Efficacy of parenteral itraconazole against disseminated Candida albicans infection in two mouse strains

Donna M. MacCallum and Frank C. Odds*

Department of Molecular and Cell Biology, Institute of Medical Sciences, Foresterhill, University of Aberdeen, Aberdeen AB25 2ZD, Scotland, UK

Received 16 October 2001; returned 4 January 2002; revised 1 February 2002; accepted 2 April 2002


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The efficacy of a new cyclodextrin intravenous formulation of itraconazole was evaluated by intraperitoneal treatment of experimental disseminated Candida albicans infections in BALB/c and DBA/2 mice. Mice were treated with doses of 5 or 20 mg/kg for 14 days and observation was continued for 28 days. Mice were randomly assigned to groups given either water or grapefruit juice as drinking fluid, since components of grapefruit juice that inhibit cytochrome P450 enzymes might influence the pharmacokinetic behaviour of itraconazole. The experiments showed that the itraconazole/cyclodextrin solution unequivocally prolonged survival and reduced weight loss over the first 4 days post-challenge, relative to placebo-treated control animals, in both mouse strains. However, the antifungal treatments did not reduce burdens of C. albicans in kidneys or brain. Placebo-treated DBA/2 mice given grapefruit juice to drink had a significantly shorter mean survival time than the equivalent group given water. In BALB/c mice the placebo-treated animals given grapefruit juice survived longer, though not significantly longer, than placebo-treated animals given water. These results give a preliminary hint that grapefruit juice alters the susceptibility of some mouse strains to C. albicans infection, regardless of any effects or non-effects it may have on itraconazole pharmacokinetics in the animals.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Itraconazole is a broad-spectrum antifungal agent currently available in formulations for oral and intravenous use. The agent is used clinically in a variety of serious fungal dis- eases in normal and immunocompromised hosts, including aspergillosis, blastomycosis, candidiasis, coccidioidomycosis, cryptococcosis, histoplasmosis, paracoccidioidomycosis, sporotrichosis and disseminated Penicillium marneffei infections.14

Previous publications on the efficacy of itraconazole in mouse models of disseminated Candida albicans infections have been based on oral administration of the drug. Its efficacy in these models has been highly variable, with doses required to achieve 50% survival determined as 5 mg/kg twice a day,5 >10 mg/kg,6 32.9 mg/kg,7 >40 mg/kg,8 80 mg/kg9 and >=100 mg/kg.10 The majority of published data therefore suggest that itraconazole is weakly active or inactive in murine models of disseminated Candida infection.

It is unclear whether the usually poor activity of itraconazole in mice is related to the pharmacokinetics of the compound in this species. Determinations of peak plasma levels of itraconazole range from <1 µg/mL to 10–20 µg/mL, depending on dose, route and formulation.8,1113 Hostetler et al.14 obtained 50% survival in experimental cryptococcosis with doses of 60 and 120 mg/kg when itraconazole was formulated in hydroxypropyl-ß-cyclodextrin for oral use, but a polyethylene glycol formulation was essentially inactive.12

Our own recent study showed significantly lower levels of itraconazole in the plasma of BALB/c mice than DBA/2 mice, regardless of whether the drug was administered orally or intraperitoneally (ip).13 The study also showed that substituting grapefruit juice for water as drinking fluid did not appear to affect the plasma levels of itraconazole in either mouse strain. However, it remains possible that the components of grapefruit juice that inhibit the activity of mammalian cytochrome P450 enzymes such as CYP3A415 may influence the behaviour of itraconazole in Candida-infected animals and affect the outcome of itraconazole treatment. Sugar & Liu16,17 showed that substitution of grapefruit juice for drinking water in their mice led to attainment of detectable levels of another triazole antifungal agent, voriconazole, and enhanced the response of mice infected experimentally with Blastomyces dermatitidis to voriconazole treatment. The present study was set up to investigate the possible role of grapefruit juice on the outcome of itraconazole treatment in experimental disseminated C. albicans infections in two mouse strains.


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

The experiments were approved by the local ethics committee and were carried out under the terms of UK Home Office licences for research on animals. Female DBA/2 and BALB/c mice (Harlan, UK) with a weight range of 17–23 g were maintained under conditions specified by the Heath and Safety Executive for level 2 biohazard containment. The animals were supplied with food ad libitum. After a 2 week period of acclimatization after delivery, half of the animals of each species and strain were randomized to receive Del Monte brand grapefruit juice, purchased from the local supermarket. Consumption of drinking fluid was monitored daily during the period of itraconazole treatment. All groups of animals were returned to water as drinking fluid on day 15 post-challenge when itraconazole treatment had been stopped.

Itraconazole and other compounds

Itraconazole in cyclodextrin for injection was provided by Ortho Biotech Products, L.P. (Raritan, NJ, USA). Sterile 40% hydroxypropyl-ß-cyclodextrin (cyclodextrin) for injection was provided by the Janssen Research Foundation (Beerse, Belgium). Samples of the itraconazole solution were diluted in 40% cyclodextrin using full aseptic precautions to provide concentrations suitable for administration to the animals.

C. albicans challenge inoculum

C. albicans SC5314 was maintained on Sabouraud agar (Oxoid). The MIC of itraconazole for this strain was 0.063 µg/L as determined by repeated broth microdilution assay. Challenge inocula were grown overnight at 30°C in NGY medium, comprising 0.1% Neopeptone (Difco, Detroit, MI, USA), 0.4% glucose and 0.1% yeast extract (Difco). Inocula were centrifuged, washed twice with sterile distilled water and resuspended in sterile saline. The inocula were standardized by spectrophotometry and adjusted to concentrations suitable for iv injection in mice. The challenge doses were chosen on the basis of preliminary experiments to result in mean survival times of 7–10 days in untreated mice: for DBA/2 mice the challenge dose was 500 cfu/g body weight and for BALB/c mice it was 50 000 cfu/g. Samples of inoculum suspensions were plated on Sabouraud agar to confirm the accuracy of the challenge dose.

Experimental design

Three days after grapefruit juice had been substituted for drinking water for half of the experimental animals, the animals were infected intravenously (iv) with C. albicans. One hour after challenge, ip treatment of the animals was commenced with cyclodextrin placebo or itraconazole solution at a daily dose of 5 or 20 mg/kg. This treatment was repeated daily for 14 days or until the death of the animal. Each treatment group comprised six mice. The body weight of each mouse was recorded daily, and the condition of each mouse was assessed twice daily. The day of death of each animal was recorded. Animals that became incapable of reaching the food or drinking fluid were humanely killed and recorded as having died on the following day. The daily consumption of drinking fluid was monitored for each experimental group. The experiment was terminated 28 days after C. albicans challenge.

Post-mortem, the left kidney and brain were removed aseptically from the animals, weighed and homogenized, and tissue burdens of C. albicans determined by plating out dilutions of the homogenate on Sabouraud agar.

Survival rates were compared statistically by the Kaplan–Meier log rank test and organ burdens were compared statistically by the Mann–Whitney U test. The experiments with groups of six mice were powered to give 95% confidence in mean survival that differed by 7 or more days.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Consumption of water and grapefruit juice

Mean daily fluid consumption for the mice over the period of the experiment was as follows: DBA/2 mice receiving water, 3.9 mL; DBA/2 mice receiving grapefruit juice, 2.9 mL; BALB/c mice receiving water, 3.7 mL; BALB/c mice receiving grapefruit juice, 3.0 mL.

Outcome of infection and itraconazole treatment

Figures 1 and 2 illustrate the survival of the BALB/c and DBA/2 mice, respectively. Placebo-treated BALB/c mice supplied with grapefruit juice instead of drinking water survived slightly longer than their counterparts given water to drink. In both groups a single animal survived until day 28 post-challenge. The survivor that drank water had a C. albicans burden in its kidneys and brain that was lowest of the animals in this experimental group. The 28 day survivor in the grapefruit juice group had no detectable kidney burden of C. albicans but slightly higher C. albicans cfu in its brain. One or two of the six BALB/c mice in each of the itraconazole treatment groups died before the end of the experiment (Figure 1), but regardless of the nature of the drinking fluid or the itraconazole dose, survival among itraconazole-treated animals was clearly prolonged relative to placebo-treated animals.



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Figure 1. Survival curves for BALB/c mice infected iv with C. albicans SC5314. Circles, placebo-treated animals; squares, ip treatment with itraconazole at 5 mg/kg daily; triangles, ip treatment with itraconazole at 20 mg/kg daily. Filled symbols, mice given grapefruit juice to drink; open symbols, mice given water to drink.

 


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Figure 2. Survival curves for DBA/2 mice infected iv with C. albicans SC5314. Circles, placebo-treated animals; squares, ip treatment with itraconazole at 5 mg/kg daily; triangles, ip treatment with itraconazole at 20 mg/kg daily. Filled symbols, mice given grapefruit juice to drink; open symbols, mice given water to drink.

 
Among the DBA/2 mice, all 12 of the placebo-treated animals succumbed before the end of the 28 day observation period, but the six animals given grapefruit juice to drink instead of water survived notably less well than those receiving water (Figure 2). All animals given water as drinking fluid and treated with itraconazole at 5 or 20 mg/kg survived to the end of the experiment, while one each of the itraconazole-treated mice given grapefruit juice to drink died before the 28 day termination.

The statistics of the outcomes of infection are summarized in Table 1. In BALB/c mice given water to drink the mean survival time among animals treated with itraconazole at 5 and 20 mg/kg was significantly increased over placebo-treated animals (log rank test, P < 0.01); however, the differences in survival time between the treated animals given grapefruit juice were not statistically significant. The difference in mean survival time between the placebo-treated BALB/c mice given water or grapefruit juice to drink was also not significant. For DBA/2 mice the mean survival time for placebo-treated animals receiving grapefruit juice was significantly lower than that of animals given water to drink (P < 0.002), and in both the water and grapefruit juice subgroups of the DBA/2 mice mean survival time of all itraconazole-treated animals was significantly prolonged over that of the corresponding placebo-treated control groups (P < 0.0001 in all cases).


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Table 1.  Statistics of C. albicans infection and ip itraconazole treatment in BALB/c and DBA/2 mice
 
Significant reductions in kidney C. albicans burdens were seen only for DBA/2 mice drinking grapefruit juice and treated with itraconazole at 20 mg/kg (Mann–Whitney U test, P < 0.01; Table 1). For all other groups the mean kidney cfu/g, when C. albicans was detectable, was similar within a 1 log range and there were no significant burden reductions. The highest kidney burden at the time of death was seen in the DBA/2 mice given grapefruit juice and treated with placebo, a finding that corresponded with the lowest mean survival time in this group. C. albicans mean log tissue burdens in Candida-positive brain samples were similar, in the range 3.9–4.8 in all groups of animals regardless of itraconazole dose or drinking fluid (Table 1). However, among DBA/2 mice given grapefruit juice and treated with itraconazole at 20 mg/kg, no fungus was detectable in culture for three of the six brains sampled.

Loss of body weight over the first 4 days after C. albicans challenge was measured as an indirect indicator of overall health of the animals. For each mouse strain and drinking fluid group the mean weight loss was consistently greatest among placebo-treated animals. The data (Table 1) showed a dose-proportional effect in all itraconazole-treated animals, with minimal weight changes (and even gains in average weight) apparent in animals receiving the drug at 20 mg/kg. The notable exception was among BALB/c mice receiving grapefruit juice to drink, where the extent of weight loss, though still itraconazole dose related, was greater than in the other mouse groups (Table 1). There was an inverse correlative trend between the mean 4 day weight loss data and the mean survival times: r2 = 0.622, with P < 0.01 by ANOVA.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Our experiments show an unequivocal activity in vivo for a cyclodextrin iv formulation of itraconazole given ip to BALB/c and DBA/2 mice with experimental disseminated C. albicans infection. Activity was manifested principally by significant prolongation of survival relative to placebo-treated control animals, and a body weight loss over the first 4 days post-infection that was reduced in proportion to the itraconazole daily dose of 5 or 20 mg/kg. Loss of body weight as an indicator of progress of infection is a well-known phenomenon. However, the finding of a significant quantitative association between mean weight loss over 4 days and mean survival time is an intriguing predictive finding that will be investigated further, particularly as an approach to reducing stress and suffering for experimentally infected animals.

Our recent study on plasma levels of itraconazole and its active metabolite, hydroxy-itraconazole, was carried out in the same two mouse strains as the present study with the same dosing regimens of the iv itraconazole formulation and the same stratification by water or grapefruit juice as drinking fluid. A major finding of that study was that itraconazole plasma concentrations were significantly lower in BALB/c than in DBA/2 mice, predicting that the therapeutic effect of itraconazole should be more evident in the latter strain than the former. However, the only evidence supporting this prediction was the finding of a significant reduction in C. albicans burdens in kidney samples removed post-mortem in DBA/2 mice drinking grapefruit juice and treated with itraconazole at 20 mg/kg as compared with controls. We do not consider that our data show any major difference in response to itraconazole between the BALB/c and DBA/2 mice, even though the pharmacokinetics of itraconazole appear to differ considerably between uninfected animals of the two strains. From our data in uninfected animals, the mice receiving itraconazole 5 mg/kg would not have sustained plasma levels of the drug in excess of the MIC for the infecting strain.13

It is normal practice to regard tissue burdens as indicators of level of infection only when the tissues have been removed prospectively from representatives of all treatment groups at the same time relative to challenge. However, we include our data since it is notable that itraconazole-treated animals surviving to day 28 had recoverable counts of C. albicans from kidneys and brain of the same order as the counts in placebo-treated animals that died within 14 days of challenge. Only future prospective studies of tissue burdens with time will show whether the cfu measured on day 28 were the result of regrowth of C. albicans in tissues with levels of cfu suppressed by the end of itraconazole treatment.

Only in the control DBA/2 mice given grapefruit juice to drink were the tissue fungal burdens of a slightly higher order than in other mice, and these animals succumbed to infection more rapidly than any other group. The trend, with untreated mice challenged with C. albicans, is for kidney burdens to increase progressively with time,18 so the finding of higher burdens at day 6 in the DBA/2 mice receiving grapefruit juice to drink hints that the juice may have augmented progression of infection in these animals (Figure 1). The same effect was not seen in BALB/c mice—indeed the placebo-treated BALB/c animals that drank grapefruit juice survived slightly longer than their counterparts receiving water (Figure 2). We interpret these findings as an indication that the influences of grapefruit juice on experimental murine infection may go beyond the effects of juice components on gastrointestinal P450 enzymes,15 possibly to the extent of augmenting a host deficiency such as the C5 complement deficit that characterizes DBA/2 mice. The observation merits further study.

Our study has shown that the itraconazole formulation for iv administration to human patients has positive effects in the prolongation of survival of experimental disseminated C. albicans infection in mice, that the prolongation of survival inversely matches the decrease in body weight over the first 4 days after challenge but occurs mainly without large decreases in renal or cerebral fungal burden, and that substitution of grapefruit juice for drinking water may produce unexpected alterations in mean survival times, dependent on the strain of mouse challenged.


    Acknowledgements
 
We are grateful to Amanda Davidson, Marlene Arthur and Steve MacBain for technical assistance. This study was supported by a grant from Ortho Biotech Products, L.P.


    Footnotes
 
* Corresponding author. Tel: +44-1224-273128; Fax: +44-1224-273144; E-mail: f.odds{at}abdn.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Denning, D. W., Lee, J. Y., Hostetler, J. S., Pappas, P., Kauffman, C. A., Dewsnup, D. H. et al. (1994). NIAID mycoses study group multicenter trial of oral itraconazole therapy for invasive aspergillosis. American Journal of Medicine 97, 135–44.[ISI][Medline]

2 . Lortholary, O., Denning, D. W. & Dupont, B. (1999). Endemic mycoses: a treatment update. Journal of Antimicrobial Chemotherapy 43, 321–31.[Abstract/Free Full Text]

3 . Sheehan, D. J., Hitchcock, C. A. & Sibley, C. M. (1999). Current and emerging azole antifungal agents. Clinical Microbiology Reviews 12, 40–79.[Abstract/Free Full Text]

4 . Sirisanthana, T., Supparatpinyo, K., Perriens, J. & Nelson, K. E. (1998). Amphotericin B and itraconazole for treatment of disseminated Penicillium marneffei infection in human immunodeficiency virus-infected patients. Clinical Infectious Diseases 26, 1107–10.[ISI][Medline]

5 . Valentin, A., Le Guennec, R., Rodriguez, E., Reynes, J., Mallie, M. & Bastide, J. M. (1996). Comparative resistance of Candida albicans clinical isolates to fluconazole and itraconazole in vitro and in vivo in a murine model. Antimicrobial Agents and Chemotherapy 40, 1342–5.[Abstract]

6 . Hata, K., Kimura, J., Miki, H., Toyosawa, T., Nakamura, T. & Katsu, K. (1996). In vitro and in vivo antifungal activities of ER-30346, a novel oral triazole with a broad antifungal spectrum. Antimicrobial Agents and Chemotherapy 40, 2237–42.[Abstract]

7 . Uchida, K., Yamaguchi, H. & Shibuya, K. (1991). The therapeutic effects of itraconazole, a new triazole antifungal agent, for experimental fungal infections. Japanese Journal of Antibiotics 44, 588–99.[Medline]

8 . Van’t Wout, J., Mattie, H. & Van Furth, R. (1989). Comparison of the efficacies of amphotericin B, fluconazole and itraconazole against a systemic Candida albicans infection in normal and neutropenic mice. Antimicrobial Agents and Chemotherapy 33, 147–51.[ISI][Medline]

9 . Polak, A. (1990). In vitro and in vivo activity of antifungal agents in combination with fleroxacin, a new quinolone. Mycoses 33, 173–8.[ISI][Medline]

10 . Sugar, A. M. & Liu, X. P. (1998). Interactions of itraconazole with amphotericin B in the treatment of murine invasive candidiasis. Journal of Infectious Diseases 177, 1660–3.[ISI][Medline]

11 . Denning, D. W., Hall, L., Jackson, M. & Hollis, S. (1995). Efficacy of D0870 compared with those of itraconazole and amphotericin B in two murine models of invasive aspergillosis. Antimicrobial Agents and Chemotherapy 39, 1809–14.[Abstract]

12 . Hostetler, J. S., Hanson, L. H. & Stevens, D. A. (1992). Effect of cyclodextrin on the pharmacology of antifungal oral azoles. Antimicrobial Agents and Chemotherapy 36, 477–80.[Abstract]

13 . MacCallum, D. M. & Odds, F. C. (2002). Influence of grapefruit juice on itraconazole plasma levels in mice and guinea pigs. Journal of Antimicrobial Chemotherapy 50, 219–224.[Abstract/Free Full Text]

14 . Hostetler, J., Hanson, L. & Stevens, D. (1993). Effect of hydroxypropyl-ß-cyclodextrin on efficacy of oral itraconazole in disseminated murine cryptococcosis. Journal of Antimicrobial Chemotherapy 32, 459–63.[Abstract]

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

16 . Sugar, A. M. & Liu, X. P. (2000). Effect of grapefruit juice on serum voriconazole concentrations in the mouse. Medical Mycology 38, 209–12.[ISI][Medline]

17 . 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]

18 . Odds, F. C. (1988). Candida and Candidosis (2nd edn), pp. 253–4. Bailliere Tindall, London.





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