Vol. 35, No. 7
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, JUlY 1991, P. 1460-1463
0066-4804/91/071460-04$02.00/0 Copyright C 1991, American Society for Microbiology
Treatment of Experimental Cryptococcosis with SCH 39304 and Fluconazole R. NEGRONI,1 M. R. I. DE ELIAS COSTA,' J. L. FINQUELIEVICH,l C. IOVANNITTI,1 I. AGORIO,1 I. N. TIRABOSCHI,l AND D. LOEBENBERG2* Centro de Micologia del Departamento de Microbiologia, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, Piso 11, C.P. 1121, Buenos Aires, Argentina,1 and Schering-Plough Research Corporation, Bloomfield, New Jersey 070032 Received 2 April 1990/Accepted 10 April 1991
The efficacy of two triazoles, SCH 39304 and fluconazole, in the treatment of disseminated cryptococcosis in Wistar rats was determined. A total of 160 rats were inoculated intracardiacally with 2 x 105 cells of Cryptococcus neoformans. Both drugs were administered by gavage once daily, at three doses (8, 16, and 32 mg/ kg/day). Two treatment schedules were followed: (i) treatment began 1 week after infection and continued for 3 weeks and (ii) prophylaxis treatment began 3 days before infection and continued an additional 3 weeks. Evaluation was based on (i) macroscopic examination of lungs, (ii) microscopic examination of brains and lungs, (iii) histopathology of brains and lungs, and (iv) determination of number of CFU in brains. The number of CFU was the best measure of activity. SCH 39304 was more active than fluconazole in both regimens, and, prophylactically, SCH 39304 was able to achieve biological cures.
study was to compare the efficacies of SCH and FLZ in this experimental animal model of disseminated cryptococcosis.
Cryptococcosis is a systemic mycosis produced by a capsulated yeastlike fungus, Cryptococcus neoformans. This infection is an emerging problem in immunocompromised patients, especially in those suffering from lymphomas, sarcoidosis, and AIDS, and those undergoing steroid treatments (2, 6, 11-14, 19). The present treatment of choice in cases of cryptococcosis is a combination of intravenous amphotericin B and oral 5-fluorocytosine for at least 6 weeks (1, 19). The use of this treatment sometimes causes severe side effects such as hypokalemia, anemia, impaired renal function, and leukopenia (12, 13). These alterations are even more severe in immunodeficient patients. In addition to these problems, relapses are often observed in patients with AIDS (13, 19). The azoles have been used successfully in cases of cryptococcosis without central nervous system involvement. They do not penetrate the blood-brain barrier well because of their high molecular weights and binding to serum proteins (2, 13). Results with itraconazole in the treatment of cryptococcosis, both in experimental models and in human beings, have been variable (5, 8, 21, 22). Fluconazole (FLZ) is a fluorinated bis-triazole watersoluble compound which can be administered orally or parenterally. It is a broad-spectrum antifungal agent and is active against C. neoformans. FLZ achieves 80% of its serum drug concentration in spinal fluid and has been successfully used in human beings (3, 4, 10, 20). SCH 39304 (SCH) is a new triazole derivative which is very active against several experimental systemic mycoses, has a very long serum half-life, and penetrates the bloodbrain barrier well (16, 17). In recent studies, we showed that Wistar rats inoculated intracardiacally with C. neoformans developed a subacute disseminated and fatal disease (15). This experimental model was used for a comparative evaluation of amphotericin B and itraconazole. Both drugs were unable to produce a biological cure in this infection (8). The aim of the present *
MATERIALS AND METHODS In vitro studies. The Ribas strain of C. neoformans (from the Mycology Center Collection), which is known for its pathogenicity for Wistar rats, was used (8, 15). MICs for SCH and FLZ were established in Sabouraud broth with 2% dextrose by performing twofold dilutions from 100 to 0.01 ,ug/ml. SCH was dissolved in polyethylene glycol 200 (PEG) and FLZ was dissolved in distilled water to a final concentration of 1 mg/ml for each. PEG was not active in vitro. These solutions were sterilized by heating at 70°C for 1 h on three consecutive days. Sterilization controls were carried out in brain heart infusion broth and agar at 37°C (9). Tubes were incubated at 28°C for 15 days. After completion of MIC incubation times, minimal fungicidal concentrations were determined. Sediments in MIC tubes were centrifuged, washed twice with sterile distilled water, suspended in 0.2 ml of sterile distilled water, and seeded in Sabouraud dextrose agar. Incubation was at 28°C for 15 days (18). In vivo studies. A total of 160 Wistar rats of both sexes, weighing approximately 250 g each, were used. Animals (10 per group, 5 of each sex) were anesthetized with ethyl ether and inoculated (0.1 ml) by the intracardiac route with a suspension of 2 x 105 C. neoformans Ribas in an isotonic saline solution. The inoculum was from a 48-h culture incubated at 37°C in Sabouraud honey agar. SCH was dissolved in PEG at pH 3 to a final concentration of 8 mg/ml. FLZ was prepared in dimethyl formamide at a ratio of 1 g: 10 ml, as used in previous experiments (8). The FLZ-dimethyl formamide solution was added to 90 ml of distilled water containing 5% dextrose and 0.2% agar. Controls contained only the vehicles. Two treatment schedules were followed. (i) Treatment began 7 days after infection and continued for 21 days. Both drugs were administered by gavage once daily (on the basis of proposed clinical usage) at 8, 16, or 32 mg/kg/day. Control groups received either PEG at pH 3.0 or the dimethyl
Corresponding author. 1460
TABLE 3. Prophylactic efficacy of SCH and FLZ in disseminated cryptococcosis in rats as shown by residual lesions in lungs % of animals with residual lesions (in lungs)
TABLE 1. Treatment efficacy of SCH and FLZ in disseminated cryptococcosis in rats as shown by presence of residual lesions in lungs % of animals with residual lesions (in lungs) evaluated by: Group
SCH SCH SCH FLZ FLZ FLZ
Dose (mg/kg/day)
8 16 32 8 16 32
SCH-vehicle FLZ-vehicle
Macroscopic exam (granulomas)
0 0 0 20 30 20 100 100
evaluated by:
Histopathological exam Wet mount Giant-cell Small granulo- granulomasb masa
30 40 70 60 100 50 100 100
60C 70c 40c 80C 100C 70c 0 0
0 0 0 10 0 0 100 100
Group
m(granulomas) SCH SCH SCH FLZ FLZ FLZ SCH-vehicle FLZ-vehicle
formamide vehicle. (ii) Prophylaxis treatment began 3 days before infection and continued for an additional 3 weeks. SCH and FLZ were dosed in the same manner as in the treatment studies. Animals were sacrificed 1 week after completion of both treatment schedules and then necropsied. The following evaluations were done: (i) macroscopic examination of lungs (for granulomas), (ii) microscopic examination of wet-mount preparations (by using a phase-contrast microscope), (iii) histopathological examination of lungs and brains, and (iv) determination of number of CFU in brains. For the histopathological studies, both lungs of each rat were fixed in 10% formaldehyde, embedded in paraffin, and cut, and tissue sections were stained with hematoxylin and eosine and mucicarmin. Histological sections were examined in double-blind fashion. Homogeneous suspensions of brain tissue were prepared in sterile mortars containing distilled water at a concentration of 100 mg/ml. A 0.1-ml aliquot of a 1:500 dilution of these suspensions was cultured in petri dishes containing Sabouraud honey agar to determine the number of CFU (limit of sensitivity, 2 x 105/g). Aliquots (0.2 ml) of undiluted suspensions of brain tissue were also cultured to detect the presence of any viable cryptococci (limit of sensitivity, 2 x 102/g). A statistical
SCH SCH SCH FLZ FLZ FLZ
SCH-vehicle FLZ-vehicle
Dose
evaluated by:
8 16 32 8 16 32
Wet
Histopathological exam
30 70 100 60 100 100 100 100
60 70 60 20 90 80 100 100
Small granu-
granulomasa
lomasb
10 0 0 0 0 10 100 100
0 0 10 20 10 10 0 0
RESULTS The results are summarized in Tables 1 through 4, with the statistical analysis shown in Table 5. No deaths occurred before termination of the experiment. Control groups exhibited typical disseminated cryptococcosis, with giant-cell granulomas containing capsulated yeast cells in lungs, pseudocystic lesions with large numbers of cryptococci in brains, and brain cultures with large numbers of CFU. Both triazoles were able to change the evolution of the experimental disease. They reduced the size of the lesions, the number of capsulated yeast cells, and the number of CFU in the brain cultures. Histopathological examination of lungs from treated rats showed only small granulomas with few epithelioid cells surrounding the few remaining morphologically altered cryptococci. In addition, in the alveolar septae, a few altered yeast cells which had evoked a weak inflammatory reaction were seen. In the treatment studies, undiluted suspensions of brain TABLE 4. Prophylactic efficacy of SCH and FLZ in disseminated cryptococcosis in rats as shown by residual lesions in brains
Group
Dose (mg/kg/day)
of brains
4.4 1.3 0.5 6.3 5.3 1.7
2.7 2.3
x
X x
i07 107 107
x
107 107
x
107
X
x x
109 109
% of animals with residual lesions (in brains) evaluated by: Wet mount
Avg CFU/ g
mount
mount Giant-cell
0 0 0 0 0 10 100 100
0 0 0 0 0 10 100 100
Histopathological exam
comparison was done by using variance analysis and Student's t test (7).
TABLE 2. Treatment efficacy of SCH and FLZ in disseminated cryptococcosis in rats as shown by residual lesions in brains
(mg/kg/day)
8 16 32 8 16 32
Wet
Giant-cell granulomas with typical cryptococci. b Small granulomas or weak inflammatory reaction.
b
% of animals with residual lesions (in brains)
Dose Macroscopic (mg/kg/day)
a
Giant-cell granulomas with typical cryptococci. Small granulomas or weak inflammatory reaction. c Altered cryptococci.
a
Group
1461
TREATMENT OF EXPERIMENTAL CRYPTOCOCCOSIS
VOL. 35, 1991
SCH
8
SCH 16 SCH 32 8 FLZ 16 FLZ 32 FLZ SCH-vehicle FLZ-vehicle a Altered cryptococci. b
CFU/g Avgbrains of
Histopathological exam
0
20 0 30'a 90
60
60 20 100 100
30 10 100 100
0 0
Twenty percent of rats were culture negative (
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, JUlY 1991, P. 1460-1463
0066-4804/91/071460-04$02.00/0 Copyright C 1991, American Society for Microbiology
Treatment of Experimental Cryptococcosis with SCH 39304 and Fluconazole R. NEGRONI,1 M. R. I. DE ELIAS COSTA,' J. L. FINQUELIEVICH,l C. IOVANNITTI,1 I. AGORIO,1 I. N. TIRABOSCHI,l AND D. LOEBENBERG2* Centro de Micologia del Departamento de Microbiologia, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, Piso 11, C.P. 1121, Buenos Aires, Argentina,1 and Schering-Plough Research Corporation, Bloomfield, New Jersey 070032 Received 2 April 1990/Accepted 10 April 1991
The efficacy of two triazoles, SCH 39304 and fluconazole, in the treatment of disseminated cryptococcosis in Wistar rats was determined. A total of 160 rats were inoculated intracardiacally with 2 x 105 cells of Cryptococcus neoformans. Both drugs were administered by gavage once daily, at three doses (8, 16, and 32 mg/ kg/day). Two treatment schedules were followed: (i) treatment began 1 week after infection and continued for 3 weeks and (ii) prophylaxis treatment began 3 days before infection and continued an additional 3 weeks. Evaluation was based on (i) macroscopic examination of lungs, (ii) microscopic examination of brains and lungs, (iii) histopathology of brains and lungs, and (iv) determination of number of CFU in brains. The number of CFU was the best measure of activity. SCH 39304 was more active than fluconazole in both regimens, and, prophylactically, SCH 39304 was able to achieve biological cures.
study was to compare the efficacies of SCH and FLZ in this experimental animal model of disseminated cryptococcosis.
Cryptococcosis is a systemic mycosis produced by a capsulated yeastlike fungus, Cryptococcus neoformans. This infection is an emerging problem in immunocompromised patients, especially in those suffering from lymphomas, sarcoidosis, and AIDS, and those undergoing steroid treatments (2, 6, 11-14, 19). The present treatment of choice in cases of cryptococcosis is a combination of intravenous amphotericin B and oral 5-fluorocytosine for at least 6 weeks (1, 19). The use of this treatment sometimes causes severe side effects such as hypokalemia, anemia, impaired renal function, and leukopenia (12, 13). These alterations are even more severe in immunodeficient patients. In addition to these problems, relapses are often observed in patients with AIDS (13, 19). The azoles have been used successfully in cases of cryptococcosis without central nervous system involvement. They do not penetrate the blood-brain barrier well because of their high molecular weights and binding to serum proteins (2, 13). Results with itraconazole in the treatment of cryptococcosis, both in experimental models and in human beings, have been variable (5, 8, 21, 22). Fluconazole (FLZ) is a fluorinated bis-triazole watersoluble compound which can be administered orally or parenterally. It is a broad-spectrum antifungal agent and is active against C. neoformans. FLZ achieves 80% of its serum drug concentration in spinal fluid and has been successfully used in human beings (3, 4, 10, 20). SCH 39304 (SCH) is a new triazole derivative which is very active against several experimental systemic mycoses, has a very long serum half-life, and penetrates the bloodbrain barrier well (16, 17). In recent studies, we showed that Wistar rats inoculated intracardiacally with C. neoformans developed a subacute disseminated and fatal disease (15). This experimental model was used for a comparative evaluation of amphotericin B and itraconazole. Both drugs were unable to produce a biological cure in this infection (8). The aim of the present *
MATERIALS AND METHODS In vitro studies. The Ribas strain of C. neoformans (from the Mycology Center Collection), which is known for its pathogenicity for Wistar rats, was used (8, 15). MICs for SCH and FLZ were established in Sabouraud broth with 2% dextrose by performing twofold dilutions from 100 to 0.01 ,ug/ml. SCH was dissolved in polyethylene glycol 200 (PEG) and FLZ was dissolved in distilled water to a final concentration of 1 mg/ml for each. PEG was not active in vitro. These solutions were sterilized by heating at 70°C for 1 h on three consecutive days. Sterilization controls were carried out in brain heart infusion broth and agar at 37°C (9). Tubes were incubated at 28°C for 15 days. After completion of MIC incubation times, minimal fungicidal concentrations were determined. Sediments in MIC tubes were centrifuged, washed twice with sterile distilled water, suspended in 0.2 ml of sterile distilled water, and seeded in Sabouraud dextrose agar. Incubation was at 28°C for 15 days (18). In vivo studies. A total of 160 Wistar rats of both sexes, weighing approximately 250 g each, were used. Animals (10 per group, 5 of each sex) were anesthetized with ethyl ether and inoculated (0.1 ml) by the intracardiac route with a suspension of 2 x 105 C. neoformans Ribas in an isotonic saline solution. The inoculum was from a 48-h culture incubated at 37°C in Sabouraud honey agar. SCH was dissolved in PEG at pH 3 to a final concentration of 8 mg/ml. FLZ was prepared in dimethyl formamide at a ratio of 1 g: 10 ml, as used in previous experiments (8). The FLZ-dimethyl formamide solution was added to 90 ml of distilled water containing 5% dextrose and 0.2% agar. Controls contained only the vehicles. Two treatment schedules were followed. (i) Treatment began 7 days after infection and continued for 21 days. Both drugs were administered by gavage once daily (on the basis of proposed clinical usage) at 8, 16, or 32 mg/kg/day. Control groups received either PEG at pH 3.0 or the dimethyl
Corresponding author. 1460
TABLE 3. Prophylactic efficacy of SCH and FLZ in disseminated cryptococcosis in rats as shown by residual lesions in lungs % of animals with residual lesions (in lungs)
TABLE 1. Treatment efficacy of SCH and FLZ in disseminated cryptococcosis in rats as shown by presence of residual lesions in lungs % of animals with residual lesions (in lungs) evaluated by: Group
SCH SCH SCH FLZ FLZ FLZ
Dose (mg/kg/day)
8 16 32 8 16 32
SCH-vehicle FLZ-vehicle
Macroscopic exam (granulomas)
0 0 0 20 30 20 100 100
evaluated by:
Histopathological exam Wet mount Giant-cell Small granulo- granulomasb masa
30 40 70 60 100 50 100 100
60C 70c 40c 80C 100C 70c 0 0
0 0 0 10 0 0 100 100
Group
m(granulomas) SCH SCH SCH FLZ FLZ FLZ SCH-vehicle FLZ-vehicle
formamide vehicle. (ii) Prophylaxis treatment began 3 days before infection and continued for an additional 3 weeks. SCH and FLZ were dosed in the same manner as in the treatment studies. Animals were sacrificed 1 week after completion of both treatment schedules and then necropsied. The following evaluations were done: (i) macroscopic examination of lungs (for granulomas), (ii) microscopic examination of wet-mount preparations (by using a phase-contrast microscope), (iii) histopathological examination of lungs and brains, and (iv) determination of number of CFU in brains. For the histopathological studies, both lungs of each rat were fixed in 10% formaldehyde, embedded in paraffin, and cut, and tissue sections were stained with hematoxylin and eosine and mucicarmin. Histological sections were examined in double-blind fashion. Homogeneous suspensions of brain tissue were prepared in sterile mortars containing distilled water at a concentration of 100 mg/ml. A 0.1-ml aliquot of a 1:500 dilution of these suspensions was cultured in petri dishes containing Sabouraud honey agar to determine the number of CFU (limit of sensitivity, 2 x 105/g). Aliquots (0.2 ml) of undiluted suspensions of brain tissue were also cultured to detect the presence of any viable cryptococci (limit of sensitivity, 2 x 102/g). A statistical
SCH SCH SCH FLZ FLZ FLZ
SCH-vehicle FLZ-vehicle
Dose
evaluated by:
8 16 32 8 16 32
Wet
Histopathological exam
30 70 100 60 100 100 100 100
60 70 60 20 90 80 100 100
Small granu-
granulomasa
lomasb
10 0 0 0 0 10 100 100
0 0 10 20 10 10 0 0
RESULTS The results are summarized in Tables 1 through 4, with the statistical analysis shown in Table 5. No deaths occurred before termination of the experiment. Control groups exhibited typical disseminated cryptococcosis, with giant-cell granulomas containing capsulated yeast cells in lungs, pseudocystic lesions with large numbers of cryptococci in brains, and brain cultures with large numbers of CFU. Both triazoles were able to change the evolution of the experimental disease. They reduced the size of the lesions, the number of capsulated yeast cells, and the number of CFU in the brain cultures. Histopathological examination of lungs from treated rats showed only small granulomas with few epithelioid cells surrounding the few remaining morphologically altered cryptococci. In addition, in the alveolar septae, a few altered yeast cells which had evoked a weak inflammatory reaction were seen. In the treatment studies, undiluted suspensions of brain TABLE 4. Prophylactic efficacy of SCH and FLZ in disseminated cryptococcosis in rats as shown by residual lesions in brains
Group
Dose (mg/kg/day)
of brains
4.4 1.3 0.5 6.3 5.3 1.7
2.7 2.3
x
X x
i07 107 107
x
107 107
x
107
X
x x
109 109
% of animals with residual lesions (in brains) evaluated by: Wet mount
Avg CFU/ g
mount
mount Giant-cell
0 0 0 0 0 10 100 100
0 0 0 0 0 10 100 100
Histopathological exam
comparison was done by using variance analysis and Student's t test (7).
TABLE 2. Treatment efficacy of SCH and FLZ in disseminated cryptococcosis in rats as shown by residual lesions in brains
(mg/kg/day)
8 16 32 8 16 32
Wet
Giant-cell granulomas with typical cryptococci. b Small granulomas or weak inflammatory reaction.
b
% of animals with residual lesions (in brains)
Dose Macroscopic (mg/kg/day)
a
Giant-cell granulomas with typical cryptococci. Small granulomas or weak inflammatory reaction. c Altered cryptococci.
a
Group
1461
TREATMENT OF EXPERIMENTAL CRYPTOCOCCOSIS
VOL. 35, 1991
SCH
8
SCH 16 SCH 32 8 FLZ 16 FLZ 32 FLZ SCH-vehicle FLZ-vehicle a Altered cryptococci. b
CFU/g Avgbrains of
Histopathological exam
0
20 0 30'a 90
60
60 20 100 100
30 10 100 100
0 0
Twenty percent of rats were culture negative (
