Pre- and Postnatal Lung Development: An Updated Species Comparison.

The purpose of this review is to give an outline of respiratory tract morphological and functional development with an emphasis on perinatal and postnatal maturational processes. In view of the rising need for qualitative and quantitative data for the development of pediatric pharmaceuticals, a comparison of the human situation to experimental animal models is made, and functional data as well as suitable models for human airway diseases and functional testing are presented. Birth Defects Research 109:1519-1539, 2017. © 2017 Wiley Periodicals, Inc.

Comparative assessment of the timing of sexual maturation in male Wistar Han and Sprague-Dawley rats.

Given the increasing use of Wistar Han (WH) rats in regulatory toxicology studies, these studies were performed to characterize the onset of sexual maturation in maturing WH rats as compared to Sprague-Dawley (SD) rats. Beginning on postnatal day (PND) 38 through PND 91 groups (n=8) of untreated WH rats were evaluated for maturation of the male reproductive system. Testicular spermatid head counts increased beginning on PND 42 until PND 70. Sperm were detected in the caput, corpus, and cauda epididymis on PND 45, 49, and 49, respectively, and counts increased through PND 91. Sperm motility was at adult levels by PND 63. The morphology of the testis/epididymis of all animals at day 70 or older was consistent with qualitative sexual maturity. Based on these endpoints, WH rats were determined to be sexually mature at PND 70, and many of these endpoints evaluated in SD rats exhibited nearly identical trends.

Object discrimination reversal as a method to assess cognitive impairment in nonhuman primate enhanced pre- and postnatal developmental (ePPND) studies: statistical power analysis.

An important aspect of the enhanced pre- and postnatal developmental (ePPND) toxicity study in nonhuman primates (NHP) is that it combines in utero and postnatal assessments in a single study. However, it is unclear if NHP ePPND studies are suitable to perform all of the evaluations incorporated into rodent PPND studies. To understand the value of including cognitive assessment in a NHP ePPND toxicity study, we performed a power analysis of object discrimination reversal task data using a modified Wisconsin General Testing Apparatus (ODR-WGTA) from two NHP ePPND studies. ODR-WGTA endpoints evaluated were days to learning and to first reversal, and number of reversals. With α = 0.05 and a one-sided t-test, a sample of seven provided 80% power to predict a 100% increase in all three of the ODR-WGTA endpoints; a sample of 25 provided 80% power to predict a 50% increase. Similar power analyses were performed with data from the Cincinnati Water Maze (CWM) and passive avoidance tests from three rat PPND toxicity studies. Groups of 5 and 15 in the CWM and passive avoidance test, respectively, provided 80% power to detect a 100% change. While the power of the CWM is not far superior to the NHP ODR-WGTA, a clear advantage is the routine use of larger sample size, with a group of 20 rats the CWM provides ~90% power to detect a 50% change. Due to the limitations on the number of animals, the ODR-WGTA may not be suitable for assessing cognitive impairment in NHP ePPND studies.

Sensitive windows of skeletal development in rabbits determined by hydroxyurea exposure at different times throughout gestation.

The critical periods of axial skeletal development in rats and mice have been well characterized, however the timing of skeletal development in rabbits is not as well known. It is important to have a more precise understanding of this timing of axial skeletal development in rabbits due to the common use of this species in standard nonclinical studies to assess embryo-fetal developmental toxicity. Hydroxyurea, a teratogen known to induce a variety of fetal skeletal malformations, was administered to New Zealand White rabbits as a single dose (500 mg/kg) on individual days during gestation (gestation day, GD 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 19) and fetal external, visceral, and skeletal morphology was examined following cesarean sections on GD 29. A wide range of fetal skeletal effects was observed following hydroxyurea treatment, with a progression of malformations from anterior to posterior structures over time, as well as from proximal to distal structures over time. The sensitive window of axial skeletal development was determined to be GD 8 to 13, while disruption of appendicular and cranio-facial skeletal development occurred primarily from GD 11 to 16 and GD 11 to 12, respectively. The results of this study provide a better understanding of the critical developmental window for different segments of the rabbit skeleton, which will aid in the design of window studies to investigate teratogenicity in rabbits.

Developmental toxicity of lersivirine in rabbits when administered throughout organogenesis and when limited to sensitive windows of axial skeletal development.

BACKGROUND: Lersivirine is a second-generation nonnucleoside reverse transcriptase inhibitor undergoing clinical development for the treatment of human immunodeficiency virus-1. An embryo-fetal development study was performed to evaluate the potential for maternal and developmental toxicity of lersivirine. METHODS: Pregnant New Zealand White rabbits were administered 0, 100, 250, and 500 mg/kg lersivirine by oral gavage once daily on gestation days (GDs) 7 to 19, followed by cesarean section on GD 29 and fetal evaluation. RESULTS: Maternal toxicity was noted at all dose levels (decreased food consumption and body weight gain), with fetal toxicity at 500 mg/kg (decreased fetal weights, increased postimplantation loss). Equivocal findings for axial skeletal malformations were observed in three fetuses at 500 mg/kg. To better understand if these malformations were related to treatment with lersivirine, a follow-up rabbit embryo-fetal development study was performed with 1000 mg/kg/day lersivirine (500 mg/kg BID, 12-hr interdose interval) for two different 3-day windows, GDs 8 to 10 or GDs 11 to 13, which represent the sensitive windows of axial skeletal development in rabbits. Control rabbits were administered vehicle following the same dosing regimen from GDs 8 to 13. Cesarean sections were performed on GD 29, and fetal skeletons were examined for the potential of lersivirine to cause skeletal malformations in rabbits. At maternal exposure levels higher than the initial study, lersivirine did not induce fetal skeletal malformations when administered in the sensitive windows of axial skeletal development. CONCLUSION: The results of these studies indicate that lersivirine did not exhibit any evidence of teratogenicity in rabbits.

Developmental toxicity study of lersivirine in mice.

Lersivirine is a second-generation nonnucleoside reverse transcriptase inhibitor undergoing clinical development for the treatment of HIV-1. An embryo-fetal developmental toxicity study was performed to evaluate the maternal and developmental toxicity of lersivirine in pregnant mice. Mated Crl:CD1(ICR) mice were administered 0, 150, 350, and 500 mg/kg lersivirine once daily by oral gavage on gestation days 6 to 17, followed by cesarean section on gestation day 18. The first 2 days of dosing for the high-dose group were done at 250 mg/kg to allow induction of hepatic metabolizing enzymes, after which the dose was increased to 500 mg/kg/day. This dosing paradigm allowed for maintenance of exposure in the high-dose group despite the considerable autoinduction that occurs in rodents following lersivirine treatment. Lersivirine did not cause an increase in external, visceral, or skeletal malformations. Intrauterine growth retardation, demonstrated by reduced fetal body weights and increased variations associated with delayed skeletal ossification, was noted at 350 and 500 mg/kg/day. The results of these studies indicate that lersivirine is not teratogenic in mice.

The placenta, transfer of immunoglobulins, and safety assessment of biopharmaceuticals in pregnancy.

Anatomical and developmental differences of the parental-offspring interface among experimental animals and humans throughout gestation are reviewed focusing on biodistribution of immunoglobulins (IgG). The formation of the extraembryonic membranes, uteroplacental circulation, and characteristics of the placenta (gross shape, modes of implantation, surface modifications that increase surface area, and extent of embryonic invasion into maternal tissue) are reviewed. Placental physiology and function are covered with attention to transfer of xenobiotics. Placental transfer of immunoglobulins in the human, non-human primate (NHP), rodent, and rabbit is discussed and the transfer of human fragment crystallizable (Fc)-containing biopharmaceuticals and potential impact on developmental toxicity risk assessment are specifically addressed. Safety assessment is often limited to the NHP as the only pharmacologically relevant model, despite poor statistical power as employed in current experimental designs. Although data are limited, the gestational timing of placental IgG transfer in rabbits appears to be more consistent with that of humans (i.e. occurring at the very end and after completion of organogenesis) than that of rodents, making the rabbit a reasonable choice assuming it is pharmacologically relevant. The rodent is not considered the most appropriate model for human placental transfer of Fc-containing biopharmaceuticals because it is currently believed to overestimate exposure during organogenesis. Nevertheless, the rodent may provide a conservative approach for hazard identification. It is clear that additional experimentation is needed to further clarify the timing of prenatal transfer of Fc-containing biopharmaceuticals in various species.

Toxicology and toxicokinetics of oral pantoprazole in neonatal and juvenile dogs.

BACKGROUND: Pantoprazole is an irreversible inhibitor of H(+) /K(+) adenosine triphosphatase proton pump. This study encompassed the period of postnatal stomach development to determine whether immature animals are uniquely sensitive to progression of PPI-induced enterochromaffin-like cell hyperplasia. METHODS: Pantoprazole was administered to beagle dogs at 3, 10, or 30 mg/kg/day (10/sex/group) from PND 1 for 13 weeks, subsets of animals had a 13-week recovery period. Clinical signs, body weights, growth, clinical chemistry, and neurobehavioral endpoints were assessed. Selected organs were weighed and histologically examined. RESULTS: There were no effects on body weights, growth, landmarks of physical and reproductive development, or sensory and neurobehavioral function. Cholesterol and triglyceride levels were increased at 10 and 30 mg/kg/day, but resolved during the recovery period. Stomach weight was increased at all doses, but after recovery the differences in stomach weights resolved for females although male stomach weights remained slightly increased. Pantoprazole-related microscopic findings in the stomach consisted of increased mucosal height, glandular necrosis, and glandular dilation at all doses; and ECL cell hyperplasia, parietal cell vacuolation, and atrophy of chief cells are noted at 10 and/or 30 mg/kg/day. There was a partial recovery of these microscopic changes indicated by a decreased incidence and/or severity of increased mucosal height, glandular necrosis, ECL cell hyperplasia, and chief cell atrophy, and complete resolution of other microscopic observations. CONCLUSION: Pantoprazole administered to beagles from PND 1 for 13 weeks resulted in findings similar to those in adult dogs and juvenile dogs, which showed no increase in severity or progression of ECL hyperplasia.

Impaired reproduction in adult male, but not female, rats following juvenile treatment with the aromatase inhibitor, exemestane.

BACKGROUND: Exemestane is an irreversible steroidal inhibitor of cytochrome-P450 aromatase required for estrogen synthesis. The safety of the drug in the pediatric population, particularly in males, has not previously been evaluated. Given the increased interest in treating children with aromatase inhibitors, we undertook a study in rats to assess the potential for exemestane to alter reproductive development and function when administered to juveniles. METHODS: Male and female rats were treated with exemestane at doses anticipated to produce exposures approximately 2- and 35-fold the expected clinical plasma exposure in young adult males during the period of reproductive maturation. After maturation, treated rats were mated to evaluate the potential impact on reproductive function. RESULTS AND CONCLUSION: There were no effects on sexual maturation in either sex or on female reproductive function. Treatment of juvenile male rats caused increased cohabitation time and decreased copulation rates; pregnancy rates and litter size were not affected in rats that mated. Decreased testis (10-15%) and epididymis (20-30%) weights, and decreased Sertoli cell numbers were noted at all doses. This indicates that exemestane can reduce Sertoli cell proliferation during maturation. The sensitive window for this effect is expected to be limited to the period of Sertoli cell proliferation, which is completed by around postnatal day 15 in rats and before puberty in humans. Treatment beginning at a later time relative to the window for Sertoli cell proliferation or for a longer duration is not expected to have additional adverse effect as the effect was not shown to be degenerative.

Toxicity study in juvenile rats with the α4ß2 nicotinic acetylcholine receptor partial agonist CP-601,927.

BACKGROUND: CP-601927 is a selective α(4) ß(2) nicotinic acetylcholine receptor (nAChR) partial agonist. The objective of this study was to assess the potential effects persisting into adulthood when CP-601,927 was administered to neonatal/juvenile rats. Since the juvenile toxicity study was being performed early in the development program and this study would represent the longest dosing period yet evaluated, the study design incorporated standard endpoints typically evaluated in a general toxicity screening study. METHODS: CP-601,927 was administered to Sprague-Dawley rats from postnatal day (PND) 7-70 by oral gavage at doses of 0.3, 1, or 3 mg/kg. During treatment animals were evaluated for growth, development, and sexual maturation. At the end of the treatment period general toxicity screening endpoints were collected (e.g., organ weights, histology, clinical chemistry). Following a 2-week latency period, animals were evaluated for CNS function in a comprehensive behavioral training battery consisting of a functional observational battery, motor activity, acoustic startle response, and learning and memory evaluations. Reproductive competency was evaluated by mating treated rats and allowing pregnant dams to deliver and rear their litters until PND 10. RESULTS AND CONCLUSIONS: Treatment-related findings included the death of 2 males receiving 3 mg/kg CP-601,927, and transient reductions in body weight for both males and females during the third week of dosing which quickly recovered to control levels. The only treatment-related alteration in behavior was decreased motor activity, which occurred only in females at the highest dose tested. CP-601,927 had no effect on acoustic startle response, learning and memory, sexual maturation, reproductive capacity, or general toxicity endpoints.

Juvenile animal toxicity study designs to support pediatric drug development.

The objective of juvenile animal toxicity studies of pharmaceuticals is to obtain safety data, including information on the potential for adverse effects on postnatal growth and development. Studies in juvenile animals may assist in identifying postnatal developmental toxicities or other adverse effects that are not adequately assessed in the routine toxicity evaluations and that cannot be safely or adequately measured in pediatric clinical trials. Unlike the traditional reproductive and developmental toxicology studies that have been discussed in the accompanying reports, the design requirements for toxicity studies in juvenile animals are not explicitly defined in regulatory guidance. However, studies in juvenile animals can be useful in providing safety information necessary to enable pediatric clinical trials in pediatric patients or when there are special concerns for toxicities that cannot be safely or adequately measured in clinical trials. These juvenile animal toxicity studies are designed on a case-by-case basis. General design considerations and examples of study designs for assessment of juvenile animal toxicity are discussed.

Comparative juvenile safety testing of new therapeutic candidates: relevance of laboratory animal data to children.

Differences in drug response in patients of various ages including children and the elderly are common, often leading to challenges in optimizing dosages and duration of use. For example, developmental changes in renal function can dramatically alter the plasma clearance of compounds with extensive renal elimination and thus can enhance renal and systemic toxicity of these drugs. Preclinical and clinical research of new therapeutics is initially focused on adults, and provides little relevant information for children especially those who are still going through skeletal and organ development. The organ systems in the pediatric population that can be most susceptible are lungs, brain, kidneys, immune, skeletal, and reproductive systems. Considering that significant differences can exist between adult and juvenile populations that may affect drug safety, major regulatory agencies around the world are encouraging and sometimes requiring companies to generate preclinical juvenile animal data to predict for potential drug toxicity in children. However, data generated from such studies are useful only if obtained using the most appropriate species at the most relevant age considering comparability of specific organ system development in question. Other factors in the design of juvenile safety studies should include the indication, existing toxicological data and likely route of human exposure. This report will discuss these factors with a focus on reviewing species-specific developmental schedules for specific target organs and relevance of preclinical data in the design and conduct of clinical pediatric studies. Specific examples will be used to discuss the relationship of preclinical juvenile toxicity observations to risk assessment in humans.

Postnatal growth and morphological development of the brain: a species comparison.

The objective of this report is to summarize the available literature regarding the postnatal growth and morphological development of the brain and compare the timelines for these events between humans and experimental species. While not the primary focus of this report, in acknowledgement of the evident role of maturation of neurotransmitter systems in development, a brief description of the comparative development of the NMDA receptor is included. To illustrate the challenges faced in estimating developmental toxicity potential in humans, the importance of postnatal experience in CNS development is also briefly reviewed. This review is part of the initial phase of a project undertaken by the Developmental and Reproductive Toxicology Technical Committee of the ILSI Health and Environmental Sciences Institute (HESI) to bring together information on a selected number of organ systems and compare their postnatal development across several species (Hurtt and Sandler: Birth Defects Res Part B 68:307-308, 2003).

Reproductive toxicity assessment of lasofoxifene, a selective estrogen receptor modulator (SERM), in male rats.

BACKGROUND: Lasofoxifene is a nonsteroidal selective estrogen receptor modulator (SERM) with greater than 100-fold selectivity against all other steroid receptors and is a potentially superior treatment for postmenopausal osteoporosis. The purpose of this study was to evaluate the effects of lasofoxifene on male reproduction in rats in light of the known effects of estrogen modulating compounds on male reproductive ability. METHODS: Lasofoxifene was administered to adult male rats at doses of 0.1, 1, 10, and 100 mg/kg for 66-70 consecutive days. After 28 days of dosing, male rats were cohabited with untreated female rats. Female rats were euthanized on gestation day 14 and a uterine examination was carried out for evaluation of reproductive parameters and embryo viability. Male rats were euthanized after 66-70 days of dosing and epididymal sperm motility and concentration were assayed. The testes, epididymides, prostate, and seminal vesicles were weighed and microscopically examined. RESULTS: The duration of cohabitation was increased for 100 mg/kg males by 0.7 days. The number of males copulating and the number of implantation sites produced per copulation were reduced in the 10 and 100 mg/kg groups. Weights of the seminal vesicles and epididymides were reduced for all groups, although the testes weight and epididymal sperm motility and concentration were not affected by treatment. There were no microscopic findings in the male reproductive tissues. CONCLUSION: The changes in male fertility and reproductive tissue weights after exposure to lasofoxifene are consistent with those previously described for estrogen receptor-modulating compounds.

Postnatal closure of membranous ventricular septal defects in Sprague-Dawley rat pups after maternal exposure with trimethadione.

BACKGROUND: Congenital membranous ventricular septal defects (VSD) have been shown to close during postnatal development in rats [Solomon et al., Teratology 55:185-194, 1997]. Although they may differ in size, spontaneous and treatment-related VSD are histologically similar; however, the postnatal fate of treatment-induced VSD is not known. The objective of this study was to determine if treatment-induced VSD persist throughout postnatal development. METHODS: Groups of 40 female rats were given oral doses of trimethadione (TMD) at 400 mg/kg/day (200 b.i.d.) or 600 mg/kg/day (300 b.i.d.) on Gestation Days (GD) 9 and 10. Twenty dams in each group were designated for Cesarean section and 20 were allowed to deliver and rear their offspring to Postnatal Day (PND) 21. The integrity of the ventricular septum was evaluated in fetuses (GD 21) and pups (PND 21). RESULTS: The incidence of membranous VSD was 0.6, 7.6, and 49.8% per litter in the Control, 400, and 600 mg/kg groups, respectively, on GD 21. Both the incidence and severity of VSD increased with dose. The VSD at 400 mg/kg were small in size and initially detected by the presence of blood flowing through the defect from the closed right ventricle. In the 600 mg/kg dose group, the VSD, although still membranous, were larger and more readily detected without the need to examine the blood flow. At 600 mg/kg, not only were the VSD larger than those in the Control or the 400 mg/kg group, 10.1% per litter of the affected fetuses had other vessel anomalies associated with the VSD, which were incompatible with pup survival. On PND 21, VSD was noted in 0.3, 0, and 6.4% per litter evaluated in the Control, 400, and 600 mg/kg groups, respectively. This demonstrates that the small, isolated treatment-related VSD can resolve postnatally; however, the closure of the larger or more severe VSD may be prolonged or may not occur at all. Although TMD exposure reduced group mean fetal weights at both dose levels, there was no difference between the mean weight of fetuses with VSD and those fetuses without VSD in the same group. CONCLUSION: Treatment-induced VSD close postnatally, and appears to be a delay in cardiac development not associated with fetal weight. The timing of closure and survivability during closure is dependent on the severity of the VSD. Further characterization of the two sizes of VSD may provide diagnostic clarity; however, the current data support the smaller VSD as a variation with no significant impact on viability and growth, and the more severe VSD to be a malformation.

Developmental toxicity study of dimethylpiperidone.

The potential maternal and developmental toxicity of dimethylpiperidone (DMPD) was assessed in rats. Groups of 25 mated female Crl:CD (SD)IGS BR rats were exposed by inhalation (whole-body exposures) for approximately six hours per day over days 7-21 of gestation (G); day 1G was the day of copulation plug detection. The exposure levels were 0, 52, 260, or 340 (vapor plus aerosol) mg/m3 DMPD. During the in-life portion, body weights, food consumption, and clinical observation data were collected. On day 22G, the dams were euthanized and examined for gross external and internal alterations. The uterine contents were described and the fetuses were weighed and examined for external, visceral, and skeletal alterations. Maternal toxicity was seen at both 260 and 340 mg/m3. At 340 mg/m3, evidence of maternal toxicity included mortality, increased clinical observations, and decreased body weight and food consumption. At 260 mg/m3, maternal toxicity was limited to increased clinical observations and decreased food consumption. Developmental toxicity was also produced at 260 and 340 mg/m3. At 340 mg/m3, evidence of developmental toxicity included decreased fetal weight, increased embryofetal lethality with concomitant reductions in litter size, and increased fetal malformations and variations. At 260 mg/m3, effects in fetuses were limited to slightly decreased fetal weight and increased fetal variations; additionally, one litter from this level consisted entirely of resorptions. There were no compound-related effects in either dams or fetuses at 52 mg/m3. It was, therefore, concluded that DMPD was not selectively toxic to the rat conceptus.