Vol. 52, No. 2 Printed in U.S.A.

T H E AMERICAN JOURNAL OF CLINICAL PATHOLOGY

Copyright © 1969 by The Williams & Wilkins Co.

AMPHOTERICIN B AND OTHER POLYENIC ANTIFUNGAL ANTIBIOTICS J. OLIVER LAMPEN, P H . D . Institute of Microbiology, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08903 ABSTRACT

The polyenic antifungals are a large group of antibiotics which first attracted attention because they were highly effective in inhibiting the growth of a wide variety of fungi, but they were inactive against bacteria. They are relatively toxic when administered parenterally. There is usually little, if any, adsorption from the gastrointestinal tract; thus, they can be given orally for yeast and fungal infections of the intestinal tract, and they are also of value as topical agents for treatment of moniliasis, especially vaginal infections. A few can be used systemically: for example, amphotericin B for the treatment of acute histoplasmosis and blastomycosis.26 For general information, the reviews by Kinsky,11 Lampen,14 and Waksman and Lechevalier28 can be consulted. The general characteristics of the polyenic antifungals are summarized in Table 1. They are active against fungi, algae, protozoa, and mammalian cells, but not against bacReceived March 24, 1969.

teria (with a few exceptions). High level resistance is rare either in the clinic or in the laboratory. The polyenes are fungicidal to resting cells; under appropriate conditions they inhibit respiration and glycolysis and prevent various energy-requiring reactions, but do not inhibit the isolated enzymes. Sensitive organisms bind polyenes; insensitive ones do not.15 Finally, protoplasts have the same sensitivity (or lack of it) as do the intact organisms.24 Thus, the polyenes do not affect fungi by inhibiting wall formation with subsequent lysis of the cells, nor are the bacteria potentially sensitive but shielded from the antibiotic by the cell wall. The structure of two typical polyenes is illustrated in Figure 1. Both filipin and nystatin have the characteristic conjugated double bond system, but the macrolide ring of the large group (nystatin) usually contains 37 to 38 atoms, whereas the ring of the small group (filipin) generally has 27 to 28. The structure of amphotericin B has not been completely defined, but it is known to have seven conjugated double bonds and is rela138

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Lampen, J. Oliver: Amphotericin B and other polyenic antifungal antibiotics. Am. J. Clin. Path., 52: 13S-146, 1969. The polyenic antifungal antibiotics are a large group of related macrolides whose ring contains a rigid planar lipophilic portion and a flexible hydrophilic portion. These inhibit growth of fungi (but not of bacteria) and have significant value in treatment of intestinal or topical monilial infections. Amphotericin B is used systemically for the treatment of acute histoplasmosis and blastomycosis. After the initial demonstration of therapeutic utility, attention became focused on the in vitro biologic action of the polyenes. The critical step was identified as formation of a complex with the membrane sterol of sensitive organisms. The consequences of this binding are being examined using natural and artificial membranes. The surprising feature that has emerged is the wide variation in ultimate physiologic effects produced by the individual polyenes. Investigation of this problem should provide important information on membrane function as well as on the nature of the specific membrane complexes. Finally, and of great interest at the moment, physiologic and pharmacologic effects on sterol and steroid metabolism in animals have been observed. These changes are consistent with available information on the mechanism of the microbial action of the polyenes. The possible practical application of these discoveries is of great interest.

Aug. 1969

POLYENE ANTIFUNGAL ANTIBIOTICS

TABLE 1 PROPERTIES OF POLYENIC ANTIFUNGALS

1. Active against fungi, algae, protozoa, mammalian cells; not against bacteria 2. Fungicidal to resting cells 3. Eesistance rare 4. Inhibit respiration and glycolysis 5. Bound by sensitive, not insensitive cells 6. Yeasts or fungi: protoplasts sensitive Bacteria: protoplasts insensitive

NATURE OF BINDING SITE

Since the ability of a cell to bind polyenes is probably the critical factor in determining

its sensitivity, the nature of this binding site was examined by a number of workers and eventually identified as the sterol of the cell membrane. 10 ' 16 First, only sensitive organisms contain sterols. The bacteria generally do not, and they are insensitive. The mycoplasma can, however, be grown so as to contain membrane sterol; their growth is now sensitive to polyenes. Second, one can demonstrate sterol-polyene complexes in vitro, and there is at least a rough relationship between the tendency of various polyenes to complex with sterol and their binding by sensitive cells. Third, polyenes bind to the plasma membrane of the protoplast in an amount roughly proportional to the sterol content. Digitonin, which complexes tenaciously with sterols, will release the polyene from protoplast membranes (osmotic lysis does not). Finally, membranes that have been extracted with solvents to remove sterol do not bind polyenes. If the membrane residue is now incubated with sterol, its ability to bind polyenes can be

OH OH F1LIPIN (C 3 5 H 5 8 0 M )

NYSTATIN (C 48 . 47 H 7S . 75 0, e N)

FIG. 1. Structure of a small (filipin) and a large (nystatin) polyene. The portion of the nystatin molecule enclosed in brackets is not entirely characterized. The arrangement shown illustrates the relative size of the filipin and nystatin rings.

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tively similar to nystatin. Individual polyenes may have a number of ring substituents, such as amino sugars, carboxyl groups, and aliphatic side chains, but no one of these groups appears to be essential. One may suggest that the active structure is the macrolide ring itself, containing both a rigid planar lipophilic portion and a flexible hydrophilic hydroxylated portion.

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LAMPEN

At high polyene levels, the internal enzymes become freely accessible to their substrates, yet many enzymes are still retained within the cell. Polyenes can be divided into two relatively distinct groups by determining the effectiveness of a supplement of K + and NH4+ salts in preventing or nullifying the inhibition of glycolysis caused by the polyene. This criterion is a measure of the degree of damage to the membrane, since the K + + NH4+ supplement will prevent the inhibition if leakage has been restricted to monovalent cations. The polyenes with which no reversal could be demonstrated were all members of the "small" group (Fig. 1). With the "large" polyenes, inhibition of glycolysis (produced by K + leakage) could readily be reversed, although often not at high polyene levels. It should be noted that the small polyenes are generally less active on a molar or weight ACTION OF POLYENES ON WASHED CELLS basis than are the large ones in inhibiting the The maimer in which one polyene, nysta- growth or glycolysis of fungi and particularly tin, kills a susceptible cell is illustrated of the yeasts. Thus the distinction between schematically in Figure 2. In the first step, the two polyene groups does not lie in their the polyene combines with the membrane relative antibiotic effectiveness but appears sterol to yield a complex which (in ways to to reflect a difference in the type or degree of be discussed subsequently) alters membrane damage done to the cell membrane. permeability. One of the systems most sensiAlthough two major types of polyenes tive to this membrane reorganization is that based on ring size can be defined, it is for the transport of monovalent cations. If probably best to consider that these antithe external pH is less than 5.5 (as com- biotics show a graded series of actions; filipin monly used for in vitro studies with fungi), and N-acetylcandidin (or N-succinyl perithere is a K+