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Antimicrobial Agents and Chemotherapy, July 1999, p. 1716-1718, Vol. 43, No. 7
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Activities of Sordarins in Murine
Histoplasmosis
John R.
Graybill,1,2,*
Laura
Najvar,1
Annette
Fothergill,2
Rosie
Bocanegra,1 and
Federico
Gomez
de las Heras3
University of Texas Health Science
Center1 and Veterans Administration
Hospital,2 San Antonio, Texas, and
Glaxo Wellcome SA, Tres Cantos, Madrid,
Spain3
Received 30 December 1998/Returned for modification 26 February
1999/Accepted 23 April 1999
 |
ABSTRACT |
Sordarins are new antifungals which inhibit fungal protein
synthesis by blocking elongation factor 2. Three compounds were evaluated in a murine model of histoplasmosis. Immune-competent mice
were infected intravenously with 106 to 108 CFU
of Histoplasma capsulatum yeast cells. Mice were treated either orally with sordarins or fluconazole from day 2 through 8 after
infection or intraperitoneally with amphotericin B during the same
period. Protection was measured by increased rates of survival for 30 days after infection or reduction of lung or kidney tissue counts 9 days after infection. All three of the antifungal drugs tested were
protective compared with controls. Sordarins were effective at doses as
low as 2 mg/kg of body weight/day. This novel class of drugs compared
favorably with amphotericin B and fluconazole for the treatment of histoplasmosis.
| |
INTRODUCTION |
Histoplasmosis has been recognized
as a life-threatening opportunistic infection associated with AIDS
(9, 10, 12, 14, 16). Treatment with amphotericin B is
effective, but toxicity is a significant side effect (10).
Treatment with triazoles is also effective, but because antifungal drug
action is slower, these drugs are reserved for less critically ill
patients (13, 15). There remains an interest in identifying
new classes of antifungal drugs that are rapidly acting, potent, and
well tolerated. Sordarins have the potential of being such a class of
agents. They interfere with protein synthesis in fungi through a novel mechanism of inhibiting protein elongation factor 2 (4-7,
17). Sordarins have been shown to inhibit a variety of fungal
pathogens, including Candida species and Pneumocystis
carinii (1, 2, 8). In the present studies, we evaluated
the activities of three compounds against infection with
Histoplasma capsulatum.
| |
MATERIALS AND METHODS |
Pathogen.
H. capsulatum clinical isolate 93-255 was
obtained from the Fungus Testing Laboratory at the University of Texas
Health Science Center, San Antonio. The organism was maintained in the
yeast phase on Sabouraud's agar at 37°C for infection of mice. In
vitro testing of conidia was performed by the National Committee for Clinical Laboratory Standards macrobroth method but with incubation continued until the control tube showed visible growth and an end point
of 80% inhibition could be determined (11).
Antifungals.
Three sordarins, GM211676A (GM1), GM237354A
(GM2), and GM193663A (GM3), were obtained from Glaxo Wellcome, Tres
Cantos, Madrid, Spain, and were prepared by dissolving in water.
Fluconazole (Pfizer, New York, N.Y.) was dissolved in 0.3% Noble agar.
Amphotericin B (Bristol-Myers Squibb, Wallingford, Conn.) was suspended
in sterile water.
Animals.
Outbred immune-competent ICR male mice, 25 to
30 g, were obtained from Charles River Laboratories (Boston,
Mass.). Prior to infection, H. capsulatum colonies were
scraped from the agar, agitated, washed three times in sterile saline,
and suspended in sterile saline for infection. Cells were counted in a
hemacytometer for an estimate of the inoculum, and the counts were
confirmed by serial colony count dilutions in the yeast phase. Inocula
are reported as viable cells. Unanesthetized mice were infected
intravenously with 106 to 108 CFU.
Treatment.
The infection was allowed to become established
for 2 days before the mice were randomized for treatment. Groups of
mice were treated either by gavage with water (controls), fluconazole
at 5 or 10 mg/kg of body weight twice daily, or one of the GM
compounds, in doses ranging from 0.06 to 50 mg/kg twice daily, or
intraperitoneally with amphotericin B at 0.06, 0.3, 1.25, 2.5, or 5 mg/kg once daily. Drugs were administered in 0.2-ml volumes per dose.
Protection measures and statistics.
For survival studies,
groups of 10 mice were treated from day 2 through 8 after infection and
observed through day 30. The log rank and Wilcoxon tests of life tables
were used for comparisons. Because of multiple comparisons, a
P value of <0.001 was considered significant for survival
studies. For tissue burden studies, groups of 7 mice were treated from
day 2 through 8 and sacrificed on day 9 after infection. The lungs and
kidneys were harvested by an aseptic technique. The organs were
homogenized, and serial 10-fold colony count dilutions were plated on
brain heart infusion agar plates supplemented with 10% sheep blood.
These were incubated at 37°C for 2 weeks, and yeast cell colonies
were counted. Dunnett's one-tailed t test was used
for comparisons of tissue counts, with a P value of
<0.05 determining significance.
| |
RESULTS |
In vitro, at 7 days of incubation, the drug concentrations to
which H. capsulatum was found to be susceptible were <0.1
µg/ml for GM1, GM2, and GM3; 0.125 µg/ml for amphotericin B; and 4 µg/ml for fluconazole.
For each drug, a toxicity study was performed, in which 10 uninfected
mice were treated with 50 mg/kg twice daily for 7 days. In the group
treated with GM1, 1 mouse died, but the other 9 mice showed no evidence
of illness. There was no mortality or apparent illness in the mice
treated with the other two drugs. Three studies of survival were done.
In the first screening study (Table 1), where high doses of a drug were administered orally twice daily, all
three drugs markedly prolonged survival beyond that of controls. In two
subsequent studies, two sordarins were compared with amphotericin B and
fluconazole (Table 2). In this study, GM2
was highly effective in prolonging survival at doses down to the lowest
tested, 2 mg/kg, while GM3 was ineffective at 5 mg/kg and below.
However, the inoculum in the GM3 study was much larger than that for
GM2.
An additional study was done to measure the tissue burden of H. capsulatum after treatment with GM3. In this study, mice were infected with a relatively small inoculum, 3.4 × 106
CFU. The lungs and kidneys were cultured. As shown in Table
3, GM3 was effective in reducing cell
counts at 5 or 10 mg/kg but not 1.25 mg/kg. The positive control drugs,
fluconazole and amphotericin B, were also effective.
| |
DISCUSSION |
The sordarins are representative of a class of antifungal agents
with a target independent of the cell membrane (targeted by polyenes
and triazoles) or the cell wall (targeted by pneumocandins). At a
lethal infective dose of approximately 106 CFU of inoculum,
GM1 was effective in prolonging survival at 50 and 10 mg/kg. At the
same infecting dose, GM2 was effective to the lowest dose tested, 2 mg/kg. At the higher infecting dose of 108 CFU, GM3 was
effective at 5 mg/kg but not 1.25 mg/kg. These results were confirmed
in studies of the reduction of tissue burden, in which GM3 was
effective in reducing tissue counts at 5 mg/kg but not 1.25 mg/kg.
In terms of relative potency, amphotericin B administered at doses of
>1 mg/kg prolonged survival and reduced tissue counts much more than
the other agents. The sordarins were generally active at 5 mg/kg and
higher. In contrast, fluconazole was variably effective at 5 and 10 mg/kg. Fluconazole was also fungistatic, with much less effect on
kidney and lung tissue burdens than either the sordarins or
amphotericin B. These results suggest that the sordarins are quite
potent antifungals with excellent activity against H. capsulatum. On a milligram-for-milligram basis, they may be less
potent than amphotericin B, but they are more potent than fluconazole.
Further, they can be given orally, unlike amphotericin B.
Due to the limited availability of drugs, the present studies were not
sufficient to distinguish among the three sordarins or to permit
comparison of all three sordarins with amphotericin B and fluconazole.
In the absence of a bioassay or high-pressure liquid chromatography
assay, we were not able to study the pharmacokinetics of the drugs.
However, others have done so and have reported a correlation between
the area under the concentration-time curve and a therapeutic benefit
in mice (3). The present studies indicate that sordarins are
highly effective against H. capsulatum in this murine model
and suggest that they may be further evaluated in vivo for antifungal
potency and spectrum. The particular compounds studied herein were not
highly active against Aspergillus, but other analogues are
under development.
| |
ACKNOWLEDGMENT |
Glaxo Wellcome supported these studies.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Infectious
Diseases Service, South Texas Veterans Administration Hospital, 7400 Merton Minter Blvd., San Antonio, TX 78284. Phone: (210) 617-5111. Fax: (210) 614-6197. E-mail: graybill{at}uthSCSA.edu.
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Antimicrobial Agents and Chemotherapy, July 1999, p. 1716-1718, Vol. 43, No. 7
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
