Anti-Candida activity and in vitro toxicity screening of antifungals complexed with ß-cyclodextrin.

The emergence of resistant fungal species and the toxicity of currently available antifungal drugs are relevant issues that require special consideration. Cyclodextrins inclusion complexes could optimize the antimicrobial activity of such drugs and create a controlled release system with few side effects. This study aimed to assess the in vitro toxicity and antifungal effectiveness of nystatin (Nys) and chlorhexidine (Chx) complexed or not with ß-cyclodextrin (ßCD). First, a drug toxicity screening was performed through the Artemia salina bioassay. Then, the minimum inhibitory concentrations (MICs) against Candida albicans were determined with the broth microdilution test. After MICs determination, the cytotoxicity of the drugs was evaluated through the methyl-thiazolyl-tetrazolium (MTT) and neutral red (NR) assays and through cell morphology analysis. The PROBIT analysis was used to determine the median lethal concentration (LC50), and the cell viability values were submitted to one-way analysis of variance(ANOVA)/Tukey (α = 0.05). Overall, the ßCD-complexed antifungals were less toxic against A. salina than their raw forms, suggesting that inclusion complexes can reduce the toxicity of drugs. The MICs obtained were as follows: Nys 0.5 mg/L; Nys:ßCD 4 mg/L; Chx 4 mg/L; and Chx:ßCD 8 mg/L. Chx showed significant cytotoxicity (MTT: 12.9 ± 9.6%; NR: 10.6 ± 12.5%) and promoted important morphological changes. Cells exposed to the other drugs showed viability above 70% with no cellular damage. These results suggest that antifungals complexed with ßCD might be a biocompatible option for the treatment of Candida-related infections.

Molecular Umbrellas Modulate the Selective Toxicity of Polyene Macrolide Antifungals.

Antifungal polyene macrolide antibiotics Amphotericin B (AmB) and Nystatin (NYS) were conjugated through the ω-amino acid linkers with diwalled "molecular umbrellas" composed of spermidine-linked deoxycholic or cholic acids. The presence of "umbrella" substituents modulated biological properties of the antibiotics, especially their selective toxicity. Some of the AmB-umbrella conjugates demonstrated antifungal in vitro activity comparable to that of the mother antibiotic but diminished mammalian toxicity, especially the hemolytic activity. In contrast, antifungal in vitro activity of NYS-umbrella conjugates was strongly reduced and all these conjugates demonstrated poorer than NYS selective toxicity. No correlation between the aggregation state and hemolytic activity of the novel conjugates was found.

Synthesis, antifungal activity of caffeic acid derivative esters, and their synergism with fluconazole and nystatin against Candida spp.

We tested the antifungal potential of caffeic acid and 8 of its derivative esters against Candidaalbicans ATCC 90028 and 9 clinical isolatesand carried out a synergism assay with fluconazole and nystatin. Propyl caffeate (C3) showed the best antifungal activity against the tested strains. When in combination, C3 markedly reduced the MIC of fluconazole and nystatin with synergistic effect up to 64-fold. Finally, C3 showed a high IC50 value and selective indexagainst oral keratinocytes, demonstrating low toxicity against this cell type and selectivity for yeast cells. Further research should confirm its antifungal potential for development of combined therapy to treat C. albicans infections.

The global effect of exposing bakers' yeast to 5-fluoruracil and nystatin; a view to Toxichip.

A genome-wide screen of a haploid deletion library of bakers' yeast (Saccharomyces cerevisiae) was conducted to document the phenotypic and transcriptional impact of exposure to each of the two pharmaceutical products 5-fluorouracil (an anti-tumor agent) and nystatin (an anti-fungal agent). The combined data set was handled by applying a systems biology perspective. A Gene Ontology analysis identified functional categories previously characterized as likely targets for both compounds. Induced transcription profiles were well correlated in yeast and human HepG2 cells. The identified molecular targets for both compounds were used to suggest a small set of human orthologues as appropriate for testing on human material. The yeast system developed here (denoted "Toxichip") has likely utility for identifying biomarkers relevant for health and environmental risk assessment applications required as part of the development process for novel pharmaceuticals.

Bioadhesive vaginal drug delivery of nystatin using a derivatized polymer: Development and characterization.

Increasing incidence of resistance to azole antifungals has highlighted the importance of the use of alternative therapeutic agents such as nystatin for the treatment of vulvovaginal candidiasis. The aim of the present study was to develop and characterize locally acting, film formulation for the treatment of candidiasis using a derivatized natural polymer. Derivatization of natural polymer was carried out in order to introduce anionic character to an otherwise neutral polymer, so as to enhance its interaction with vaginal mucous membrane along with inheriting the biocompatibility and nonirritant characteristics of its parent polymer. A carboxymethyl derivative of fenugreek gum (CMFG) was prepared, and characterized by DSC, FTIR and X-ray diffraction studies. The derivatized gum was found to possess bioadhesive and film forming properties. A 3(2) factorial design was employed to formulate vaginal films and a response surface methodological approach was used to study the effect of formulation variables on film properties. Films containing 5% w/v polymer and 2% v/v glycerol exhibited optimum properties in vitro. The optimized drug loaded formulation was able to release 100% drug over a period of 5h and followed Korsmeyer-Peppas kinetics. It was found to be non-irritant and nontoxic to vaginal mucosa and showed appropriate antifungal properties in vivo.

A novel in vitro assay for assessing efficacy and toxicity of antifungals using human leukaemic cells infected with Candida albicans.

AIMS: This study describes a novel in vitro assay that simultaneously determines antifungal efficiency and host cell toxicity using suspensions of human leukaemic cells (HL-60) infected with Candida albicans. METHODS AND RESULTS: The effect of Candida infection on host cell viability was evaluated by the microscopy of trypan blue-stained cells and lactate dehydrogenase (LDH) activity. The in vitro 'drug potency assay' utilized the Cell Counting Kit-8 and measured post-antifungal treatment viability of Candida-infected HL-60 cells and the ability of the antifungal treatment to prevent infection. LDH activity showed that 42% ± 4·0 and 85·3% ± 7·40 of HL-60 cells were killed following Candida infection at the multiplicity of infection (MOI) of 1 : 1 and 1 : 5, respectively. The antifungal nystatin (0·78-25 µmol l(-1) ) was found to inhibit C. albicans infection as seen by the significantly increased viability of HL-60 cells. Cytotoxicity of nystatin towards infected HL-60 cells was evident at higher concentrations and this was also confirmed by propidium iodide staining. CONCLUSIONS: An assay using undisturbed cell suspension conditions was successfully developed for assessing the selectivity of the antifungal therapy in the host-Candida environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay employing Candida infection of host cell suspensions represents a promising method for testing interactions of antifungal compounds with both fungal and host cells.

Novel Nystatin A1 derivatives exhibiting low host cell toxicity and antifungal activity in an in vitro model of oral candidosis.

Opportunistic oral infections caused by Candida albicans are frequent problems in immunocompromised patients. Management of such infections is limited due to the low number of antifungal drugs available, their relatively high toxicity and the emergence of antifungal resistance. Given these issues, our investigations have focused on novel derivatives of the antifungal antibiotic Nystatin A1, generated by modifications at the amino group of this molecule. The aims of this study were to evaluate the antifungal effectiveness and host cell toxicity of these new compounds using an in vitro model of oral candidosis based on a reconstituted human oral epithelium (RHOE). Initial studies employing broth microdilution, revealed that against planktonic C. albicans, Nystatin A1 had lower minimal inhibitory concentration than novel derivatives. However, Nystatin A1 was also markedly more toxic against human keratinocyte cells. Interestingly, using live/dead staining to assess C. albicans and tissue cell viability after RHOE infection, Nystatin A1 derivatives were more active against Candida with lower toxicity to epithelial cells than the parent drug. Lactate dehydrogenase activity released by the RHOE indicated a fourfold reduction in tissue damage when certain Nystatin derivatives were used compared with Nystatin A1. Furthermore, compared with Nystatin A1, colonisation of the oral epithelium by C. albicans was notably reduced by the new polyenes. In the absence of antifungal agents, confocal laser scanning microscopy showed that C. albicans extensively invaded the RHOE. However, the presence of the novel derivatives greatly reduced or totally prevented this fungal invasion.

Predictive genomic biomarkers for drug-induced nephrotoxicity in mice.

The present study aimed to establish candidate biomarker genes for the early detection of nephrotoxicity in mice, with a particular focus on nephrotoxicity caused by polyene macrolides. Comprehensive gene expression changes were evaluated using microarrays in a mouse model in which acute nephrotoxicity was induced by amphotericin B deoxycholate, trade name Fungizone. The upregulated genes identified through microarray analysis of kidney tissue of Fungizone-treated mice included several genes that have been reported as nephrotoxicity biomarkers in rats, and 14 genes were selected as candidate nephrotoxicity biomarkers. The usefulness of these genes as nephrotoxicity biomarkers in mice was evaluated further through expression profiling under several experimental conditions using real time RT-PCR. Expression of genes encoding kidney injury molecule 1, lipocalin 2, tissue inhibitor of metalloproteinase 1, and secreted phosphoprotein 1 was highly upregulated by Fungizone, nystatin, natamycin, amphotericin B methyl ester, and liposomal amphotericin B, and their area under the ROC curve values were more than 0.95. These genes were more sensitive at detecting nephrotoxicity than traditional clinical chemistry and histopathology parameters. This study provides novel evidence that these nephrotoxicity biomarker genes identified are translatable to mice, and that they are useful for early and sensitive detection of nephrotoxicity.

Is ototopical nystatin ototoxic? A chinchilla model.

In this prospective controlled animal study, the authors investigated the potential ototoxic effects of ototopical application of nystatin through a tympanostomy tube, using their established chinchilla animal model. Each of the 10 animals used had ventilation tubes inserted in both ears; 1 ear was randomly assigned to receive nystatin suspension, whereas the other ear did not receive any medication, serving as control. Distortion product otoacoustic emissions (DPOAEs) were measured in each animal before application of nystatin and at 45 and 60 days after application. Each cochlea was also processed for scanning electron microscopy (SEM) at the end of the experiment. There was no significant difference in the DPOAEs and SEM appearances of the experimental and control ears over the 60-day period of the experiment. The authors conclude that transtympanic nystatin did not produce any long-term ototoxic effects detectable by DPOAEs or SEM.

New nystatin-related antifungal polyene macrolides with altered polyol region generated via biosynthetic engineering of Streptomyces noursei.

Polyene macrolide antibiotics, including nystatin and amphotericin B, possess fungicidal activity and are being used as antifungal agents to treat both superficial and invasive fungal infections. Due to their toxicity, however, their clinical applications are relatively limited, and new-generation polyene macrolides with an improved therapeutic index are highly desirable. We subjected the polyol region of the heptaene nystatin analogue S44HP to biosynthetic engineering designed to remove and introduce hydroxyl groups in the C-9-C-10 region. This modification strategy involved inactivation of the P450 monooxygenase NysL and the dehydratase domain in module 15 (DH15) of the nystatin polyketide synthase. Subsequently, these modifications were combined with replacement of the exocyclic C-16 carboxyl with the methyl group through inactivation of the P450 monooxygenase NysN. Four new polyene macrolides with up to three chemical modifications were generated, produced at relatively high yields (up to 0.51 g/liter), purified, structurally characterized, and subjected to in vitro assays for antifungal and hemolytic activities. Introduction of a C-9 hydroxyl by DH15 inactivation also blocked NysL-catalyzed C-10 hydroxylation, and these modifications caused a drastic decrease in both antifungal and hemolytic activities of the resulting analogues. In contrast, single removal of the C-10 hydroxyl group by NysL inactivation had only a marginal effect on these activities. Results from the extended antifungal assays strongly suggested that the 9-hydroxy-10-deoxy S44HP analogues became fungistatic rather than fungicidal antibiotics.

Effects and mechanisms of action of polyene macrolide antibiotic nystatin on Babesia gibsoni in vitro.

Nystatin is a membrane-active polyene macrolide antibiotic and a channel-forming ionophore. Nystatin exhibits in vitro activity against Babesia gibsoni infecting normal canine erythrocytes containing low potassium (LK) and high sodium concentrations, i.e., LK erythrocytes. The calculated IC(50) value of nystatin against B. gibsoni infecting LK erythrocytes was 31.96 µg/ml. The anti-babesial activity of nystatin disappeared when B. gibsoni in LK erythrocytes were incubated in culture media containing high potassium concentrations (HK). Moreover, when the parasites were harbored in canine HK erythrocytes, which contained high potassium and low sodium concentrations as a result of high Na-K-ATPase activity, the in vitro anti-babesial activities of nystatin also disappeared, apparently due to protection by HK erythrocytes. This suggested that nystatin could show in vitro anti-babesial activity against B. gibsoni by its ionophorous activity, the same as other ionophores such as valinomycin. Subsequently, the effects of nystatin on the host cells were observed. Nystatin could not modify the intracellular concentrations of potassium, sodium, adenosine triphosphate, or glucose in either LK or HK erythrocytes, although it caused weak hemolysis in HK erythrocytes. In addition, nystatin did not affect the survival of canine peripheral polymorphonuclear leukocytes. In conclusion, nystatin destroyed B. gibsoni by ionophorous activity but did not affect either canine erythrocytes or leukocytes in vitro.

Improved antifungal polyene macrolides via engineering of the nystatin biosynthetic genes in Streptomyces noursei.

Seven polyene macrolides with alterations in the polyol region and exocyclic carboxy group were obtained via genetic engineering of the nystatin biosynthesis genes in Streptomyces noursei. In vitro analyses of the compounds for antifungal and hemolytic activities indicated that combinations of several mutations caused additive improvements in their activity-toxicity properties. The two best analogs selected on the basis of in vitro data were tested for acute toxicity and antifungal activity in a mouse model. Both analogs were shown to be effective against disseminated candidosis, while being considerably less toxic than amphotericin B. To our knowledge, this is the first report on polyene macrolides with improved in vivo pharmacological properties obtained by genetic engineering. These results indicate that the engineered nystatin analogs can be further developed into antifungal drugs for human use.

Maitotoxin induces two dose-dependent conductances in Xenopus oocytes. Comparison with nystatin effects as a pore inductor.

Maitotoxin (MTX)-induced conductances in Xenopus oocytes were thoroughly characterized using the two-electrode voltage clamp technique with a hyperpolarizing voltage protocol. MTX 5-100pM induced an inward current with maximal amplitude between 0.1 and 10microA. The kinetics of this current had rising and decaying phases, which were non-voltage dependent. Its reversal potential (Erev) was close to 0mV in high K+ or Na+ external solution, indicating the participation of non-selective cation channels (NSCC). A second conductance was developed at MTX doses higher than 200pM whose amplitude increased continuously. This current showed a large instantaneous component and a voltage-independent decay, as well as similar selectivity for Na+ and K+ ions (Erev approximately 0 mV). Moreover, the maximal current amplitude was about 34% bigger in high K+ than in high Na+. The MTX effect was reversible at all doses in pM range. All the properties found are similar to those of NSCC. The differences in the current kinetics suggest that the MTX-elicited currents reflect the activation of two sets of voltage-independent NSCC. As MTX has been proposed to act by forming pores directly into the plasma membrane, we compared its effects with those of nystatin, a well-known membrane pore inductor. We found strong differences between the effects of both substances suggesting different mechanisms for these drugs.

Liposomal polyene antibiotics.

Polyene antibiotics (i.e., amphotericin B and nystatin) have been incorporated into lipid-based delivery systems to decrease their toxicity and enhance their therapeutic index, the most common being liposomes. This chapter describes the protocols for preparing liposomal amphotericin B and determining the efficacy and toxicity of the formulations in animals. Furthermore, methods for determining the pharmacokinetics and drug distribution after administration of amphotericin B in lipid-based delivery systems are discussed. Procedures for comparing the toxicity of different amphotericin B formulations in cell culture studies are also elucidated.

[Comparative analysis of in vitro antifungal activity and in vivo acute toxicity of the nystatin analogue S44HP produced via genetic engineering].

New polyene macrolide S44HP was purified from the culture of recombinant Streptomyces noursei strain with engineered nystatin polyketide synthase. S44HP, nystatin (NYS), and amphotericin B (Amph-B) were tested against 19 clinical fungal isolates in agar diffusion assay, which demonstrated clear differences in antifungal activities of these antibiotics. Sodium deoxycholate suspensions of all three antibiotics were subjected to acute toxicity studies in vivo upon intravenous administration in mice. NYS exhibited the lowest acute toxicity in mice in these experiments, while both Amph-B and S44HP were shown to be 4 times more toxic as judged from the LD50 values. While the acute toxicity of S44HP was higher than that of Amph-B, the data analysis revealed a significantly increased LD10 to LD50 dose interval for S44HP compared to Amph-B. The data revealed structural features of polyene macrolides, which might have an impact on both the activity and toxicity profiles of these antibiotics. These results represent the first example of preclinical evaluation of an "engineered" polyene macrolide, and can be valuable for rational design of novel antifungal drugs with improved pharmacological properties.

Protection by tetracyclines against ion transport disruption caused by nystatin in human airway epithelial cells.

Polyene antifungal antibiotics like nystatin form monovalent cation pores on the plasma membrane that perturb the intracellular electrolyte milieu, resulting in cell damage. In the present study, we investigated the effects of tetracyclines (minocycline and tetracycline) on ion transporters disrupted by nystatin in cultured human airway Calu-3 cells. Apical application of nystatin (50 microM) on a monolayer of the cells stimulated Na(+)-K(+) pump activity as estimated by ouabain (1 mM)-sensitive short-circuit current (I(sc)). The nystatin-potentiated I(sc) was inhibited by minocycline (IC(50) = 25 microM) or tetracycline (IC(50) = 150 microM) applied only from the apical (nystatin-treated) side. Nystatin increased monolayer conductance that was reversed by the application of tetracyclines. In contrast, ouabain potentiated the nystatin-induced change in the conductance. Further, Na(+)-glucose transport affected by nystatin was also normalized by tetracyclines from the nystatin-treated side of the membrane. These data suggest that tetracyclines may lower the cell permeability potentiated by nystatin, protecting cells against damage.

Polyene antibiotics: relative degrees of in vitro cytotoxicity and potential effects on tubule phospholipid and ceramide content.

Polyene antibiotic administration is limited by dose-dependent nephrotoxicity. The latter is believed to be mediated by polyene anchoring to plasma membrane cholesterol, resulting in pore formation, abnormal ion/solute flux, adenosine triphosphate (ATP) declines, and, ultimately, a loss of tubule viability. The relative nephrotoxicity of these agents and their liposomal preparations has remained poorly defined. Thus, freshly isolated mouse proximal tubules or cultured human proximal tubule (HK-2) cells were exposed to either nystatin, amphotericin B, or three different polyene liposomal preparations (Nyotran, AmBisome, or Abelcet; 4 to 64 microg/mL). The impact of these agents on (1) plasma membrane injury (sodium-driven ATP consumption, assessed by ATP-adenosine diphosphate [ADP] ratios); (2) cellular susceptibility to superimposed injury (chemical hypoxia or ferrous ammonium sulfate-mediated oxidative stress; assessed by lactate dehydrogenase release); and (3) membrane cholesterol, phospholipid, and ceramide expression was assessed. Amphotericin B was more cytotoxic than nystatin (approximately 25% to 50% greater ATP-ADP ratio declines). Most of this toxicity could be eliminated by polyene liposomal formulation. Nevertheless, the liposomal polyenes still fully sensitized tubule cells to superimposed chemical hypoxic (antimycin/deoxyglucose), but not oxidant, attack. Nystatin and amphotericin B caused acute increments in tubule sphingomyelin-phosphatidylcholine ratios and ceramide content (indicating an impact on the plasma membrane extending beyond the classic view of pore formation with ion flux). In conclusion, (1) nystatin is seemingly less cytotoxic than amphotericin B (in contrast to the prevailing clinical view); (2) liposomal formulation markedly decreases this cytotoxicity; (3) despite this reduced toxicity, liposomal polyenes are still able to render tubule cells more vulnerable to selected forms of superimposed injury; and (4) acute alterations in plasma membrane phospholipid and ceramide expression are previously unrecognized consequences and potential mediators of polyene-mediated tubular cell attack.

Ototoxicity of common topical antimycotic preparations.

OBJECTIVE: To determine the ototoxic effects of five commonly used topical antimycotic agents-clotrimazole, miconazole, nystatin, tolnaftate, and gentian violet-in the guinea pig. DESIGN: A controlled animal study in which the ototoxicity of commonly used topical antifungal agents was investigated by measurement of hair cell loss. METHODS: Several readily available topical antimycotic preparations were instilled into the middle ears of female Hartley guinea pigs over a 1-week period. Two weeks after the last instillation, the animals were euthanized. An active control group was treated with neomycin to confirm the adequacy of the treatment in delivering a known ototoxin; an untreated control group defined the normal distribution of hair cells. The temporal bones were removed, and the cochleas were fixed and dissected. The basilar membranes were examined under the scanning electron microscope. A map of hair cell survival was made for each row in segments of each turn. RESULTS: The untreated control animals had no discernible hair cell loss in the two lower turns. In the apical turn and sometimes the third turn, loss of hair cells was a common finding, this is a known effect of aging in this species. The animals treated with neomycin had damage consistently in the basal turn, sometimes extending into the second turn, as well as the expected hair cell loss in the apical turn. Clotrimazole, miconazole, or tolnaftate did not cause any hair cell loss in the first two turns. Hair cell loss in the third and fourth turns was similar to that of the untreated control group. Likewise, nystatin exhibited no evidence of ototoxicity. Of note, however, the preparation used in this study left a persistent residue in the round window niche. Of the first four animals treated with gentian violet, three developed pronounced behavioral signs of vestibular damage, and three demonstrated extensive middle ear inflammation and extensive new bone growth. Hair cell counts were not attempted because the extreme bone growth interfered with successful perfusion and dissection. CONCLUSIONS: Extrapolating from guinea pigs to humans requires caution. However, it is likely that guinea pigs are, if anything, more susceptible to topical ototoxins than are humans. The specific antimycotics clotrimazole, miconazole, and tolnaftate appear to be safe. Gentian violet has the potential for severe damage. The persistent residue left by the nystatin preparation is cause for concern and is a reminder that both the active ingredient and vehicle must be considered in evaluation of safety.

The reproductive and developmental toxicity of the antifungal drug Nyotran (liposomal nystatin) in rats and rabbits.

Nyotran is a liposomally encapsulated i.v. formulation of the antifungal polyene nystatin. This drug was evaluated in a series of reproductive toxicity studies, according to the guidelines outlined by the International Conference on Harmonization (ICH). A fertility and early embryonic development study (SEG I) and a prenatal and postnatal development (SEG III) study were conducted in rats, and embryo-fetal development (SEG II) studies were conducted in rats and rabbits. Nyotran was administered iv in all studies. In SEG I and SEG III, rats were administered daily doses of 0.5, 1.5, or 3.0 mg/kg Nyotran. In both studies, parental mortality and toxicity in the 3.0 mg/kg dose group necessitated the lowering of the high dose to 2.0 mg/kg/day. Parental toxicity, in the form of decreased body weights, decreased food consumption, and piloerection were also observed at the 1.5 mg/kg/day dose level in the SEG I and SEG III studies. Despite the parentally toxic doses in the SEG I study, there was no effect of Nyotran on F0 male or female fertility or early embryonic development of F1 offspring. In the SEG III study, lactational body weights of the F1 generation were decreased at all Nyotran dose levels. There was no effect on pre-wean developmental landmarks, but post-wean development was affected by Nyotran administration at all dosage levels. Preputional separation was delayed in the 1.5 and 3.0/2.0 mg/kg/day F1 offspring, auditory startle function was decreased in F1 females at all dose levels, and motor activity was decreased in male F1 offspring at all dose levels. However, there were no treatment-related effects on the subsequent mating of the F1 generation and resulting F2 offspring. In SEG II studies, rats and rabbits were also administered 0.5, 1.5, or 3.0 mg/kg/day of Nyotran during gestation. The high dose in these SEG II studies was not lowered, as the maternal animals were able to tolerate the shorter duration of dosing. Maternal effects in rabbits were observed only in the high-dose group and were limited to decreased food consumption and decreased absolute and relative liver weight. Decreased food consumption in high-dose dams and clinical weight loss in some animals at the mid- and high-dose levels evidenced maternal toxicity in rats. Nyotran did not have any effect on Caesarian section parameters in either rats or rabbits and no effect on the incidence of fetal malformations in rabbits. A statistically significant increase in mild hydrocephaly, observed in 4 rat fetuses, was seen at the highest dose level of 3.0 mg/kg/day. The biological significance and relationship to Nyotran treatment of this finding is not clear. This finding may represent a change in the background incidence or a change in the pattern of responsiveness of this strain of rat fetus to the test chemical. Toxicokinetic data were also collected in the SEG II rabbit and rat studies for comparison to human exposures. In both species, systemic exposure to the nystatin at effective antifungal concentrations was demonstrated. The systemic exposures in rats and rabbits were, however, considerably less than have been reported in humans administered clinical doses of 2 or 4 mg/kg/day Nyotran. Thus, humans tolerate higher dosages and systemic exposures of Nyotran relative to rats and rabbits and there is no margin of safety in either dosage level or systemic exposure to drug. Given this lack of a margin of safety and the effects on postnatal development in F1 rats, caution should be exercised when using this drug in females of childbearing potential.