William H. Beggs

Action of imidazole-containing antifungal drugs.

Abstract During the past few years several promising new antifungal drugs (e.g. miconazole and ketoconazole) have emerged from a large and diverse group of synthetic imidazole-containing compounds. Potentially, these agents could provide the first major break-through in the management of systematic mycoses in over 20 years. Although this review briefly traces the historical development of antifungal imidazoles and summarizes pertinent information regarding chemical characteristics, biological properties, and growth inhibitory activities, it is primarily concerned with experimental findings and current ideas with respect to mode of action. Physiological, biochemical, and cytological studies have established that the primary adverse effect of imidazoles on fungal cells is disorganization of the plasma membrane. Normal structural and permeability characteristics of the membrane are altered to the extent that the fungal cell cannot adequately govern cytoplasmic levels of essential ions and low molecular weight metabolites. This much seems clear, but the biochemical mechanisms involved in membrane disorganization have not been resolved. There are currently two major hypotheses, each of which is based on substantial experimental evidence. The first suggests that imidazoles interfere with one or more enzymatic steps in the biosynthesis of ergosterol, an integral sterol component of fungal cell membranes. In the second hypothesis, membrane disorganization is explained in purely physicochemical terms. It is postulated that imidazoles and unsaturated fatty acid components of the membrane undergo hydrophobic interactions that are the direct cause of structural alterations and loss of normal permeability controls.

Influence of Antioxidants on the Bioactivity of Amphotericin B

Four antioxidants, propyl gallate, butylated hydroxyanisole, butylated hydroxytoluene, and d-α-tocopherol acid succinate were found to stabilize amphotericin B and to prolong its antifungal activity against Candida albicans. Although each of the antioxidants was effective in this respect, propyl gallate and butylated hydroxyanisole were better than butylated hydroxytoluene and d-α-tocopherol acid succinate. None of the antioxidants alone adversely affected normal cell growth. It is suggested that amphotericin B instability is due, at least in part, to lability of the carbon-carbon double bonds of the polyene moiety toward autoxidation. By protecting the drug molecule with an antioxidant, it is possible to significantly lower the quantity of AB necessary to obtain particular antifungal effects.

Isoniazid uptake and growth inhibition of Mycobacterium tuberculosis in relation to time and concentration of pulsed drug exposures.

We have examined the relationships of drug concentration and exposure time to drug uptake and growth inhibition of tubercle bacilli following single pulsed isoniazid exposures. Isoniazid-sensitive strain H37Ra of Mycobacterium tuberculosis was grown in Sautons medium with aeration at 37C. Cells were suspended in fresh medium at 0·6 mg. dry wt./ml., isoniazid or 14C-isoniazid was added and incubation continued. Pulses were terminated by two methods. Samples were either diluted l:200 in drug-free medium for turbidimetric growth assay or filtered and cell-bound 14C. activity determined. When isoniazid was varied from 0-l to 0·5 ug./ml. and the period of exposure varied accordingly to give a constant time-concentration product (TCP) of exposure equal to 0·5 μg. hr., a transient growth inhibition resulted followed by rapid recovery. At six days pulsed cells were inhibited approximately 25% A more pronounced effect occurred at a TCP of l-0 μg. hr. and at six days inhibition was about 50%. Populations arising from treated cells retained isoniazid sensitivity. At TCPs of 0·5 and 1·0 μg. hr., 14C. activity corresponding to 20–26 and 37–47 nanograms isoniazid/mg. cells was bound respectively. Since drug carry-over became a problem in growth assays with isoniazid pulses in excess of 0·5 μg./ml., filtration was substituted for dilution to terminate exposures. At a TCP of 1·0 μg. hr. over a range of 0·25 to 4·0 μg./ml., growth inhibition was about 50%. Our data indicate that the degree or extent of inhibition following short pulsed isoniazid exposures is rather precisely governed by the time-concentration product of exposure and is closely correlated with the amount of drug bound by the cells.

Liquid chromatographic assay of ketoconazole.

A reverse-phase, high-pressure liquid chromatographic method for the rapid and quantitative determination of ketoconazole has been developed. Drug levels from 0.5 to 10 microgram/ml can be determined in either yeast nitrogen base medium or human serum by using an octadecylsilane column. A retention time of 4.9 +/- 0.1 min resulted when the drug was eluted from a column with 75% methanol-25% 0.02 M (pH 7.5) phosphate buffer at a flow rate of 2 ml/min. Optimum sensitivity was obtained at a wavelength of 231 nm.

Synergistic action of amphotericin B and antioxidants against certain opportunistic yeast pathogens.

Results of earlier turbidimetric growth experiments showed that certain antioxidants prolonged the antifungal activity of amphotericin B (AB) against Candida albicans, presumably by retarding autoxidative destruction of the drug. Viability studies were designed to examine this in more detail. Subinhibitory concentrations of either butylated hydroxyanisole, n-propyl gallate, or nordihydroguaiaretic acid in combination with fungistatic levels of AB exerted synergistic fungicidal activity against two strains of C. albicans and one of C. parapsilosis. Although synergism was not seen in tests with a strain of Torulopsis glabrata, antioxidants prolonged the inhibitory action of AB against this organism. On the basis of these findings and other considerations that are discussed, it is suggested that stabilization of AB and the ability to act synergistically with AB represent two distinctly different effects of the antioxidants.

Inhibition of Dihydrostreptomycin Binding to Mycobacterium smegmatis by Monovalent and Divalent Cation Salts

To better understand salt antagonism of dihydrostreptomycin (DSM) action on Mycobacterium smegmatis, the effects of monovalent and divalent cation salts on drug uptake were studied in relation to the lethal activity of DSM. In Sauton liquid medium NaCl, MgCl2, and SrCl2 inhibited the initial instantaneous binding of [3H]DSM to the organism and suppressed secondary uptake. These data correlated well with the capacity of each salt to prevent the lethal activity of DSM. It was concluded that monovalent and divalent cation salt antagonism of DSM action on M. smegmatis involves nonspecific interference with drug uptake.

Further evidence for sequential action of amphotericin B and 5-fluorocytosine against Candida albicans.

Experiments were designed to determine the nature of amphotericin B (AMB)-5-fluorocytosine (5-FC) synergism against strains of Candida albicans susceptible to either drug alone. AMB strongly inhibited cell uptake of 14C-labeled 5-FC during incubation in a synthetic liquid medium. After several hours, however, this inhibition was dramatically released. Onset of 14C-5-FC uptake correlated with the known kinetics of AMB decay. These findings support the concept of sequential drug action proposed earlier. AMB apparently acts first and essentially alone until it approaches depletion. Experiments with a 5-FC-resistant mutant indicated that AMB interferes with transport of 5-FC to the cell interior.

Inhibition of Potentially Pathogenic Yeastlike Fungi by Clotrimazole in Combination with 5-Fluorocytosine or Amphotericin B

Clotrimazole (CTM) has a doubtful future with respect to use in treatment of the systemic mycoses. To assess the potential of CTM in combined drug regimens, antifungal effects of CTM together with 5-fluorocytosine (5-FC) or amphotericin B (AMB) were tested in a synthetic liquid medium against Candida albicans, Candida tropicalis, and Torulopsis glabrata. Viable counts were monitored over a 48-h incubation period. Weak inhibitory concentrations of CTM were tested in combination with levels of 5-FC or AMB that alone produced transient antifungal effects followed by rapid recovery of proliferative capacity. Results were similar for each of the organisms studied. Between 24 and 48 h, when cultures containing 5-FC or AMB alone were in the recovery phase, CTM plus 5-FC and CTM plus AMB continued to markedly suppress cell multiplication. It would appear that weak inhibitory concentrations of CTM can act together with 5-FC or AMB to produce antifungal effects greater than that obtained with either of the latter two drugs alone.

Nonspecific Ionic Inhibition of Ethambutol Binding by Mycobacterium smegmatis

Magnesium sulfate and spermidine were tested for their effects on binding of 14C-ethambutol by Mycobacterium smegmatis. Concentrations were used that protected the organism from ethambutol inhibition. Sodium salts were examined as possible ethambutol antagonists to test the previously reported specificity of the divalent cation salt effect. Consistent with growth-protection experiments, 20 mM MgSO4 or 2.0 mM spermidine prevented and reversed 14C binding by cells shaken with 0.2 μg of 14C-ethambutol per ml of Sauton medium at 37 C. Sodium salts were not effective ethambutol antagonists when tested at 20 mM, but at concentrations equivalent in ionic strength (μ) to that provided by 20 mM MgSO4 they were effective. Thus, 20 mM MgSO4, 80 mM NaCl, or 27 mM Na2SO4 (μ = 0.08) all gave similar results in growth protection and binding experiments, suggesting that MgSO4 antagonism is a nonspecific ionic effect. Because spermidine (μ ≤ 0.012) antagonized ethambutol at an ionic strength substantially less than that required for the metal salts, its effect may hinge on structural similarity to ethambutol rather than its cationic character. Drug and polyamine may compete for one site or a heterogeneous group of binding sites involving adsorption, transport, and intracellular target reactions. Until we know at which of these levels spermidine antagonizes ethambutol binding, the relationship between polyamines and ethambutol action will remain obscure. However, these studies have weakened the earlier argument for a divalent cation-requiring system as a specific ethambutol target site.

Kinetics of amphotericin B decay in a liquid medium and characterization of the decay process

A turbidimetric microbiological assay procedure was used to study the kinetics of amphotericin B decay in a pH 7 liquid medium incubated at 37°C with aeration (shaking at 150 rpm). Disappearance of biologically active drug showed characteristics of a first-order reaction with a half-life of 4–5 h under these conditions. Decay was not as rapid at either 27°C or when the system was not aerated. Inactivation was slowed even further when both of these environmental conditions were used. The antioxidantn-propyl gallate preserved bioactivity of the drug during either 37°C-aerated or 27°C-nonaerated incubation. It is suggested that amphotericin B decay is primarily an autoxidative process in this system.