The Reproductive and Developmental Toxicity of the Antifungal Drug Nyotran® (Liposomal Nystatin) in Rats and Rabbits

Jeffrey L. Larson*,1, Thomas L. Wallace*, Rochelle W. Tyl{dagger}, Melissa C. Marr{dagger}, Christina B. Myers{dagger} and Paul A. Cossum*

* Aronex Pharmaceuticals, Inc., 8707 Technology Forest Place, The Woodlands, Texas 77381; and {dagger} Reproductive and Developmental Toxicology Laboratory, Center for Life Sciences and Toxicology, Research Triangle Institute, Research Triangle Park, North Carolina 27709

Received May 17, 1999; accepted September 28, 1999


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nyotran is a liposomally encapsulated iv 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.

Key Words: Nyotran; nystatin; antifungal; reproductive toxicity; developmental toxicity.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polyene antifungal antibiotics, derived from fermentation by Streptomyces, are among the drugs used to combat fungal infections. Nystatin is one of the compounds in this class and has been used for the treatment of cutaneous, vaginal, and oral fungal infections since the 1950s (Pace and Schantz, 1956Go; Physicians Desk Reference, 1998Go; Stark, 1967Go). Topical and oral administration are not associated with significant toxicities since the compound is not absorbed through the skin or from the gastrointestinal tract (Newcomer et al., 1955Go; Physicians Desk Reference, 1998Go). Early attempts to administer nystatin parenterally in the clinic resulted in sclerosing of the veins and initial injections were accompanied by severe shaking, chills, fever, and malaise; these reactions were severe enough to preclude its use (Newcomer et al., 1955Go). With administration of nystatin limited to non-toxic routes, the toxicity of nystatin in animal models given the compound systemically has not been evaluated. To circumvent the problems associated with systemic administration of free nystatin, Aronex Pharmaceuticals has developed Nyotran, a liposomal formulation of nystatin.

Nystatin is closely related structurally to amphotericin B, another of the polyene antifungal drugs. Amphotericin B is currently the gold standard for iv treatment of invasive fungal infections, with deoxycholate, colloidal, and liposomal formulations available (Hughes et al., 1997Go; Walsh and Lee, 1993Go). The toxicity profile of amphotericin B has been fully described in laboratory animals and man. Acute reactions of amphotericin B in man include fever, shaking chills, nausea, vomiting, and headache (Butler, 1966Go; Edwards et al., 1978Go). Nephrotoxicity, due primarily to renal tubule necrosis, is the predominant and most prominent feature of amphotericin B therapy, and can be dose-limiting in both man and laboratory animals (Butler, 1966Go; Joly et al, 1989Go; Longuet et al., 1991Go; Medoff et al., 1983Go;). This kidney damage is observed with both acute and repeated doses of amphotericin B. In addition, hepatotoxicity has been seen in rats (Dayan and Working, 1994Go; Lee et al., 1994Go; Proffitt et al., 1991Go) and dogs (Fielding et al., 1991Go). Moderate hepatocellular necrosis and transitional-cell hyperplasia of the kidneys, ureters, and urinary bladder were observed in rats given liposomal amphotericin B by the iv route for 30 days (Boswell et al., 1998Go). Results from nonclinical studies with Nyotran indicate that the kidney was the target organ in rats, rabbits, and dogs, consistent with renal injury induced by amphotericin, although hepatocellular necrosis was not seen in rats or dogs administered Nyotran (manuscript in preparation).

Development of Nyotran for iv administration provides an additional tool with which the clinician would be able treat systemic fungal infections. Numerous studies demonstrate that while the fungicidal activities of nystatin and amphotericin B overlap, they are not identical. For example, Candida albicans strains exist that are markedly more sensitive to nystatin than to amphotericin B (Broughton et al., 1991Go; Hebeka and Solotorovsky, 1965Go). Nystatin was found to be effective against Geotrichum, Torulopsis, Candida krusei, and Beauvaria, whereas amphotericin B had no activity against these fungal organisms (Stern et al., 1988Go). Candida albicans from patients with mycotic vaginitis had approximately equal sensitivity to nystatin and amphotericin B, but Torulopsis glabrata obtained from these patients was much more sensitive to nystatin compared to amphotericin B (Guaschino et al., 1986Go). These results support the clinical development of Nyotran to complement current antifungal therapy.

As part of the nonclinical regulatory submission for Nyotran, the recommended ICH battery of reproductive toxicity bioassays was conducted in rats and rabbits (ICH 1994ICH 1996) and the results provided in this report. For comparative purposes, the Fungizone® formulation of amphotericin B increased the number of stillborn fetuses when administered to pregnant rats and rabbits; however, no teratogenic effects were observed (Physicians Desk Reference, 1998Go). There were no effects of liposomal formulations of amphotericin B (Ablecet®, Amphotec®) on fertility parameters or developmental toxicity in either rats or rabbits (Physicians Desk Reference, 1998Go).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals and husbandry.
All animal dosing and toxicology were conducted at Research Triangle Institute (Research Triangle Park, NC) under contract to Aronex Pharmaceuticals, Inc. (The Woodlands, TX). Rats were Sprague-Dawley (Crl:CD® BR) obtained from Charles River (Raleigh, NC). At study initiation, rats were approximately 8, 9, and 11 weeks of age for the SEG I, SEG II, and SEG III studies, respectively. Following an acclimation period, the rats were individually housed (except for cohabitation) in solid bottom polycarbonate cages with stainless steel wire lids and Sani-Chip® cage litter (SEG I and III studies; P. J. Murphy Forest Products Corp., Montville, NJ) or Ab-Sorb-Dri® cage litter (SEG II study; Laboratory Products, Garfield, NJ). With the exception of minor variations, animal rooms were maintained at 68–76°F and 35–70% humidity with a 12-h light/dark cycle. Food (#5002 Purina Certified Rodent Chow, PMI, St. Louis, MO) and tap water were available ad libitum throughout the study. For mating, individual females were placed in the home cages of singly-housed males. On the following morning and each morning thereafter, the females were examined for the presence of vaginal sperm or a vaginal copulation plug. The day on which the sperm or copulation plug was found was designated as gestational day (gd) 0.

Timed-mated New Zealand White (NZW) rabbits were supplied by Covance (Denver, PA), were approximately 6 months of age at study initiation, and weighed 3.3–4.4 kg. Rabbits were individually housed in stainless steel cages with wire-mesh flooring. Food (#5322 Purina Certified Rabbit Chow®) was rationed at 65 g for the first 24 h for those females at gd 1, at 125 g for those females at gd 2, and available ad libitum for all females from gd 3 to study termination. This gradual feeding to move the animals to ad libitum was done to prevent the animals from overeating, with possible development of mucoid enteropathy, after having been on food restriction during travel. Tap water was available ad libitum. Animal rooms were maintained at 64–71°F and 44–64% humidity with a 12-h light/dark cycle.

All animals were checked daily for clinical signs, mortality and evidence of abortion. Body weights and food consumption were measured at predetermined intervals throughout the course of the studies.

Test article.
Nyotran is a sterile, lyophilized powder containing nystatin and lipids at a drug:lipid ratio of 1:10. Dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG) at a ratio of 7:3, respectively, made up the liposomes. Nyotran (Lot No. 503–33–008) was shipped to RTI in clear bottles, each containing 50 mg of the active drug nystatin (CAS No. 1400–61–9). Dosing solutions were prepared by reconstitution with 50-ml saline to give a final nystatin concentration of 1 mg/ml. Different dose levels were obtained by altering the administered dose volume. Doses were administered by bolus injections over 5–15 s.

Fertility and early embryonic development study in rats (ICH 4.1.1, SEG I).
Nyotran was administered iv to male and female rats at 0.5, 1.5, or 3.0 mg/kg/day into the lateral tail vein, as a bolus injection. Control group animals in this study, as in the other studies, received a volume of 0.9% saline equal to the dose volume administered to the Nyotran high-dose group; in this study the dose volume was 3.0 ml/kg. There were 25 animals/sex/dosage group. Male rats (F0) were dosed daily for 4 consecutive weeks and females (F0) for 2 consecutive weeks prior to breeding. Due to parental mortality at 3.0 mg/kg/day, the high group dosage was lowered to 2.0 mg/kg/day, beginning on study days 9 and 23, for females and males, respectively. Animals were examined daily and clinical signs were recorded. Body weight and food consumption data were collected throughout the study for both males and females. For a 3-week mating period, animals were randomly mated on the basis of 1 male to 1 female, within treatment groups, to produce the F1 generation. Treatment for both sexes continued throughout mating until the F0 females reached gestation day (gd) 6. F0 males were sacrificed within 24 h after the last dose. The testes and epididymides were collected at necropsy. Sperm number was evaluated immediately at necropsy, and epididymal sperm concentration (the number of sperm per g cauda epididymis) was determined using fixed sperm. F0 females were terminated on gd 15 and necropsied with evaluation of uterine contents. The ovarian corpora lutea were counted and the uterine contents recorded. No histopathological assessment was performed on the reproductive organs of either sex.

Pre- and postnatal study in rats (ICH 4.1.2, SEG III).
Timed-mated female rats (F0) were divided into 4 groups of 25 to receive Nyotran at 0.5, 1.5, or 3.0 mg/kg/day or the saline control vehicle. The study began with 25 females per group and mated 1 male to 1 female, to yield at least 20 pregnant females/group at or near term. Treatment was administered via bolus iv injections into the lateral tail vein on gd 6 through postnatal day (pnd) 20, a duration of approximately 37 days. Maternal deaths in the 3.0 mg/kg/day dose group reduced the number of surviving pregnant rats to 18 and necessitated the lowering of the daily dose to 2.0 mg/kg, beginning on gd 19–22. F0 females were allowed to raise their offspring (F1) until weaning on pnd 21, at which time F0 dams were necropsied. Clinical signs, body weight, and food consumption were collected for F0 dams throughout the duration of the study. At necropsy, the thoracic and abdominal organs were examined grossly; there were no macroscopic findings and no tissues were therefore saved.

F1 pups were counted, weighed, sexed, and examined externally as soon as possible on the day of birth. On pnd 4, the size of each litter was adjusted by eliminating extra pups randomly to give 4 males and 4 females per litter. The decision to cull was made to avoid confounding of the study data by different litter sizes between and within treatment groups. Litters with 8 or fewer pups were not culled. Pups were observed daily and body weight was collected thoughout the duration of the study. During the pre-weaning period (up to pnd 21), pups were observed for developmental landmarks: age at acquisition of pinna detachment (pnd 1–4), incisor eruption (pnd 8–13), and eye opening (pnd 11–16). Pups were observed daily and the number of pups achieving each landmark was recorded until all pups of the same sex in a litter had the response.

At weaning on pnd 21, at least 1 male and 1 female (whenever possible) from each F1 litter, for a total of 20/sex/dose group, was randomly selected to become parents of the next generation (F2). Because of the excess mortality in the high dose group, 5 litters had 2 males and 2 females selected. F1 offspring not selected were grossly examined for external abnormalities, and euthanized without further examination. The randomly selected 20/sex/group F1 pups were held on the study, without dosing, for a minimum of 49 days until all pups were at least 70 days old. During this post-weaning period, F1 pups were weighed weekly. The following assessments were also performed: auditory function (startle reflex and habituation) in pups 23–32 days old, acquisition of vaginal patency beginning at 22 days of age, acquisition of preputial separation beginning at 35 days of age, motor activity, assessed in residential cages, in pups 34–35 days of age, and learning and memory, using a water-filled Morris maze in pups 41–50 days of age (described in Morris, 1981Go). For the last 12 days of the post-wean holding period, all F1 females were evaluated daily for estrous cyclicity.

F1 animals were mated (1 male to 1 female, with brother-sister mating avoided) within groups for 14 days. F1 dams were weighed during pregnancy and allowed to deliver their F2 litters. All F2 rats were counted, sexed, weighed and examined grossly as soon as possible on the day of birth, and again at study termination on pnd 4. In addition, all F1 males and females were necropsied on pnd 4 and thoracic and abdominal cavities examined; there were no gross lesions and no organs or tissues were therefore saved.

Developmental toxicity study in rats (ICH 4.1.3, SEG II).
Timed-pregnant rats, 25 sperm-positive females per group, were administered Nyotran at dose levels of 0.5, 1.5, or 3.0 mg/kg/day. Control group rats received 3.0 ml/kg 0.9% saline. Bolus iv injections were administered daily into the lateral tail vein on gd 6 through gd 15. Clinical signs, body weight, and food consumption were assessed throughout the course of the study. Also included in this study was an analysis of toxicokinetics (TK), with 20 of the 25 rats/sex/group randomly assigned to 1 of 4 bleeding schedules, and with samples collected on the last day of dosing (gd 15). Thus, 5 rats/sex/group were bled at pre-dose and at-8-h post-dosing, 5/sex/group were bled at 0.25 and 4 h post-dose, 5/sex/group at 0.5 h and 8 h post-dose, and 5/sex/group at 1 and 24 h post-dosing.

On gd 20, all dams were necropsied and evaluated for body, liver and gravid uterine weights. Ovarian corpora lutea were counted and the status of uterine implantation sites was recorded. Fetuses were dissected from the uterus, counted, weighed and examined for external abnormalities. Approximately one-half of the live fetuses in each litter were examined for visceral malformations and variations (Stuckhardt and Poppe, 1984Go). These fetuses were decapitated and the heads fixed in Bouin's solution; serial free-hand sections of the heads were examined for soft tissue craniofacial malformations and variations. All fetuses in each litter were eviscerated, fixed in ethanol, and stained with alizarin red S/alcian blue (Marr et al., 1988Go). Intact fetuses (not decapitated) in each litter were examined for skeletal malformations and variations.

Developmental toxicity study in rabbits (ICH 4.1.3, SEG II).
Presumed-pregnant New Zealand White rabbits, 20 females per group, were administered Nyotran at dose levels of 0.5, 1.5, or 3.0 mg/kg/day. Control group rabbits received 3.0 ml/kg 0.9% saline. Doses were administered by bolus iv injection into the marginal ear vein on gd 6 through 18. Clinical signs, body weight, and food consumption were assessed throughout the course of the study. Also included was an analysis of TK parameters using 5 does per group, bled on the last day of dosing at 0, 0.25, 0.5, 1, 2, 4, 8 and 24 h post-dosing.

At scheduled sacrifice on gd 30, does were necropsied and body, liver, and gravid uterine weights collected. Ovarian corpora lutea were counted and the status of the implantation sites determined. Fetuses were dissected from the uterus, counted, weighed and examined for external abnormalities. All live fetuses were sexed and examined for visceral malformations and variations. Approximately one-half of the live fetuses per litter were decapitated and the Bouin's fixed heads were serially sectioned for examination of soft tissue craniofacial malformations and variations. All fetuses in each litter were examined for skeletal malformations and variations after staining with alizarin red S/alcian blue.

Toxicokinetic analyses.
Sample preparation consisted of an extraction of nystatin from blood with methanol and subsequent analysis with an HPLC system. The method was specific for the nystatin A1 isomer. The samples were extracted by adding 2.5 ml of methanol (HPLC-grade) to every 1.0 ml blood sample. The mixture was then vortexed for 3 h at room temperature and then centrifuged to obtain a supernatant. An aliquot (400–500 µl) of the supernatant was then filtered through a 0.22-micron filter (Durapore Centrifugal Filter, Millipore Corp.) to provide a 100 µl sample for subsequent injection onto the HPLC system. A reverse-phase column (Waters µBondapak C18, 125 Å, 10 µm, 3.9 x 300 mm) was equilibrated and run with 10 mM monobasic sodium phosphate, 1 mM EDTA, 30% methanol and 30% acetonitrile, pH 6.0. A column heater set to 30°C was employed to minimize the retention time shifts of nystatin A1. The flow rate was 1.8 ml/minute and the injection volume of the sample was 100 µl. The total run time was 20 min. Nystatin eluted with an approximate retention time of 5.5 min ± 1.5 min. The use of a photo diode array detector afforded positive identification of nystatin spectra in the pharmacokinetic samples as compared to a library spectra of the standard. Pharmacokinetic parameters were determined using non-compartmental modeling with the WinNonlin (Scientific Consulting, Lexington, KY) computer program. For rats, the mean of each blood concentration time point was used as input into the model since blood samples were collected from different rats at different time points. For rabbits, the blood concentration from each individual rabbit was used as input into the model. The following pharmacokinetic parameters were calculated: peak blood concentration (Cmax), areas under the mean blood concentration-time curve calculated to infinity (AUC), half-life (T1/2), clearance (CL), and volume of distribution at steady state (Vd).

Statistical analyses.
Quantitative continuous data were compared by the use of Bartlett's test for homogeneity of variances. If Bartlett's test indicated lack of homogeneity of variances, then nonparametric statistical tests were employed for the continuous variables (Winer, 1962Go). If Bartlett's test indicated homogeneous variances, then parametric statistical tests were employed for the continuous variable. Appropriate General Linear Models (GLM) procedures (SAS Institute Inc., 1989) for ANOVA were used. Prior to GLM analysis, an arcsine-square root transformation was performed on all litter-derived percentage data (Snedecor and Cochran, 1967Go) to allow use of parametric methods. For these litter-derived percentage data, the ANOVA was weighted according to litter size. GLM analysis was used to determine whether significant dosage effects had occurred for selected measures (ANOVA). When a significant (p < 0.05) main effect for dosage occurred, Dunnett's Multiple Comparison Test (Dunnett, 1964Go) was used to compare each treatment group to the vehicle control group for that measure. A one-tailed test was used for all pair-wise comparisons to the vehicle control group, except that a two-tailed test was used for maternal body and organ weight parameters, maternal feed consumption, fetal body weight, and percent males per litter. Nonparameteric tests included the Kruskal-Wallis Test to determine if significant differences were present among the groups, followed by the Mann-Whitney U test for pair-wise comparisons to the vehicle control group, if the Kruskal-Wallis test was significant (Siegel, 1956Go). Jonckheere's test for k independent samples (Jonckheere, 1954Go) was used to identify dose-response trends for nonparametric continuous data. Nominal scale measures were analyzed by the Chi-square test for independence for differences among treatment groups (Snedecor and Cochran, 1967Go), and by the Cochran-Armitage Test for Linear Trend on Proportions (Armitage, 1955Go; Cochran, 1954Go). When Chi-square revealed significant differences (p < 0.05) among groups, then a two-tailed Fisher's Exact Probability Test, with appropriate adjustments for multiple comparisons, was used for pairwise comparisons between treated and control groups. For developmental landmarks in SEG III, group means were compared to the control by Mann-Whitney U test (Siegel, 1956Go). Values from functional assessment, including behavioral tests, were compared to control values by ANOVA, followed by individual pairwise comparisons (Dunnett, 1964Go).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fertility and Early Embryonic Development Study in Rats (ICH 4.1.1; Segment I)
The study was initiated in 25 rats/sex/group at Nyotran dosage levels of 0, 0.5, 1.5, or 3.0 mg/kg/day. The summary of observed toxicities is provided in Table 1Go. After 22 days of dosing for males and 8 days of dosing of females, 1 female exhibited seizures and died, and 1 additional female had difficulty breathing and lay in a prone position. One male had also died after dosing on day 12. This mortality led to the decision to lower the daily dose in the high-dose group to 2.0 mg/kg and, accordingly, the volume in the control-dose group to 2.0 ml/kg beginning on day 23 of male dosing and day 9 of female dosing. In addition to these 2 deaths in the high-dose group, deaths were also observed in 1 control group, an F0 male, and 1 F0 female in the 1.5-mg/kg/day dose group.


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TABLE 1 Summary of Toxicity in a SEG I Fertility and Early Embryonic Development Study in Rats
 
Additional clinical signs observed included piloerection in 5/25 males at 1.5 mg/kg/day and 13/25 males at 3.0/2.0 mg/kg/day. Rapid respiration, prone positioning, and lethargy were observed in 3/25 males at 3.0/2.0 mg/kg/day. In females given 3.0/2.0 mg/kg/day, piloerection was seen in 3/25 rats. Significant decreases in group-mean body weight attributed to treatment were observed during the first week of treatment and continued for the duration of dosing for the 1.5-mg/kg/day males (92–96% of control) and 3.0/2.0 mg/kg/day males (92–94% of control). Decrements in food consumption were observed along with these decreased body weights. The group-mean body weight change of females in the 3.0/2.0-mg/kg/day group was also significantly decreased (64% of control mean). A gross necropsy of all F0 animals revealed no gross findings, either in the animals that died prematurely or in those that survived to the scheduled end of study.

There was no evidence of treatment-related effects on F0 male reproduction. There were no differences among groups for male mating or fertility indices and no differences in epididymal sperm number or motility. Similarly, there were no treatment effects on F0 female reproductive parameters. There were no differences among groups for female mating or fertility indices. Embryonic development of the F1 generation was not affected by treatment with Nyotran. There were no treatment effects on the number of corpora lutea or implantation sites per dam, the number or percent of resorptions per litter, the number of litters with resorptions or the percent litters with resorptions, the number or percent of non-live implants per litter, the number or percent of litters with non-live implants, the number of live implants per litter, or the percent preimplantation or postimplantation loss per litter.

Pre- and Postnatal Development Study in Rats (ICH 4.1.2; Segment III)
A summary of the toxicities observed in this study is presented in Table 2Go. Maternal toxicity in the F0 generation occurred at the 1.5 and 3.0 mg/kg dose levels. Seven females in the 3.0 mg/kg/day dose group died during the period corresponding to gd 18–21. Hence, the dose was decreased for this group to 2.0 mg/kg/day at gd 19–22. At this lowered dose of 2.0 mg/kg/day, an additional maternal death was observed during lactation and the F1 litter was euthanized. Two additional F1 litters in the 2.0 mg/kg/day dose group were also moribund, necessitating the euthanasia of 2 dams. Two females at the 1.5 mg/kg/day were observed to be moribund during lactation and were euthanized along with their litters. Maternal gestational body weights were approximately 95 and 90% of control means for the mid- and high-dose groups, respectively. Piloerection was observed during the course of the study in 13/25 F0 rats at 1.5 mg/kg/day and in all rats at the 3.0 mg/kg/day dosage group. In addition, 3 F0 rats in the high dosage group exhibited ataxia, labored respiration, seizures, and/or prostration. Treatment-related gross necropsy findings were present only in the 7 high-dose females found dead during the gestational period; hemorrhagic lungs in 6/7 females and pale livers in 4/7 females.


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TABLE 2 Summary of Toxicity in a SEG III Pre- and Postnatal Development Study in Rats
 
There were no effects of Nyotran treatment on any F0 maternal reproductive, gestational, or lactational indices. This conclusion includes no differences among groups for mating, fertility, or gestational indices; for gestational length; for post-implantational loss; or for total, live, or dead pups per litter on pnd 0, nor for stillbirth or live-birth indices or for survival indices throughout lactation. The average number of F1 pups per litter was significantly decreased only at the top dose and only on pnd 4 pre-cull. Pup body weight per litter was significantly reduced at 0.5 mg/kg/day only on pnd 7 and 1.5 mg/kg/day on pnd 7 and 21 (about 95 and 88% of the control mean weight, respectively, on pnd 7). The pup weight per litter at 3.0/2.0 mg/kg was decreased on pnd 7 (89% of control). The absence of milk bands (stomach filled with milk visualized through the abdominal wall) with an increased incidence at 1.5 and 3.0/2.0mg/kg/day was the only clinical observation of F1 pups during lactation. There were no effects of treatment on pre-wean developmental landmarks, including acquisition of pinna detachment, incisor eruption, eye opening or vaginal patency. There were no treatment-related findings for pups that died or were euthanized moribund on pnd 0–20.

Treatment-related effects on post-wean development were observed at all dosage levels. Acquisition of preputial separation in F1 males was significantly delayed at the 1.5 and 3.0/2.0 mg/kg/day dose levels; although the delay was less than 2 days and there were no consequences in terms of mating of these animals. The auditory startle response was affected in females at all Nyotran dosage levels, with a decreased force, but not duration, of jump. No differences in auditory startle response were observed in males. Male pups exhibited a significant downward trend for total activity in the motor-activity assessment, but with no significant pairwise comparisons to the vehicle control group. There were no differences in the motor activity of female pups.

During the post-weaning period of F1 pups, there were no decrements in group mean body weight, no treatment-related clinical observations, and no effect of treatment on estrous cycling. There were no treatment-related effects on body weight during F1 gestation and lactation periods. No treatment-related effects were seen in male or female reproductive indices of the F1 generation. The F1 female and male mating indices were high and equivalent (95–100%) for all dose groups. The fertility and pregnancy indices were also high and equivalent (90–100%), and there were no effects of Nyotran on gestational length, number of implantation sites per litter, or the percent postimplantation loss per litter. There were no treatment-related findings in F2 progeny; lactational indices were similar across groups and there were no differences in average pup weights. Necropsy of F1 males and females found no effect of Nyotran administration.

Developmental Toxicity Evaluation in Rats (ICH 4.1.3; Segment II)
No females aborted, delivered early, were removed from the study, or died. A summary of the results is presented in Table 3Go. Pregnancy was high and equivalent (96%) across all treatment groups and all pregnant animals had 1 or more live fetuses at scheduled necropsy on gd 20. Maternal body weights were equivalent across all groups for all time points. The group-mean maternal body weight change was significantly reduced only in the high-dose group, i.e., 2 g versus 12 g in control, for the first interval of dosing, gd 6–9. Maternal feed consumption was significantly reduced at 3.0 mg/kg/day for gd 6–9 and gd 6–15 (dosing periods). The only treatment-related maternal clinical observation was clinical weight loss (defined as >=5.0 g) observed in 2 dams given 1.5 mg/kg/day and in 3 dams administered 3.0 mg/kg/day on gd 9. There were no treatment-related effects on maternal gravid uterine or liver weights.


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TABLE 3 Summary of Toxicity in a SEG II Developmental Study in Rats
 
There were no significant effects of treatment on preimplantation or postimplantation loss, the number of total implants, resorptions, non-live (dead plus resorbed) or affected (non-live plus malformed) implants per litter, on fetal sex ratio (% males) per litter or on fetal body weight (all fetuses or sexes separately) per litter.

There were no significant changes among treatment groups in the incidence of pooled external, visceral, skeletal, or total fetal malformations or variations. External malformations were limited to 1 fetus at 3 mg/kg with anophthalmia. Visceral malformations included mild hydrocephaly (discussed below), hydronephrosis in all Nyotran-treated groups with no dose-response incidence, and hydroureter in all groups. Hydronephrosis and hydroureter are common findings in this laboratory for CD® rat fetuses. Fetal skeletal malformations were limited to effects in the thoracic centra in the control and low- and mid-dose groups. There were no fetal external variations. Fetal visceral variations included enlarged nasal sinuses in 1 fetus at 0.5 mg/kg/day and in 2 fetuses in 2 litters at 3.0 mg/kg/day, enlarged lateral ventricles of the cerebrum in all groups, and distended ureter in all groups; the latter 2 findings are common in term CD® rat fetuses. Fetal skeletal variations were limited to an extra rib (rudimentary or full) on Lumbar I in all groups, a short thirteenth rib at 0.5 and 3.0 mg/kg/day, a wavy rib at 0 and 1.5 mg/kg/day, and reduced ossification in thoracic centra in all groups.

The incidence of mild hydrocephaly (a visceral malformation defined as dilation of the dorsal midline portion of the lateral ventricles of the cerebrum) was significantly increased at the 3.0-mg/kg/day dosage level, involving 4 fetuses in 4 litters. Mild hydrocephaly was present (not statistically significantly increased) in 1 fetus in 1 litter at 1.5 mg/kg/day.

The TK values in pregnant rats administered Nyotran are provided in Table 4Go. Systemic exposure was demonstrated at all dosage levels. Because different rats are used at different time points, there are no estimates of error; that is, no standard deviations around the mean values.


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TABLE 4 TK Parameters for Nystatin A1 in Blood of Rats and Rabbits Administered Nyotran in SEG II Studies
 
Developmental Toxicity Evaluation in Rabbits (ICH 4.1.3; Segment II)
No female rabbits aborted or died during the study. Two does, 1 control doe and 1 doe administered 1.5 mg/kg/day Nyotran, delivered early. Only 1 female was not pregnant at scheduled sacrifice, a female given 3 mg/kg/day. A summation of the results is presented in Table 5Go. Pregnancy was high and equivalent across all treatment groups and all pregnant animals had 1 or more live fetuses at scheduled necropsy on gd 20. Maternal body weights were the same across all groups for all time points. The group mean maternal body weight change was significantly reduced only at 3.0 mg/kg/day for gd 9–12 (71 g in control compared to –15 g in treated). Maternal feed consumption was significantly reduced at the 3.0-mg/kg/day dose level. Maternal liver weights were decreased by 17% at 3.0 mg/kg. There were no treatment-related effects on maternal gravid uterine weights.


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TABLE 5 Summary of Toxicity in a SEG II Developmental Study in Rabbits
 
There were no significant effects from the treatment on the number of ovarian corpora lutea, preimplantation, or postimplantation loss. The number of total implants, resorptions, non-live (dead plus resorbed) or affected (non-live plus malformed) implants per litter, on fetal sex ratio (% males) per litter or on fetal body weight (all fetuses or sexes separately) per litter. Sex ratio per litter did exhibit a dose-related significant upward trend across groups with no significant pairwise comparisons to the concurrent control group mean. However, there was no increase in non-live implants nor any preferential loss of fetuses of a particular sex that might imply a preferential loss of one sex, as one might expect in the case of a biologically meaningful change in sex ratio.

There were no significant treatment-related changes in the incidence of pooled external, visceral, skeletal, or total fetal malformations or variations in this study. No external malformations were observed. Visceral malformations included mild hydrocephaly in 1 control-group fetus, and bilateral hydronephrosis in 1 fetus in the 1.5-mg/kg/day dosage group. Fetal skeletal malformations were limited to effects in the sternum. These effects were a fused sternebrae in 1 fetus and a hole in the cartilage of the sternum in 2 fetuses in 2 litters at 3.0 mg/kg/day and an extra rib cartilage attached to cartilage of Rib III in 1 fetus at 1.5 mg/kg/day. Fetal external variations were seen as clubbed limb without bone change in 1 control fetus and 2 fetuses in 2 litters at 0.5 mg/kg/day. Fetal visceral variations included agenesis of the innominate artery in 1 control-group fetus and in 2 fetuses in 1 litter at 1.5 mg/kg/day, an abnormal number of papillary muscles of the heart valves in all groups, and gall bladder findings in all groups; e.g. liver-like tissue attached to the gall bladder. Fetal skeletal variations were limited to an extra rib(s), either rudimentary or full, on Lumbar I in all groups, with no relationship to treatment, and to extra ossification sites between sternebrae in 2 fetuses in 1 litter at 0.5 mg/kg/day.

The TK values are presented in Table 4Go. Dose-proportional systemic exposures were observed across the range of dose levels.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nystatin has been used as an antifungal agent for over 40 years. Nevertheless, since systemic exposures are not observed in the current clinical routes of administration, there is a lack of data regarding the safety of nystatin in laboratory animals or in human pregnancy. In support of the regulatory submission of Nyotran, a liposomal formulation of nystatin, we have addressed the potential reproductive and developmental toxicity of this drug in laboratory animals.

The battery of tests recommended in the ICH guidelines for the detection of toxicity to reproduction was conducted in rats and rabbits. The dose levels of Nyotran utilized were 0.5, 1.5, and 3.0 mg/kg/day in all studies. In the SEG I study of fertility and early embryonic development (ICH 4.1.1) and the SEG III pre- and postnatal development (ICH 4.1.2), rats did not tolerated extended dosing with 3.0 mg/kg/day Nyotran. This daily dosage proved to be toxic to the parental animals and resulted in our lowering the daily dose to 2.0 mg/kg in both of these studies. Maternal toxicity, in the absence of mortality, was also seen in the SEG II studies (ICH 4.1.3) in rats and rabbits. Hence, the dose levels were adequate to assess reproductive toxicity at doses that produced significant parental toxicity.

There was no effect of Nyotran on male and female reproductive parameters or on the development of the F1 generation in the SEG I fertility and early embryonic development study. The NOAEL for parental toxicity in the SEG I study was 0.5 mg/kg/day, whereas the NOAEL for reproductive toxicity was greater than the highest dose of 2.0 mg/kg/day. Nyotran also had no effect on F0 or F1 reproductive performance in the SEG III study of pre- and postnatal development. In that SEG III study, the NOAEL for parental toxicity was again 0.5 mg/kg/day and the NOAEL for reproductive performance again was greater than the highest dose used in the study, 2.0 mg/kg/day. Nyotran did not affect reproductive parameters or change the incidence of fetal malformations in the rabbit SEG II study. In the SEG II rabbit study, the maternal NAOEL was 1.5 mg/kg/day, while the NOAEL for F1 toxicity was greater than the highest dose level in the study, 3.0 mg/kg/day.

Nyotran was found to have caused some effects on the F1 generation at all dose levels in the SEG III study of pre- and postnatal development in rats, specifically on lactational body weights and in several post-wean developmental tests. There was a delay in the acquisition of preputial opening in F1 males at 1.5 and 3.0/2.0 mg/kg dose groups. However, the delay was less than 2 days and there were no consequences in terms of mating of these animals. The U.S. EPA's Reproductive Toxicity Risk Assessment Guidelines states that "Biological relevance of a change in these measures (preputial separation or vaginal patency) of a day or two is unknown" (U.S. EPA, 1996, p. 56295). Post-wean development was also affected in terms of auditory startle response in females and of motor activity in males. Females in all Nyotran treatment groups exhibited a decreased force, but not duration, of jump, and males in all Nyotran treatment groups exhibited a significant downward trend for total motor activity. The biological relevance of these effects is not clear. Reproductive performance of this F1 generation was not affected by treatment and the F2 generation was unaffected by parental exposure to Nyotran. Nonetheless, the NOAEL for effects on post-wean development in rats was less than 0.5 mg/kg/day Nyotran.

The SEG II early embryonic development study in rats found no evidence of any treatment-related effects on Caesarian section parameters, although there was some evidence of maternal toxicity at the 1.5- and 3.0-mg/kg/day dose levels. Developmental effects were limited to a statistically significant increased incidence of mild hydrocephaly among offspring in the 3.0-mg/kg/day-dose group. The NOAEL for maternal toxicity in this SEG II rat study was 0.5 mg/kg/day and 1.5 mg/kg/day was the NOAEL for developmental toxicity.

Mild hydrocephaly had not been seen in the historical control data for this rat strain in over 6000 term fetuses in the performing contract laboratory. However, enlarged lateral ventricles, which may represent the low end of a continuum through mild to full hydrocephaly is the most common finding, designated a variation in this rat strain. In a developmental toxicity study performed in the same contract laboratory shortly after this study, with a different drug by a different route of administration, using rats of the same strain and from the same supplier, mild hydrocephaly was also observed. Mild hydrocephaly in that study was not seen in the control group, but was seen, with no relationship to dose, in 6 fetuses in the low-dose group, 7 fetuses in the mid-dose group, and 3 fetuses in the high-dose group. Thus, mild hydrocephaly might represent a change in the background pattern of spontaneous malformations and variations (i.e., genetic drift), or a change in the pattern of responsiveness to exposure to test materials in utero in the term CD® rat fetuses. With this said, the incidence of hydrocephaly and the relationship to Nyotran treatment, in the absence of any other treatment-related developmental effects, is not clear.

Systemic exposures of animals to the active nystatin A1 isomer were evaluated in the rat and rabbit SEG II studies. Both of the studies demonstrated blood concentrations that were within the range of concentrations that are pharmacologically active in vitro. Antifungal activity has been demonstrated at concentrations of 1–8 µg/ml Nyotran, where the concentration is that of the active drug nystatin, against a wide variety of fungi (Johnson et al., 1998Go). The pharmacokinetics of nystatin in rats and rabbits administered Nyotran were quite different and not strictly correlative with maternal toxicity. The systemic exposure was much higher in rabbits, but rabbits were no more sensitive than rats to the toxic effects of Nyotran.

Preliminary results in humans show that systemic exposures at comparable mg/kg dosage levels are even higher than in rabbits or rats. At clinical dosages of 2- and 4-mg/kg Nyotran, respectively, the pharmacokinetics of nystatin A1 in the blood of patients were as follows: Cmax = 5 and 24 µg/ml; AUC = 15 and 80 µg•h/ml; t1/2 = 3.5 and 3.5 h (Cossum et al., 1996Go). In the clinic, a daily dosage of 4 mg/kg is well tolerated, whereas this dose exceeds the highest tolerated dose in these reproductive toxicity studies, especially in rats in the SEG I and SEG III studies. That humans are resistant cannot be explained by systemic exposures, since these too, are much higher in humans compared to laboratory animals.

In conclusion, this package of reproductive toxicity tests showed some effects on postnatal development of F1 rats. Given the lack of a margin of safety extrapolated to the clinic based on dosage or systemic exposure, caution should be exercised when using this drug in females of childbearing potential.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (281) 367-1676. E-mail: jlarson{at}aronex-pharm.com. Back


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