Therapy of Murine Aspergillosis with Amphotericin B in, Combination ...

ANTrMCROBIAL AGaNTs AND CHEMOTHERAPY, Jan. 1977, P. 21-25 Copyright C 1977 American Society for Microbiology

Vol. 11, No. 1 Printed in U.S.A.

Therapy of Murine Aspergillosis with Amphotericin B in, Combination with Rifampin or 5-Fluorocytosine JULIO ARROYO, GERALD MEDOFF,* Pam GEORGE S. KOBAYASHI Divisions ofInfectious Diseases,* Dermatology, and Laboratory Medicine, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110

Received for publication 19 July 1976

Suboptimal doses of amphotericin B in combination with either rifampin or 5-

fluorocytosine were better than single-drug therapy in the treatment of disseminated Aspergillus fumigatus infection in mice. Despite the increased effectiveof combination therapy, none of the therapeutic regimens we used completely eradicated infections in the mice when evaluated by mycological culture, even in long-term survivors.. ness

The combined use of amphotercin B (AmB) plus 5-fluorocytosine (5FC) has been shown to be more effective than either agent alone against Candida albicans both in vitro (14, 15) and in vivo (18). Similar results were observed when this combination was tested against Cryptococcus neoformans in vitro (15) and when used in the treatment of murine and hu-man cryptococcosis (5, 19). AmB used together with rifampin has been found to be more effective than either drug alone when tested against C. albicans (16) and Aspergillus (10) in vitro and against Histoplasma capsulatum and Blastomyces dermatitidis in vitro (12, 13) and in mice (9). Based on our observations on the in vitro effectiveness of both of these drug combinations against species of Aspergillus (10, 11), we have studied the in vitro efficacy of AmB plus rifampin and AmB plus 5FC in mice infected with a clinical isolate of A. fumigatus.

spores. The spore concentration was determined by hemocytometer counts, and the number of viable spores per milliliter was assessed by colony counts. Drugs. AmB (Fungizone) was purchased from E. R. Squibb and Sons, Inc., Princeton, N.J. Rifampin (Rifadin) was obtained from Dow Chemical Co., Zionsville, Ind. 5FC (Ancobon) was obtained from Hoffman-LaRoche, Inc., Nutley, N.J. Treatment groups. All animals were inoculated by the intravenous (i.v.) route with 0.1 ml of a freshly prepared spore suspension. The mice were then randomly distributed into cages in groups of five. In each experiment the inoculating dose of spores varied, but was always greater than the mean lethal dose. The mean lethal dose was 2 x 105 for AKR mice and for CD1 mice it was 1 x 106; the virulence of the organism remained constant throughout these experiments. Antifungal treament was started 2 days after inoculation and was continued for 28 days thereafter. All of the drugs were freshly prepared just before use. AmB was dissolved in sterile 5% dextrose in water to a concentration of 0.10 mg/ml. A 10-,g (= 0.5 mg/kg) dose of AmB was injected through a lateral tail vein on Mondays, Wednesdays, and Fridays for 4 weeks. Rifampin (8 mg) was dissolved in 0.5 ml of 50% ethanol and dilhited with sterile water to the desired concentrations. Rifampin and 5FC were administered daily by mouth using a curved, round-tip, 20-gauge gavage needle. Treatment groups usually consisted of 10 mice each. Control mice received sterile 5% dextrose in water and/or sterile water without antibiotics i.v. and orally, respectively. In all of the experiments, the efficacy of drug treatment was measured by the number of surviving mice at the cessation of therapy, which was 30 days after infection. In the different experiments the periods of observation after 30 days were variable. Histology and culture of organs. A number of treated and untreated mice were necropsied, and sections of various organs Were cultured on Mycosel agar. Brain and kidney sections were fixed in formalin and stained with hematoxylin and eosin and methanamine silver dyes.

MATERIALS AND METHODS Animals. AKR female mice weighing 18 to 20 g, obtained from Jackson Laboratories, Bar Harbor, Me., were used in some experiments to determine whether strain differences occurred in response to therapy. In all subsequent experiments, female CD1 mice weighing 18 to 22 g, purchased from Charles River, Wilmington, Mass., were employed as experimental animals since their responses were equivalent and they were more readily available. All animals were housed in plastic cages in groups of five and received food and water ad libitum. Organism. A. fumigatus no. 1 (10, 11), a clinical isolate and part of our permanent laboratory collection, was maintained at 37C and transferred weekly on Sabouraud agar slants. Sporulating subcultures of this organism were rinsed with sterile saline to collect the spores. Vigorous shaking of the resulting suspension with small glass beads yielded a uniform suspension consisting mainly of single 21

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ARROYO, MEDOFF, AND KOBAYASHI

ANTimiCROB. AGENTS CHEMOTHER.

RESULTS Treatment groups. Figure 1 shows the combined results of two separate experiments with AKR mice. The administration of AmB and rifampin in combination was significantly better than either drug alone in preventing deaths from aspergillosis (P < 0.05) (P values calculated by simple chi-square analysis). The infecting dose in these experiments was 5 x 103 spores. AmB, at a dose of 10 ug, was minimally effective in increasing the survival as compared with controls. Rifampin alone, at 50 or 100 ,ug, had no beneficial effect and actually appeared detrimental in these and other experiments. Since the curves for rifampin, 50 and 100 ,ug, were similar, only the former concentration is represented in the figures. A similar experiment using CD1 mice and an inoculum of 2 x 106 spores is shown in Fig. 2. The combination of AmB plus rifampin, 50 or 100 Am8 - I0ag

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FIG. 4. (a) Percentage of mice surviving 30 da,ys after infection ofgroups of 10 CD1 mice infected wiith increasing numbers of spores of A. fumigatus anLd treated with the regimens shown. The groups receiIVing 10 pg of AmB alone and 50 pg of rifampin alot had no survivors at the end of 30 days (data n ot shown). (b) Percentage of mice surviving 30 da,ys after infection ofgroups of 10 CD1 mice infected wilth 2 x 106 spores and treated with the regimens show)n.

ments, 5FC alone at a dose of 5 mg was Xas effective as the lower dose combination groulp (10 ,ug of AmB and 1.25 mg of 5FC), and both of these groups differed significantly from contr ol mice (P < 0.006). In addition, the differen ce between the groups treated with 10 ,g of AmLB and 5 mg of 5FC and the 5-mg 5FC group wfas significant at P < 0.001. Histology and organ culture. A total of 77 mice surviving the course of combination the,rapy were sacrificed 30 to 60 days after infecticIn and selected organs were cultured and exannined histologically. Twenty-eight of the 77 heid aspergilli cultured from kidneys. One had a positive spleen culture and another had a pos3itive liver culture. No organisms were culturE d from the brains or the lungs of these long-ter: m

fumigatus. Since rifampin alone had no therapeutic value and AmB at a dose of 10 ,g was only marginally active, we believe that the in vivo interaction of these two drugs is synergistic. Whereas 5FC alone at the 5-mg dose was effective against this organism, at equivalent doses the combination of AmB and 5FC was better than either agent alone. These data indicate that the effectiveness of combined drug therapy was synergistic. In the previous report in which we showed that these same combinations were synergistic in vitro against this strain of Aspergillus, the levels of 5FC and rifampin required for synergism were higher than clinically achievable concentrations when measurements of synergism were based on visual turbidity (10, 11). But, when the effects of the drugs were measured by the inhibition of ribonucleic acid synthesis or dry weight increase, much lower concentrations were effective. The results we report here, which show an

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FIG. 5. CD1 mice were infected on day 0 with 2 x 106 spores ofA. fumigatus. Treatment with AmB was started on day 2 and was given i.v. on Monday, Wednesday, and Friday. 5FC was given orally every dway for 28 days. There were 10 mice in each treatment group, and the results shown are the average of two separate experiments.

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ARROYO, MEDOFF, AND KOBAYASHI

enhanced effectiveness of these two drug combinations at clinically achievable blood levels against Aspergillus infection in mice, indicate that the use of effects on ribonucleic acid synthesis and dry weight as measures of susceptibility to rifampin and 5FC may be a more accurate determinant of in vivo susceptibility than the usual methods that use growth on agar or turbidity. The cause of death in the mice dying during therapy was disseminated infection involving many organ systems. This confirms other studies which showed that early in the course of infection aspergilli could be cultured from many different organs. These studies also show that as the animals survived for longer periods aspergilli disappeared, first from the lungs and followed by the spleen, liver, and brain, but the kidneys in these studies usually remained culture positive (17). Our histopathological data for the long-term survivors also confirm these latter observations, and the deaths that occurred several weeks or months after the infection of our animals appeared to be related to the persistence of aspergilli in the kidneys. Because there was no evidence of late dissemination in these mice, we think that obstructive nephropathy and uremia was a major cause of death. Using a similar experimental model, Corbel and Eades (7) also found that mice chronically infected with Absidia ramosa suffered moderate to severe uremia. The reason for the failure of therapy to eradicate kidney infection is unknown, but local conditions in the kidney, such as depressed activity of phagocytic cells (6) or inactivation of complement (3), might account for the persistence of infection. In regard to drug levels, serum levels of 5FC in our mice (S. A. Garding and H. M. Solomon, Prog. Abstr. Intersci. Conf. Antimicrob. Agents Chemother., 15th, Washington, D.C., Abstr. 416, 1975) were 92 and 43 ,ug/ml at 1 and 6 h after a single oral dose of 5 mg. These levels are in the high therapeutic range. Rifampin serum levels were not measured, but we feel we used maximum dosages because the 100 ,ug of rifampin was no better, and was perhaps worse, than the 50-,ug dose (2). However, according to serum measurements, the dose of AmB we used in our experiments was suboptimal. AmB serum levels at 1 and 24 h after a 10-,ug i.v. dose were 0.2 ,ug/ml (4, 8, 9). Therefore, it is possible that by using higher doses of AmB together with 5FC or rifampin in the treatment ofAspergillus infection, mycological sterility in the kidneys might be achieved. In summary, AmB in combination with

ANTIMICROB. AGENTS CHEMOTHER.

either rifampin or 5FC was better than singledrug therapy in the treatment of disseminated A. fumigatus infection in mice. Our observation that 5FC alone at a daily dose of 5 mg (250 mg/kg) was also effective confirms the clinical experience of others (1). Because none of the therapeutic regimens we used completely eradicated infections, even in long-term survivors, higher doses of AmB in the combinations should be tried. ACKNOWLEDGMENTS The AmB serum level determinations were performed by John E. Bennet, National Institute of Allergy and Infectious Diseases, Bethesda, Md., and the 5-fluorocytosine levels were performed by Harvey M. Solomon, Johns Hopkins Hospital, Baltimore, Md. We are grateful to Sung C. Choi for furnishing the statistical analysis of our data. This work was supported by Public Health Service grants AI 10622 and AI 06213 and training grants AI 00459 and AI 07015 from the National Institute of Allergy and Infectious Diseases, grants from John A. Hartford Foundation, Inc., a Brown-Hazen grant, and a special Public Health Service training fellowship, AI 00199, for Julio Arroyo from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Atkinson, G. W., and H. L. Israel. 1973. 5-Fluorocytosine treatment of meningeal and pulmonary aspergillosis. Am. J. Med. 55:496-505. 2. Bassi, L., L. DiBerardino, V. Arioli, L. G. Silvestri, and E. L. Cherie-Ligniere. 1973. Conditions for immunosuppression by rifampicin. J. Infect. Dis. 128:736-744. 3. Beeson, P. B., and D. Rowley. The anticomplementary effect of kidney tissue: its association with ammonia production. J. Exp. Med. 110:685. 4. Bindschadler, D. D., and J. E. Bennett. 1969. A pharmacologic guide to the clinical use of amphotericin B. J. Infect. Dis. 120:427-436. 5. Block, E. R., and J. E. Bennett. 1973. The combined effect of 5-fluorocytosine and amphotericin B in the therapy of murine cryptococcosis. Proc. Soc. Exp. Biol. Med. 142:476-480. 6. Charnew, I., and A. I. Braude. 1962. Depression of phagocytosis by solutes in concentrations found in the kidney and urine. J. Clin. Invest. 41:1945. 7. Corbel, M. J., and S. M. Eades. 1975. Factors determining the susceptibility of mice to experimental phycomycosis. J. Med. Microbiol. 8:551-574. 8. Green. W. R., J. E. Bennett, and R. D. Goos. 1965. Ocular penetration of amphotericin B. Arch. Ophthalmol. 73:769-775. 9. Kitahara, M., G. S. Kobayashi, and G. Medoff. 1976. Enhanced efficacy of amphotericin B and rifampicin combined treatment of murine histoplasmosis and blastomycosis. J. Infect. Dis. 133:663-668. 10. Kitahara, M., V. K. Seth, G. Medoff, and G. S. Kobayashi. 1976. Activity of amphotericin B, 5-fluorocytosine, and rifampin against six clinical isolates ofAspergillus. Antimicrob. Agents Chemother. 9:915-919. 11. Kitahara, M., V. K. Seth, G. Medoff, and G. S. Kobayashi. 1976. Antimicrobial susceptibility testing of six clinical isolates of Aspergillus. Antimicrob. Agents Chemother. 9.908-914. 12. Kobayashi, G. S., S. C. Cheung, D. Sehlessinger, and G. Medoff. 1974. Effects of rifamycin derivatives, alone and in combination with amphotericin B, against Histoplasma capsulatum. Antimicrob.

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THERAPY OF MURINE ASPERGILLOSIS WITH AmB

Agents Chemother. 5:16-18. 13. Kobayashi, G. S., G. Medoff, D. Schlessinger, C. N. Kwan, and W. E. Musser. 1972. Amphotericin B potentiation of rifampin as an antifungal agent against the yeast phase of Histoplasma capsulatum. Science 177:709-710. 14. Kwan, C. N., G. Medoff, G. S. Kobayashi, D. Schiessinger, and H. J. Raskas. 1972. Potentiation of the antifungal effects of antibiotics by amphotericin B. Antimicrob. Agents Chemother. 2:61-65. 15. Medoff, G., M. Comfort, and G. S. Kobayashi. 1971. Synergistic action of amphotericin B and 5-fluorocytosine against yeast-like organisms. Proc. Soc. Exp. Biol. Med. 13&8571-574. 16. Medoff, G., G. S. Kobayashi, C. N. Kwan, D. Schlessin-

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and P. Venkov. 1972. Potentiation of rifampicin and 5-fluorocytosine as antifungal antibiotics by amphotericin B. Proc. Natl. Acad. Sci. U.S.A. 69:196199. 17. Smith, G. R. Experimental aspergillosis in mice: aspects of resistance. J. Hyg. 70:741-754. 18. Titsworth, E., and E. Grunberg. 1973. Chemotherapeutic activity of 5-fluorocytosine and amphotericin B against Candida albicans in mice. Antimicrob. Agents Chemother. 4:306-308. 19. Utz, J. P., I. L. Garriques, M. A. Sande, J. F. Warner, G. L. Mandell, R. F. McGehee, R. J. Duma, and S. Shadomy. 1975. Therapy of cryptococcosis with a combination of fluorocytosine and amphotericin B. J. Infect. Dis. 132:368-373. ger,