ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 1990, p. 896-898
Vol. 34, No. 5
0066-4804/90/050896-03$02.00/0 Copyright © 1990, American Society for Microbiology
NOTES Treatment of Murine Pulmonary Blastomycosis with SCH 39304, New Triazole Antifungal Agent
a
ALAN M. SUGAR,* MICHELE PICARD, AND LIZANNE NOBLE Evans Memorial Department of Clinical Research and Department of Medicine, Boston University Medical Center, Boston, Massachusetts 02118 Received 21 November 1989/Accepted 31 January 1990
SCH 39304, a broad-spectrum azole derivative, was evaluated in an experimental mouse model of blastomycosis pneumonia. Five days after being inoculated with Blastomyces dermattdis, infected mice were treated with either oral SCH 39304, fluconazole, or intraperitoneal amphotericin B. A dose response protective effect was observed with SCH 39304 at 5 to 100 mg/kg of body weight per day, with 5 mg of SCH 39304 per kg per day providing activity similar to that of 100 mg of fluconazole per kg per day. Colony counts of yeasts in the lungs of mice sacrificed while on therapy with SCH 39304 were consistently below those of controls, and several lungs were sterile. We conclude that SCH 39304 is effective in murine blastomycosis treatment and deserves to be evaluated in the treatment of human blastomycosis.
dermatitidis and continued for 3 weeks. SCH 39304 and fluconazole were given by oral gavage once daily, and amphotericin B was given by intraperitoneal injection 5 days per week. Cages were observed twice daily for deaths. Mann-Whitney U test and Fisher's exact tests were performed when appropriate using a commercially available statistics program suitable for use on IBM PC-compatible equipment (NCSS, Kaysville, Utah). In the first two experiments, mice weighing 22.5 and 19.1 g were infected with 6,600 and 6,800 CFU, respectively. The times to achieve the 90% lethal dose for these experiments were 14 and 19 days, respectively. In contrast, while mice in the third experiment were noted by the supplier to be the same age as those used in the other experiments, they weighed significantly less than the other mice, 8.3 g (P < 0.05). These mice were infected with 4,376 CFU, and a 90% lethal dose was reached in 13 days. In experiment 1, untreated mice developed typical signs and symptoms of pulmonary blastomycosis, and all were dead by day 20 (Fig. lb). Examination of the lungs of selected dead mice revealed the typical lesions of blastomycosis. In contrast, mice treated with 25, 50, or 100 mg of fluconazole per kg of body weight per day had 70 or 100% survival on day 20. By the end of the experiment (day 45), survival rates at these doses were 20, 10, and 30%, respectively. Therapy with SCH 39304 provided better survival than that seen with fluconazole: 90 or 100% of mice treated with 5, 25, or 50 mg of SCH 39304 per kg per day were alive on day 20 (Fig. 1; P < 0.05). On day 45, survival rates were 30, 70, and 100%, respectively. Similar results were obtained in the second experiment. The survival rate (day 45) of mice treated with SCH 39304 (5 mg/kg per day) was comparable to that of mice treated with fluconazole (100 mg/kg per day): 30 versus 0%o, respectively. In experiment 3, all untreated mice were dead by day 20 (Fig. 2). On day 45, 8% of mice treated with fluconazole (100 mg/kg per day) were alive compared with 0, 7, and 31% of those treated with 25, 50, or 100 mg of SCH 39304 per kg per day for 3 weeks and 62% of those treated with 100 mg of
SCH 39304 is a new azole antifungal agent with broadspectrum activity against many pathogenic fungi (4-6). Interesting features of this drug include a long half-life (60 to 100 h in humans and approximately 6 h in mice), good absorption following oral administration, and activity against many common and uncommon fungal pathogens (Investigator's brochure, Schering Corp.). Since the activity of SCH 39304 in experimental blastomycosis has not been previously described, we tested this agent in a well-described model of murine blastomycosis (1), comparing the activity of SCH 39304 to that of fluconazole and amphotericin B (3). (Part of this research was presented at the 29th Interscience Conference on Antimicrobial Agents and Chemotherapy, Houston, Tex., 17 to 20 September 1989.) Four-week-old male BALB/cByJ mice were obtained from Jackson Laboratory (Bar Harbor, Maine). Mice were housed, 10 per cage, in filter-topped cages and were fed standard mouse chow and water ad libitum. Blastomyces dermatitidis ATCC 26199, also referred to as V, was obtained from the American Type Culture Collection and maintained in the yeast phase in a synthetic medium (2) on a rotary shaker at 37°C. A 1-ml sample of the yeast suspension was subcultured onto enriched blood agar (GIBCO Laboratories, Grand Island, N.Y.) and incubated for 3 days at 37°C. Fungi were harvested by being washed twice in sterile physiologic saline, were counted with a hemacytometer, and were adjusted to the desired concentration for mouse inoculation. SCH 39304 was provided as sterile powder from Schering Corp. It was suspended in Emulphor EL-719P and prepared every 2 to 3 days at the appropriate concentration for administration to the mice. Fluconazole was obtained from Pfizer Inc. (Groton, Conn.) as a sterile powder and was also prepared as a suspension in Emulphor EL-719P. Amphotericin B (Sigma Chemical Co., St. Louis, Mo.) was prepared fresh daily in sterile water. Therapy was begun 5 days after inoculation with B. *
Corresponding author.
896
VOL. 34, 1990
NOTES
A a
B
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897
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26
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45
DAYS
FIG. 1. Survival curves of mice infected with B. dermatitidis in experiment 1. (A) Effects-of 25 (A), 50 (0), and 100 (A) mg of fluconazole per kg per day and amphotericin B (A); (B) effects of 5 (A), 25 (0), and 50 (-) mg of SCH 39304 per kg per day and controls (+). All treatments resulted in better survival compared with controls (P < 0.0001). Fluconazole (100 mg/kg per day) was less effective than SCH 39304 (5 mg/kg per day) (P = 0.054) and both higher doses of SCH 39304 (P = 0.02). Amphotericin was significantly more effective than 5 and 25 mg of SCH 39304 per kg per day (P < 0.0001) but not more effective than 100 mg of SCH 39304 per kg per day (P = 0.069).
SCH 39304 per kg per day for 4 weeks. Attempts to improve posttreatment survival by extending therapy with the highest dose of SCH 39304 from 3 to 4 weeks were not successful. Mice began to die once therapy was discontinued, and on day 45, the last day of the experiment, many of the surviving mice began to look ill. Thus, although therapy for 4 weeks did not appear to consistently eradicate the fungus from infected animals, it appears that as long as the animals are being given SCH 39304 they will survive. The ability of the two azoles and amphotericin B to sterilize infected lungs was also evaluated. Fluconazole (100 mg/kg per day) and SCH 39304 (5 mg/kg per day) effectively suppressed fungal growth but were unable to decrease the
A
number of yeasts recovered from the lungs (Fig. 3). On the other hand, the two highest doses of SCH 39304 (50 and 100 mg/kg per day) markedly decreased colony counts during the treatment period. Once therapy was discontinued, fungal growth resumed, and by the end of the experiment, counts equalled or exceeded the original inoculum size. Similar results were obtained in studies performed in experiment 3 (data not shown). In both experiments, only occasional mice had sterile lung cultures by the end of the experiment, and most animals demonstrated regrowth of the fungus once therapy was stopped. We have shown that SCH 39304 is an effective agent in the treatment of experimental murine pulmonary blastomycosis.
B
I
80 >60
'40
35
25 35 45 DAYS FIG. 2. Survival curves of mice infected with B. dermatitidis in experiment 3. (A) Effects of fluconazole (100 mg/kg per day) (A) and SCH 39304 (25 mg/kg per day (0), compared with control (+) and amphotericin B-treated (U) mice. (B) Effects of mice treated with 50 (E) or 100 (V) mg of SCH 39304 per kg per day for 3 weeks or 100 mg/kg per day for 4 weeks (0). Fluconazole (100 mg/kg per day) provided protection against death equal to that of 25 and 50 mg of SCH 39304 per kg per day (P < 0.5). However, both 3 and 4 weeks of treatment with SCH 39304 resulted in increased survival compared with the fluconazole-treated group (P < 0.0001). Amphotericin B provided the best protection of any of the groups (P < 0.001). 5
1S
25 DAYS
45
5
15
898
ANTIMICROB. AGENTS CHEMOTHER.
NOTES
106
1 07 106
10' 101
6
11
16 21
26 47
50
compared with subcutaneously in the previous study. However, in the earlier study, even though mice survived for 24 days while receiving 50 mg of fluconazole per kg per day, all lungs were heavily infected with the fungus. Our current results confirm that lungs are not sterilized by either drug, although SCH 39304 was more effective than fluconazole as determined by fungal counts in infected lungs during treatment. In fact, colony counts in lungs recovered from mice treated with SCH 39304 were similar to those from amphotericin B-treated mice. At the dosages used, none of the drugs consistently resulted in sterilization of the lungs. In conclusion, we have demonstrated that SCH 39304 is more active than fluconazole in an experimental model of murine pulmonary blastomycosis. Other drugs active in this model, such as amphotericin B, ketoconazole, itraconazole, and fluconazole, have been shown to be active in human blastomycosis as well. Therefore, we suggest that further studies in human blastomycosis are indicated.
Day FIG. 3. Colony counts from lungs cultured during and after therapy (experiment 2). Therapy was begun on experiment day 5 and continued for 21 consecutive days. Symbols: +, controls; A, SCH 39305, 5 mg/kg per day; 0, SCH 39305, 25 mg/kg per day; a, SCH 39305, 50 mg/kg per day; A, fluconazole, 50 mg/kg per day; *, fluconazole, 100 mg/kg per day. On day 21, colony counts of the high-dose SCH 39304 were significantly lower than those from all other groups (P < 0.05). Each point represents the mean of counts obtained from two animals. CFU obtained from each mouse at each time point were within 1 log of each other.
Compared with fluconazole, SCH 39304 was approximately 20 times more active on a weight basis, and if treatment was extended to 4 weeks, 100 mg/kg per day resulted in survival similar to that seen with amphotericin B. Our results with fluconazole in this study are not directly comparable to those in our earlier experiments (3) because the results reported here were obtained from mice infected with higher inocula (>4,000 CFU versus 800 to 1,000 CFU). In addition, mice were observed for a longer period of time (45 versus 24 days), and fluconazole was administered orally
1.
2. 3.
4.
5.
6.
LITERATURE CITED Harvey, R. P., E. S. Schmid, C. C. Carrington, and D. A. Stevens. 1978. Mouse model of pulmonary blastomycosis: utility, simplicity, and quantitative parameters. Am. Rev. Respir. Dis. 117: 695-703. Hoeprich, P. D., and P. D. Finn. 1972. Obfuscation of the activity of antifungal antimicrobics by culture media. J. Infect. Dis. 126:353-361. Lyman, C. A., A. M. Sugar, and R. D. Diamond. 1986. Comparative activities of UK-49,858 and amphotericin B against Blastomyces dermatitidis infections in mice. Antimicrob. Agents Chemother. 29:161-162. McIntyre, K. A., and J. N. Galgiani. 1989. In vitro susceptibilities of yeasts to a new antifungal triazole, SCH 39304: effects of test conditions and relation to in vivo efficacy. Antimicrob. Agents Chemother. 33:1095-1100. Perfect, J. R., K. A. Wright, M. M. Hobbs, and D. T. Durack. 1989. Treatment of experimental cryptococcal meningitis and disseminated candidiasis with SCH 39304. Antimicrob. Agents Chemother. 33:1735-1740. Restrepo, B. I., J. Ahrens, and J. R. Graybili. 1989. Efficacy of SCH 39304 in murine cryptococcosis. Antimicrob. Agents Chemother. 33:1242-1246.
Vol. 34, No. 5
0066-4804/90/050896-03$02.00/0 Copyright © 1990, American Society for Microbiology
NOTES Treatment of Murine Pulmonary Blastomycosis with SCH 39304, New Triazole Antifungal Agent
a
ALAN M. SUGAR,* MICHELE PICARD, AND LIZANNE NOBLE Evans Memorial Department of Clinical Research and Department of Medicine, Boston University Medical Center, Boston, Massachusetts 02118 Received 21 November 1989/Accepted 31 January 1990
SCH 39304, a broad-spectrum azole derivative, was evaluated in an experimental mouse model of blastomycosis pneumonia. Five days after being inoculated with Blastomyces dermattdis, infected mice were treated with either oral SCH 39304, fluconazole, or intraperitoneal amphotericin B. A dose response protective effect was observed with SCH 39304 at 5 to 100 mg/kg of body weight per day, with 5 mg of SCH 39304 per kg per day providing activity similar to that of 100 mg of fluconazole per kg per day. Colony counts of yeasts in the lungs of mice sacrificed while on therapy with SCH 39304 were consistently below those of controls, and several lungs were sterile. We conclude that SCH 39304 is effective in murine blastomycosis treatment and deserves to be evaluated in the treatment of human blastomycosis.
dermatitidis and continued for 3 weeks. SCH 39304 and fluconazole were given by oral gavage once daily, and amphotericin B was given by intraperitoneal injection 5 days per week. Cages were observed twice daily for deaths. Mann-Whitney U test and Fisher's exact tests were performed when appropriate using a commercially available statistics program suitable for use on IBM PC-compatible equipment (NCSS, Kaysville, Utah). In the first two experiments, mice weighing 22.5 and 19.1 g were infected with 6,600 and 6,800 CFU, respectively. The times to achieve the 90% lethal dose for these experiments were 14 and 19 days, respectively. In contrast, while mice in the third experiment were noted by the supplier to be the same age as those used in the other experiments, they weighed significantly less than the other mice, 8.3 g (P < 0.05). These mice were infected with 4,376 CFU, and a 90% lethal dose was reached in 13 days. In experiment 1, untreated mice developed typical signs and symptoms of pulmonary blastomycosis, and all were dead by day 20 (Fig. lb). Examination of the lungs of selected dead mice revealed the typical lesions of blastomycosis. In contrast, mice treated with 25, 50, or 100 mg of fluconazole per kg of body weight per day had 70 or 100% survival on day 20. By the end of the experiment (day 45), survival rates at these doses were 20, 10, and 30%, respectively. Therapy with SCH 39304 provided better survival than that seen with fluconazole: 90 or 100% of mice treated with 5, 25, or 50 mg of SCH 39304 per kg per day were alive on day 20 (Fig. 1; P < 0.05). On day 45, survival rates were 30, 70, and 100%, respectively. Similar results were obtained in the second experiment. The survival rate (day 45) of mice treated with SCH 39304 (5 mg/kg per day) was comparable to that of mice treated with fluconazole (100 mg/kg per day): 30 versus 0%o, respectively. In experiment 3, all untreated mice were dead by day 20 (Fig. 2). On day 45, 8% of mice treated with fluconazole (100 mg/kg per day) were alive compared with 0, 7, and 31% of those treated with 25, 50, or 100 mg of SCH 39304 per kg per day for 3 weeks and 62% of those treated with 100 mg of
SCH 39304 is a new azole antifungal agent with broadspectrum activity against many pathogenic fungi (4-6). Interesting features of this drug include a long half-life (60 to 100 h in humans and approximately 6 h in mice), good absorption following oral administration, and activity against many common and uncommon fungal pathogens (Investigator's brochure, Schering Corp.). Since the activity of SCH 39304 in experimental blastomycosis has not been previously described, we tested this agent in a well-described model of murine blastomycosis (1), comparing the activity of SCH 39304 to that of fluconazole and amphotericin B (3). (Part of this research was presented at the 29th Interscience Conference on Antimicrobial Agents and Chemotherapy, Houston, Tex., 17 to 20 September 1989.) Four-week-old male BALB/cByJ mice were obtained from Jackson Laboratory (Bar Harbor, Maine). Mice were housed, 10 per cage, in filter-topped cages and were fed standard mouse chow and water ad libitum. Blastomyces dermatitidis ATCC 26199, also referred to as V, was obtained from the American Type Culture Collection and maintained in the yeast phase in a synthetic medium (2) on a rotary shaker at 37°C. A 1-ml sample of the yeast suspension was subcultured onto enriched blood agar (GIBCO Laboratories, Grand Island, N.Y.) and incubated for 3 days at 37°C. Fungi were harvested by being washed twice in sterile physiologic saline, were counted with a hemacytometer, and were adjusted to the desired concentration for mouse inoculation. SCH 39304 was provided as sterile powder from Schering Corp. It was suspended in Emulphor EL-719P and prepared every 2 to 3 days at the appropriate concentration for administration to the mice. Fluconazole was obtained from Pfizer Inc. (Groton, Conn.) as a sterile powder and was also prepared as a suspension in Emulphor EL-719P. Amphotericin B (Sigma Chemical Co., St. Louis, Mo.) was prepared fresh daily in sterile water. Therapy was begun 5 days after inoculation with B. *
Corresponding author.
896
VOL. 34, 1990
NOTES
A a
B
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I
0
I
0
aI
o 0
61
o
i
e
.
I; I
0
i X
41[
co LL
897
I.
0
0
X
0
I_@
21
z
z 0
15v
6
26
35
45
1s
5
DAYS
26
35
45
DAYS
FIG. 1. Survival curves of mice infected with B. dermatitidis in experiment 1. (A) Effects-of 25 (A), 50 (0), and 100 (A) mg of fluconazole per kg per day and amphotericin B (A); (B) effects of 5 (A), 25 (0), and 50 (-) mg of SCH 39304 per kg per day and controls (+). All treatments resulted in better survival compared with controls (P < 0.0001). Fluconazole (100 mg/kg per day) was less effective than SCH 39304 (5 mg/kg per day) (P = 0.054) and both higher doses of SCH 39304 (P = 0.02). Amphotericin was significantly more effective than 5 and 25 mg of SCH 39304 per kg per day (P < 0.0001) but not more effective than 100 mg of SCH 39304 per kg per day (P = 0.069).
SCH 39304 per kg per day for 4 weeks. Attempts to improve posttreatment survival by extending therapy with the highest dose of SCH 39304 from 3 to 4 weeks were not successful. Mice began to die once therapy was discontinued, and on day 45, the last day of the experiment, many of the surviving mice began to look ill. Thus, although therapy for 4 weeks did not appear to consistently eradicate the fungus from infected animals, it appears that as long as the animals are being given SCH 39304 they will survive. The ability of the two azoles and amphotericin B to sterilize infected lungs was also evaluated. Fluconazole (100 mg/kg per day) and SCH 39304 (5 mg/kg per day) effectively suppressed fungal growth but were unable to decrease the
A
number of yeasts recovered from the lungs (Fig. 3). On the other hand, the two highest doses of SCH 39304 (50 and 100 mg/kg per day) markedly decreased colony counts during the treatment period. Once therapy was discontinued, fungal growth resumed, and by the end of the experiment, counts equalled or exceeded the original inoculum size. Similar results were obtained in studies performed in experiment 3 (data not shown). In both experiments, only occasional mice had sterile lung cultures by the end of the experiment, and most animals demonstrated regrowth of the fungus once therapy was stopped. We have shown that SCH 39304 is an effective agent in the treatment of experimental murine pulmonary blastomycosis.
B
I
80 >60
'40
35
25 35 45 DAYS FIG. 2. Survival curves of mice infected with B. dermatitidis in experiment 3. (A) Effects of fluconazole (100 mg/kg per day) (A) and SCH 39304 (25 mg/kg per day (0), compared with control (+) and amphotericin B-treated (U) mice. (B) Effects of mice treated with 50 (E) or 100 (V) mg of SCH 39304 per kg per day for 3 weeks or 100 mg/kg per day for 4 weeks (0). Fluconazole (100 mg/kg per day) provided protection against death equal to that of 25 and 50 mg of SCH 39304 per kg per day (P < 0.5). However, both 3 and 4 weeks of treatment with SCH 39304 resulted in increased survival compared with the fluconazole-treated group (P < 0.0001). Amphotericin B provided the best protection of any of the groups (P < 0.001). 5
1S
25 DAYS
45
5
15
898
ANTIMICROB. AGENTS CHEMOTHER.
NOTES
106
1 07 106
10' 101
6
11
16 21
26 47
50
compared with subcutaneously in the previous study. However, in the earlier study, even though mice survived for 24 days while receiving 50 mg of fluconazole per kg per day, all lungs were heavily infected with the fungus. Our current results confirm that lungs are not sterilized by either drug, although SCH 39304 was more effective than fluconazole as determined by fungal counts in infected lungs during treatment. In fact, colony counts in lungs recovered from mice treated with SCH 39304 were similar to those from amphotericin B-treated mice. At the dosages used, none of the drugs consistently resulted in sterilization of the lungs. In conclusion, we have demonstrated that SCH 39304 is more active than fluconazole in an experimental model of murine pulmonary blastomycosis. Other drugs active in this model, such as amphotericin B, ketoconazole, itraconazole, and fluconazole, have been shown to be active in human blastomycosis as well. Therefore, we suggest that further studies in human blastomycosis are indicated.
Day FIG. 3. Colony counts from lungs cultured during and after therapy (experiment 2). Therapy was begun on experiment day 5 and continued for 21 consecutive days. Symbols: +, controls; A, SCH 39305, 5 mg/kg per day; 0, SCH 39305, 25 mg/kg per day; a, SCH 39305, 50 mg/kg per day; A, fluconazole, 50 mg/kg per day; *, fluconazole, 100 mg/kg per day. On day 21, colony counts of the high-dose SCH 39304 were significantly lower than those from all other groups (P < 0.05). Each point represents the mean of counts obtained from two animals. CFU obtained from each mouse at each time point were within 1 log of each other.
Compared with fluconazole, SCH 39304 was approximately 20 times more active on a weight basis, and if treatment was extended to 4 weeks, 100 mg/kg per day resulted in survival similar to that seen with amphotericin B. Our results with fluconazole in this study are not directly comparable to those in our earlier experiments (3) because the results reported here were obtained from mice infected with higher inocula (>4,000 CFU versus 800 to 1,000 CFU). In addition, mice were observed for a longer period of time (45 versus 24 days), and fluconazole was administered orally
1.
2. 3.
4.
5.
6.
LITERATURE CITED Harvey, R. P., E. S. Schmid, C. C. Carrington, and D. A. Stevens. 1978. Mouse model of pulmonary blastomycosis: utility, simplicity, and quantitative parameters. Am. Rev. Respir. Dis. 117: 695-703. Hoeprich, P. D., and P. D. Finn. 1972. Obfuscation of the activity of antifungal antimicrobics by culture media. J. Infect. Dis. 126:353-361. Lyman, C. A., A. M. Sugar, and R. D. Diamond. 1986. Comparative activities of UK-49,858 and amphotericin B against Blastomyces dermatitidis infections in mice. Antimicrob. Agents Chemother. 29:161-162. McIntyre, K. A., and J. N. Galgiani. 1989. In vitro susceptibilities of yeasts to a new antifungal triazole, SCH 39304: effects of test conditions and relation to in vivo efficacy. Antimicrob. Agents Chemother. 33:1095-1100. Perfect, J. R., K. A. Wright, M. M. Hobbs, and D. T. Durack. 1989. Treatment of experimental cryptococcal meningitis and disseminated candidiasis with SCH 39304. Antimicrob. Agents Chemother. 33:1735-1740. Restrepo, B. I., J. Ahrens, and J. R. Graybili. 1989. Efficacy of SCH 39304 in murine cryptococcosis. Antimicrob. Agents Chemother. 33:1242-1246.
