JAC Advance Access originally published online on May 4, 2007
Journal of Antimicrobial Chemotherapy 2007 60(1):162-165; doi:10.1093/jac/dkm123
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Efficacy of voriconazole in a murine model of cryptococcal central nervous system infection
Unitat de Microbiologia, Facultat de Medicina i Ciències de la Salut, Universitat Rovira i Virgili, Reus, Spain
* Corresponding author. Tel: +34-977-759359; Fax: +34-977-759322; E-mail: josep.guarro{at}urv.cat
Received 5 December 2006; returned 16 February 2007; revised 15 March 2007; accepted 5 April 2007
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Abstract |
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Objectives: We studied the efficacy of voriconazole compared with amphotericin B in a murine model of central nervous system infection by Cryptococcus neoformans. Infection was established intracerebrally.
Methods: Mice were treated with voriconazole at 10, 40 or 60 mg/kg/day orally or with amphotericin B at 1.5 mg/kg/day intraperitoneally. Treatment began 1 day after infection and continued for 10 days post-infection. Tissue burden studies were performed on day 7 and 1 day after the treatment finished.
Results: Both drugs were able to significantly prolong survival with respect to the control group. The highest tissue burden reduction was achieved with voriconazole at 60 mg/kg/day.
Conclusions: We have developed a murine model of cryptococcal central nervous system infection and demonstrated that voriconazole has a potential for the treatment of cryptococcosis.
Keywords: Cryptococcus neoformans , intracranial murine model , amphotericin B , voriconazole
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Introduction |
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In spite of aggressive antifungal therapy, cryptococcosis is still an important cause of morbidity and mortality in immunocompromised patients, especially those with AIDS.1–3 The treatment of choice for severe cryptococcosis, including CNS disease, is amphotericin B alone or combined with flucytosine, and for less severe disease fluconazole could be an alternative.4 The toxicity of amphotericin B and flucytosine and the increasing isolation of fluconazole-resistant strains5,6 underline the need for improved treatments and the use of new strategies. Voriconazole has demonstrated excellent in vitro activity against Cryptococcus neoformans,7 achieves good levels in CSF8 and there is some evidence of clinical efficacy.9–11 The aim of the current study was to evaluate the activity of voriconazole in a murine model of cryptococcal CNS infection.
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Materials and methods |
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Three clinical isolates were used in this study, two belonging to C. neoformans var. neoformans [strains FMR 8398 (strain A) and FMR 8411 (strain B), both with serotype AD, from the CSF of an HIV-negative patient and an HIV-positive patient, respectively] and one belonging to C. neoformans var. grubii [FMR 8409 (strain C), serotype A, isolated from the CSF of an HIV-negative patient]. They were subcultured on Sabouraud dextrose agar (SDA) plates and incubated at 35ºC for 48 h. In vitro susceptibilities of the three strains to the antifungals were tested using a microdilution reference method.12 For azoles and flucytosine, the MICs were defined as the lowest concentrations at which there was 50% inhibition of growth when compared with a drug-free control. For amphotericin B, the MIC was defined as the lowest concentration resulting in 100% inhibition of growth.
Male OF-1 mice weighing 18–20 g were briefly anaesthetized with fluothane and challenged intracerebrally with ~700 cfu of strains A or B, or 2500 cfu of strain C. Preliminary experiments with the three strains (data not shown) demonstrated that these concentrations of fungal elements were the optimal dose for producing an acute infection, with 100% of the animals dying within 15 days of infection. The inoculum was delivered in a volume of 0.05 mL through a 27-gauge needle by direct puncture through the cranial vault, ~6 mm posterior to the orbit. The procedure was approved by the Animal Welfare Committee of the Rovira i Virgili University.
For survival studies, groups of 10 mice were randomly established for each strain and treatment before starting the study. Treatment began 1 day after infection and continued for 10 days post-infection. Amphotericin B purchased as Fungizone was given at 1.5 mg/kg/day intraperitoneally in a volume of 0.2 mL; voriconazole purchased as Vfend was given at 10, 40 or 60 mg/kg/day orally by gavage in a volume of 0.1 mL. Control animals received no treatment. From 3 days prior to infection, the mice that received voriconazole and the control group were given grapefruit juice (Hero, Spain) instead of water. Mice were checked daily for 30 days after challenge.
For tissue burden studies, groups of 10 mice were randomly established for each strain and treatment and one group as control. Five animals from each group, randomly chosen before starting the study, were sacrificed on day 7 post-infection and the remaining five 1 day after the treatment finished (day 11 post-infection). Brain and lungs were aseptically removed and homogenized in 1 mL of sterile saline. Serial 10-fold dilutions of the homogenates were plated on SDA, incubated at 35°C and examined daily for 3 days. The numbers of cfu per gram of tissue were calculated.
Mean survival times were estimated by the Kaplan–Meier method and compared among groups by using the log-rank test. cfu counts were analysed by the Mann–Whitney U-test using SPSS for Windows, version 11.5.
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Results and discussion |
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MICs of amphotericin B and voriconazole were similar for the three strains, the differences among them never being higher than one dilution. Voriconazole showed very low MIC values for the strains A, B and C (0.03, 0.03 and 0.06 mg/L, respectively). MICs of amphotericin B for the strains A, B and C were 0.5, 0.5–1 and 1 mg/L, respectively.
All treatments significantly prolonged survival of mice infected with the three strains of C. neoformans tested with respect to the control group (P < 0.05) (Figure 1). The survival prolongation was markedly evident in animals that received voriconazole at 60 mg/kg/day, being 100% at day 30 for mice infected with the strains A and B and 80% for those infected with strain C. Voriconazole administered at lower doses (10 and 40 mg/kg/day) had a moderate but significant effect in prolonging survival when compared with the control group. These results agree with those obtained by other authors who obtained a partial response with voriconazole at doses lower than 40 mg/kg/day in an experimental fusariosis in mice.13
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Amphotericin B significantly prolonged the survival of infected mice in all groups with respect to the control group. Our results suggest that the efficacy of amphotericin B could be dependent on the serotype of C. neoformans or that it is at least strain-dependent. However, due to the small number of strains tested here, further studies with more strains are warranted to confirm this. No significant differences in survival were observed between mice treated with amphotericin B and voriconazole at 40 and 60 mg/kg.
All therapies reduced the fungal load versus controls in at least one organ at day 7 after infection for the three strains (Table 1). Amphotericin B significantly reduced the tissue burden with respect to the control group in brain and lungs (P = 0.007) of the animals infected with strains A and B but not in lungs of those inoculated with strain C (P = 0.055). Voriconazole at 60 mg/kg/day significantly reduced the fungal load with respect to the control group in brain and lungs for the three strains tested (P = 0.008). Voriconazole at lower doses (10 and 40 mg/kg/day) also significantly reduced the fungal loads with respect to the control group in brain and lungs for the three strains tested, except for lungs of mice infected with strain C. Voriconazole at 60 mg/kg significantly reduced fungal burden in brain and lungs with respect to amphotericin B in mice infected with strain A.
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On the seventh day, amphotericin B and voriconazole at high doses reduced the fungal load similarly, with the exception of those mentioned above. However, this tendency changed on day 12 post-infection, at which voriconazole at 60 mg/kg/day was the most effective treatment, significantly reducing fungal load with respect to amphotericin B in both brain and lungs of mice infected with strains A and B and in lungs of those infected with strain C. Moreover, only voriconazole at 60 mg/kg/day cleared fungal burden of both organs in practically all the animals infected with strain A.
Our results agree in part with Mavrogiorgos et al.14 who demonstrated, although only testing one strain of C. neoformans var. grubii, using a murine model of pulmonary cryptococcal infection, that voriconazole was able to reduce serum fungal polysaccharide levels and prolong survival. However, in that study, fungal burden in lungs was not reduced. In spite of the differences between the experimental models used in both studies, it is clear that voriconazole is active against C. neoformans and that this drug has potential efficacy for the treatment of CNS cryptococcosis.
It is well known that voriconazole is quickly metabolized in mice. Therefore, in the present study and in order to avoid its rapid clearance, the animals that received voriconazole and the control group were given grapefruit juice instead of water. Grapefruit juice blocks the voriconazole metabolism and increases its serum concentration in mice to suitable levels for performing treatment studies.15 Autoinduction of voriconazole metabolism is not observed in humans, plasma steady-state concentrations remaining constant with time at standard dosing16 and showing significant transport across the blood–brain barrier.17 Although data are still scarce, voriconazole efficacy has also been demonstrated in humans. Lutsar et al.11 demonstrated that a linear relationship existed between doses of voriconazole and CSF concentration in two patients with cryptococcal meningitis. They showed a partial and a stable response, respectively, to voriconazole treatment. In another clinical study, involving 18 patients with cryptococcal meningitis who were treated with voriconazole, a satisfactory and a stable response was obtained in 7 and 10 of them, respectively.10
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None to declare.
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Acknowledgements |
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We thank Marcia dos Santos Lazéra from the Instituto de Pesquisa Clínica Evandro Chagas, FIOCRUZ, Rio de Janeiro, Brazil, for providing the strains used in this study. This work was supported by a grant from Fondo de Investigaciones Sanitarias from the Ministerio de Sanidad y Consumo of Spain (PI 050031).
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References |
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1 de Hoog GS, Guarro J, Gené J, et al. Atlas of Clinical Fungi (2000) Second Edition. Utrecht, The Netherlands: Centraalbureau voor Schimmelcultures. and University Rovira i Virgili, Reus, Spain.
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Sabatelli F, Patel R, Mann PA, et al. In vitro activities of posaconazole, fluconazole, itraconazole, voriconazole, and amphotericin B against a large collection of clinically important molds and yeasts. Antimicrob Agents Chemother (2006) 50:2009–15.
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Graybill JR, Najvar LK, Gonzalez GM, et al. Improving the mouse model for studying the efficacy of voriconazole. J Antimicrob Chemother (2003) 51:1373–6.
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Sugar AM, Liu X. Efficacy of voriconazole in treatment of murine pulmonary blastomycosis. Antimicrob Agents Chemother (2001) 45:601–4.
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Roffey SJ, Cole S, Comby P, et al. The disposition of voriconazole in mouse, rat, rabbit, guinea pig, dog, and human. Drug Metab Dispos (2003) 31:731–41.
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