* Aronex Pharmaceuticals, Inc., 8707 Technology Forest Place, The Woodlands, Texas 77381; and
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
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ABSTRACT |
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Key Words: Nyotran; nystatin; antifungal; reproductive toxicity; developmental toxicity.
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INTRODUCTION |
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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., 1997; Walsh and Lee, 1993
). 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, 1966
; Edwards et al., 1978
). 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, 1966
; Joly et al, 1989
; Longuet et al., 1991
; Medoff et al., 1983
;). 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, 1994
; Lee et al., 1994
; Proffitt et al., 1991
) and dogs (Fielding et al., 1991
). 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., 1998
). 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., 1991; Hebeka and Solotorovsky, 1965
). 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., 1988
). 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., 1986
). 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, 1998). 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, 1998
).
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MATERIALS AND METHODS |
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Timed-mated New Zealand White (NZW) rabbits were supplied by Covance (Denver, PA), were approximately 6 months of age at study initiation, and weighed 3.34.4 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 6471°F and 4464% 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. 50333008) was shipped to RTI in clear bottles, each containing 50 mg of the active drug nystatin (CAS No. 1400619). 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 515 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 1922. 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 14), incisor eruption (pnd 813), and eye opening (pnd 1116). 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 2332 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 3435 days of age, and learning and memory, using a water-filled Morris maze in pups 4150 days of age (described in Morris, 1981). 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, 1984). 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., 1988
). 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 (400500 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, 1962). 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, 1967
) 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, 1964
) 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, 1956
). Jonckheere's test for k independent samples (Jonckheere, 1954
) 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, 1967
), and by the Cochran-Armitage Test for Linear Trend on Proportions (Armitage, 1955
; Cochran, 1954
). 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, 1956
). Values from functional assessment, including behavioral tests, were compared to control values by ANOVA, followed by individual pairwise comparisons (Dunnett, 1964
).
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RESULTS |
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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 2. 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 1821. Hence, the dose was decreased for this group to 2.0 mg/kg/day at gd 1922. 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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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 (95100%) for all dose groups. The fertility and pregnancy indices were also high and equivalent (90100%), 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 3. 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 69. Maternal feed consumption was significantly reduced at 3.0 mg/kg/day for gd 69 and gd 615 (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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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 4. 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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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 4. Dose-proportional systemic exposures were observed across the range of dose levels.
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DISCUSSION |
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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 18 µg/ml Nyotran, where the concentration is that of the active drug nystatin, against a wide variety of fungi (Johnson et al., 1998). 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 µgh/ml; t1/2 = 3.5 and 3.5 h (Cossum et al., 1996). 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.
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NOTES |
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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Boswell, G. W., Bekersky, I., Buell, D., Hiles, R., and Walsh, T. J. (1998). Toxicological profile and pharmacokinetics of a unilamellar liposomal vesicle formulation of amphotericin B in rats. Antimicrob. Agents Chemother. 42, 263268.
Broughton, M. C., Bard, M., and Lees, N. D. (1991). Polyene resistance in ergosterol producing strains of Candida albicans. Mycoses 34, 7583.[ISI][Medline]
Butler, W. T. (1966). Pharmacology, toxicology, and therapeutic usefulness of amphotericin B. J. Am. Med. Assoc. 195, 371375.[Medline]
Cochran, W. (1954). Some methods for strengthening the common 2 tests. Biometrics, 10, 417451.[ISI]
Cossum, P. A., Wyse, J., Simmons, V., Wallace, T. L., and Rios, A. (1996). Pharmacokinetics of Nyotran (liposomal nystatin) in human patients. 36th Interscience Conference on Antimicrobial Agents and Chemotherapy. Abstract No. A88.
Dayan, A. D., and Working, P. K. (1994). Non-clinical studies of the efficacy, pharmacokinetic, and safety of amphotericin B colloidal dispersion (ABCD). R. Soc. Med. Press, 32, 1226.
Dunnett, C. W. (1964). New tables for multiple comparisons with control. Biometrics, 20, 482491.[ISI]
Edwards, J. E., Jr., Lehrer, R. I., Stiehm, E. R., Fischer, T. J., and Young, L. S., (1978). Severe candidal infections: Clinical perspective, immune defense mechanism, and current concepts of therapy. Ann. Intern. Med. 89, 91106.[ISI][Medline]
Fielding, R. M., Smith, P. C., Wang, L. H., Porter, J., and Guo, L. S. (1991). Comparative pharmacokinetics of amphotericin B after administration of a novel colloidal delivery system, ABCD, and a conventional formulation to rats. Antimicrob. Agents Chemother. 35, 12081213.[ISI][Medline]
Guaschino, S., Michelone, G., Stola, E., Lombardi, G., Spinillo, A., and Viale, P. (1986). Mycotic vaginitis in pregnancy: A double evaluation of the susceptibility of the main antimycotic drugs of isolated species. Biol. Res. Pregnancy Perinatol. 7, 2022.[ISI][Medline]
Hebeka, E. K., and Solotorovsky M. (1965). Development of resistance to polyene antibiotics in Candida albicans. J. Bacteriol. 89, 15331539.[ISI]
Hughes, W. T., Armstrong, D., Bodey, G. P., Brown, A. E., Edwards, J. E., Feld, R., Rizzo, P., Rolston, K. V., Shenep, J. L., and Young, L. S. (1997). 1997 guidelines for the use of antimicrobial agents in neutropenic patients with unexplained fever. Clin. Infect. Dis. 25, 551573.[ISI][Medline]
International Conference on Harmonization (ICH) (1994). Department of Health and Human Services. Guideline on Detection of Toxicity to Reproduction for Medicinal Products; Notice. Federal Register 59 (183), 4874648752. (September 22, 1994).
International Conference on Harmonisation (ICH) (1996). Department of Health and Human Services. Guideline on Detection of Toxicity to Reproduction for Medicinal Products: Addendum on Toxicity to Male Fertility; Availability. Federal Register 61(67), 1535915361. (April 5, 1996).
Johnson, E. M., Ojwang, J. O., Szekely, A., Wallace, T. L., and Warnock, D. W. (1998). Comparison of in vitro antifungal activities of free and liposome-encapsulated nystatin with those of four amphotericin B formulations. Antimicrob. Agents Chemother. 42, 14121416.
Joly, V., Dromer, F., Barge, J., Yeni, P., Seta, M., Molas, G., and Carbon, C. (1989). Incorporation of amphotericin B (AMB) into liposomes alters the AMB-induced nephrotoxicity in rabbits. 251, 311316.
Jonckheere, A. R. (1954). A distribution-free k-sample test against ordered alternatives. Biometrika, 41, 133145.[ISI]
Lee, J. W., Amantea, M. A., Francis, P. A., Navarro, E. E., Bacher, J., Pizzo, P. A., and Walsh, T. J. (1994). Pharmacokinetics and safety of a unilamellar liposomal formulation of amphotericin B (AmBisome) in rabbits. Antimicrob. Agents Chemother. 38, 713718.[Abstract]
Longuet, P., Joly, V., Amirault, P., Seta, M., Carbon, C., and Yeni, P. (1991). Limited protection by small unilamellar liposomes against the renal tubular toxicity induced by repeated amphotericin B infusions in rats. Antimicrob. Agents Chemother. 35, 13031308.[ISI][Medline]
Marr, M. C, Myers, C. B., George, J. D., and Price, C. J. (1988). Comparison of single and double staining for evaluation of skeletal development: The effects of ethylene glycol (EG) in CD rats. Teratology, 37, 476.[ISI]
Medoff, G., Brajtburg, J., Kobayashi, G. S., and Bolard, J. (1983). Antifungal agents useful in the therapy of systemic fungal infections. Ann. Rev. Pharmacol. Toxicol. 23, 303304.[ISI][Medline]
Morris, R. G. M. (1981). Spatial localization does not require the presence of local cues. Learning and Motivation, 12, 239260.[ISI]
Newcomer, V. D., Wright, E .T., Sternberg, T. H., Graham, J. H., Wier, R. H., and Egeberg, R.. O. (1955). Evaluation of Nystatin in the treatment of coccidioimycosis in man. In Therapy of Fungal Diseases (T. H. Sternberg and V. D. Newcomer, Eds.), pp. 260267. Little, Brown, Boston.
Pace, H. R., and Schantz, S. I. (1956). Nystatin (Mycostatin) in the treatment of monilial and nonmonilial vaginitis. JAMA 162, 268271.[ISI]
Physicians Desk Reference, 52nd ed. (1998). Medical Economics, Montvale, NJ.
Proffitt, R.T., Sartorius, A., Chiang, S.M., Sullivan, L., and Adler-Moore, J.P. (1991). Pharmacology and toxicology of a liposomal formulation of amphotericin B (AmBisome) in rodents. J. Antimicrob. Chemother. 28(Suppl. B), 4961.[ISI][Medline]
SAS Institute (1989). SAS/STAT® Users' Guide, Ver. 6, 4th ed., Vols. 1 and 2, SAS Institute, Cary, NC.
Siegel, S. (1956). Nonparametric Statistics for Behavioral Sciences. McGraw-Hill, New York.
Snedecor, G. W., and Cochran, W. G. (1967). Statistical Methods. 6th ed., Iowa State University Press, Ames, IA.
Stark, J. E. (1967). Allergic pulmonary aspergillosis successfully treated with inhalations of nystatin. Dis. Chest. 51, 9699.[ISI][Medline]
Stern, J. C., Shah, M. K., and Lucente, F. E. (1988). In vitro effectiveness of 13 agents in otomycosis and review of the literature. Laryngoscope, 98, 11731177.[ISI][Medline]
Stuckhardt, J. L., and Poppe, S. M. (1984). Fresh visceral examination of rat and rabbit fetuses used in teratogenicity testing. Teratog. Carcinog. Mutagen. 4, 181188.[ISI][Medline]
U.S. EPA (1996). Part II. Environmental Protection Agency. Reproductive Toxicity Risk Assessment Guideline; Notice. Federal Register 61(212), 5627456322 (October 31, 1996).
Walsh, T. J., and Lee, J.W. (1993). Prevention of invasive fungal infections in patients with neoplastic disease. Clin. Infect. Dis. 17(suppl 2), 468480.
Winer, B. J. (1962). Statistical Principles in Experimental Design. McGraw-Hill, New York.