ANTIMICROBIAL AGENTS
AND
CHEMOTHERAPY, Dec. 1992,
p.
2681-2685
Vol. 36, No. 12
0066-4804/92/122681-05$02.00/0 Copyright © 1992, American Society for Microbiology
Saperconazole Therapy in a Rabbit Model of Invasive Aspergillosis THOMAS F. PATTERSON,* DAVID GEORGE, PEGGY MINITER, AND VINCENT T. ANDRIOLE Department of Medicine, Section of Infectious Diseases, Yale University School of Medicine, New Haven, Connecticut 06510 Received 1 June 1992/Accepted 2 October 1992
The efficacy of orally and intravenously administered saperconazole against Aspergilius fumigatus was assessed in an immunosuppressed temporarily leukopenic rabbit model of invasive aspergillosis and compared with that of amphotericin B. Oral saperconazole at dosages of 5, 10, and 15 mg/kg of body weight per day improved survival compared with that of controls. In addition, saperconazole at 10 and 15 mg/kg/day reduced the tissue burden and reduced levels of circulating antigen, which correlated with increasing dosages of saperconazole. Intravenous saperconazole produced levels in serum more than 10-fold that of oral therapy. Intravenous saperconazole not only improved survival and reduced antigen levels but also significantly eradicated A. fumigatus from tissues compared with those of controls and was as effective as amphotericin B in these studies. Saperconazole was effective in the treatment of experimental invasive aspergillosis and demonstrates the potential of the newer azoles in therapy for invasive aspergillosis.
dose of cyclophosphamide (200 mg) given intravenously on the first day of the model and triamcinolone acetonide (10 mg; Westwood Pharmaceuticals, Buffalo, N.Y.), which was given subcutaneously each day. With this immunosuppressive regimen, the rabbits had reduced total leukocyte counts through day 7, as previously reported (14). Twenty-four hours after immunosuppression, groups of five to eight rabbits were challenged intravenously with a lethal inoculum of 106 Aspergillus fumigatus conidia. Antifungal therapy was given as described below. Each group contained at least one untreated control rabbit. Blood was obtained daily for determining total leukocyte counts and serum aspergillus antigen. Ceftazidime (200 mg; SmithKline Beecham, Philadelphia, Pa.) was administered intramuscularly daily beginning on the day of challenge to prevent intercurrent bacterial infection. Amphotericin B (Fungizone; Bristol-Myers Squibb, Princeton, N.J.) or saperconazole (Ortho Pharmaceuticals, Raritan, N.J.) therapy was begun 24 h after challenge. In initial experiments, saperconazole was administered orally after it was first dissolved in concentrated HCl with 0.01 ml of polyethylene glycol 200 (Sigma Chemical Co., St. Louis, Mo.) per ml of HCl and then 4 mg of hydroxypropyl-pcyclodextrin (9) (American Maize Products, Hammond, Ind.) per mg of saperconazole was added, for a saperconazole concentration of 1 mg/ml in distilled water. This saperconazole suspension was administered orally via a gastric gavage tube (American Pharmaseal Company, Valencia, Calif.) at a dosage of 5, 10, or 15 mg/kg/day for 5 days. In subsequent experiments, saperconazole was administered intravenously and compared with amphotericin B. Saperconazole was dissolved in dimethyl sulfoxide (Sigma) at a saperconazole concentration of 22 mg/ml. This intravenous saperconazole solution was administered over 5 min through a lateral ear vein at a dosage of 5.0 or 7.5 mg/kg/day for 6 days. Amphotericin B was diluted with 5% dextrose in sterile water at a ratio of 1 mg of drug to 10 ml of diluent and was given intravenously over 30 to 60 min through a lateral ear vein at a dosage of 1.5 mg/kg/day for a mean (+ standard error) of 5.1 (+ 0.3) days (range, 4 to 7 days). Organ cultures. Cultures and histopathology were performed at the time of autopsy or when the animals were
Invasive aspergillosis is associated with significant morbidity and mortality despite therapy with amphotericin B (2, 4). Management of invasive aspergillosis is further complicated by toxicity associated with amphotericin B therapy (6). Saperconazole is a triazole antifungal compound with excellent in vitro activity against Aspergillus spp. (3, 10). These newer azoles offer the potential for oral as well as intravenous therapy and reduced acute toxicity compared with amphotericin B (6). Saperconazole has been shown to have activity in a guinea pig model and in a murine model of aspergillosis (8, 18), but its limited activity in another murine model (15) could be related to the low levels in serum achieved with orally administered drug in that model. Absorption of related triazole compounds, such as ketoconazole and itraconazole, has been reported to be erratic in some immunosuppressed hosts (5, 6), so that adequate delivery of drug via oral administration is likely essential for successful therapy. In this immunosuppressed model, rabbits are made leukopenic and are further immunocompromised with steroid therapy (13, 14). Extensive infection develops in the liver, kidneys, lungs, and brain, similar to clinical dissemination of invasive aspergillosis (11, 12). Efficacy of therapy is assessed by mortality, semiquantitative organ cultures, and Asperillus antigen measurement. In this study, we assessed the activity of orally and intravenously administered saperconazole in a lethal model of experimental infection compared with that of amphotericin B. (This work was presented in part at the 90th Annual Meeting of the American Society for Microbiology, Anaheim, Calif., 13 to 17 May 1990 [abstract F-71] and the 30th Interscience Conference on Antimicrobial Agents and Chemotherapy, Atlanta, Ga., 21 to 24 October 1990 [abstract 603].)
MATERIALS AND METHODS Rabbit model. New Zealand White rabbits were immunosuppressed as previously described (13, 14) with a single *
Corresponding author. 2681
2682
PATTERSON ET AL.
sacrificed (24 to 48 h after completion of therapy for the treated rabbits). Rabbits were sacrificed, following anesthesia with ketamine (35 mg/kg; Bristol Laboratories, Syracuse, N.Y.) and xylazine (10 mg/kg; Mobay Corp., Shawnee, Kans.), by lethal exsanguination. Cultures were taken by placing minced organ samples directly on blood agar and on Sabouraud dextrose agar plates. Samples were considered positive when two or more colonies of A. fimigates were present on 21 g of minced organ tissues plated directly on Sabouraud dextrose and blood agar plates or when semiquantitative cultures of tissue homogenates contained 20 or more CFU/g of tissue (11, 14). The tissue burden of A.
finigatus
was evaluated with a modification (11) of the semiquantitative culture technique of Graybill and Kaster (7). Samples of liver, kidney, lung, and brain tissues were manually chopped, weighed, diluted 1:10 (wt/vol) with sterile saline, and homogenized for 25 s in an electric tissue homogenizer (TRI-R Instruments, Rockville Center, N.Y.). Then, 1.0 and 0.1 ml of each organ homogenate were plated in duplicate on Sabouraud dextrose and blood agar. Plates were incubated for 48 h at 37C, and colonies were counted. These methods combined detect from 2 to 20,000 CFU/g of tissue. For each animal, only a single kidney or lung was sampled at random. Drug levels. Levels of saperconazole in serum and tissue were measured by bioassay by using modifications of previously reported techniques (10, 11, 17). Briefly, Candida kefyr (kindly provided by Patrick Robinson) was grown overnight in Sabouraud dextrose broth at 37C and diluted
with saline to a concentration of 5 x 105 cells with a hemacytometer. A 1:50 dilution of cells was made with High Resolution media (Pfizer Central Research, Groton, Conn.), prepared according to the manufacturer's specifications. Forty-five milliliters of the inoculated media was poured into plastic plates (150 by 25 mm), and wells were cut with a 4-mm punch. A standard curve was made by using known concentrations of drug. Samples and standards were placed in duplicate into wells, and plates were incubated at 30'C for 24 h. Inhibition zones were measured, and sample zones were compared with known standards. Blood for determining drug levels in serum was drawn at 45 and 60 min for oral and intravenous saperconazole, respectively. Trough levels in serum were obtained 24 h after six doses of therapy (which coincided with the time of sacrifice). Levels in lung tissue were measured by using a 1:10 (wt/vol) dilution of lung homogenate made as described above. Standards were made by using normal rabbit lung and known quantities of saperconazole. This method could detect 0.05 p,g of saperconazole per ml in serum and 0.5 iag/ml in lung homogenates. Inhibition EUSA for serum aWerglus antigen. The procedures for the inhibition enzyme-linked immunosorbent assay (ELISA) and for its required antigen and antibody
preparation were performed 16).
as
-~
ANTiMICROB. AGENTS CHEMOTHER.
previously reported (13, 14,
Statistical analysis. The Fisher exact test and the Wilcoxon rank sum test were used where appropriate. The method of least squares for linear regression and Mantel-Haenszel chi-square test were used to test for trend of increasing dosages of drug (SAS Institute Inc. Statistical Software, Cary, N.C.). Statistical significance was defined as P of
AND
CHEMOTHERAPY, Dec. 1992,
p.
2681-2685
Vol. 36, No. 12
0066-4804/92/122681-05$02.00/0 Copyright © 1992, American Society for Microbiology
Saperconazole Therapy in a Rabbit Model of Invasive Aspergillosis THOMAS F. PATTERSON,* DAVID GEORGE, PEGGY MINITER, AND VINCENT T. ANDRIOLE Department of Medicine, Section of Infectious Diseases, Yale University School of Medicine, New Haven, Connecticut 06510 Received 1 June 1992/Accepted 2 October 1992
The efficacy of orally and intravenously administered saperconazole against Aspergilius fumigatus was assessed in an immunosuppressed temporarily leukopenic rabbit model of invasive aspergillosis and compared with that of amphotericin B. Oral saperconazole at dosages of 5, 10, and 15 mg/kg of body weight per day improved survival compared with that of controls. In addition, saperconazole at 10 and 15 mg/kg/day reduced the tissue burden and reduced levels of circulating antigen, which correlated with increasing dosages of saperconazole. Intravenous saperconazole produced levels in serum more than 10-fold that of oral therapy. Intravenous saperconazole not only improved survival and reduced antigen levels but also significantly eradicated A. fumigatus from tissues compared with those of controls and was as effective as amphotericin B in these studies. Saperconazole was effective in the treatment of experimental invasive aspergillosis and demonstrates the potential of the newer azoles in therapy for invasive aspergillosis.
dose of cyclophosphamide (200 mg) given intravenously on the first day of the model and triamcinolone acetonide (10 mg; Westwood Pharmaceuticals, Buffalo, N.Y.), which was given subcutaneously each day. With this immunosuppressive regimen, the rabbits had reduced total leukocyte counts through day 7, as previously reported (14). Twenty-four hours after immunosuppression, groups of five to eight rabbits were challenged intravenously with a lethal inoculum of 106 Aspergillus fumigatus conidia. Antifungal therapy was given as described below. Each group contained at least one untreated control rabbit. Blood was obtained daily for determining total leukocyte counts and serum aspergillus antigen. Ceftazidime (200 mg; SmithKline Beecham, Philadelphia, Pa.) was administered intramuscularly daily beginning on the day of challenge to prevent intercurrent bacterial infection. Amphotericin B (Fungizone; Bristol-Myers Squibb, Princeton, N.J.) or saperconazole (Ortho Pharmaceuticals, Raritan, N.J.) therapy was begun 24 h after challenge. In initial experiments, saperconazole was administered orally after it was first dissolved in concentrated HCl with 0.01 ml of polyethylene glycol 200 (Sigma Chemical Co., St. Louis, Mo.) per ml of HCl and then 4 mg of hydroxypropyl-pcyclodextrin (9) (American Maize Products, Hammond, Ind.) per mg of saperconazole was added, for a saperconazole concentration of 1 mg/ml in distilled water. This saperconazole suspension was administered orally via a gastric gavage tube (American Pharmaseal Company, Valencia, Calif.) at a dosage of 5, 10, or 15 mg/kg/day for 5 days. In subsequent experiments, saperconazole was administered intravenously and compared with amphotericin B. Saperconazole was dissolved in dimethyl sulfoxide (Sigma) at a saperconazole concentration of 22 mg/ml. This intravenous saperconazole solution was administered over 5 min through a lateral ear vein at a dosage of 5.0 or 7.5 mg/kg/day for 6 days. Amphotericin B was diluted with 5% dextrose in sterile water at a ratio of 1 mg of drug to 10 ml of diluent and was given intravenously over 30 to 60 min through a lateral ear vein at a dosage of 1.5 mg/kg/day for a mean (+ standard error) of 5.1 (+ 0.3) days (range, 4 to 7 days). Organ cultures. Cultures and histopathology were performed at the time of autopsy or when the animals were
Invasive aspergillosis is associated with significant morbidity and mortality despite therapy with amphotericin B (2, 4). Management of invasive aspergillosis is further complicated by toxicity associated with amphotericin B therapy (6). Saperconazole is a triazole antifungal compound with excellent in vitro activity against Aspergillus spp. (3, 10). These newer azoles offer the potential for oral as well as intravenous therapy and reduced acute toxicity compared with amphotericin B (6). Saperconazole has been shown to have activity in a guinea pig model and in a murine model of aspergillosis (8, 18), but its limited activity in another murine model (15) could be related to the low levels in serum achieved with orally administered drug in that model. Absorption of related triazole compounds, such as ketoconazole and itraconazole, has been reported to be erratic in some immunosuppressed hosts (5, 6), so that adequate delivery of drug via oral administration is likely essential for successful therapy. In this immunosuppressed model, rabbits are made leukopenic and are further immunocompromised with steroid therapy (13, 14). Extensive infection develops in the liver, kidneys, lungs, and brain, similar to clinical dissemination of invasive aspergillosis (11, 12). Efficacy of therapy is assessed by mortality, semiquantitative organ cultures, and Asperillus antigen measurement. In this study, we assessed the activity of orally and intravenously administered saperconazole in a lethal model of experimental infection compared with that of amphotericin B. (This work was presented in part at the 90th Annual Meeting of the American Society for Microbiology, Anaheim, Calif., 13 to 17 May 1990 [abstract F-71] and the 30th Interscience Conference on Antimicrobial Agents and Chemotherapy, Atlanta, Ga., 21 to 24 October 1990 [abstract 603].)
MATERIALS AND METHODS Rabbit model. New Zealand White rabbits were immunosuppressed as previously described (13, 14) with a single *
Corresponding author. 2681
2682
PATTERSON ET AL.
sacrificed (24 to 48 h after completion of therapy for the treated rabbits). Rabbits were sacrificed, following anesthesia with ketamine (35 mg/kg; Bristol Laboratories, Syracuse, N.Y.) and xylazine (10 mg/kg; Mobay Corp., Shawnee, Kans.), by lethal exsanguination. Cultures were taken by placing minced organ samples directly on blood agar and on Sabouraud dextrose agar plates. Samples were considered positive when two or more colonies of A. fimigates were present on 21 g of minced organ tissues plated directly on Sabouraud dextrose and blood agar plates or when semiquantitative cultures of tissue homogenates contained 20 or more CFU/g of tissue (11, 14). The tissue burden of A.
finigatus
was evaluated with a modification (11) of the semiquantitative culture technique of Graybill and Kaster (7). Samples of liver, kidney, lung, and brain tissues were manually chopped, weighed, diluted 1:10 (wt/vol) with sterile saline, and homogenized for 25 s in an electric tissue homogenizer (TRI-R Instruments, Rockville Center, N.Y.). Then, 1.0 and 0.1 ml of each organ homogenate were plated in duplicate on Sabouraud dextrose and blood agar. Plates were incubated for 48 h at 37C, and colonies were counted. These methods combined detect from 2 to 20,000 CFU/g of tissue. For each animal, only a single kidney or lung was sampled at random. Drug levels. Levels of saperconazole in serum and tissue were measured by bioassay by using modifications of previously reported techniques (10, 11, 17). Briefly, Candida kefyr (kindly provided by Patrick Robinson) was grown overnight in Sabouraud dextrose broth at 37C and diluted
with saline to a concentration of 5 x 105 cells with a hemacytometer. A 1:50 dilution of cells was made with High Resolution media (Pfizer Central Research, Groton, Conn.), prepared according to the manufacturer's specifications. Forty-five milliliters of the inoculated media was poured into plastic plates (150 by 25 mm), and wells were cut with a 4-mm punch. A standard curve was made by using known concentrations of drug. Samples and standards were placed in duplicate into wells, and plates were incubated at 30'C for 24 h. Inhibition zones were measured, and sample zones were compared with known standards. Blood for determining drug levels in serum was drawn at 45 and 60 min for oral and intravenous saperconazole, respectively. Trough levels in serum were obtained 24 h after six doses of therapy (which coincided with the time of sacrifice). Levels in lung tissue were measured by using a 1:10 (wt/vol) dilution of lung homogenate made as described above. Standards were made by using normal rabbit lung and known quantities of saperconazole. This method could detect 0.05 p,g of saperconazole per ml in serum and 0.5 iag/ml in lung homogenates. Inhibition EUSA for serum aWerglus antigen. The procedures for the inhibition enzyme-linked immunosorbent assay (ELISA) and for its required antigen and antibody
preparation were performed 16).
as
-~
ANTiMICROB. AGENTS CHEMOTHER.
previously reported (13, 14,
Statistical analysis. The Fisher exact test and the Wilcoxon rank sum test were used where appropriate. The method of least squares for linear regression and Mantel-Haenszel chi-square test were used to test for trend of increasing dosages of drug (SAS Institute Inc. Statistical Software, Cary, N.C.). Statistical significance was defined as P of
