Journal of Antimicrobial Chemotherapy (2002) 49, 353-357
© 2002 The British Society for Antimicrobial Chemotherapy
Efficacy of ravuconazole (BMS-207147) in a guinea pig model of disseminated aspergillosis
a Department of Medicine, Division of Infectious Diseases, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, Mail Code 7881, San Antonio, TX 78229-3900; b Audie Murphy Division, South Texas Veterans Health Care System, San Antonio, TX 78284, USA
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Abstract |
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Ravuconazole (BMS-207147, ER-30346), an oral triazole, was evaluated in an immunosuppressed temporarily neutropenic guinea pig model of invasive aspergillosis. In this model, guinea pigs were immunosuppressed with triamcinolone 20 mg/kg sc od beginning 4 days before challenge and made neutropenic with cyclophosphamide 300 mg/kg ip 1 day before challenge. Treatments of ravuconazole 5, 10 and 25 mg/kg po od were compared with itraconazole 2.5 and 5.0 mg/kg po bd and amphotericin B 1.25 mg/kg ip od. Treatment began 24 h after lethal intravenous challenge with Aspergillus fumigatus and continued for 5 days. Mortality occurred in eight of eight untreated control animals versus none of eight treated with ravuconazole 5 or 10 mg/kg/day or itraconazole 10 mg/kg/day. Mortality occurred in one of eight animals treated with ravuconazole 25 mg/kg/day, one of eight with amphotericin B and two of eight treated with itraconazole 5 mg/kg/day. Compared with controls, each of the antifungals examined significantly reduced the tissue burden in liver and brain, although only the highest doses of the azole drugs and amphotericin B significantly reduced tissue burden in the kidney. All three doses of ravuconazole improved survival and also reduced the tissue burden of Aspergillus. In this model of invasive aspergillosis, ravuconazole showed significant activity and may be a useful compound in human disease.
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Introduction |
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Invasive aspergillosis in immunocompromised patients continues to be a significant life-threatening disease with high morbidity and mortality.1–5 Amphotericin B and itraconazole, each with shortcomings, are the antifungal agents used most often against Aspergillus spp., yet they continue to be insufficient therapeutic choices, with successful treatment seen in less than one-half of patients.1–3,5,6 The drug of choice for the treatment of disseminated aspergillosis is amphotericin B, but drug toxicity persists as a problem with standard formulation amphotericin B and the treatment may be ineffective, particularly in immunosuppressed patients.2,6 Itraconazole is less toxic than amphotericin B but has variable bioavailability in certain patient groups; recently, itraconazole resistance in Aspergillus fumigatus has been described.2,7–9 Newer formulations of these two drugs may address the problems of the toxicity of amphotericin B and the absorption of itraconazole.1,3 An important approach to improving antifungal therapies continues to be the development of newer azoles that offer several potential advantages over amphotericin B, including oral therapy, reduced toxicity and a broad therapeutic index.2,10 One of these newer azoles is ravuconazole, an oral triazole compound with an effective spectrum of activity against several major common groups of pathogenic fungi, including Aspergillus spp., Candida spp., Cryptococcus neoformans and dermatophytes.3,11,12 Ravuconazole has a long half-life and is effective following oral administration.4,13,14
We used a guinea pig model of invasive aspergillosis to assess the effectiveness of antifungal therapy in this disseminated disease.5,15 In this lethal model, guinea pigs were made leucopenic and additional immune-system suppression was induced with concomitant steroid administration. The development of pervasive infection in the liver, kidney, lung and brain was comparable to clinically disseminated invasive aspergillosis.16,17 We evaluated the activity of ravuconazole in experimental invasive aspergillosis and compared its efficacy with that of amphotericin B and itraconazole.
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Materials and methods |
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Guinea pig model
Male Hartley guinea pigs (0.5 kg) were immunosuppressed with triamcinolone acetonide 20 mg/kg sc od (Steris Laboratories, Phoenix, AZ, USA) beginning 4 days before challenge and rendered neutropenic with cyclophosphamide 300 mg/kg ip (Pharmacia, Kalamazoo, MI, USA) 1 day before challenge. With this temporarily immunosuppressive regimen, the guinea pigs had reduced total white blood cell counts through day 7. Twenty-four hours after induction of neutropenia, groups of eight to 10 guinea pigs were sedated with ketamine HCl 44 mg/kg (Fort Dodge Laboratories, Fort Dodge, IA, USA), atropine 0.04 mg/kg (Elkin-Sinn, Cherry Hill, NJ, USA) and xylazine 5 mg/kg (Bayer Corporation Agriculture Division, Shawnee Mission, KS, USA), and challenged intravenously through the saphenous vein with a lethal inoculum of 106 A. fumigatus conidia. Each group contained at least one untreated control guinea pig, for which the lethal challenge was fatal within 6 days, with a mean survival of 4.8 ± 0.4 days (range 3–6 days) after challenge. The administration of ceftazidime 100 mg/kg im od (SmithKline Beecham Pharmaceuticals, Philadelphia, PA, USA) began on the day of challenge to prevent intercurrent bacterial infection. All animal research procedures were approved by the Institutional Animal Care and Use Committee at the University of Texas Health Science Center (San Antonio, TX, USA).
Antifungal agents
Antifungal treatment comprised amphotericin B (Fungizone; Bristol-Myers Squibb Co., Princeton, NJ, USA), ravuconazole (Bristol-Myers Squibb Co.) or itraconazole cyclodextrin solution (Janssen Research Foundation, Beerse, Belgium), and began 24 h after challenge with A. fumigatus conidia and continued for 5 days. Amphotericin B was diluted with 5% dextrose in sterile water at a ratio of 1.25 mg/mL of diluent and was administered at 1.25 mg/kg ip od. Ravuconazole was suspended in polyethylene glycol (PEG) 400 (Sigma Chemical, St Louis, MO, USA) and administered as a 10 mg/mL suspension at 5, 10 or 25 mg/kg po od. Itraconazole (10 mg/mL) was administered at 2.5 or 5.0 mg/kg po bd for a total dose of 5 mg/ kg/day or 10 mg/kg/day, respectively.
Organ cultures
Organ cultures were carried out post-mortem [after the death of the animal during treatment (n = 12) or 96 h after completion of treatment in the remaining treated guinea pigs (n = 44)]. Guinea pigs were humanely killed by terminal exsanguination after being anaesthetized with ketamine HCl 44 mg/kg and xylazine 10 mg/kg. Organs (brain, lung, liver and kidneys) were removed aseptically and were cultured to determine the degree of infection with A. fumigatus. Organs were considered positive when three or more colonies of A. fumigatus were present on minced tissues placed directly on Sabouraud Dextrose Agar (SDA) plates (Becton Dickinson and Co., Cockeysville, MD, USA) or when semi-quantitative cultures of tissue homogenates contained over 30 cfu/g of tissue.5 Tissue burdens of Aspergillus were evaluated with semi-quantitative cultures that could detect 30–20 000 cfu/g of tissue.18 Samples of each organ were finely chopped (manually), weighed, diluted 1:10 (w/v) with sterile saline and homogenized for 25 s with an electric tissue homogenizer (IKA-Works, Cincinnati, OH, USA). Duplicate 0.1 and 1.0 mL samples of the organ homogenate were plated on SDA plates, incubated at 37°C for 48 h, and colonies were counted. In combination, these two methods detected A. fumigatus at 3–20 000 cfu/g of tissue.
Organisms
A. fumigatus isolate P171, a clinical isolate that had been used in previous animal studies, was grown on SDA slants at 37°C for 24 h. The 24/48 h MICs of ravuconazole for this isolate were 0.25/1 mg/L. For injection into the guinea pigs, conidia were harvested by a sterile saline wash of the slant surface, with conidia being dislodged by gentle rubbing with a sterile glass rod. The resultant conidia suspension was adjusted to the desired concentration of 106 conidia/mL by haemocytometer count, which was verified by duplicate serial plating on SDA plates for colony counts.
Statistical analysis
The Fisher exact test and the Wilcoxon rank sum test were used where appropriate. Statistical significance was defined as P < 0.05, but adjustments were made for multiple dose comparisons for each organ evaluated so that the level of significance was P < 0.003.
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Results |
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In sequential experiments, antifungal treatment with amphotericin B, itraconazole and ravuconazole, beginning 24 h after lethal challenge with A. fumigatus conidia, enhanced survival when compared with the untreated infected controls (Figure
). By the sixth day after challenge, guinea pigs treated with ravuconazole 5, 10 or 25 mg/kg/day or itraconazole 10 mg/kg/day showed a modest increase in survival over those treated with amphotericin B or itraconazole 5 mg/kg/day (not significant) versus controls. Death occurred during the course of the experiment in all eight control animals, one of eight (13%) treated with ravuconazole 25 mg/kg/day and amphotericin B and two of eight (25%) treated with itraconazole 5 mg/kg/day (Table 1). Compared with controls, all regimens decreased mortality by the sixth day after challenge (P < 0.003). Additionally, all antifungal regimens used in these trials increased the mean days of survival (P < 0.003) for these guinea pigs (Table 1).
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) received no antifungal treatment. Guinea pigs were treated with amphotericin B 1.25 mg/kg/day (
) or 10 (
) mg/kg/day or ravuconazole 5 (
), 10 (
) or 25 (
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Semi-quantitative organ cultures of liver, lung, kidney and brain are shown in Table 2
. The brain, kidneys, liver and lungs of all untreated control animals were extensively infected. Compared with controls, ravuconazole 5, 10 and 25 mg/kg/day and itraconazole 10 mg/kg/day reduced the tissue burden of Aspergillus in brain and liver nearly 1000-fold and also showed up to 10-fold improvement over other treatments. Ravuconazole 25 mg/kg/day, itraconazole 5 and 10 mg/kg/day and amphotericin B also significantly reduced the colony counts in the kidneys. In lung tissue, reductions in colony counts were obtained with ravuconazole 10 and 25 mg/kg/day, itraconazole 10 mg/kg/ day and amphotericin B, yet neither ravuconazole 5 mg/ kg/day nor itraconazole 5 mg/kg/day differed from controls in this tissue.
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Culture results from guinea pigs treated with ravuconazole, itraconazole and amphotericin B are shown in Table 3
. Compared with controls, ravuconazole 10 and 25 mg/kg/day and itraconazole 10 mg/kg/day were more effective than the other agents at sterilizing brain and lung tissues. Additionally, ravuconazole 10 mg/kg/day was more effective in reducing positive cultures (A. fumigatus was undetectable in six of eight cultures) in liver tissue than were itraconazole 5 or 10 mg/kg/day or amphotericin B. In kidney tissue, ravuconazole 25 mg/kg/day was equal to itraconazole 10 mg/kg/day and superior to any of the other doses or agents used in these experiments, with only half of the cultures yielding growth. Culture results from guinea pigs treated with itraconazole 5 mg/kg/day showed only slight improvement versus controls in kidney, liver and lung tissue.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Ravuconazole was evaluated in our lethal guinea pig model of infectious aspergillosis and was compared with similar doses of itraconazole and with a high dose of conventional amphotericin B. Compared with untreated controls, ravuconazole treatment significantly decreased mortality and was comparable to either dose of itraconazole or to amphotericin B. Ravuconazole treatment at either dose prolonged mean days of survival in this model. All untreated guinea pigs and one of eight (13%) of those treated with ravuconazole 25 mg/kg/day or amphotericin B died before the planned termination of the experiment (the eighth day after infection), as did two of eight (25%) of the animals treated with itraconazole 5 mg/kg/day. Those treated with ravuconazole 5 or 10 mg/kg/day or itraconazole 10 mg/kg/ day showed 100% survival. Aspergillus tissue burden in brain and liver tissue was reduced as much as 1000-fold by ravuconazole, which proved more effective than the maximally tolerated dose of amphotericin B at reducing tissue burden in this experimental model. Additionally, 10-fold to nearly 100-fold reductions in kidney tissue burden were seen with the two highest doses of ravuconazole. Ravuconazole 10 mg/kg/day and itraconazole 10 mg/kg/day were virtually indistinguishable in reducing tissue burden in the liver, lung and brain, but kidney counts were reduced almost 100-fold by itraconazole 10 mg/kg/day when compared with ravuconazole 10 mg/kg/day. Ravuconazole 25 mg/kg/day and itraconazole 10 mg/kg/day were found to sterilize brain and lung tissues more effectively than the lower doses of ravuconazole, the low dose of itraconazole or amphotericin B. In terms of producing sterile cultures in liver, lung and brain, all three doses of ravuconazole were equal or superior to the high dose of amphotericin B chosen for these experiments. In kidney tissues, a dose response was seen with ravuconazole, with the 25 mg dose being equal or superior to all other regimens examined. However, one guinea pig treated with ravuconazole 25 mg/ kg/day died during treatment and was found to have tissue burdens in the kidney and liver that were similar to those of the control animals. This may be related to absorption difficulties such as those documented for itraconazole that have been reported for ravuconazole in dogs.19 Also, during the course of these experiments, serum levels of the antifungals were not measured. However, absorption issues during treatment with itraconazole are well known; therefore, the performance of itraconazole may have been affected by decreased serum levels in some of the animals. The ability of ravuconazole to exceed or approximate the ability of the high-dose itraconazole and amphotericin B to reduce fungal burden in this model, coupled with the reduced mortality attributed to ravuconazole treatment, indicates that this compound may have clinical benefit in invasive aspergillosis.
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Acknowledgements |
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We thank the Fungus Testing Laboratory at UTHSCSA for performing antifungal susceptibility testing. This work was supported by a grant from Bristol-Myers Squibb Co. This study was presented in part at the 101st General Meeting of the American Society for Microbiology, Orlando, FL, USA, May 20–24, 2001 (Abstract F-89).
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Notes |
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* Corresponding author. Tel: +1-210-567-4823; Fax: +1-210-567-3303; E-mail: kirkpatrick{at}uthscsa.edu
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Received 14 May 2001; returned 17 July 2001; revised 23 August 2001; accepted 31 October 2001
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