The repeat dose toxicity of various liposomal formulations containing amphotericin B has been determined in mice. In general, small liposomes (e.g. 100-150 nm) were found to be more toxic than their large counterparts (e.g. about 2000 nm). However, the repeat dose toxicity of small liposomes could be diminished substantially by the inclusion of sterol (i.e. ergosterol) into the liposomal membranes. Tissue accumulation studies of amphotericin B after repeat dosing may be a useful adjunct to formulation development.

The in vitro activities of 5-chloro-8-hydroxyquinoline (CHQ) against single strains of 12 different species of mycoplasma and the impacts ofrepeated exposure of these strains to CHQ on their susceptibility to this agent have been studied. On initial exposure, the minimal inhibitory concentrations for these strains ranged from 0.24 to 1.92 ,ug of CHQ per ml of test medium; activities remained unchanged during 10 serial transfers in CHQ-containing medium.Previous reports (1-5) have shown that 5-chloro-8-hydroxyquinoline (CHQ) has significant activity in vitro against a wide variety of bacteria, fungi, and protozoa. This report summarizes the results of in vitro studies on the activity of CHQ against both pathogenic and saprophytic species of mycoplasma and the impact of repeated exposure to this agent on such activity.The species and strains of mycoplasma used in this study and their origins are listed in Table 1. The organisms were grown and tested for their susceptibility to CHQ in modified Chanock medium (20 ml of inactivated horse serum, 10 ml of yeast extract, 1 ml of 10% glucose solution, 1 ml of 1% nicotinamide adenine dinucleotide solution, 1 ml of 10% arginine solution, 1 ml of 5% thallous acetate solution, 100,000 U of benzylpenicillin, 2 ml of 0.1% phenol red, 1.47 g of PPLO broth [Difco Laboratories], and 70 ml of distilled water).CHQ (Halquinol, E. R. Squibb & Sons Ltd.) was dissolved in dimethylformamide at a concentration of 8,000 jig/ml. Doubling dilutions were made in dimethylformamide, and 0.05 ml of each dilution was added to 6-ml portions of sterile medium in bijou bottles (0.25 ounce, ca. 7.5 ml). A 0.5-ml amount of a 24-h actively growing culture of mycoplasma was added to each bijou. The final concentrations of CHQ in the medium ranged from 61.5 to 0.12 ug/ml. Control cultures, with 0.05 ml of dimethylformamide, were included with each minimal inhibitory concentration (MIC) determination. The cultures were incubated at 37°C for 48 h, and the MIC was determined. Portions (0.5 ml) were

A simple, rapid (30 min) microbiological assay for nystatin is discussed. It is based on the efflux of Rb+ ions from nystatin treated yeast cells which had been grown in a medium enriched with this element. Results obtained with this method and the conventional agar diffusion method for nystatin in raw materials and finished products compare favourably both in accuracy and reproducibility. For simplicity and reproducibility, a cryogenically‐stored inoculum is advocated; its use gives a confidence interval (P= 0·05) on six results of < 5%.

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A bioassay method for the polyene antibiotics nystatin and amphotericin B is proposed based on the measurement of the efflux of rubidium ions from a rubidium-loaded yeast culture challenged with the antibiotics. For this purpose a major proportion of the intracellular K+ ions in a Saccharomyces cerevisiae culture has been substituted by Rb+ ions. The rubidium leakage is measured by atomic absorption spectrophotometry, and a straight-line, dose-response correlation has been obtained for both antibiotics.It has been recognized since the early 1960s that one of the principal effects of polyene antibiotic action on susceptible microorganisms is the leakage of vital cytoplasmic constituents from the cells (8-11). The work of Sutton et al. (11) and Marini et al. (10) highlighted the leakage of K+ ions and NH4+ ions particularly. Since these early papers, many authors have examined induced potassium efflux from yeast cells by polyenes (4,5,7,(12)(13)(14).From some of the literature referred to above a correlation between polyene concentration and potassium efflux can be identified, and the prognosis for a possible rapid bioassay of this class of antibiotics, by measuring ion release from cells, is good.Smithler To overcome this problem, an alternative ion, which could be introduced into the yeast cells without affecting their viability or membrane function but which had a very low level of natural occurrence, was sought.To reproduce as closely as possible the characteristics of K+ ion leakage across the nystatin-impaired yeast cell membrane, it is necessary to find an ion of like charge and ionic radius. K+ ions exist in solution in a hydrated form, and the hydrated ionic radii of K+ (0.232 nm) and Rb+ (0.228 nm) are very similar, as are the ionic mobilities of these two ions.Rubidium ions are known to be well tolerated by erythrocytes, and an examination of the literature revealed a number of papers reporting the uptake of Rb+ ion by the yeast S. cerevisiae (1-3). Rubidium can also be determined at low levels with precision by atomic absorption spectrophotometry. It was therefore decided to examine the feasibility of using Rb+ uptake and subsequent release from yeast cells by the action of the polyene antibiotics nystatin and amphotericin as a bioassay system. The work reported here clearly shows such a system to be feasible in that a linear dose-response relationship can be obtained. MATERIALS AND METHODSYeast culture. S. cerevisiae SQ.1600 (from the Squibb Culture Collection) was used throughout.