Journal of Antimicrobial Chemotherapy (1991) 28, Suppl. B, 27-38
Amphotericin B: an introduction David W. Warnock
Amphotericin B has a broad spectrum of action that includes most of the major fungal pathogens of man. This drug binds to the membrane stcrols of fungal cells, causing impairment of their barrier function and loss of cell constituents. Metabolic disruption and cell death are consequent upon membrane alterations. Investigations of the sterol content of mutant strains of Candida albicans and Cryptococcus neoformans has demonstrated that resistance is often associated with alterations in membrane sterol composition. Treatment failure due to the development of amphotericin B resistance is an uncommon problem. It has tended to occur in patients receiving treatment with cytotoxk drugs. Interactions between amphotericin B and a number of other antimicrobial drugs have been observed in tests in vitro and in vivo. However, apart from one report that the combination withflucytosineis superior to amphotericin B on its own in the treatment of cryptococcal meningitis, there have been no controlled trials to support the use of drug combinations in human infections.
Introduction
Amphotericin A and B are natural fermentation products of an actinomycete recovered from soil collected in Venezuela in 1953. The organism was later named Streptomyces nodosus (Trejo & Bennett, 1963). Both compounds were found to possess a broad spectrum of antifungal action (Steinberg, Jambor & Suydam, 1956), but amphotericin A was not developed. Amphotericin B remains the drug of choice for most forms of deep-seated fungal infection, despite its harmful side effects and the continuing development of new compounds. The chemical structure of amphotericin B was elucidated in 1970 (Mechlinski et al., 1970). The molecule consists of a large macrolide lactone ring of 37 carbon atoms (Figure). One side of the macrolide ring is composed of a rigid lipophilic chain of seven conjugated double bonds and on the opposite side there are a similar number of hydroxyl groups. Thus, the molecule is amphipathic and this feature of its structure is believed to be important in its biological mechanism of action. The macrolide ring also contains a six-membered ketalic ring and the amino sugar mycosamine is bonded to the ring through an a-glycosidic linkage. In most patients, administration of amphotericin B is associated with harmful sideeffects and unpleasant reactions which often limit the amount that can be given. These problems have stimulated attempts to develop less toxic chemical modifications of the drug. The free carboxylic acid group on the macrolide ring has formed the basis for the preparation of esters and the free amino group of the amino sugar mycosamine has 27 0305-7453/91/28B027 + 12 $02.00/0
© 1991 The British Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on May 29, 2015
Regional Mycology Laboratory, Department of Microbiology, Bristol Royal Infirmary, Bristol BS2 8HW, UK
28
D. W. Wunock Ampltottrlcin B: R » H Amphoitrlctn B mathyl t»Ur-.R=CH 3
CH
• CH,
Figure. Chemical itructure of amphotericin B and amphotericin B methyl ester.
been used to prepare N-acyl, N-methylated, N-glycosylated, N-aminoacylated and N-guanidino derivatives of amphotericin B (Schaffner, 1987). Esterification produced derivatives that were active in vitro and in vivo, but less nephrotoxic and better tolerated than amphotericin B. However, clinical testing was discontinued following reports of progressive neurological dysfunction and diffuse white matter degeneration in patients receiving prolonged high-dose treatment with the methyl ester (Ellis, Sobel & Nielsen, 1982). Other derivatives have also been prepared, most of which were less toxic, but also less active than amphotericin B (Schaffner, 1987). None has achieved clinical importance. Mechanism of action The major action of amphotericin B is to damage the membrane of fungal cells. The drug binds to ergosterol, the principal membrane sterol, causing an impairment of barrier function that results in the loss of protons and cations from the cell. The precise mechanism involved has not been elucidated, but a number of molecular models have been proposed (Bolard, 1986; Kerridge, 1986). Treatment with amphotericin B also results in the loss of other cell constituents, but this is due to dissipation of the proton gradient. At low concentrations, loss of cell constituents is restricted to small molecules or cations such as sodium and potassium. At higher concentrations or after prolonged incubation, other cell constituents are lost and this leads to metabolic disruption and cell death. The action of amphotericin B on fungal cells is now believed to involve more than one mechanism (Brajtburg et al., 1990). It has been reported that the lethal effects of higher concentrations of the drug on Candida albicans are not a simple consequence of its membrane-permeabilizing effects, but involve oxidative damage to the cell (Sokol-Anderson, Brajtburg & Medoff, 1986a, b). The drug appears to induce a cascade of oxidative reactions linked to its own oxidation, but the actual mechanism involved remains to be clarified. In addition to its antifungal effects, amphotericin B has potent immunomodulating effects in the host. Again, oxidation-dependent events appear to be involved (Brajtburg et al., 1990). Stimulation or suppression of cell-mediated and humoral immunological function can occur, depending on the drug concentration and the timing of its
Downloaded from http://jac.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on May 29, 2015
CH
Ampboteridn B introduced
29
administration (Hauser & Remington, 1982). The clinical importance of such effects has not been determined. Spectrum of action
Most strains of C. albicans are inhibited from growth in vitro at amphotericin B concentrations ranging from 0-05 to 1-0 mg/L. Nine hundred C. albicans strains tested in one investigation (Athar & Winner, 1971) all had MICs of 1-0 mg/L or less, while 250 strains of Candida tropicalis had MICs of 2-0 mg/L or less. Amphotericin B-resistant strains have seldom been isolated before treatment with the drug, although Grillot et al. (1975) described two C. albicans strains with MICs greater than 50 mg/L
Downloaded from http://jac.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on May 29, 2015
Tests designed to ascertain the lowest concentration of drug needed to inhibit the growth of organisms in vitro (minimum inhibitory concentration or MIC) are often used in attempts to predict the spectrum of action of an antimicrobial drug. As with other antifungal drugs, the results of MIC determinations with amphotericin B must be interpreted with caution, because the conditions under which the tests are performed can have an effect on the results obtained. Medium composition, inoculum concentration and length of incubation are factors that can affect amphotericin B MICs (Doern et al., 1986). Their influence is reflected in the divergent results that have been obtained when MICs of particular strains have been determined with different methods (Galgiani et al., 1987). Even when identical methods have been used, discrepant results have been obtained (Calhoun et al., 1986), emphasizing the need for careful standardization of test conditions if MICs are to be used to predict the outcome of treatment. There has been one formal attempt to test the extent of the correlation between MIC tests with amphotericin B and the clinical results of its administration (O'Day et al., 1987). This assessment was performed with an animal model of ocular infection with C. albicans. It demonstrated a good correlation between the MICs of 17 strains and the response of infected animals to topical treatment with the drug. It is often difficult to assess the clinical results of treatment with amphotericin B (Dismukes et al., 1980) and this has hampered efforts to establish the extent of their correlation with the results of MIC tests. The suggestion that deep-seated fungal infections can be managed if amphotericin B is administered in sufficient amounts to obtain in the patient serum concentrations twice the MIC of the causative fungus (Drutz et al., 1968) has seldom been adopted. This is at best a rough correlation because there is no clear relationship between blood and tissue levels of the drug. The growth of most strains of most of the principal fungal pathogens of man (Blastomyces dermatitidis, Coccidioides immitis, Cryptococcus neoformans, and Histoplasma capsulation) is inhibited in vitro at concentrations of amphotericin B ranging from (M)5 to 1-Omg/L (Bennett, 1967; Hoeprich & Huston, 1975; Shadomy et al., 1978). These concentrations are similar to blood levels attained during parenteral treatment with the drug, but it cannot be assumed that all patients infected with these organisms will respond to amphotericin B. Other factors, such as the immunological status of the host and the location of the infection, must be taken into account. The range of MICs reported for different strains of these organisms is more limited than those obtained with certain other fungal pathogens. That said, this variation could be significant: Hoeprich & Huston (1975) observed a definite correlation between MIC test results and clinical outcome among patients receiving amphotericin B for coccidioidomycosis.
30
D. W. Wunock
Mechanisms of resistance Mutant strains of C. albicans and C. neoformans resistant to amphotericin B and nystatin have seldom emerged during treatment of human infections, but have proved much easier to obtain under artificial conditions. Comparison of the lipid composition of cells of such strains with those of the parental strains has shown that resistance is often associated with alterations in the nature of the membrane sterols or in the amount of sterols present. Mutations that affect the lipid composition of the fungal cell often impair other aspects of cell function. Thus, most resistant mutants of C. albicans have been less vigorous than their parental strains (Pesti, Paku & Novak, 1982) and have proved less pathogenic (Athar & Winner, 1971). Athar & Winner (1971) obtained amphotericin B-resistant strains of C. albicans and C. tropicalis following serial sub-culture with increasing concentrations of the drug. The resistant strains had a reduced ergosterol content. HsuChen & Feingold (1974) observed that the amount of ergosterol was reduced in mutagen-induced nystatin- and amphotericin B-resistant strains of C. albicans while Subden et al. (1977) showed that nystatin- and amphotericin B-resistant strains contained increased amounts of methylated sterols. The most detailed account of membrane sterol alterations in mutant
Downloaded from http://jac.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on May 29, 2015
and Safe et al. (1977) obtained a resistant strain of Candida parakrusei (now reclassified as Candida parapsilosis) from a patient who had not received antifungal treatment A recent report has noted an interesting relationship between amphotericin B MICs (read after 48 h incubation) and the outcome of treatment with the drug among 26 patients with candidosis (Powderly et al., 1988). Ten cases in which the causative fungus had an MIC greater than 0-8 mg/L all proved fatal, in comparison with eight of 17 cases involving organisms with MICs of 04 or 0-8 mg/L. Further work is needed to define more clearly the implications of these findings. Most strains of Aspergillus fumigatus are inhibited from growth at amphotericin B concentrations of less than 2-0 mg/L (Brandsberg & French, 1972; Shadomy et al., 1978). Other Aspergillus spp. have had MICs ranging from 01 to greater than 100 mg/L. Nevertheless, there have been no convincing demonstrations that treatment failure in patients with aspergillosis can be attributed to the development of amphotericin B resistance. MICs ranging from 0-1 to 0-5 mg/L have been reported for the aetiological agents of mucormycosis (Howarth, Tewari & Solotorowsky, 1975). However, up to 50 mg/L is often required for fungicidal action. Most strains of Pseudallescheria boydii are resistant to amphotericin B in vitro with MICs in excess of 2-0 mg/L (Lutwick et al., 1976; Shadomy et al., 1978) and infections with this organism are best treated with other antifungal drugs. Likewise, some of the aetiological agents of chromoblastomycosis and phaeohyphomycosis have MICs of 4-0 mg/L or greater and must be considered insensitive (Shadomy et al., 1978). It has been reported that some strains of Trichosporon beigelii (T. cutaneum) from patients who died with disseminated trichosporonosis despite amphotericin B treatment were insensitive to the drug in vivo (Walsh et al., 1990). Strains of this organism are inhibited at concentrations of amphotericin B ranging from 0-3 to 90mg/L, and minimum fungicidal concentrations (MFCs) have ranged from 0-6 to greater than 18 mg/L (Walsh et al., 1990). The suggestion that the latter measurement might be more useful in predicting clinical outcome in granulocytopenic patients deserves further investigation.
Ampbotefidn B introduced
31
Acquisition of resistance daring ctinkal me
Treatment failure attributable to the development of amphotericin B resistance has remained an uncommon problem. It has been documented in six patients, all of whom were being treated for candidosis: two cases of C. tropicalis infection (Drutz & Lehrer, 1978; Merz & Sandford, 1979), three cases of Candida lusitaniae infection (Pappagianis et al., 1979; Guinet et al., 1983; Mere, 1984), and one case of Candida guilliermondii infection (Dick et al., 1985). In most of these cases the organisms were recovered from compromised patients who had received amphotericin B for prolonged periods. The strains of C. tropicalis from the case of Drutz & Lehrer (1978) had MICs of the order of 100 to 500 mg/L as did the strain from the case of Merz & Sandford (1979). In both cases the strains were ergosterol deficient. It is unusual to isolate resistant strains from patients receiving prophylactic oral treatment with nystatin or amphotericin B (although it must be said that MIC testing is seldom done as a routine procedure). However, Safe et al. (1977) isolated one strain each of Candida krusei and C. tropicalis resistant to amphotericin B and nystatin from patients receiving prophylactic treatment with the latter drug. Further tests showed that both strains contained much reduced amounts of ergosterol. Clinical resistance to amphotericin B could be more common than has been suspected. Dick, Mere & Saral (1980) compared the MICs of Candida spp. from 70 neutropenic patients with the MICs of similar organisms recovered from other patients. None of the 625 strains from the control group were classified as 'resistant' to the drug (MIC ^ 2-0 mg/L). In contrast, 55 strains originating from six of the 70 neutropenic patients were resistant: 27 strains of C. albicans (from three patients), three strains of C. tropicalis (one patient), and 25 strains of C. glabrata (two patients). The resistant strains all contained reduced amounts of ergosterol.
Downloaded from http://jac.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on May 29, 2015
strains of C. albicans is that of Pierce et al. (1978). These authors found that the higher the drug concentration needed to inhibit growth, the greater the shift in lipid composition from ergosterol to 4- and 14-methylated sterols. It is clear that, although membrane sterols are involved in the disruptive interaction of amphotericin B with sensitive fungal cells, alterations in sterol content are insufficient to account for drug resistance in all strains of C. albicans and C. neoformans. Mutant strains of C. albicans, resistant to amphotericin B, have sometimes been found to possess elevated levels of membrane ergosterol (Hamilton-Miller, 1972). Moreover, resistant strains of C. neoformans with normal ergosterol levels and sensitive strains with reduced levels have both been observed (Kim et al., 1975). In these strains some other resistance mechanism must operate. Elevated catalase levels have been detected in several amphotericin B-resistant strains of C. albicans (Sokol-Anderson et al., 1988). The enhanced resistance that this confers against oxidation-dependent damage to the fungal cell might well be another important mechanism of amphotericin B resistance, but this has still to be confirmed. To gain access to the fungal cell membrane, amphotericin B must first be transported across the rigid cell wall. In C. albicans alterations in the /M,3 glucan content of the cell wall after cessation of growth prevent the drug from reaching the membrane and result in the cells becoming more resistant to amphotericin B (Gale, 1986). However, this 'phenotypic' resistance is lost as soon as growth is resumed. Its clinical significance is unclear.
32
D. W. Wtmock
Interactions between ampboteridn B and other antimicrobial drugs The first report of a favourable interaction between amphotericin B and another antifungal drug appeared in 1971. Medoff, Comfort & Kobayashi (1971) observed synergistic effects when combinations of amphotericin B and flucytosine (5-fluorocytosine) were tested against strains of C. albicans, C. tropicalis and C. neoformans in vitro. Synergism was defined as a four-fold or greater reduction in the MIC of flucytosine in the presence of amphotericin B at concentrations below its MIC for each test strain. Other conflicting reports followed these initial observations, perhaps because of the differing test conditions, strains and drug concentrations used (Beggs, Sarosi & Andrews, 1974a; Shadomy et al., 1975; Polak, 1978). Odds (1982) concluded that for most strains of C. albicans the drug interaction is, at best, additive.
Downloaded from http://jac.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on May 29, 2015
These results suggest that it might be useful to screen for amphotericin B resistance in certain groups of patients. The individuals from whom the resistant strains were isolated had all received parenteral treatment with amphotericin B and five had also received oral nystatin (Dick et al., 1980). All had earlier harboured sensitive strains of the same organism, all had been neutropenic for long periods and all had received treatment with cytotoxic drugs. Four patients developed deep-seated infections with the resistant strains, of which two were fatal. The findings of a second report support the suggestion that acquisition of resistance could be a potential problem in neutropenic patients undergoing amphotericin B treatment. Powderly et al. (1988) observed that the amphotericin B MICs of Candida spp. isolated from 26 transplant patients were higher than those of similar organisms obtained from non-compromised patients. Although 15 transplant patients had received empirical treatment with amphotericin B, the MICs of their strains were similar to those of strains from transplant patients who had not received such treatment. This suggests that use of the drug was not the sole factor contributing to the selection of less sensitive organisms in this patient group. The transplant patients had all received prophylactic oral treatment with clotrimazole and it is possible that use of this imidazole led to the development of amphotericin B resistance as a result of the selection of organisms with a reduced membrane ergosterol content (Sud & Feingold, 1983). Another potential explanation for the development of amphotericin B resistance in patients undergoing cytotoxic treatment is that such treatment leads to the generation of mutant strains. Treatment with amphotericin B then leads to the selection of mutants that are also drug resistant. Because cytotoxic treatment injures cells through oxidative mechanisms, its use could lead to the selection of fungal cells that are resistant to oxidation-dependent damage. Such cells might well be resistant to the lethal oxidation-dependent effects of amphotericin B (Sokol-Anderson et al., 1986a). Most of the amphotericin B resistant strains that have been recovered during treatment have so far belonged to species other than C. albicans, in particular C. lusitaniae and C. tropicalis. The potential for resistance also appears to be high in C. guilliermondii—which, being haploid (Magee & Magee, 1987), can be induced to produce resistant mutants more readily than C. albicans which is diploid—and in C. parapsilosis, which is inhibited from growth at similar concentrations of amphotericin B to other members of the genus, but which is much less susceptible to its lethal effects (Seidenfeld et al., 1983).
Ampboteridn B introduced
33
Downloaded from http://jac.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on May 29, 2015
Conflicting > results have also been obtained from tests in vitro for interactions between amphotericin B and fiucytosine against Aspergillus spp. Kitahara et al. (1976/>) detected synergism, but subsequent work has failed to confirm their findings (Lauer, Reller & Schroter, 1978; Hughes et al., 1984a). One explanation for the synergism detected with combinations of amphotericin B and fiucytosine is that the membrane-permeabilizing effects of low concentrations of the former drug facilitate the penetration of the latter to the cell interior, potentiating its deleterious effects on cell metabolism (Medoff et al., 1972). However, Beggs, Andrews & Sarosi (1977) observed that amphotericin B can inhibit the uptake of fiucytosine into C. albicans cells. Beggs & Sarosi (1982) suggested that synergism results from sequential, rather than combined action of the two drugs. In this hypothesis, amphotericin B acts first and alone until its gradual oxidation results in its depletion. At this point flucytosine acts on the surviving fungal cells. It is difficult to detect and assess drug interactions in vivo unless the effects are pronounced. Moreover, reports of favourable interactions are often based on too lenient interpretation of test results. That said, it has been claimed that combination treatment with amphotericin B and flucytosine is more effective than amphotericin B alone in animal models of aspergillosis, candidosis and cryptococcosis (Block & Bennett, 1973; Titsworth & Grunberg, 1973; Arroyo, Medoff & Kobayashi, 1977; Polak, Scholer & Wall, 1982). However, with the exception of the work of Bennett et al. (1979) demonstrating that the combination is superior to amphotericin B alone in the treatment of cryptococcal meningitis, there are no results from controlled clinical trials to support the use of the combination in other fungal infections. In spite of this, the combination has become a popular choice for the treatment of many forms of deepseated candidosis. In addition, there are a number of anecdotal reports of its use in patients with aspergillosis and mucormycosis. The antifungal azoles inhibit the 14-demethylation step in the biosynthesis of ergosterol (Vanden Bossche, 1985; Kerridge, 1986). The consequent depletion of ergosterol and accumulation of lanosterol and other 14-methylsterols leads to alterations in a number of membrane-associated functions. Because depletion of ergosterol should lead to a reduction in cell membrane sites for the binding of amphotericin B, it is reasonable to expect that prior treatment with an azole might antagonize the effects of amphotericin B (Sud & Feingold, 1983; Wilson & Peacock, 1985). Tests with combinations of amphotericin B and miconazole, ketoconazole, econazole or clotrimazole in vitro have given conflicting results with indications both of antagonism (Cosgrove, Beezer & Miles, 1978; Dupont & Drouhet, 1979) and synergism (Beggs, Sarosi & Steele, 1976; Odds, 1982) against Candida spp. and C. neoformans. It appears that the nature of the interaction detected depends on the test conditions employed, the order of drug addition and the length of incubation (Brajtburg et al., 1982; Smith, McFadden & Miller, 1983). Tests in animal models of candidosis have revealed both antagonistic and synergistic interactions between amphotericin B and the azoles (Polak et al., 1982). Tests in vitro with combinations of amphotericin B and ketoconazole against Aspergillus spp. have given differing results. Hughes et al. (1984a) observed no interactions in tests in which both drugs were added together. However, Schaffner & Frick (1985) showed that prior treatment with ketoconazole resulted in a rise in amphotericin B MFCs from 0-6 mg/L or less to greater than 2-5 mg/L. Moreover, prior treatment of infected animals with ketoconazole abolished the protective effect of subsequent
34
D. W. Waroock
References Arroyo, J., Medoff, G. & Kobayashi, G. S. (1977). Therapy of murine aspergillosis with amphotericin B in combination with rifampin or 5-fluorocytosine. Antimicrobial Agents and Chemotherapy 11, 21-5. Athar, M. A. & Winner, H. I. (1971). The development of resistance by Candida species to polyene antibiotics in vitro. Journal of Medical Microbiology 4, 505-17. Beggs, W. H., Andrews, F. A. & Sarosi, G. A. (1977). Evidence for sequential action of amphotericin B and 5-fluorocytosine on Candida albicans ATCC 11651. Research Communications in Chemical Pathology and Pharmacology 16, 557-60. Beggs, W. H. & Sarosi, G. A. (1982). Further evidence for sequential action of amphotericin B and 5-fluorocytosine against Candida albicans. Chemotherapy (Basel) 28, 341-4. Beggs, W. H., Sarosi, G. A. & Andrews, F. A. (1974a). Inhibition of Candida albicans by amphotericin B in combination with 5-fluorocytosine. Research Communications in Chemical Pathology and Pharmacology 8, 559-62. Beggs, W. H., Sarosi, G. A. & Andrews, F. A. (1974A). Synergistic action of amphotericin B and rifampin on Candida albicans. American Review of Respiratory Disease 110, 671-3. Beggs, W. H., Sarosi, G. A. & Steele, N. M. (1976). Inhibition of potentially pathogenic yeastlike fungi by clotrimazole in combination with 5-fluorocytosine or amphotericin B. Antimicrobial Agents and Chemotherapy 9, 863-5. Bennett, J. E. (1967). Susceptibility of Cryptococcus neoformans to amphotericin B. Antimicrobial Agents and Chemotherapy—1966, 405-10. Bennett, J. E., Dismukes, W. E., Duma, R. J., Medoff, G., Sande, M. A., Gallis, H. et al. (1979). A comparison of amphotericin B alone and combined with flucytosine in the treatment of cryptococcal meningitis. New England Journal of Medicine 301, 126-31. Block, E. R. & Bennett, J. E. (1973). The combined effect of 5-fluorocytosine and amphotericin B in the therapy of murine crYptococcosis. Proceedings of the Society for Experimental Biology and Medicine 142, 476-80.
Downloaded from http://jac.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on May 29, 2015
amphotericin B against murine aspergillus infection. Further work is needed to define the clinical importance of these findings. The membrane-damaging effects of low concentrations of amphotericin B alter the fungal cell membrane in a manner that renders it permeable to drugs that would not otherwise be taken up. This might explain the favourable interactions between amphotericin B and the antibacterial drug rifampicin that have been demonstrated in tests in vivo with strains of Aspergillus spp. (Kitahara et al., 19766; Hughes et al., 1984fl), Candida spp. (Beggs, Sarosi & Andrews, 19746; Edwards et al., 1980), C. immitis (Huppert et al., 1976), C. neoformans (Fujita & Edwards, 1981), H. capsulation (Kobayashi et al., 1974) and Rhizopus spp. (Christenson et al., 1987). These effects were detected at achievable concentrations for both drugs. Tests in animal models of infection have indicated that treatment with amphotericin B and rifampicin in combination has at least an additive effect on infections with A.fumigatus. B. derma t it idis and H. capsulatum (Kitahara, Kobayashi & Medoff, 1976
Amphotericin B: an introduction David W. Warnock
Amphotericin B has a broad spectrum of action that includes most of the major fungal pathogens of man. This drug binds to the membrane stcrols of fungal cells, causing impairment of their barrier function and loss of cell constituents. Metabolic disruption and cell death are consequent upon membrane alterations. Investigations of the sterol content of mutant strains of Candida albicans and Cryptococcus neoformans has demonstrated that resistance is often associated with alterations in membrane sterol composition. Treatment failure due to the development of amphotericin B resistance is an uncommon problem. It has tended to occur in patients receiving treatment with cytotoxk drugs. Interactions between amphotericin B and a number of other antimicrobial drugs have been observed in tests in vitro and in vivo. However, apart from one report that the combination withflucytosineis superior to amphotericin B on its own in the treatment of cryptococcal meningitis, there have been no controlled trials to support the use of drug combinations in human infections.
Introduction
Amphotericin A and B are natural fermentation products of an actinomycete recovered from soil collected in Venezuela in 1953. The organism was later named Streptomyces nodosus (Trejo & Bennett, 1963). Both compounds were found to possess a broad spectrum of antifungal action (Steinberg, Jambor & Suydam, 1956), but amphotericin A was not developed. Amphotericin B remains the drug of choice for most forms of deep-seated fungal infection, despite its harmful side effects and the continuing development of new compounds. The chemical structure of amphotericin B was elucidated in 1970 (Mechlinski et al., 1970). The molecule consists of a large macrolide lactone ring of 37 carbon atoms (Figure). One side of the macrolide ring is composed of a rigid lipophilic chain of seven conjugated double bonds and on the opposite side there are a similar number of hydroxyl groups. Thus, the molecule is amphipathic and this feature of its structure is believed to be important in its biological mechanism of action. The macrolide ring also contains a six-membered ketalic ring and the amino sugar mycosamine is bonded to the ring through an a-glycosidic linkage. In most patients, administration of amphotericin B is associated with harmful sideeffects and unpleasant reactions which often limit the amount that can be given. These problems have stimulated attempts to develop less toxic chemical modifications of the drug. The free carboxylic acid group on the macrolide ring has formed the basis for the preparation of esters and the free amino group of the amino sugar mycosamine has 27 0305-7453/91/28B027 + 12 $02.00/0
© 1991 The British Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on May 29, 2015
Regional Mycology Laboratory, Department of Microbiology, Bristol Royal Infirmary, Bristol BS2 8HW, UK
28
D. W. Wunock Ampltottrlcin B: R » H Amphoitrlctn B mathyl t»Ur-.R=CH 3
CH
• CH,
Figure. Chemical itructure of amphotericin B and amphotericin B methyl ester.
been used to prepare N-acyl, N-methylated, N-glycosylated, N-aminoacylated and N-guanidino derivatives of amphotericin B (Schaffner, 1987). Esterification produced derivatives that were active in vitro and in vivo, but less nephrotoxic and better tolerated than amphotericin B. However, clinical testing was discontinued following reports of progressive neurological dysfunction and diffuse white matter degeneration in patients receiving prolonged high-dose treatment with the methyl ester (Ellis, Sobel & Nielsen, 1982). Other derivatives have also been prepared, most of which were less toxic, but also less active than amphotericin B (Schaffner, 1987). None has achieved clinical importance. Mechanism of action The major action of amphotericin B is to damage the membrane of fungal cells. The drug binds to ergosterol, the principal membrane sterol, causing an impairment of barrier function that results in the loss of protons and cations from the cell. The precise mechanism involved has not been elucidated, but a number of molecular models have been proposed (Bolard, 1986; Kerridge, 1986). Treatment with amphotericin B also results in the loss of other cell constituents, but this is due to dissipation of the proton gradient. At low concentrations, loss of cell constituents is restricted to small molecules or cations such as sodium and potassium. At higher concentrations or after prolonged incubation, other cell constituents are lost and this leads to metabolic disruption and cell death. The action of amphotericin B on fungal cells is now believed to involve more than one mechanism (Brajtburg et al., 1990). It has been reported that the lethal effects of higher concentrations of the drug on Candida albicans are not a simple consequence of its membrane-permeabilizing effects, but involve oxidative damage to the cell (Sokol-Anderson, Brajtburg & Medoff, 1986a, b). The drug appears to induce a cascade of oxidative reactions linked to its own oxidation, but the actual mechanism involved remains to be clarified. In addition to its antifungal effects, amphotericin B has potent immunomodulating effects in the host. Again, oxidation-dependent events appear to be involved (Brajtburg et al., 1990). Stimulation or suppression of cell-mediated and humoral immunological function can occur, depending on the drug concentration and the timing of its
Downloaded from http://jac.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on May 29, 2015
CH
Ampboteridn B introduced
29
administration (Hauser & Remington, 1982). The clinical importance of such effects has not been determined. Spectrum of action
Most strains of C. albicans are inhibited from growth in vitro at amphotericin B concentrations ranging from 0-05 to 1-0 mg/L. Nine hundred C. albicans strains tested in one investigation (Athar & Winner, 1971) all had MICs of 1-0 mg/L or less, while 250 strains of Candida tropicalis had MICs of 2-0 mg/L or less. Amphotericin B-resistant strains have seldom been isolated before treatment with the drug, although Grillot et al. (1975) described two C. albicans strains with MICs greater than 50 mg/L
Downloaded from http://jac.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on May 29, 2015
Tests designed to ascertain the lowest concentration of drug needed to inhibit the growth of organisms in vitro (minimum inhibitory concentration or MIC) are often used in attempts to predict the spectrum of action of an antimicrobial drug. As with other antifungal drugs, the results of MIC determinations with amphotericin B must be interpreted with caution, because the conditions under which the tests are performed can have an effect on the results obtained. Medium composition, inoculum concentration and length of incubation are factors that can affect amphotericin B MICs (Doern et al., 1986). Their influence is reflected in the divergent results that have been obtained when MICs of particular strains have been determined with different methods (Galgiani et al., 1987). Even when identical methods have been used, discrepant results have been obtained (Calhoun et al., 1986), emphasizing the need for careful standardization of test conditions if MICs are to be used to predict the outcome of treatment. There has been one formal attempt to test the extent of the correlation between MIC tests with amphotericin B and the clinical results of its administration (O'Day et al., 1987). This assessment was performed with an animal model of ocular infection with C. albicans. It demonstrated a good correlation between the MICs of 17 strains and the response of infected animals to topical treatment with the drug. It is often difficult to assess the clinical results of treatment with amphotericin B (Dismukes et al., 1980) and this has hampered efforts to establish the extent of their correlation with the results of MIC tests. The suggestion that deep-seated fungal infections can be managed if amphotericin B is administered in sufficient amounts to obtain in the patient serum concentrations twice the MIC of the causative fungus (Drutz et al., 1968) has seldom been adopted. This is at best a rough correlation because there is no clear relationship between blood and tissue levels of the drug. The growth of most strains of most of the principal fungal pathogens of man (Blastomyces dermatitidis, Coccidioides immitis, Cryptococcus neoformans, and Histoplasma capsulation) is inhibited in vitro at concentrations of amphotericin B ranging from (M)5 to 1-Omg/L (Bennett, 1967; Hoeprich & Huston, 1975; Shadomy et al., 1978). These concentrations are similar to blood levels attained during parenteral treatment with the drug, but it cannot be assumed that all patients infected with these organisms will respond to amphotericin B. Other factors, such as the immunological status of the host and the location of the infection, must be taken into account. The range of MICs reported for different strains of these organisms is more limited than those obtained with certain other fungal pathogens. That said, this variation could be significant: Hoeprich & Huston (1975) observed a definite correlation between MIC test results and clinical outcome among patients receiving amphotericin B for coccidioidomycosis.
30
D. W. Wunock
Mechanisms of resistance Mutant strains of C. albicans and C. neoformans resistant to amphotericin B and nystatin have seldom emerged during treatment of human infections, but have proved much easier to obtain under artificial conditions. Comparison of the lipid composition of cells of such strains with those of the parental strains has shown that resistance is often associated with alterations in the nature of the membrane sterols or in the amount of sterols present. Mutations that affect the lipid composition of the fungal cell often impair other aspects of cell function. Thus, most resistant mutants of C. albicans have been less vigorous than their parental strains (Pesti, Paku & Novak, 1982) and have proved less pathogenic (Athar & Winner, 1971). Athar & Winner (1971) obtained amphotericin B-resistant strains of C. albicans and C. tropicalis following serial sub-culture with increasing concentrations of the drug. The resistant strains had a reduced ergosterol content. HsuChen & Feingold (1974) observed that the amount of ergosterol was reduced in mutagen-induced nystatin- and amphotericin B-resistant strains of C. albicans while Subden et al. (1977) showed that nystatin- and amphotericin B-resistant strains contained increased amounts of methylated sterols. The most detailed account of membrane sterol alterations in mutant
Downloaded from http://jac.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on May 29, 2015
and Safe et al. (1977) obtained a resistant strain of Candida parakrusei (now reclassified as Candida parapsilosis) from a patient who had not received antifungal treatment A recent report has noted an interesting relationship between amphotericin B MICs (read after 48 h incubation) and the outcome of treatment with the drug among 26 patients with candidosis (Powderly et al., 1988). Ten cases in which the causative fungus had an MIC greater than 0-8 mg/L all proved fatal, in comparison with eight of 17 cases involving organisms with MICs of 04 or 0-8 mg/L. Further work is needed to define more clearly the implications of these findings. Most strains of Aspergillus fumigatus are inhibited from growth at amphotericin B concentrations of less than 2-0 mg/L (Brandsberg & French, 1972; Shadomy et al., 1978). Other Aspergillus spp. have had MICs ranging from 01 to greater than 100 mg/L. Nevertheless, there have been no convincing demonstrations that treatment failure in patients with aspergillosis can be attributed to the development of amphotericin B resistance. MICs ranging from 0-1 to 0-5 mg/L have been reported for the aetiological agents of mucormycosis (Howarth, Tewari & Solotorowsky, 1975). However, up to 50 mg/L is often required for fungicidal action. Most strains of Pseudallescheria boydii are resistant to amphotericin B in vitro with MICs in excess of 2-0 mg/L (Lutwick et al., 1976; Shadomy et al., 1978) and infections with this organism are best treated with other antifungal drugs. Likewise, some of the aetiological agents of chromoblastomycosis and phaeohyphomycosis have MICs of 4-0 mg/L or greater and must be considered insensitive (Shadomy et al., 1978). It has been reported that some strains of Trichosporon beigelii (T. cutaneum) from patients who died with disseminated trichosporonosis despite amphotericin B treatment were insensitive to the drug in vivo (Walsh et al., 1990). Strains of this organism are inhibited at concentrations of amphotericin B ranging from 0-3 to 90mg/L, and minimum fungicidal concentrations (MFCs) have ranged from 0-6 to greater than 18 mg/L (Walsh et al., 1990). The suggestion that the latter measurement might be more useful in predicting clinical outcome in granulocytopenic patients deserves further investigation.
Ampbotefidn B introduced
31
Acquisition of resistance daring ctinkal me
Treatment failure attributable to the development of amphotericin B resistance has remained an uncommon problem. It has been documented in six patients, all of whom were being treated for candidosis: two cases of C. tropicalis infection (Drutz & Lehrer, 1978; Merz & Sandford, 1979), three cases of Candida lusitaniae infection (Pappagianis et al., 1979; Guinet et al., 1983; Mere, 1984), and one case of Candida guilliermondii infection (Dick et al., 1985). In most of these cases the organisms were recovered from compromised patients who had received amphotericin B for prolonged periods. The strains of C. tropicalis from the case of Drutz & Lehrer (1978) had MICs of the order of 100 to 500 mg/L as did the strain from the case of Merz & Sandford (1979). In both cases the strains were ergosterol deficient. It is unusual to isolate resistant strains from patients receiving prophylactic oral treatment with nystatin or amphotericin B (although it must be said that MIC testing is seldom done as a routine procedure). However, Safe et al. (1977) isolated one strain each of Candida krusei and C. tropicalis resistant to amphotericin B and nystatin from patients receiving prophylactic treatment with the latter drug. Further tests showed that both strains contained much reduced amounts of ergosterol. Clinical resistance to amphotericin B could be more common than has been suspected. Dick, Mere & Saral (1980) compared the MICs of Candida spp. from 70 neutropenic patients with the MICs of similar organisms recovered from other patients. None of the 625 strains from the control group were classified as 'resistant' to the drug (MIC ^ 2-0 mg/L). In contrast, 55 strains originating from six of the 70 neutropenic patients were resistant: 27 strains of C. albicans (from three patients), three strains of C. tropicalis (one patient), and 25 strains of C. glabrata (two patients). The resistant strains all contained reduced amounts of ergosterol.
Downloaded from http://jac.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on May 29, 2015
strains of C. albicans is that of Pierce et al. (1978). These authors found that the higher the drug concentration needed to inhibit growth, the greater the shift in lipid composition from ergosterol to 4- and 14-methylated sterols. It is clear that, although membrane sterols are involved in the disruptive interaction of amphotericin B with sensitive fungal cells, alterations in sterol content are insufficient to account for drug resistance in all strains of C. albicans and C. neoformans. Mutant strains of C. albicans, resistant to amphotericin B, have sometimes been found to possess elevated levels of membrane ergosterol (Hamilton-Miller, 1972). Moreover, resistant strains of C. neoformans with normal ergosterol levels and sensitive strains with reduced levels have both been observed (Kim et al., 1975). In these strains some other resistance mechanism must operate. Elevated catalase levels have been detected in several amphotericin B-resistant strains of C. albicans (Sokol-Anderson et al., 1988). The enhanced resistance that this confers against oxidation-dependent damage to the fungal cell might well be another important mechanism of amphotericin B resistance, but this has still to be confirmed. To gain access to the fungal cell membrane, amphotericin B must first be transported across the rigid cell wall. In C. albicans alterations in the /M,3 glucan content of the cell wall after cessation of growth prevent the drug from reaching the membrane and result in the cells becoming more resistant to amphotericin B (Gale, 1986). However, this 'phenotypic' resistance is lost as soon as growth is resumed. Its clinical significance is unclear.
32
D. W. Wtmock
Interactions between ampboteridn B and other antimicrobial drugs The first report of a favourable interaction between amphotericin B and another antifungal drug appeared in 1971. Medoff, Comfort & Kobayashi (1971) observed synergistic effects when combinations of amphotericin B and flucytosine (5-fluorocytosine) were tested against strains of C. albicans, C. tropicalis and C. neoformans in vitro. Synergism was defined as a four-fold or greater reduction in the MIC of flucytosine in the presence of amphotericin B at concentrations below its MIC for each test strain. Other conflicting reports followed these initial observations, perhaps because of the differing test conditions, strains and drug concentrations used (Beggs, Sarosi & Andrews, 1974a; Shadomy et al., 1975; Polak, 1978). Odds (1982) concluded that for most strains of C. albicans the drug interaction is, at best, additive.
Downloaded from http://jac.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on May 29, 2015
These results suggest that it might be useful to screen for amphotericin B resistance in certain groups of patients. The individuals from whom the resistant strains were isolated had all received parenteral treatment with amphotericin B and five had also received oral nystatin (Dick et al., 1980). All had earlier harboured sensitive strains of the same organism, all had been neutropenic for long periods and all had received treatment with cytotoxic drugs. Four patients developed deep-seated infections with the resistant strains, of which two were fatal. The findings of a second report support the suggestion that acquisition of resistance could be a potential problem in neutropenic patients undergoing amphotericin B treatment. Powderly et al. (1988) observed that the amphotericin B MICs of Candida spp. isolated from 26 transplant patients were higher than those of similar organisms obtained from non-compromised patients. Although 15 transplant patients had received empirical treatment with amphotericin B, the MICs of their strains were similar to those of strains from transplant patients who had not received such treatment. This suggests that use of the drug was not the sole factor contributing to the selection of less sensitive organisms in this patient group. The transplant patients had all received prophylactic oral treatment with clotrimazole and it is possible that use of this imidazole led to the development of amphotericin B resistance as a result of the selection of organisms with a reduced membrane ergosterol content (Sud & Feingold, 1983). Another potential explanation for the development of amphotericin B resistance in patients undergoing cytotoxic treatment is that such treatment leads to the generation of mutant strains. Treatment with amphotericin B then leads to the selection of mutants that are also drug resistant. Because cytotoxic treatment injures cells through oxidative mechanisms, its use could lead to the selection of fungal cells that are resistant to oxidation-dependent damage. Such cells might well be resistant to the lethal oxidation-dependent effects of amphotericin B (Sokol-Anderson et al., 1986a). Most of the amphotericin B resistant strains that have been recovered during treatment have so far belonged to species other than C. albicans, in particular C. lusitaniae and C. tropicalis. The potential for resistance also appears to be high in C. guilliermondii—which, being haploid (Magee & Magee, 1987), can be induced to produce resistant mutants more readily than C. albicans which is diploid—and in C. parapsilosis, which is inhibited from growth at similar concentrations of amphotericin B to other members of the genus, but which is much less susceptible to its lethal effects (Seidenfeld et al., 1983).
Ampboteridn B introduced
33
Downloaded from http://jac.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on May 29, 2015
Conflicting > results have also been obtained from tests in vitro for interactions between amphotericin B and fiucytosine against Aspergillus spp. Kitahara et al. (1976/>) detected synergism, but subsequent work has failed to confirm their findings (Lauer, Reller & Schroter, 1978; Hughes et al., 1984a). One explanation for the synergism detected with combinations of amphotericin B and fiucytosine is that the membrane-permeabilizing effects of low concentrations of the former drug facilitate the penetration of the latter to the cell interior, potentiating its deleterious effects on cell metabolism (Medoff et al., 1972). However, Beggs, Andrews & Sarosi (1977) observed that amphotericin B can inhibit the uptake of fiucytosine into C. albicans cells. Beggs & Sarosi (1982) suggested that synergism results from sequential, rather than combined action of the two drugs. In this hypothesis, amphotericin B acts first and alone until its gradual oxidation results in its depletion. At this point flucytosine acts on the surviving fungal cells. It is difficult to detect and assess drug interactions in vivo unless the effects are pronounced. Moreover, reports of favourable interactions are often based on too lenient interpretation of test results. That said, it has been claimed that combination treatment with amphotericin B and flucytosine is more effective than amphotericin B alone in animal models of aspergillosis, candidosis and cryptococcosis (Block & Bennett, 1973; Titsworth & Grunberg, 1973; Arroyo, Medoff & Kobayashi, 1977; Polak, Scholer & Wall, 1982). However, with the exception of the work of Bennett et al. (1979) demonstrating that the combination is superior to amphotericin B alone in the treatment of cryptococcal meningitis, there are no results from controlled clinical trials to support the use of the combination in other fungal infections. In spite of this, the combination has become a popular choice for the treatment of many forms of deepseated candidosis. In addition, there are a number of anecdotal reports of its use in patients with aspergillosis and mucormycosis. The antifungal azoles inhibit the 14-demethylation step in the biosynthesis of ergosterol (Vanden Bossche, 1985; Kerridge, 1986). The consequent depletion of ergosterol and accumulation of lanosterol and other 14-methylsterols leads to alterations in a number of membrane-associated functions. Because depletion of ergosterol should lead to a reduction in cell membrane sites for the binding of amphotericin B, it is reasonable to expect that prior treatment with an azole might antagonize the effects of amphotericin B (Sud & Feingold, 1983; Wilson & Peacock, 1985). Tests with combinations of amphotericin B and miconazole, ketoconazole, econazole or clotrimazole in vitro have given conflicting results with indications both of antagonism (Cosgrove, Beezer & Miles, 1978; Dupont & Drouhet, 1979) and synergism (Beggs, Sarosi & Steele, 1976; Odds, 1982) against Candida spp. and C. neoformans. It appears that the nature of the interaction detected depends on the test conditions employed, the order of drug addition and the length of incubation (Brajtburg et al., 1982; Smith, McFadden & Miller, 1983). Tests in animal models of candidosis have revealed both antagonistic and synergistic interactions between amphotericin B and the azoles (Polak et al., 1982). Tests in vitro with combinations of amphotericin B and ketoconazole against Aspergillus spp. have given differing results. Hughes et al. (1984a) observed no interactions in tests in which both drugs were added together. However, Schaffner & Frick (1985) showed that prior treatment with ketoconazole resulted in a rise in amphotericin B MFCs from 0-6 mg/L or less to greater than 2-5 mg/L. Moreover, prior treatment of infected animals with ketoconazole abolished the protective effect of subsequent
34
D. W. Waroock
References Arroyo, J., Medoff, G. & Kobayashi, G. S. (1977). Therapy of murine aspergillosis with amphotericin B in combination with rifampin or 5-fluorocytosine. Antimicrobial Agents and Chemotherapy 11, 21-5. Athar, M. A. & Winner, H. I. (1971). The development of resistance by Candida species to polyene antibiotics in vitro. Journal of Medical Microbiology 4, 505-17. Beggs, W. H., Andrews, F. A. & Sarosi, G. A. (1977). Evidence for sequential action of amphotericin B and 5-fluorocytosine on Candida albicans ATCC 11651. Research Communications in Chemical Pathology and Pharmacology 16, 557-60. Beggs, W. H. & Sarosi, G. A. (1982). Further evidence for sequential action of amphotericin B and 5-fluorocytosine against Candida albicans. Chemotherapy (Basel) 28, 341-4. Beggs, W. H., Sarosi, G. A. & Andrews, F. A. (1974a). Inhibition of Candida albicans by amphotericin B in combination with 5-fluorocytosine. Research Communications in Chemical Pathology and Pharmacology 8, 559-62. Beggs, W. H., Sarosi, G. A. & Andrews, F. A. (1974A). Synergistic action of amphotericin B and rifampin on Candida albicans. American Review of Respiratory Disease 110, 671-3. Beggs, W. H., Sarosi, G. A. & Steele, N. M. (1976). Inhibition of potentially pathogenic yeastlike fungi by clotrimazole in combination with 5-fluorocytosine or amphotericin B. Antimicrobial Agents and Chemotherapy 9, 863-5. Bennett, J. E. (1967). Susceptibility of Cryptococcus neoformans to amphotericin B. Antimicrobial Agents and Chemotherapy—1966, 405-10. Bennett, J. E., Dismukes, W. E., Duma, R. J., Medoff, G., Sande, M. A., Gallis, H. et al. (1979). A comparison of amphotericin B alone and combined with flucytosine in the treatment of cryptococcal meningitis. New England Journal of Medicine 301, 126-31. Block, E. R. & Bennett, J. E. (1973). The combined effect of 5-fluorocytosine and amphotericin B in the therapy of murine crYptococcosis. Proceedings of the Society for Experimental Biology and Medicine 142, 476-80.
Downloaded from http://jac.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on May 29, 2015
amphotericin B against murine aspergillus infection. Further work is needed to define the clinical importance of these findings. The membrane-damaging effects of low concentrations of amphotericin B alter the fungal cell membrane in a manner that renders it permeable to drugs that would not otherwise be taken up. This might explain the favourable interactions between amphotericin B and the antibacterial drug rifampicin that have been demonstrated in tests in vivo with strains of Aspergillus spp. (Kitahara et al., 19766; Hughes et al., 1984fl), Candida spp. (Beggs, Sarosi & Andrews, 19746; Edwards et al., 1980), C. immitis (Huppert et al., 1976), C. neoformans (Fujita & Edwards, 1981), H. capsulation (Kobayashi et al., 1974) and Rhizopus spp. (Christenson et al., 1987). These effects were detected at achievable concentrations for both drugs. Tests in animal models of infection have indicated that treatment with amphotericin B and rifampicin in combination has at least an additive effect on infections with A.fumigatus. B. derma t it idis and H. capsulatum (Kitahara, Kobayashi & Medoff, 1976
