H.J. Scholer

Mode of Action of 5-Fluorocytosine and Mechanisms of Resistance

Mode of action of 5-fluorocytosine (5-FC) and mechanisms of resistance to the drug are discussed on the basis of experiments performed with Candida albicans ATCC 26790 and with 50 selected clinical isolates of C. albicans belonging to serological type A or B and representing various degrees and models of 5-FC resistance (sensitivity). Incorporation of 5-fluorouridylic acid into RNA appeared as a prerequisite to antifungal activity, although at a given incorporation rate, growth inhibition varied considerably from one strain to the other. The amino acid pool was unbalanced, and there was evidence for disturbance of protein synthesis. These dysfunctions of RNA probably account for growth inhibition and cell death, whereas up to the present, there was no proof of formation of 5-fluorodeoxyuridylic acid nor of subsequent inhibition of thymidylate synthetase. Incorporation of fluorinated pyrimidine into RNA was lower in normally sensitive type B strains than in normally sensitive ones of type A, whereas the frequency of 5-FC-resistant mutants was the same. The two serological types did not differ in the activity of cytosine permease nor in that of cytosine deaminase. Among 29 clinical isolates with 6-FC resistance (or impaired sensitivity) no instance of cytosine permease deficiency was found. Two isolates (belonging to the serological type A) were deficient in cytosine deaminase, whereas the majority was probably deficient in uridine monophosphate pyrophosphorylase or had a surplus of de novo synthesis of pyrimidines. Relative 5-FC resistance was more common than complete resistance.

Pharmacokinetic Studies on the Oral Antimycotic Agent 5-Fluorocytosine in Individuals with Normal and Impaired Kidney Function

Pharmacokinetic studies were performed on the systemic antimycotic agent 5-fluorocytosine (5-FC) by giving 2 g orally to 10 healthy adult volunteers and to 40 patients with renal impairment of various degree (serum creatinine concentration varying from 1.0 to 16.4 mg/l00 ml) including 5 nephrectomized or anuric patients. The microbiological method used for the measurement of the serum and urinary concentrations of the drug is described in detail. In 20 patients endogenous creatinine clearance was determined in addition to the serum creatinine concentration. The average biological half-life of 5-FC in serum was 2.89 h in the healthy individuals (range 2.36–3.99 h) but in the patients, it was significantly prolonged with increasing serum creatinine concentration and decreasing creatinine clearance. The average half-life in the five nephrectomized or anuric patients was 85.0 h (29.9–250 h). A linear correlation was found to exist between the elimination rate constant of the drug and creatinine clearance, and based on this, a 5-FC dosage schedule has been proposed for patients with impaired kidney function. 5-FC is eliminated almost exclusively by the kidneys but has no renal toxicity. Provided that dosage is adapted accordingly, the drug is appropriate for chemotherapeutic treatment of patients with renal impairment which is a relative contraindication to amphotericin B.

Metabolic Studies with 5-Fluorocytosine-6-14C in Mouse, Rat, Rabbit, Dog and Man

Metabolism of 5-fluorocytosine-6-14C (5-FC) was studied in mice, rats, rabbits and dogs after oral and subcutaneous, single and repeated administration. In the urines of all species, intact 5-FC accounted for more than 90% of the total radioactivity at any time of the various treatment schedules. The average proportion of the urinary metabolites was around 5% in dogs, 3% in rabbits, 2.5% in rats, and 2% in mice of the total radioactivity. At repeated dosage, there was an increase of metabolites in mice but a decrease in rats treated subcutaneously. Neither increase nor decrease was observed in rabbits (treated orally) and dogs. Two metabolites were identified, alpha-fluoro-beta-ureido-propionic acid (FUPA) and alpha-fluoro-beta-alanine, the latter occurring mainly after oral treatment. These compounds represent probably that part of 5-FC which was deaminated to 5-fluorouracil (5-FU) or directly to 5-fluorodihydrouracil. FUPA was the only metabolite found in the urines collected from 4 out of 5 human volunteers during the first 12 h after single oral administration of 3.5 g of the radiolabelled drug. Its maximum proportion was 1.1% of the total radioactivity. No metabolites were detected in the urine neither of the 5th volunteer nor in those of 3 mycosis patients who were given the radioactive dose after they had received regular chemotherapy with unlabelled 5-FC (150 mg/kg/day) for at least 2 weeks. The sensitivity threshold of the method was 0.1-0.4% of the total radioactivity. One of the patients had developed thrombocytopenia which was probably due to 5-FC chemotherapy. The symptoms of 5-FC intolerance were in most of the examined species similar to those observed with 5-FU [9]. However, no quantitative correlation between proportion of metabolites and 5-FC toxicity is apparent except that man is the species in which both metabolism and toxicity are the lowest. It has not been proved yet that 5-FC intolerance occurring in a small percentage of patients receiving 5-FC chemotherapy (mainly leukopenia, thrombocytopenia) results in fact from conversion to 5-FU.

Fungistatic Activity, Uptake and Incorporation of 5-FIuorocytosine in Candida albicans, as influenced by Pyrimidines and Purines

Uptake of the radiolabelled antimycotic 5-fluorocytosine (5-FC) by Candida albicans and distribution of radioactivity in the cell fractions, expandable pool, internal pool, and RNA,

Correlation of Susceptibility Test Results in vitro with Response in vivo: Ketoconazole Therapy in a Systemic Candidiasis Model

In a previous study with flucytosine (5-FC) susceptibility of 40 Candida albicans isolates in vitro proved statistically correlated with response in systemic murine candidiasis in vivo, although exceptions occurred with individual isolates. For the present analogous study with ketoconazole, 58 C. albicans isolates were used of which 38 were from the 5-FC study and 20 were added to equalize the numbers of serotype A (n = 30) and B (n = 28) and to make the range of susceptibility in vitro to ketoconazole continuous and wide. The widest range of ketoconazole susceptibility was noted for the minimal inhibitory concentrations on Kimmig and Casitone agars (0.015-256 micrograms/ml) and disk zone diameters on YNB agar (0-54 mm), whereas with disk tests on other media, the range of 50% inhibitory concentrations, relative inhibition factors and MICs on serum agar remained narrow and/or showed strong ties. The Spearmans rank correlation between the in vitro activities determined with the various parameters showed wide variation consistent with p values from less than 0.001 to greater than 0.05. The serotype B isolates generally were more susceptible than the A isolates (p less than 0.02 for the majority of parameters). Evaluation of response in vivo was hampered by the low activity of ketoconazole on the murine infection with any of the isolates, the range of the ED50s being only 10- greater than 100 mg/kg. The serotype B infections exhibited significantly better response (p less than 0.05) than the serotype A infections. The overall correlation (Spearmans rank) of the susceptibility test results in vitro with the response in vivo was poor (p less than 0.05 for almost all parameters) suggesting very limited if any precise predictive values of the susceptibility tests in vitro with ketoconazole against C. albicans. However, the narrow range of the ED50 suggests relatively little variation in the response of the different isolates in vivo and similarly small variation was also noted in some of the tests in vitro.

COMBINATION OF AMPHOTERICIN B AND 5-FLUOROCYTOSINE

Moderate synergism of fungistasis was demonstrated in all normally sensitive Cryptococcus neoformans and in the partially 5-fluorocytosine (5-FC)-resistant Candida albicans strains. Synergism of the fungicidal effect was found throughout. It was more pronounced in Sabouraud broth than in an antagonist-free medium. In all strains the appearance of 5-FC-resistant mutants was significantly reduced by subinhibitory concentrations of amphotericin B (amph. B).

Fungistatic Activity, Uptake and Incorporation of 5-Fluorocytosine in Candida albicans, as Influenced by Pyrimidines and Purines. I. Reversal Experiments

The fungistatic activity of fluorinated pyrimidines, particularly 5-fluorocytosine (5-FC), and the reversal power of bases and nucleosides of the pyrimidines and purine classes were studied, chiefly in Candida albicans. 5-FC was the most potent antifungal agent, followed by 5-fluorouridine. Cytosine, uridine and adenine were the strongest antagonists of 5-FC activity. A direct correlation, independent of the chemical nature of the antagonists, was found between radiolabelled 5-FC taken up in the yeast cells and inhibition of growth. Except for the anatagonistic effect of some purines, particularly adenine, both antifungal and reversal patterns were compatible with the suggested pathway and mechanism of action of 5-FC (formation of 5-fluorouridylic acid after intracellular deamination of the drug to 5-fluorouracil).

Stellung und Bedeutung der Mykosen unter den menschlichen Infektionskrankheiten

When considering the indigenous deep-seated mycoses, candidiasis, cryptococcosis, aspergillosis and mucormycosis, as being ‘modern’, one should keep in mind that most of them were f