6',7'-dihydroxybergamottin, both of which inhibit gut mucosal cyp 3A4. This in turn raises blood levels of drugs normally metabolized by cyp 3A4.4,5 Therefore, we considered that grapefruit juice might inhibit voriconazole metabolism by the mucosal cytochrome enzymes and thus increase intestinal absorption and systemic delivery of voriconazole. Although this mechanism has not been demonstrated specifically for voriconazole, as per Sugar & Liu,2 grapefruit juice does cause a prolonged rise in voriconazole serum concentrations.

Fusariosis is an infection associated with severe immune suppression. Treatment is difficult, as the organisms are often resistant to amphotericin B.6 In vitro studies show that Fusarium isolates tend to be susceptible to voriconazole, genaconazole and fluconazole, but not itraconazole.7 Some responses to fluconazole have been noted in mice with fusariosis, and responses to voriconazole have been noted in humans infected with Fusarium (Pfizer company files). Accordingly, we considered that grapefruit juice treatment might slow the clearance of voriconazole sufficiently for the drug to be effective in murine fusariosis.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Pathogens

F. solani isolate 95-2478 was obtained from the Fungus Testing Laboratory, University of Texas Health Science Center, San Antonio. The MIC of voriconazole for F. solani was 2 mg/L at 48 h incubation.8 Fungi were grown on potato flake agar at room temperature until used. Fusarium mycelial colonies were harvested by scraping the plates with sterile isotonic saline and filtering the suspension through sterile glass wool to remove hyphal fragments. Conidia were counted in a haemocytometer and suspended at the desired concentration in isotonic saline. The inocula were prepared in volumes of 0.2 mL per mouse, and confirmed by serial colony count dilutions. The viable inoculum counts are reported.

Animals

Outbred ICR mice, weighing ~25–30 g, were housed four per cage and given food and water ad libitum. On the day before Fusarium infection, mice were given a single dose of 5-fluorouracil 150 mg/kg intravenously.

Treatment

Voriconazole (obtained from Pfizer) was dissolved in polyethylene glycol. Controls were given polyethylene glycol in 0.2 mL volume once daily and also grapefruit juice. Commercially obtained bottled grapefruit juice was administered by gavage at 0.2 or 0.5 mL once daily. Administration of grapefruit juice was started 3 days before the beginning of the voriconazole therapy, or on the day voriconazole therapy began, and was continued during voriconazole administration. Treatment with voriconazole, in a volume of 0.2 mL by gavage once daily, was started 1 day before or 1 day after infection. For survival studies, the regimen was continued for 10 days after infection. Survival was observed to day 15, by which time deaths had stopped. For tissue burden studies, mice were treated to day 7. One day later, tissues were removed aseptically, homogenized and plated for serial colony count dilutions. Effort was made to keep trauma to the tissue constant.

Statistics

The logrank test was used to compare survival between treated and control animals. The Mann–Whitney test was used for comparisons of tissue burden. P <= 0.05 determined significance.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In a preliminary study, at up to 40 mg/kg daily, in the absence of grapefruit juice, voriconazole did not reduce mortality in mice infected intravenously with F. solani. As shown in Figure 1, administration of grapefruit juice 0.5 mL/day was begun either 3 days before or on the same day voriconazole therapy commenced. Voriconazole 20 mg/kg was begun 1 day after infection. In this study of mice infected with a high inoculum, voriconazole did not prolong survival over controls. In Figure 2, mice were infected with a lower dose of Fusarium. Grapefruit juice administration was begun 3 days before the start of voriconazole therapy, and voriconazole was begun 1 day before infection. Voriconazole 10 and 20 mg/kg (P = 0.004 and 0.005, respectively) significantly prolonged survival over controls.



View larger version (11K):
[in this window]
[in a new window]
 
Figure 1. Survival of mice infected with F. solani and treated with voriconazole 20 mg/kg. Groups of 12 mice were infected with 2 x 106 cfu/mouse of Fusarium and treated for 1–10 days. Squares demonstrate survival of controls administered grapefruit juice starting 3 days before the beginning of polyethylene glycol administration. Diamonds represent late grapefruit juice administration that began on the same day as voriconazole therapy. Triangles indicate early grapefruit juice administration that began 3 days before the start of voriconazole therapy.

 


View larger version (11K):
[in this window]
[in a new window]
 
Figure 2. Survival of groups of 12 mice infected with F. solani at 8 x 105 cfu/mouse and treated with voriconazole at 10 or 20 mg/kg. Grapefruit juice administration (0.2 mL/day) began 3 days before the start of voriconazole therapy. Control (squares); voriconazole 10 mg/kg (diamonds); voriconazole 20 mg/kg (triangles).

 
In tissue burden studies, mice were infected and treated with voriconazole and grapefruit juice. As shown in Figure 3, at an infecting dose of 8 x 105 cfu, kidney counts of Fusarium were significantly reduced by voriconazole 10 and 20 mg/kg (P = 0.0025 and 0.005, respectively). Spleen counts (Figure 4) were very low and not reduced further by voriconazole.



View larger version (7K):
[in this window]
[in a new window]
 
Figure 3. Tissue burden of kidneys of groups of 5–7 mice infected intravenously with 8 x 105 cfu/mouse of F. solani and treated from days 1–7 post-challenge with voriconazole, and grapefruit juice (0.2 mL/day) beginning 3 days before the start of voriconazole therapy. Controls; voriconazole 10 mg/kg; voriconazole 20 mg/kg.

 


View larger version (7K):
[in this window]
[in a new window]
 
Figure 4. Tissue burden of spleens of groups of 5–7 mice infected intravenously with 8 x 105 cfu/mouse of F. solani and treated from days 1–7 with voriconazole, and grapefruit juice (0.2 mL/day) 3 days before the start of voriconazole therapy. Controls; voriconazole 10 mg/kg; voriconazole 20 mg/kg.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present study shows that voriconazole is effective in treating fusariosis in neutropenic mice. Protection was modest, but the doses we explored were limited to 10 and 20 mg/kg. MacCallum & Odds9 found recently that grapefruit juice substantially raises the concentration of itraconazole in mice, with levels persisting in blood for >7 h. The effect was pronounced in DBA mice, but not in BALB/c mice. This supports a specificity of mouse strain used. The effect was not observed after parenteral dosing, supporting the cause as decreased gut mucosal metabolism of itraconazole. At similar doses, Sugar & Liu2 have found that voriconazole persists >4 h in the blood. Although we did not measure voriconazole in the bloodstream of our grapefruit juice recipients, it is very likely that the delayed clearance of voriconazole accounted for the protective effect. Results in several studies comparing 10 and 20 mg/kg were similar, suggesting that the presence of grapefruit juice, more than the dose of voriconazole, accounts for its antifungal activity. Furthermore, the benefit may be overwhelmed by a large fungal inoculum, as no protection was shown in mice infected with 2 x 106 cfu, but protection was evident at 8 x 105 cfu.

In addition to an effect on murine cytochrome enzymes, it is possible that grapefruit juice inhibited fungal cytochrome enzymes directly. Whereas we did not test this directly in vitro, we included controls treated with grapefruit juice. It is also possible that the grapefruit juice stimulated some protective host immune defence. However, the timing of grapefruit juice administration (begun on the same day as, or 3 days before, voriconazole treatment) would seem very short to generate such an effect. In any case, the controls given grapefruit juice succumbed at the same rate as controls with no grapefruit juice.

These studies open the possibility of using the mouse model to evaluate voriconazole treatment in a variety of systemic mycoses. This model should also permit comparison of voriconazole with other triazoles and echinocandins, all of which are effective in some murine mycoses.1,1014 As the mouse has been used most commonly for studies of systemic mycoses, and is the least costly and least sentient of the laboratory animals commonly employed, there should additionally be an impetus to use this simple model for comparison of multiple pharmaceuticals and combinations of antifungal drugs.


    Footnotes
 
* Corresponding author. Tel: +1-210-567-0990; Fax: +1-210-567-0962; E-mail: jrgraybill{at}aol.com Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Miller, G. H., Loebenberg, D., Antonacci, B., Cacciapuoti, A., Moss, E. L., Menzel, F. et al. (1996). In vitroin vivo correlations for the azole antifungals SCH56592 (56592), SCH39304 (39304), fluconazole (FCZ) and itraconazole (ITZ) in candidiasis and aspergillosis. In Programs and Abstracts of the Thirty-sixth Interscience Conference on Antimicrobial Agents and Chemotherapy, New Orleans, LA, 1996. Abstract F94, p. 116. American Society for Microbiology, Washington, DC, USA.

2 . Sugar, A. M. & Liu, X. P. (2000). Effect of grapefruit juice on serum voriconazole concentrations in the mouse. Medical Mycology 38, 209–13.[ISI][Medline]

3 . Sugar, A. M. & Liu, X. P. (2001). Efficacy of voriconazole in treatment of murine pulmonary blastomycosis. Antimicrobial Agents and Chemotherapy 45, 601–4.[Abstract/Free Full Text]

4 . Evans, A. M. (2000). Influence of dietery compounds on the gastrointestinal metabolism and transport of drugs. Therapeutic Drug Monitoring 22, 131–6.[CrossRef][ISI][Medline]

5 . Singh, B. N. (1999). Effects of food on clinical pharmacokinetics. Clinical Pharmacokinetics 37, 213–55. [ISI][Medline]

6 . Walsh, T. J. & Pizzo, P. A. (1988). Nosocomial fungal infections. Annual Reviews of Microbiology 42, 517–45.[CrossRef]

7 . Arikan, S., Lozano-Chiu, M., Paetznick, V., Nangia, S. & Rex, J. H. (1999). Microdilution susceptibility testing of amphotericin B, itraconazole, and voriconazole against clinical isolates of Aspergillus and Fusarium species. Journal of Clinical Microbiology 37, 3946–51.[Abstract/Free Full Text]

8 . National Committee for Clinical Laboratory Standards. (1998). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Conidium-forming Filamentous Fungi. Proposed Standard M38-P. NCCLS, Wayne, PA, USA.

9 . MacCallum, D. M. & Odds, F. C. (2002). Influence of grapefruit juice on itraconazole levels in mice and guinea pigs. Journal of Antimicrobial Chemotherapy 50, 219–24.[Abstract/Free Full Text]

10 . Abruzzo, G. K., Flattery, A. M., Gill, C. J., Kong, L., Smith, J. G., Pikounis, V. B. et al. (1997). Evaluation of the echinocandin antifungal MK-0991 (L-743,872) efficacies in mouse models of disseminated aspergillosis, candidiasis, and cryptococcosis. Antimicrobial Agents and Chemotherapy 41, 2333–8.[Abstract]

11 . Abruzzo, G. K., Gill, C. J., Flattery, A. M., Kong, L., Leighton, C., Smith, J. G. et al. (2000). Efficacy of the echinocandin caspofungin against disseminated aspergillosis and candidiasis in cyclophosphamide-induced immunosuppressed mice. Antimicrobial Agents and Chemotherapy 44, 2310–8.[Abstract/Free Full Text]

12 . Graybill, J., Bocanegra, R., Luther, M., Fothergill, A. & Rinaldi, M. G. (1997). Treatment of murine Candida krusei or Candida glabrata infection with L-743,872. Antimicrobial Agents and Chemotherapy 41, 1937–9.[Abstract]

13 . Graybill, J. R., Bocanegra, R., Luther, M. & Loebenberg, D. (1998). SCH56592 treatment of murine invasive aspergillosis. Journal of Antimicrobial Chemotherapy 42, 539–42.[Abstract]

14 . Lutz, J. E., Clemons, K. V., Aristizabal, B. H. & Stevens, D. A. (1997). Activity of the triazole SCH56592 against disseminated murine coccidioidomycosis. Antimicrobial Agents and Chemotherapy 41, 1558–61.[Abstract]