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Ann. Rev. Microbiol. 1977. 31:291-308 Copyright © 1977 by Annual Reviews Inc. All rights reserved

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ANTIFUNGAL AGENTS:

-:-1706

RECENT DEVELOPMENTS1 George S. Kobayashi and Gerald Medoff Department of Medicine, Washington University School of Medicine, St Louis, Missouri 63130

CONTENTS INTRODUCTION

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POLYENE ANTIBIOTICS

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Chemical and Physical Properties of the Polyene Antibiotics Mechanisms ofAction of the Polyene Antibiotics . . . . . In Vitro Antibiotic Effects of the Polyenes . . . . . . . ' In Vivo Effects of the Polyenes .. . .. . . .

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S-FC

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Clinical Use of 5-FC COMBINED THERAPY GRISEOFULVIN

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TOPICAL AGENTS Tolnajiate Haloprigin Clotrimazole Miconazole

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CONCLUDING REMARKS

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291 292 292 294 296 297 298 299 300 301 304 304 304 304 305 305

INTRODUCTION Unlike therapy for bacterial infections, there have been relatively few therapeutic agents developed for the treatment of infections caused by fungi. There are several reasons for this. (a) Fungal infections are much less common than bacterial infec­ tions, so the impetus for the development of agents has been small. (b) The most frequent fungal infections, the superficial dermatophytoses, are nonlife threatening and in most cases trivial, again decreasing the incentive for developing new agentS. (c) Fungi are eukaryotic, and it has been difficult to develop antifungal agents that 'The survey of the literature pertaining to this review was concluded in December 1976. 291

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have the specificity for fungal structures and macromolecular synthesis that antibac­ terial agents have for bacteria. The most important antifungal agents are those that affect cell membranes. The polyene macrolides and the more recently discovered imidizole derivatives are the two groups of antibiotics in this class. The other important antifungal agents are 5-fluorocytosine, which affects RNA synthesis, and griseofulvin, which interacts with microtubules and inhibits cell division in a variety of different types of cells. In this review, we summarize what is known about the most important antifungal agents and cover only those that have been useful in clinical infections. We do not intend to present a complete review of all of the properties of these agents because several recent articles have covered the chemical and biological properties of the polyene antibiotics (105), the biology of action of polyenes in natural and artificial membrane systems (78), the pharmacology of 5-fluorocytosine and its use with amphotericin B (93), and the absorption, distribution, metabolism, and excretion of griseofulvin (70). These should be referred to for information not covered in this article. POLYENE ANTIBIOTICS Among the long list of polyenes that have been isolated and described are some of the most effective antifungal agents known. Problems associated with solubility, stability, absorption, and toxicity, however, have made only a few of these agents therapeutically useful. Table 1 lists the polyene antibiotics that have been the most useful clinically, some of which will be covered in detail in this review.

Chemical and Physical Properties of the Polyene Antibiotics All of the polyene antibiotics analyzed to date have certain common structural features in addition to a conjugated double-bond system (Figure 1). They are all characterized by a macrolide ring of carbon atoms closed by the formation of an internal ester or lactone. The presence of the lactone confers a highly characteristic peak on the infrared spectra of these compounds. The ring sizes of the various polyenes vary from 12-14 up to 35-37 carbon atoms. The conjugated double-bond system is contained exclusively withi n the cyclic lactone. The analysis of the charac­ teristic ultraviolet (UV) spectra of the polyene antibiotics and also the X-ray crystalTable

1 Clinically useful polyene antibiotics Chemical

Name

Amphotericin B Nystatin

Pimaricin Candicidin

Producing organism

Streptomyces nodosus S. albulus or S. noursei S. natalensis S. griseus

composition

Molwt

924 926 666 1200

ANTIFUNGAL AGENTS OH

OH

OH

OH

0

OH

293

0

HOOC 0 CHa

Annu. Rev. Microbiol. 1977.31:291-308. Downloaded from www.annualreviews.org by Brigham Young University - Idaho on 04/29/13. For personal use only.

HO

:

~ NH2

H

0

/'V�A/'V/'VA" amphotericin B

CH3

0

OH

OH

OH

OH CHa

OH

HOOC 0 CH3 HO

:

"P NH 2

H

0

/ �/'V'V"V�/ nystatin

CH3

OH CH3

HO

pimaricin

Figure 1

Chemical structures of amphotericin B, nystatin, and pimaricin.

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lographic analysis of an N-iodoacetyl derivative of amphotericin B (32) indicate that all the double bonds are in the trans conformation. Another highly characteristic feature of the polyene antibiotics is the large num­ ber of hydroxyls present on the molecule. The number varies between 6-14, and they are usually distributed along the macrolide ring on alternate carbon atoms. The presence of such a large number of polar hydroxyl groups and the multiple hydro­ phobic double bonds on the opposite side of the macrolide ring confer upon the polyene antibiotics the additional characteristic chemical property of being am­ phapathic. It is likely that this amphapathic feature plays an important role in the mode of action of these substances as they interact in various biological systems. The most characteristic physical property of the polyene antibiotics is the UV absorption spectrum. The UV spectra of all the polyenes have a regular series of sharp peaks of absorption, which are separated by sharp troughs, all in the range of 400-280 nm. Oroshnik & Mebane (80) have given an extensive tabulation of the exact absorption maxima for many of the polyene antibiotics and have shown that the characteristic UV absorbing pattern is due to the conjugated double bonds. Careful analysis of the UV spectra indicates that there are several distinct classes of chromophores: tetraenes, pentaenes, hexaenes, and heptaenes. The molar extinc­ tion coefficients of the various absorption maxima range from 2-8 X 104, but it is not a simple matter to accurately measure the absorption of aqueous solutions of polyene antibiotics because there is a difference in the molar extinction coefficients when the antibiotics are dissolved in an organic or aqueous solvent. It is suspected that the differences are due to micelle or aggregate formation in water.

Mechanisms of Action of the Polyene Antibiotics In the early 1960s, several laboratories independently presented evidence that poly­ ene antibiotics could increase the cell membrane permeability of a number of organ­ isms, thereby promoting a leakage of important cellular constituents and ultimately lysis and death of the cell (55, 56). Many other studies have substantiated these findings and have reinforced the belief that the other metabolic effects of the pol­ yenes (inhibition of aerobic and anaerobic metabolism) were due to the leakage of vital cytoplasmic constituents resulting from the alteration of cell membrane perme­ ability rather than a direct inhibition of glycolysis or some other critical metabolic events. The antifungal effects of the polyene antibiotics are dependent upon a binding of the drugs to the cell membranes. Bacteria and protoplasts did not bind polyenes and were unaffected by them. These results led to the notion that the polyene worked by binding to membrane sterols, which are constituents of eukaryotic cells but which are not present in bacterial cell walls or membranes. Among the experiments sug­ gesting a relationship between sterols and the action of polyenes, two series have been most convincing. First, all organisms susceptible to polyenes contain sterols (e.g. yeasts, algae, protozoa, flatworms, and mammalian cells) and all resistant organisms do not (30, 41, 114). Second, sterols added to the medium could protect polyene-susceptible

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cells from these antibiotics (12, 36). This reversal of polyene effect was secondary to a physiological interaction between the polyene antibiotics and the added sterols, which prevented the antibiotic from interacting with the sterols of the organisms under study. In addition to the indirect evidence cited above for the interaction between sterols and polyene antibiotics, direct evidence based on spectrophotometry data is also available. We have already emphasized that polyene antibiotics have characteristic UV absorption spectra with large molar extinction coefficients. Lampen, Arnow & Safferman (67) reported that when sterols were added to aqueous solutions of polyene antibiotics the UV absorbance values decreased. This suggested that a direct interaction had occurred between the added sterol and the polyene. Several other groups have confirmed these findings (62, 76, 77, 94), and more recently, Schroeder, Holland & Bieber (95) have presented strong direct evidence for binding between sterols and polyenes by use of a fluorometric technique involving the measurement of partial quantum efficiencies. The nature of the membrane lesion resulting from the polyene-sterol interaction leading to the increase in permeability has been studied extensively by using natural and artificial membranes. Freeze-etch electron microscopy, on erythrocytes and Acholeplasma laidlawii treated with polyenes, have shown structural alterations such as pits, doughnut-shaped craters, and protrusions in the membranes, but there was no indication that these structural changes had resulted in holes or pores through the membrane. It has been assumed, but not proven, that these structural changes are responsible for the enhanced permeability of the membranes. Studies have also been carried out using several different model membranes, including liposomes, black film bilayers, and monolayer systems, and have essen­ tially confirmed the work in natural membranes. The effects of the polyenes were found to be dependent upon the presence of sterols in the membranes, and it was suggested that lipid rearrangement (58, 59) or fragmentation of the membrane caused by the presence or formation of polyene-sterol complexes was responsible for the molecular action of polyene antibiotics (22). Freeze-etch electron microscopy of Epidermophyton floccosum (79) showed that amphotericin B induced profound ultrastructural changes in this fungus, which consisted of aggregation of membrane-associated particles, 85A in size, and the formation of depressions or craters on the inner faces of the membranes. No pores or holes were observed, which is in agreement with the other studies using A. laidlawii and artificial membranes. The absence of pores or holes in the surface membranes after polyene treatment has supported the notion that the interaction of the polyene antibiotics with the membrane increases permeability by inducing dramatic changes in the physical properties of the membranes by the avid binding of the polyenes to the membrane sterols, whose presence stabilizes membrane func­ tion and affects membrane permeability. The binding of the polyenes to membrane sterols would then mediate a phase transition from an ordered state to a melted or random state, resulting in an increased permeability (77). Although there is no direct evidence for this sequence of events, there is strong circumstantial support (31, 65, 76).

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Work in our own laboratory on the nature of the membrane lesion induced by polyenes supports the idea that the changes produced are dependent upon the kind of membranes being treated and the type and concentration of polyene used. Yeast are more susceptible to amphotericin B than other polyene antibiotics because amphotericin B binds more avidly to ergosterol, the principal sterol in fungal membranes, than cholesterol, the main sterol in animal cell membranes ( 12). The increased susceptibility of animal cells to the other polyenes compared to amphoteri­ cin B is due to the increased binding of the former to cholesterol. Each cell type (animal cell or fungus) also varies in susceptibility to the polyenes. For example, 3T3 and L cells are much more susceptible to amphotericin B than are HeLa cells (12). Neither the absolute cellular content of free sterol nor the amount of polyene bound to cells correlates completely with this type of differential susceptibility. Rather, we have found that the cells more susceptible to the polyene effects have a lower cholesterol: phospholipid molar ratio than the more resistant cell lines. This indicates that the sterol content in relation to phospholipid levels in cell membranes determines polyene susceptibility and is in agreement with this suggestion first made by Kinsky, Luse & Van Deenen (58) and Van Zutphen, Van Deenen & Kinsky (109). Other factors may also play a role in the susceptibility of cells to polyenes. Hsu Chen & Feingold (50) have shown that the fatty acid composition of the phospholip­ ids in cells is a determinant of susceptibility to amphotericin B. Because of all of these variables, it is clear that the mechanism of selective effects of the polyenes is complex.

In Vitro Antibiotic Effects of the Polyenes The polyenes are inactive against bacteria. They exert various degrees of inhibition against many species of fungi such as yeasts (Candida, Cryptococcus), dimorphic fungi (e.g. Histoplasma, Blastomyces, and Coccidioides), dermatophytes (e.g. Tri­ chophyton, Microsporum, and Epidermophyton), and molds (e.g. Asperigillus and Penicillium ) In addition to antimycotic activities, many polyenes also are toxic to protozoa of medical importance, such as trichomonads, Entamoeba, Naeglaria, and trypano­ somes. Nystatin and amphotericin B are toxic to Leishmania donovanii. This effect has been shown to be a result of the loss of intracellular constituents because of its effects on membrane permeability and eventual cell lysis. Nystatin is rapidly bound to these cells and is preferentially bound to those fractions containing the highest sterol content. As with fungi, the binding is inhibited by adding digitonin or choles­ terol to the incubation medium. Seneca & Bergendahl (97) and Johnson, Miller & Brumbaugh (53) have found that polyenes are toxic to snails and planaria. As a general rule, the antifungal activity of the polyenes increases as the number of double bonds increases. This assertion is made despite the fact t;hat accurate figures to support this widely held opinion are difficult to come by because experi­ mentally determined minimum inhibitory concentrations (MIC) vary so much among different laboratories. Among the factors that may affect the MIC are inoculum size, temperature, and duration of incubation and medium composition .

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ANTIFUNGAL AGENTS

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(61). Therefore, only if strictly standardized conditions are adhered to can suscepti­ bility data from different centers be compared. Utahara et al (106) were among the first to show that tetraenes and a pentaene (eurocidin) had about one quarter of the activity, on a weight-for-weight basis, of a hexaene (mediocidin) and heptaenes against Candida albicans. Table 2 is based on MIC data obtained by Athar (3) for clinically isolated strains of C. albicans. The heptaenes are more than one order of magnitude more active against Candida than filipin and pimaricin, whereas nystatin is about twice as active as the latter two compounds. Because effective therapy for systemic fungal infection is essentially limited to the polyenes, development of resistance to these agents in fungi poses a major problem. Although fungi are intrinsically capable of giving rise to polyene-resistant variants, fortunately, the laboratory phenomenon of polyene resistance has not spread to clinical situations. The reason for this is not known, but it is probably related to the observed defectiveness of the resistant fungi. One explanation for this is that the membrane changes responsible for polyene resistance is detrimental to overall sur­ vival of these organisms in the natural environment.

In Vivo Effects of the Polyenes The only polyene antibiotic sufficiently nontoxic and tolerated parenterally in exper­ imental animals is amphotericin B. One cannot easily extrapolate from human dosages or in vitro studies so that the safest procedure is to be governed by a knowledge of its toxicity and measures of drug levels in serum. By using these parameters to determine drug dosage, the in vivo experiments have generally con­ firmed the in vitro effectiveness of amphotericin B against a variety of fungi at tolerable drug levels (61). In any discussion of the medical uses of the polyene antibiotics, the first point to be emphasized is that these are very toxic drugs. This toxicity is well described in the literature and is probably based on the membrane effects of the polyenes on host cells (57). Only a few of the polyenes have been developed for clinical use. The only one that is available for parenteral administration is amphotericin B. It is marketed as a bile-salt complex that forms a colloidal dispersion when hydrated. Established Table 2 Relative activities against C. albicans of five polyene anti­ biotics Median MIC

Antib ioti c -------

Nystatin ' Pimaricin Filipin Candicidin Amphotericin B

Jl.g/ml -_.

-

3 5 5 0.5 0.5

3.3 7.5 7.4 0.42 0.54

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protocols and therapeutic regimens for its use in a variety of different systemic fungal infections have been published (25). Nystatin is the most frequently used topical or oral polyene antibiotic. Originally called fungicidin, this antibiotic is the product of a soil survey conducted by mem­ bers of the staff of the New York State Department of Health, and the present name is a derivation of this laboratory. Nystatin is made by Streptomyces noursei. Its in vitro activity is highest against yeast-like fungi, but it is also effective against Histo­ plasma capsulatum. This polyene antibiotic is too toxic to be given parenterally, and there is very little or no absorption when given orally. For these reasons, its most important use is to treat topical or superficial infections by direct application. The local action of nystatin in controlling vulvovaginal candidiasis or Candida infec­ tions involving any part of the alimentary tract from the mouth to the anus is now so generally familiar that it does not require further discussion. Several other polyene antibiotics, notably pimaricin, hamycin, trichomycin, and candicidin, have been used to a limited extent, topically, orally, or by inhalation for the treatment of localized and systemic mycotic infections. A colloidal preparation of hamycin when given orally (8) was found to be effective against some systemic fungal infections in animals and humans. However, the absorption of this polyene from the gastrointestinal tract has not been consistent enough to depend on it as reliable systemic chemotherapeutic agent. Pimaricin, trichomycin, and candicidin have been used to treat local fungal infections of skin, eye, and vagina by topical application or injection into infected tissue. The fact that certain of these agents combine activity against yeasts and trichomonas makes them particularly attractive antibiotics for use in vaginitis, but it is difficult to judge if they offer any real advantages over amphotericin B or nystatin. Other uses of the polyenes are as food preservatives to prevent the growth of molds, especially on the surface of fruit, and in tissue culture media where am­ photericin B is widely used with penicillin and streptomycin to suppress the growth of microorganisms. Recently, Schaffner & Mechlinski (92) and Lawrence & Hoeprich (68) have shown that esterification of the free carboxyl group of amphotericin B or its N­ acylated derivatives caused no loss of biological activity. Furthermore, the hydro­ chlorides of the methyl esters of amphotericin B, nystatin, pimaricin, mediocidin, candicidin, and trichomycin were highly water soluble and retained the full activity of the respective parent compounds while being of greatly decreased toxicity. It is too early to know if these preparations of polyenes will offer a therapeutic advantage, but considering the detailed information about the structure of the polyenes, it is surprising that only a few such attempts at improving the drug by molecular manipulation have been successful. 5-FC 5-Fluorocytosine (5-FC) is a synthetic oral antimycotic agent that is effective in the treatment of some deep-seated fungal infections, in particular candidiasis, cryp­ tococcosis, and chromomycosis. It was first synthesized by Duschinsky, Pleven &

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Heidelberger (26), and unlike the other compounds of this series, including 5fluorouracil (5-FU), its riboside and deoxyriboside, as well as the riboside and de­ oxyriboside of 5-FC, 5-FC itself was found to lack significant cytostatic activity and toxicity in animals. In contrast to the other fluorinated pyrimidines, 5-FC exhibited practically no bacteriostatic activity in vitro but marked and rather selective antifun­ gal activity against Candida albieans in vitro and in vivo. These properties suggested the clinical usefulness of 5-FC as an antifungal antibiotic. As expected from its structure, and supported by the reversal of the in vitro inhibitory effects by cytosine, 5-FC has been considered an antagonist of cytosine. This direct function of 5-FC as an antimetabolite is probably of less importance, in regard to its antifungal properties, than a more indirect effect resulting from conver­ sion to the antimetabolite 5-FU by organisms possessing the necessary deaminase. The evidence for this is that the antifungal effects of 5-FC correlate directly with the amount of replacement of uracil by 5-FU in rR NA and tR NA of the sensitive organisms. Polak & Scholer (88), by using many isolates of C. albicans with various degress and models of 5-FC sensitivity and resistance, have worked out a scheme of the pathways of 5·FC and its metabolites, which are probably responsible for its antifungal properties (Figure 2). The incorporation rates found with the various sensitive or resistant clinical isolates after exposure to 2-14C·labeled 5·FC are particularly interesting because they strongly suppon; the aformentioned hypothe­ sis of the key role of the metabolite 5·FU in the action of the drug on R NA synthesis. It is assumed on the basis of good supporting evidence that R NA heavily substituted with 5·FU functions poorly, and abnormal proteins are synthesized, which lead to cell death. The inhibitory effect of 5·FU on mammalian cells and certain bacteria may also be related to a block of thymidylate synthetase, leading to inhibition of D NA synthesis. There is no strong evidence for the existence of this mechanism in 5·FC­ susceptible fungi, although Wagner & Shadomy (110) think this may be the mecha­ nism of inhibition in aspergilli. The pathway and mode of action of 5·FC proposed by Polak & Scholer (88) also explains the different mechanisms of resistance to the drug. Resistance may be produced by lack or deficiency of an enzyme at any step of the pathway, by a sur­ plus of de novo synthesis of normal compounds competing with the fluorinated antimetabolites, or, hypothetically, by any compensation for the abnormal R NA function.

Clinical Use of 5-FC 5-FC is effective in vitro against Candida. Cryptococcus. Torulopsis. agents of chromomycosis, and some strains of Aspergillus. It has no inhibitory effects against psrmBOS(J

deominos8

5-FC " .. ..5-FC" .... 5-FU .. 5-FUMP.. 5-FUDP " 5-FUTP"RNA Figure 2

Proposed metabolic pathway and mode of action of 5-FC. after Polak et al (87). 5·FUMP, 5-ftuorouracil monophosphate; 5-FUDP, 5-ftuorouracil diphosphate; 5-FUTP, 5fluorouracil triphosphate.

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the agents of other systemic mycosis such as Histoplasma capsulatum or Blas­ tomyces dermatitidis. The drug has been used successfully in the treatment of clinical infections by some of these fungi, but in general its performance has been disappointing. This is probably a result of its strictly fungistatic effects and the high level of resistance present initially on some fungi or developing with continued use of the drug. If a MIC of 16 JLg/ml is used to define a sensitive organism, resistance to 5-FC was initially present in 4-67% of Candida species or developed in 100% of Cryp­ tococcus neoformans after 7-10 days of therapy with the drug alone (102). The variability of some of these data may be due to the different media used in sensitivity testing and in the inoculum size. It has been shown that purines and pyrimidines present in most media greatly suppress the antifungal effects of 5-FC, and this has been one of the difficulties in interpreting in vivo sensitivity testing. Toxicity of 5-FC has not been an important clinical problem. Bone marrow suppression and occasional diarrhea have been reported. The former has been thought to be secondary to conversion of 5-FC to 5-FU in the gastrointestinal tract by bacteria, but this has only been shown in animals and there is no evidence that it occurs in man (87). COMBINED THERAPY It is probable that the lethal effects of polyene antibiotics are separable from their permeabilizing properties. For example, Hsu Chen & Feingold (51) have been able to generate fungal mutants that are resistant to the permeabilizing effects of am­ photericin B but not to its lethal effects. In addition, work done in our laboratory has shown that amphotericin B has a range of effects on the same cell types. We have d�termined that there are two types of binding of amphotericin B to fungi (12). One is energy dependent and reversible, and the other is irreversible and non-energy dependent. Only the latter is associated with lethality. We have also shown that very low concentrations of amphotericin B increase the permeability of eukaryotic cells to small molecules. Higher levels increase the size of the molecules transported, and finally, very high levels are lethal to the cell (11). These low concentrations of amphotericin B, which affect membrane permeability, are nonlethal, have com­ pletely reversible effects, and can be exploited to increase the therapeutic efficacy of antibiotics and antitumor agents, which normally penetrate poorly or not at all into eukaryotic cells (74). Some of these membrane effects on specific cells of the immune system are probably responsible for the adjuvant properties of amphotericin B,. which we have descilbed elsewhere (10). We have exploited the permeabilizing nonlethal effects of amphotericin B on cells to increase the therapeutic effectiveness of several second agents (2, 60, 63, 64, 66, 72, 73). That is, at concentrations well below its MIC, amphotericin B can facilitate the entry into fungi of antibiotics that do not ordinarily affect fungi, probably because the intact cells do not take them up. Thus far, we have shown that in combination with amphotericin B the antifungal effect of 5-FC is increased against Cryptococcus neoformans and various species of Candida (72); and the effects of

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rifampin are increased against Candida albicans, Saccharomyces cerevisiae, Aspergil­ lus sp., Histoplasma capsulatum, and Blastomyces dermatitidis (63, 64, 66, 73). We have concluded that the increased activity of the drug combinations was secondary to a potentiation of the effects of the second agents by amphotericin B, since in the presence of amphotericin B, there was an increased uptake of 14C-labeled 5-FC into the fungi, and furthermore, the specificity of the second agents was maintained in the killing process. For example, when rifampin was used in combination with amphotericin B, R NA synthesis was inhibited; and when tetracycline was used, protein synthesis was inhibited. The combinations were truly synergistic because all of these effects were more than the additive effects of the two agents used alone. These in vitro synergistic effects have been confirmed in vivo in animal models of infection (2, 60) and are presently undergoing clinical trials in patients (7). Thus far, early results of these trials are encouraging. GRISEOFULVIN Griseofulvin, 7-chloro-2',4,6-trimethoxy-6'-methylspiro-[benzofuran-2(3H), I' -(2) cyclohexene]-3,4'-dione, is a metabolic product of several species of Penicillium (14, 16, 52) and has been used for the past 18 years as the systemic drug of choice in the treatment of chronic dermatophyte (tinea, ringworm) infections. It was discov­ ered and its chemistry was partially characterized in 1939 (81), and in 1947, the chemical structure, C17H17CI06 (Figure 3), was completely elucidated. The com­ pound has UV absorption maxima at 324, 291, 252, and 236 nu (38--40), and it is possible to assay this compound spectrophotometrically in biological specimens (6, 29, 115). Griseofulvin has been effectively used in the treatment of dermatophyte infections of the scalp and hair, nails, glaborous skin, groin, and interdigital areas of the feet caused by Epidermophyton floccosum, and various species in the genera Micro­ sporum and Trichophyton. Hildick-Smith, Blank & Sarkany (46) have extensively described its pharmacological, chemical, and antimicrobial properties. It is ther­ mostable and unaltered by autoclaving at 15 psi for 15 min unless it is in solution, when it may lose potency on heating at autoclave temperatures. The usual oral dose in adults is 500 mg per day, but this will vary according to the severity of the infection and may require starting doses as high as 1.0 g a day. In children weighing 30 to 50 pounds, a dosage of 10 mglkg is usually adequate. Medication must be continued until the fungus is completely eradicated, as indicated by appropriate mycologic culture of specimens taken from the affected area. The duration of therapy generally depends upon the time required for normal replacement of the infected tissues and varies from 3 weeks of therapy in uncomplicated tinea infections to 12 months in the case of toenail infections (46). In recent years, a great deal of work has been done on the absorption, distribution, metabolism, and elimination of griseofulvin in man and animals. Griseofulvin ap­ pears in the stratum corneum within 4-8 hr after oral administration (99). The degree of absorption from the gastrointestinal tract varies and depends upon particle size of the drug, fat and lipid intake, dissolution rate in intestinal fluids, and dosage

302

KOBAYASHI & MEDOFF

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griseofulvin

V::

-C-CE$C-1 H2

C1

CI

tolnoftote

clotrimazole Figure 3

'

CI

holoprigin

miconazole

Chemical structures of griseofulvin, tolnaftate, haloprigin, c1otrimazole, and

miconazole.

regimen. In man, the major metabolites of griseofulvin are 6-desmethylgriseofulvin and 6-desmethylgriseofulvin glucuronide. The absorption and metabolism of griseo­ fulvin have been extensively reviewed by Lin & Symchowicz (70). The biological properties of griseofulvin were first described by Brian and his colleagues (13-15). It was termed "curling factor" because of its ability to induce stunted and aberrant growth of mycelium of Botrytis alii. Gentles (33) demonstrated in 1958 that experimental ringworm infection in the guinea pig could be effectively treated by oral administration of this drug. Therapeutic trials in humans followed with dramatic responses reported (9, 116). The MIC of griseofulvin against various dermatophytes ranges between 0.14-0.6 JLg/ml, but the antibiotic has little or no effect against yeasts and bacteria (91). Microscopy studies on susceptible fungi have shown that, at concentrations of 0. 1-0.2 JLg/m1, grossly distorted growth of hyphae results (14).

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ANTIFUNGAL AGENTS

303

The exact mechanism by which this antibiotic produces its effect against dermato­ phytes is unknown, but addition of griseofulvin to actively growing cultures of Microsporum gypseum or Trichophyton mentagrophytes temporarily halted protein and nucleic acid synthesis (28). El- Nakeeb & Lampen (27) demonstrated that the uptake of griseofulvin by the dermatophyte M gypseum involved a two-step process similar to the process seen in plants (20). The first is an immediate uptake of small amounts of the antibiotic from the medium and does not require energy. This probably represents a simple absorption of the antibiotic to the lipids of the fungus. The second phase of uptake is prolonged and requires an energy source. Although little is known about the antifungal mechanism of griseofulvin on dermatophytes, there is a growing fund of information on the effects of griseofulvin on microtubules of animal cells. In 1958 Paget & Walpole (82) described the striking arrest of mitosis in metaphase of bone marrow cells and intestinal epithelial cells caused by intravenous injection of griseofulvin into rats. They also reported that griseofulvin caused spindle disorientation and chromosome scattering and that the drug inhibited chromosome movement at anaphase. Although these antimitotic properties are similar to antitubulins, such as colchicine, podophilotoxin, isopropyl­ N-phenyl carbamate, and vinblastine sulfate (117), the molecular action of griseoful­ vin is thought to be different from the other compounds that bind to the receptors on tubulin and inactivate the free subunits (llS). The evidence for this is that griseofulvin does not interfere with tubulin in polymerization in vitro and that HeLa cells arrested in metaphase by griseofulvin show morphologically normal mi­ crotubules (37). It was proposed that the drug altered the function of intact mi­ crotubules in cells rather than causing them to depolymerize (37). There are data that show, however, that griseofulvin interferes with tubulin polymerization in vitro and that the observed effects of griseofulvin on microtubular assembly are dependent upon concentration of the antibiotic (113). At 10-5 M concentration of griseofulvin, 3T3 cells are arrested in metaphase and exhibit normal microtubules, but when the concentration of drug is increased to 5 X 10-5 M, the cytoplasmic microtubules are destroyed. In addition to its antimitotic properties and in vitro action on tubulin, griseofulvin shares other properties with those of colchicine and the vinca alkaloids (18). 1t has been used in the treatment of gout (101, 111) and has some anti-inflammatory activity (21). In in vitro studies with Boyden chambers (4, 5), clinically achievable concentrations of griseofulvin (0.1-1.0 p.g/ml) inhibited chemotaxis of human poly­ morphonuclear leukocytes. At least two possibilities have been considered to ac­ count for this inhibition. The first is that antitubulins (e.g. griseofulvin) inactive microtubules essential for the release of chemotatic substances (S3), and the alterna­ tive hypothesis states that cytoplasmic microtubules are essential in conduction of chemotactic message from cell membrane to locomotor machinery of the cell (5). Although much information has been gained about the effects of griseofulvin on microtubular structure and function in human cells, the mechanism of its action on dermatophytes remains to be elucidated. The recent isolation of tubulin from yeast (112) and filamentous fungi (100) may provide new insights into the understanding of the action of griseofulvin on fungi. Added impetus should be given to such studies ,

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in view of reports that griseofulvin specifically inhibits the mitotic spindle in various species of fungi (19, 41-43, 54).

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TOPICAL AGENTS Several topical antifungal agents are currently available for the therapeutic manage­ ment of superficial mycoses, i.e. pityriasis versicolor, and the dermatomycoses, i.e. ringworm. In general, there is a paucity of information available concerning the mechanism of action of these drugs. In this section, the available information on the chemistry and mode of action of each agent is discussed.

Tolnaftate Tolnaftate, m, N-dimethylthiocarbanilic acid 0-2 naphthyl ester, synthesized and developed by Noguchi et al (75), is a colorless, odorless compound with the chemical formula CI9H17NOS (Figure 3). The antibiotic is applied topically twice a day, as a 1 % solution or cream, to the affected area of skin. Tolnaftate is not very effective against dermatophyte infections of hair and nails. It is soluble in polyethylene glycol and chloroform, slightly soluble in ether and alcohol, and insoluble in water. Tolnaf­ tate is effective against· Microsporum gypseum. Microsporum canis. Microsporum audouinii, Trichophyton mentagrophytes, Trichophyton rubrum, Epidermophyton

and Pityrosporum orbiculare but not against Candida albicans, Aspergil­ or Penicillium notatum (90). Although the drug has been used successfully in the treatment of cutaneous fungal infections, little has been published on the mechanism of its activity.

floccocum,

lus !umigatus,

Haloprigin Another topically active antifungal agent is haloprigin, 3-iodo-2-propynyl-2,4,5trichlorophenyl ether, which was synthesized by Seki et al (96). It is used as a 1% solution or cream in the treatment of dermatophyte infections of the skin. It is a y-iodopropargyl aryl ether with the formula C9H4 Cl3IO (Figure 3). This synthetic compound is as active against dermatophytes as tolnaftate. Haloprigin, however, has a wider spectrum of action than tolnaftate because it is effective against species of Allescheria, Alternaria, Aspergillus. Monosporium, Nigrospora. and Penicillium (44). Little is known about the mechanism of action of haloprigin, but yeast cells of C. albicans treated with it showed an inhibition of respiration and disruption of cell membrane integrity (45).

Clotrimazole Clotrimazole, CnH17N2CI (Figure 3), [I-(O-chloro-a,a-diphenylbenzyl) imida­ zole], an imidazole derivative, has an extraordinarily wide range of activity against dermatophytes, yeasts, and filamentous and dimorphic fungi (84, 85). The drug has been shown to be fungistatic at 10 p.g/ml but fungicidal at higher concentrations (17, 98). As with many other antibiotics, the MIC values of c1otrimazole depend on the organism being tested, size of inoculum, length of incubation, temperature, and type of medium employed in the assay procedure (48, 86).

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305

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The drug is rapidly eliminated in the feces and urine of rats after oral or intrave­ nous administration (104), and there is minimal absorption of the drug or its metabolites through the skin after topical application. Prolonged oral administration produces hepatic and adrenal changes (104), resulting in increased liver weight and cellular hyperplasia. Gastrointestinal intolerance is reported to be frequent and so severe that perbral therapy is not usually possible. Clotrimazole as a 1 % solution or cream is used topically in dermatophyte infections. Suitable parenteral forms of clotrimazole for intravenous use have not as yet been developed (48).

Miconazole Miconazole, 1-[2,4,dichloro-,8-(2-4-dichlorobenzyloxy)-phenyl] imidazole nitrate (Figure 3), is a white crystalline imidazole derivative with the chemical structure ClsH14N20C14' It was synthesized and shown to have antimycotic activity in 1969 (34). Miconazole nitrate is used as a 2% cream applied topically in dermatophyte infections. It is very soluble in organic solvents but practically insoluble in water. In vitro, miconazole is active against yeasts, filamentous fungi, and gram-positive bacteria (16, 34, 35, 107). It has been used in the treatment of dermatophyte infections (18, 71), candidiasis (49), aspergillosis (49), candidal vulvovaginitis (89), and coccidioidomycosis (47) with moderate to good success. In a murine model of coccidioidomycosis, Levine et al (69) have reported the effectiveness of miconazole in preventing deaths of all the infected animals whereas 60-100% of the untreated animals succumbed. Intravenous, subcutaneous, or intramuscular injection of miconazole into mice resulted in the formation of hematomas, fibrotic lesions and bleeding, and ulcerations to occur at the sites of inoculation of the drug. It is thought that miconazole works by interacting with the cell membrane of fungi and causing leakage of cytoplasmic cations, amino acids, and proteins (103, 108). Transmission (23) and scanning (24) electron microscopy studies of yeast cells treated with miconazole support the contention that its antifungal effects are caused by a membrane effect. Similar experiments done in yeast cells of C. albicans treated with clotrimazole are not as convincing (1). The recent results of the use of miconazole in the treatment of murine coccidi­ oidomycosis (69) and the treatment of coccidioidomycosis and cryptococcal menin­ gitis in humans (47) are encouraging, but much more experience will be necessary before its usefulness in the treatment of systemic fungal infection is known. CONCLUDING REMARKS It is apparent that only a small number of antibiotics are currently available for the treatment of fungus infections. Only amphotericin B is sufficiently nontoxic so that it can be given parenterally. It is presently the most useful antifungal antibiotic for systemic fungal infections. 5·FC, given orally, is most effective when given with amphotericin but its use is limited to only selected fungal infections. Griseofulvin, the other oral drug, and several different topical agents are limited to the treatment of cutaneous fungus infections.

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With the exception of the polyenes and 5-FC, there is little known about the mechanism of action of the antifungal agents. The increasing occurrence of systemic fungal infections, particularly in the compromised host situation, should add im­ petus to the search for less toxic and more effective agents and perhaps to a greater understanding of the mechanisms of action of the ones presently available.

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ACKNOWLEDGMENTS

Studies from this laboratory were supported by United States Public Health Service grants AI062 13 and AI l 0622 and training grants AIO0459 and AI07015; by the John A. Hartford Foundation, Inc.; and by the Research Corporation (Brown­ Hazen Fund). Literature Cited

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