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Ervogastat (PF-06865571) is a small molecule diacylglycerol acyltransferase 2 (DGAT2) inhibitor being developed for the oral treatment of nonalcoholic steatohepatitis (NASH) with liver fibrosis. DGAT2 is a key enzyme in triglyceride synthesis in tissues and in regulating energy metabolism. Fertility and developmental toxicity studies with ervogastat were conducted in female rats and rabbits. There were no effects on female rat fertility or rabbit embryo-fetal development. Administration of ervogastat to pregnant rats during organogenesis reduced fetal weight and caused higher incidences of bent bones in fetuses that were shown to resolve by postnatal day 28 and were therefore considered to be transient variations secondary to developmental delay. Extended dosing in rats through the end of gestation and lactation (pre- and post-natal development study) caused impaired skin development, reduced offspring viability, and growth retardation. The spectrum of developmental effects in rats is consistent with the intended pharmacology (altered triglyceride metabolism) and the transient nature of the skeletal findings, along with the late gestational window of sensitivity for the effects on skin barrier development, reduce the concern for potential adverse developmental effects following unintended early gestational exposure to ervogastat in humans where treatment can be discontinued once pregnancy is determined.

Bococizumab is an anti-PCSK9 monoclonal antibody that was intended for the treatment of hypercholesterolemia. After reviewing the 6-month rat toxicity study data, in which there was a low spontaneous tumor incidence, unrelated to bococizumab administration, the U.S. FDA granted a carcinogenicity waiver request based on a weight-of-evidence assessment of low carcinogenic risk. Subsequently, after reviewing 6-month rat toxicity study data from another anti-PCSK9 antibody, RN317, with a similar low tumor incidence (unrelated to RN317), the U.S. FDA rescinded the bococizumab carcinogenicity study waiver and requested a full 2-year rat carcinogenicity study be conducted. The resulting 2-year carcinogenicity study demonstrated no bococizumab-related increase in tumors, confirming the weight-of-evidence evaluation and alleviating concerns regarding the carcinogenic potential. Here we report the scientific and regulatory background that led to the request for a rat carcinogenicity study, the feedback on the design of the carcinogenicity study, and the results from this study which affirmed the original weight-of-evidence assessment of low carcinogenic risk.

A maternal Group B Streptococcus (GBS) six-valent polysaccharide conjugate vaccine (GBS6) is being developed to protect neonates and infants up to 3 months of age through passive transfer of antibodies from the mother to the infant. Fertility and developmental toxicity studies were conducted in female Sprague Dawley rats and New Zealand White rabbits with GBS6 (20 μg capsular polysaccharide/serotype formulated with or without AlPO4 , the highest clinical dose). Females were administered the full human dose of the GBS6 formulation intramuscularly twice prior to mating and twice during gestation, to ensure that high antibody levels were maintained throughout gestation and lactation. Approximately, half of the rats and rabbits were evaluated at the end of gestation, and the remainder were evaluated at the end of lactation. Maternal blood for GBS6 serology, to measure antibody titers to the GBS6 antigens, was collected prior to the first dose, prior to mating, and at each necropsy. Blood for serology was also collected from offspring at the end of gestation and lactation. In both species, there was no evidence of vaccine-related effects on fertility, embryo-fetal development, or postnatal development of the offspring, supporting regulatory guidance that single-species evaluation would have been sufficient. Functional serum antibodies to all six serotypes in the vaccine were confirmed in maternal animals and functional serum antibodies to one or more of the six serotypes was also confirmed in some rat offspring and most of the rabbit offspring. The results of these studies supported the safety of GBS6 vaccine administration to pregnant women.

Achondroplasia is an autosomal disorder caused by point mutation in the gene encoding fibroblast growth factor receptor 3 (FGFR3) and resulting in gain of function. Recifercept is a potential disease modifying treatment for achondroplasia and functions as a decoy protein that competes for ligands of the mutated FGFR3. Recifercept is intended to restore normal bone growth by preventing the mutated FGFR3 from negative inhibitory signaling in pediatric patients with achondroplasia. Here we evaluated the potential effects of twice weekly administration of recifercept to juvenile cynomolgus monkeys (approximately 3-months of age at the initiation of dosing) for 6-months. No adverse effects were noted in this study, identifying the high dose as the no-observed-adverse-effect-level and supporting the use of recifercept in pediatric patients from birth. Considering that juvenile toxicity studies in nonhuman primates are not frequently conducted, and when they are conducted they typically utilize animals ≥9 months of age, this study demonstrates the feasibility of executing a juvenile toxicity study in very young monkeys prior to weaning.

Acetyl-CoA carboxylase (ACC) is an enzyme within the de novo lipogenesis (DNL) pathway and plays a role in regulating lipid metabolism. Pharmacologic ACC inhibition has been an area of interest for multiple potential indications including oncology, acne vulgaris, metabolic diseases such as type 2 diabetes mellitus, and nonalcoholic fatty liver disease/nonalcoholic steatohepatitis. A critical role for ACC in de novo synthesis of long-chain fatty acids during fetal development has been demonstrated in studies in mice lacking Acc1, where the absence of Acc1 results in early embryonic lethality. Following positive predictions of developmental toxicity in the alternative in vitro assays (positive in murine embryonic stem cell [mESC] assay and rat whole embryo culture, but negative in zebrafish), developmental toxicity (growth retardation and dysmorphogenesis associated with disrupted midline fusion) was observed with the oral administration of the dual ACC1 and 2 inhibitors, PF-05175157, in Sprague Dawley rats and New Zealand White rabbits. The results of these studies are presented here to make comparisons across the assays, as well as mechanistic insights from the mESC assay demonstrating high ACC expression in the mESC and that ACC-induced developmental toxicity can be rescued with palmitic acid providing supportive evidence for DNL pathway inhibition as the underlying mechanism. Ultimately, while the battery of alternative approaches and weight-of-evidence case were useful for hazard identification, the embryo-fetal development studies were necessary to inform the risk assessment on the adverse fetal response, as malformations and/or embryo-fetal lethality were limited to doses that caused near-complete inhibition of DNL.

Acetyl-CoA carboxylase (ACC) catalyses the first step of de novo lipogenesis (DNL). Pharmacologic inhibition of ACC has been of interest for therapeutic intervention in a wide range of diseases. We demonstrate here that ACC and DNL are essential for platelet production in humans and monkeys, but in not rodents or dogs. During clinical evaluation of a systemically distributed ACC inhibitor, unexpected dose-dependent reductions in platelet count were observed. While platelet count reductions were not observed in rat and dog toxicology studies, subsequent studies in cynomolgus monkeys recapitulated these platelet count reductions with a similar concentration response to that in humans. These studies, along with ex vivo human megakaryocyte maturation studies, demonstrate that platelet lowering is a consequence of DNL inhibition likely to result in impaired megakaryocyte demarcation membrane formation. These observations demonstrate that while DNL is a minor quantitative contributor to global lipid balance in humans, DNL is essential to specific lipid pools of physiological importance. Pharmacological targeting of de novo lipogenesis is an attractive clinical target for a wide range of diseases. Kelly et al. report that de novo lipogenesis is essential for platelet production in primates, but not in dogs and rats.

Preclinical and clinical data suggest that acetyl-CoA carboxylase (ACC) inhibitors have the potential to rebalance disordered lipid metabolism, leading to improvements in nonalcoholic steatohepatitis (NASH). Consistent with these observations, first-in-human clinical trials with our ACC inhibitor PF-05175157 led to robust reduction of de novo lipogenesis (DNL), albeit with concomitant reductions in platelet count, which were attributed to the inhibition of fatty acid synthesis within bone marrow. Herein, we describe the design, synthesis, and evaluation of carboxylic acid-based ACC inhibitors with organic anion transporting polypeptide (OATP) substrate properties, which facilitated selective distribution of the compounds at the therapeutic site of action (liver) relative to the periphery. These efforts led to the discovery of clinical candidate PF-05221304 (12), which selectively inhibits liver DNL in animals, while demonstrating considerable safety margins against platelet reduction in a nonhuman primate model.

A maternal Group B Streptococcus (GBS) six-valent polysaccharide conjugate vaccine (GBS6) is being developed to protect neonates and infants up to 3 months of age through passive transfer of antibodies from the mother to the infant. Fertility and developmental toxicity studies were conducted in female Sprague Dawley rats and New Zealand White rabbits with GBS6 (20 μg capsular polysaccharide/serotype formulated with or without AlPO

BNT162b2 is a vaccine developed to prevent coronavirus disease 2019 (COVID-19). BNT162b2 is a lipid nanoparticle formulated nucleoside-modified messenger RNA (mRNA) encoding the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein locked in its prefusion conformation. A developmental and reproductive toxicity study was conducted in rats according to international regulatory guidelines. The full human BNT162b2 dose of 30 μg mRNA/dose (>300 times the human dose on a mg/kg basis) was administered intramuscularly to 44 female rats 21 and 14 days prior to mating and on gestation days 9 and 20. Half of the rats were subject to cesarean section and full fetal examination at the end of gestation, and the other half were allowed to deliver and were monitored to the end of lactation. A robust neutralizing antibody response was confirmed prior to mating and at the end of gestation and lactation. The presence of neutralizing antibodies was also confirmed in fetuses and offspring. Nonadverse effects, related to the local injection site reaction, were noted in dams as expected from other animal studies and consistent with observations in humans. There were no effects of BNT162b2 on female mating performance, fertility, or any ovarian or uterine parameters nor on embryo-fetal or postnatal survival, growth, physical development or neurofunctional development in the offspring through the end of lactation. Together with the safety profile in nonpregnant people, this ICH-compliant nonclinical safety data supports study of BNT162b2 in women of childbearing potential and pregnant and lactating women.

Traditionally, understanding potential developmental toxicity from pharmaceutical exposures has been based on the results of ICH guideline studies in two species. However, support is growing for the use of weight of evidence approaches when communicating the risk of developmental toxicity, where the intended pharmacologic mode of action affects fundamental pathways in developmental biology or phenotypic data from genetically modified animals may increasingly be included in the overall assessment. Since some concern surrounds the use of data from knockout (KO) mice to accurately predict the risk for pharmaceutical modulation of a target, a deeper understanding of the relevance and predictivity of adverse developmental effects in KO mice for pharmacological target modulation is needed. To this end, we compared the results of embryo-fetal development (EFD) studies for 86 drugs approved by the FDA from 2017 to 2019 that also had KO mouse data available in the public domain. These comparisons demonstrate that data from KO mouse models are overall highly predictive of malformations or embryo-fetal lethality (MEFL) from EFD studies, but less so of a negative outcome in EFD studies. This information supports the use of embryo-fetal toxicity data in KO models as part of weight of evidence approaches in the communication of developmental toxicity risk of pharmaceutical compounds.

We previously observed a cutaneous type IV immune response in nonhuman primates (NHP) with the mGlu5 negative allosteric modulator (NAM) 7. To determine if this adverse event was chemotype- or mechanism-based, we evaluated a distinct series of mGlu5 NAMs. Increasing the sp3 character of high-throughput screening hit 40 afforded a novel morpholinopyrimidone mGlu5 NAM series. Its prototype, (R)-6-neopentyl-2-(pyridin-2-ylmethoxy)-6,7-dihydropyrimido[2,1-c][1,4]oxazin-4(9H)-one (PF-06462894, 8), possessed favorable properties and a predicted low clinical dose (2 mg twice daily). Compound 8 did not show any evidence of immune activation in a mouse drug allergy model. Additionally, plasma samples from toxicology studies confirmed that 8 did not form any reactive metabolites. However, 8 caused the identical microscopic skin lesions in NHPs found with 7, albeit with lower severity. Holistically, this work supports the hypothesis that this unique toxicity may be mechanism-based although additional work is require...

Palbociclib is a selective inhibitor of the cyclin-dependent kinase (CDK) 4/6, approved for the treatment of breast cancer. We assessed the potential effects of oral administration of palbociclib on reproduction and development. There were no effects on female or male fertility indices; however, in the male there was seminiferous tubule degeneration in the testes and secondary findings in the epididymides, lower testicular and epididymal weights, sperm density and motility. Palbociclib was not teratogenic in rats or rabbits; however, in the presence of maternal toxicity (lower maternal body weight gain and food consumption), low fetal body weights were observed in rats and small forepaw phalanges were noted in rabbits. There were, however, no adverse effects on the F1 generation in a pre- and post-natal developmental toxicity study in the rat.

This paper provides considerations on approaches to the development of medicines initially developed for pediatric use (ie, "pediatric-first" or "pediatric-only" drugs). The most common development approach for these types of medicines involves a first-in-human (FIH) clinical trial with healthy adult volunteers to assess safety and tolerability. This approach generally requires nonclinical repeat-dose studies in adult animals; safety pharmacology and in vivo genetic toxicology studies in adult animals are also performed for small-molecule drugs. Additional studies in juvenile animals may be required prior to clinical trials in pediatric patients, on a case-by-case basis. In this paradigm, the starting dose for pediatric patients is primarily driven by modeling from the adult pharmacokinetic assessment and pharmacology data. A second development approach is where the FIH clinical trial is conducted in pediatric patients. This approach is generally supported by repeat-dose studies in juvenile animals, with the onset of dosing at ages that developmentally correlate to the age of the pediatric patients. Safety pharmacology and in vivo genetic toxicology studies are generally performed in adult animals for small-molecule drugs. To define a safe yet minimally efficacious starting dose for pediatric patients, various complementing approaches can be used, including human equivalent dose, minimal anticipated biological effect level, and physiologically based pharmacokinetic modeling. Case examples for pediatric-first drug candidates show how both drug development approaches (ie, entry into human first in adults or directly in pediatric populations) are used in the pharmaceutical industry.

To determine the sensitivity of male reproductive toxicity endpoints in NHPs we performed a power analysis of routine and triggered endpoints using control data from sexually mature Asian and Mauritian NHPs. The power to detect a 50% change from control was 13-30% for male reproductive organ weights, ∼30% for testicular volume, 6-66% for seminal analyses and 10-78% for male hormones. Overall, male reproductive endpoints have poor power (less than 80%) to detect a 50% change from control with a group size of 3 monkeys. Confidently identifying adverse male reproductive effects with these endpoints would likely require specialized study designs with larger group sizes. Triggering of non-routine endpoints in cases where there is special concern for male reproductive toxicity is unlikely to increase sensitivity to detect adverse effects.

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.

Regulatory non-clinical safety testing of human pharmaceuticals typically requires embryo-fetal developmental toxicity (EFDT) testing in two species (one rodent and one non-rodent). The question has been raised whether under some conditions EFDT testing could be limited to one species, or whether the testing in a second species could be decided on a case-by-case basis. As part of a consortium initiative, we built and queried a database of 379 compounds with EFDT studies (in both rat and rabbit animal models) conducted for marketed and non-marketed pharmaceuticals for their potential for adverse developmental and maternal outcomes, including EFDT incidence and the nature and severity of adverse findings. Manifestation of EFDT in either one or both species was demonstrated for 282 compounds (74%). EFDT was detected in only one species (rat or rabbit) in almost a third (31%, 118 compounds), with 58% (68 compounds) of rat studies and 42% (50 compounds) of rabbit studies identifying an EFDT signal. For 24 compounds (6%), fetal malformations were observed in one species (rat or rabbit) in the absence of any EFDT in the second species. In general, growth retardation, fetal variations, and malformations were more prominent in the rat, whereas embryo-fetal death was observed more often in the rabbit. Discordance across species may be attributed to factors such as maternal toxicity, study design differences, pharmacokinetic differences, and pharmacologic relevance of species. The current analysis suggests that in general both species are equally sensitive on the basis of an overall EFDT LOAEL comparison, but selective EFDT toxicity in one species is not uncommon. Also, there appear to be species differences in the prevalence of various EFDT manifestations (i.e. embryo-fetal death, growth retardation, and dysmorphogenesis) between rat and rabbit, suggesting that the use of both species has a higher probability of detecting developmental toxicants than either one alone.

INTRODUCTION Human renal development involves two basic processes, the morphological development of kidney cells and structure followed by the acquisition of function. The anatomic formation of the human kidney occurs exclusively in utero. However, the acquisition of adult-like function encompasses in utero and postnatal development, beginning concurrent with formation of the nephron and continuing with rapid attainment of mature functional capability occurring after birth. The processes occurring during morphological and functional development are quite different, but both are susceptible to toxic injury. The extended period of functional kidney development presents the potential for differing vulnerability to toxic insult compared to the adult kidney.

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.

BACKGROUND Sunitinib (SUTENT, Pfizer Inc., New York, NY) is a multitargeted inhibitor of selected receptor tyrosine kinases, which produces an antiproliferative and antiangiogenic effect by blocking pathways fundamental to tumor growth and survival. We investigated the effects of sunitinib on male and female fertility and early embryonic development in the rat. METHODS In the female fertility and early embryonic development phase, untreated males were paired with treated females dosed at 0 (control), 0.5, 1.5, and 5 mg/kg/day from 14 days premating, through mating, to gestation day 7. In the male fertility phase, the same males were then treated 58 days at doses of 0 (control), 1, 3, and 10 mg/kg/day, mated with untreated females, with continued daily dosing for a total of 74 days. RESULTS There was no systemic toxicity- or treatment-related effects on fertility in female rats. Females exposed at 5 mg/kg/day had an increase in the number of early resorptions with associated decrease in viable embryos. In the males, body weight and food consumption were decreased at 10 mg/kg/day compared to the controls. Male reproductive capacity, as assessed by copulation, fertility, and conception indices, was not impacted at any dose level. Sperm morphology, concentration, and motility were also unaffected by treatment. CONCLUSIONS There were no effects on male reproduction. An increase in corpora lutea and an increase in early resorptions with associated reduction in viable embryos was noted in the females dosed 5 mg/kg/day. Sunitinib at doses up to 1.5 and 10 mg/kg/day had no effects on female and male reproduction, respectively.

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.

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.

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 objective of this study was to determine the shortest period of time necessary to detect histologic evidence of estrous cycle disruption in Sprague-Dawley rats treated for up to 28 days with the aromatase inhibitor exemestane at 1,000 mg/kg. Rats were evaluated on day 5, 8, 15, or 29. Vaginal mucification, uterine and cervical epithelial atrophy, uterine luminal epithelial vacuolation, decreased uterine granulocytes, and hypertrophy/hyperplasia of mammary ducts and alveoli were noted by day 5 and persisted throughout the study. From day 8 to day 29, absence of recent basophilic corpora lutea, increased atresia of antral follicles, interstitial cell hyperplasia, and increased luteinized follicles were present in the ovaries of treated rats. Vaginal smears detected persistent diestrus, confirming estrous cycle disruption between days 5 and 8. Ovary and uterine weights were largely unaffected. Serum hormone levels were not useful due to the study design employed. Other effects of exemestane included decreased adrenal weights and decreased cell size in both the adrenal zona fasciculata and the pituitary pars distalis. While early histologic changes were evident on day 5, only after 8 days of treatment were findings considered sufficient to clearly identify exemestane-induced estrous cycle disruption using microscopy alone.

In order to investigate methamphetamine (MA)-induced neurotoxicity, two studies were carried out. In the first study, MA-induced neostriatal monoamine depletions in male and female Sprague-Dawley CD rats were studied under conditions in which the magnitude of MA-induced hyperthermia was comparable between the sexes. MA (5 or 10 mg/kg) or saline (3 ml/kg) was administered s.c. four times at two hr intervals. Rectal temperatures were monitored for 9 hours in a room with an ambient temperature of 23° 2°C. Animals were sacrificed three days post-treatment for the determination of dopamine (DA) and serotonin (5-HT). MA induced significant monoamine reductions but the magnitude of these reductions and of the hyperthermic responses were not significantly different between males and females. Unlike reports in mice, gender does not play a role in MA-induced monoamine reductions in rat neostriatum when MA-induced hyperthermia is comparable across sexes. In the second study, the neurotoxic effects of a single administration of MA were investigated. After a single dose of MA (10, 20, 30, or 40 mg/kg, s.c.) or saline (3 ml/kg) to Sprague-Dawley CD male rats, rectal temperatures were monitored for 9 hours. Neostriatal DA, 5HT, tyrosine hydroxylase (TH), and glial fibrillary acidic protein (GFAP) were assayed 3 days following MA treatment. MA induced significant dose-dependent hyperthermia. MA also induced dose-dependent reductions of DA, 5-HT and TH, and increases of GFAP. These results demonstrate that a single-dose of MA can be as effective as the widely used four-dose regimen, provided ambient temperature is modestly increased. A single-dose model offers advantages for mechanistic studies, especially those employing antagonists or other agents thought to be neuroprotective.

Antimuscarinics are first-line pharmacotherapy for the treatment of overactive bladder. However, because central nervous system cholinergic neurotransmission is involved in cognition, and the central nervous system-permeable antimuscarinics scopolamine and oxybutynin affect memory, cognitive impairment has been noted as a possible side effect of these drugs. We evaluated the effect of tolterodine, an antimuscarinic for overactive bladder, in a mouse passive-avoidance model of memory. Mice were chosen because like humans, mice but not rats, form the pharmacologically active 5-hydroxymethyl metabolite of tolterodine, DD01. In the passive-avoidance test, tolterodine at 1 or 3 mg/kg had no effect on memory; the latency to cross and percentage of animals crossing were comparable to controls. In contrast, scopolamine induced a memory deficit; the latency to cross was decreased, and the number of animals crossing was increased. Therefore, at a dose exceeding therapeutic exposure by six-fold, tolterodine had no effect on memory in the mouse passive-avoidance model, indicating that tolterodine does not disrupt cognitive function in this testing paradigm.

Three orally administered metabotropic glutamate receptor 5 (mGluR5) negative allosteric modulators caused skin lesions consistent with delayed type-IV hypersensitivity in cynomolgus macaques in 2- and 12-week toxicity studies. Several monkeys developed macroscopic skin lesions in multiple locations after 8 to 9 days of dosing; the most prominent effects involved the genital region of males and generalized erythema occurred in both sexes. Microscopic lesions occurred in both clinically affected and unaffected areas and were characterized by lymphocytic interface inflammation, subepidermal bullae, and individual keratinocyte vacuolation/necrosis. In the 12-week study, clinical effects in 2 animals resolved with continued dosing, whereas in others the inflammatory process progressed with 1 female exhibiting systemic lymphocytic inflammation in multiple tissues. The inflammatory infiltrate consisted of CD3 and CD4 positive T lymphocytes with minimal CD68 positive macrophages and only rare CD8 positive T lymphocytes. A subset of animals given a dosing holiday was subsequently rechallenged with similar lesions developing but with a more rapid clinical onset. These skin lesions were consistent with type-IV delayed hypersensitivity with some features comparable to bullous drug eruptions in humans. A relationship between these findings and the intended mode of action for these compounds could not be ruled out, given the occurrence across different chemotypes.

Bococizumab is a humanized monoclonal IgG2Δa antibody against proprotein convertase subtilisin/kexin type 9 (PCSK9) for the treatment of hyperlipidemia. The evaluation of potential effects on embryo-fetal development was conducted in the rat. In a pharmacokinetic/pharmacodynamic study bococizumab was administered intravenously to pregnant Sprague–Dawley (SD) rats (n = 8/group) at 0, 10, 30, and 100 mg/kg during organogenesis. Maternal and fetal bococizumab, total cholesterol and HDL concentrations were determined. Bococizumab was well tolerated and there were no effects on ovarian or uterine parameters. Maternal and fetal bococizumab exposure increased with increasing dose, with a corresponding dose-dependent decrease in fetal cholesterol levels. Maternal cholesterol levels were decreased significantly, with reductions that were of a similar magnitude regardless of dose. In the definitive embryo-fetal development study bococizumab was administered to pregnant SD rats (n = 20/group) at 0, 10, 30, and 100 mg/kg and no adverse maternal or developmental effects were observed up to 100 mg/kg. These studies have provided an appropriate and relevant safety assessment of bococizumab in pregnant rats to inform human risk assessment, demonstrating no adverse effects on embryo-fetal development at magnitudes greater than anticipated clinical exposure and in the presence of maximal reductions in maternal cholesterol and dose-dependent reductions in fetal cholesterol.

BACKGROUND: Appropriate maternal nutrition and body weight gain during pregnancy is well established as a major factor in healthy prenatal development in humans. Given the role of nutrition and body weight gain in normal development, pharmaceuticals intended to reduce appetite and promote weight loss will generate developmental toxicity data that may be challenging to interpret. To aid with this, the effects of feed restriction, and subsequent reduction in maternal body weight gain, on embryo-fetal development was investigated in the rabbit. METHODS: Groups of 15 pregnant New Zealand White rabbits were offered 150 (control), 110, 75, 55, 35, and 15 g feed/day from gestation day (GD) 7–19. Cesarean sections were carried out on GD 29 and fetuses were examined for external, visceral, and skeletal development. RESULTS: Maternal body weights at the end of the feed restriction period (GD 20) were 0.97, 0.98, 0.93, 0.94, and 0.86 × control for the 110, 75, 55, 35, and 15 g feed/day groups, respectively. Only at 15 g feed/day was there a net maternal body weight loss (the GD 20 body weight was 0.93 × the GD 6 body weight) at the end of the feed restriction period. Six does aborted in the 15 g feed/day group; there were no other abortions associated with feed restriction. Fetal body weight was significantly reduced at 75, 55, 35, and 15 g feed/day (0.95, 0.90, 0.86, and 0.84 × control, respectively). There were no external or visceral malformations or variations, and no skeletal malformations associated with feed restriction. The incidence of fetuses with sternebrae 5 or 6 unossified was increased at feed levels ≤75 g/day. At a feed level of 35 g/day there was an increase in unossified metatarsals and metacarpals, and an increase in the number of fetuses with a reduced number of caudal vertebrae ossified. Although these findings were not increased at a feed level of 15 g/day, the lack of dose response was likely due to increased abortion and subsequent decrease in fetuses available for evaluation at 15 g feed/day. CONCLUSION: These data demonstrate that feed restriction to feed levels that produce substantial reductions in maternal body weight gain can result in developmental toxicity expressed by abortion, reduced fetal weight, and alterations in ossification. Abortion only occurred when feed was restricted to an amount that produced maternal body weight loss (15 g feed/day) whereas reduced fetal weight and increased incidence of fetuses with unossified sternebrae, metatarsals, metacarpals, or caudal vertebrae were noted at feed levels of ≤75 g/day. There were no fetal malformations associated with feed restriction. Birth Defects Res B 74:424–430, 2005. © 2005 Wiley-Liss, Inc.

Aspirin (ASA), an irreversible cyclooxygenase (COX) inhibitor, induces ventricular septal defect (VSD) and diaphragmatic hernia (DH) in rat fetuses when administered on gestation days (GDs) 9-10, a critical period for cardiovascular (CV) and midline development. Evaluation of a spectrum of nonsteroidal antiinflammatory drugs (NSAIDs; reversible COX inhibitors) showed that while some NSAIDs induced VSD in rats, none of the NSAIDs evaluated produced DH. In addition to inhibiting COX, ASA also inhibits carbonic anhydrase. The purpose of this study was to determine whether concurrent inhibition of COX and carbonic anhydrase would produce a teratogenic profile that includes both VSD and DH. To inhibit both COX and carbonic anhydrase, ibuprofen (COX inhibitor) and acetazolamide (carbonic anhydrase inhibitor) were coadministered on GDs 9-10. Groups of 20 female Crl:CD(SD)IGS BR rats were given either 300 mg kg(-1) day(-1) ibuprofen, 1000 mg kg(-1) day(-1) acetazolamide, or both (combination of ibuprofen and acetazolamide). Fetuses were evaluated on GD 21 for external and visceral development. Ibuprofen induced VSD in 3.7% of fetuses per litter; no defects in appendicular skeletal development were noted. Acetazolamide induced VSD in 5.9% of the fetuses per litter and appendicular defects in 41% of the fetuses per litter. Coadministration of ibuprofen and acetazolamide produced VSD in 18.7% of the fetuses per litter and appendicular defects in 77% of the fetuses per litter; however, there were no DH. Therefore, while concurrent inhibition of COX and carbonic anhydrase did not produce DH, potentiation was noted for the induction of VSD and appendicular anomalies.

BACKGROUND: Lasofoxifene is a nonsteroidal selective estrogen receptor modulator (SERM). With high affinity to the α and β human estrogen receptors and greater potency than other SERMs, lasofoxifene is potentially a superior treatment for postmenopausal osteoporosis. In light of the known effects of estrogen-modulating compounds on female reproductive indices, two studies were conducted to evaluate the effects of lasofoxifene on female rat cyclicity, reproduction, and parturition. METHODS: One study evaluated effects of lasofoxifene on estrous cyclicity, and the second study assessed effects on implantation and parturition. In the cyclicity study, lasofoxifene was administered to female rats at doses of 0.1, 0.3, and 1.0 mg/kg/day for 14 consecutive days. After treatment, there was a 3-week reversibility phase followed by a mating phase. In the implantation study, lasofoxifene was administered to pregnant female rats at doses of 0.01, 0.03, and 0.1 mg/kg/day for 7 consecutive days (gestation day [GD] 0–6). Some animals were euthanized on GD 21, and the remainder of the group was allowed to deliver the F1 generation. Several developmental indices were evaluated in the F1 pups through post-natal day (PND) 21. RESULTS: In the cyclicity study, all lasofoxifene-treated females were anestrous by Study Day 7 (1.0 mg/kg) or 9 (0.3 and 0.1 mg/kg). The reversibility phase resulted in restoration of normal estrous cycles by the end of 1 (0.1 mg/kg) or 2 weeks (0.3 and 1.0 mg/kg). During the mating phase, no adverse effects occurred in pregnancy success or reproductive parameters. In the implantation study, all doses of lasofoxifene increased pre- and post-implantation losses, increased gestation length, and reduced litter size. None of the developmental parameters measured on the F1 generation was adversely affected. CONCLUSION: Lasofoxifene reversibly altered the estrous cycle and inhibited implantation, consistent with what would be expected from a member of the SERM class. Birth Defects Res B 71:150–160, 2004. © 2004 Wiley-Liss, Inc.

BACKGROUND: Hoshi et al. [Hoshi et al. J Toxicol Sci 10(Suppl):187–255, 1985a,b,c,d] evaluated the potential for hydroxypropyl methylcellulose acetate succinate (HPMCAS) to produce developmental and reproductive toxicity in a series of studies that included rat and rabbit teratology studies, a rat fertility study, and a rat peri- and postnatal study. The authors concluded that there were no compound-related findings. In the cesarean-section phase of the rat teratology study, however, clubfoot was reported for 0.8, 2.1, 5.5, and 4.1% of fetuses in the control, 625, 1250, and 2500 mg/kg groups, respectively. There were no significant increases in external anomalies, but the apparent dose-related increase in clubfoot was not specifically addressed. In the rabbit teratology study, the number of litters evaluated (12–13 per group) was not consistent with current regulatory guidelines. Therefore, to definitively establish the potential of HPMCAS to produce developmental toxicity, embryo/fetal development studies were carried out in rats and rabbits. METHODS: Groups of 20 pregnant Sprague–Dawley rats and New Zealand White rabbits were dosed with 0, 50, 150, 625, or 2500 mg/kg HPMCAS from gestational day (GD) 6–17 or GD 7–19 for rats and rabbits, respectively. Fetuses were collected by cesarean section and examined for external, visceral and skeletal development. RESULTS: No developmental toxicity was observed as a result of HPMCAS exposure demonstrating that maternal HPMCAS exposure during gestation does not induce developmental anomalies. There were no findings of clubfoot or other limb anomalies in these studies at dose levels equivalent to those that were previously associated with a possible increase in clubfoot. CONCLUSIONS: The conclusion of the earlier study indicating that treatment with HPMCAS at doses up to and including 2500 mg/kg did not produce developmental toxicity was confirmed with these studies. It is likely that the clubfoot noted in the earlier rat teratology study was a misdiagnosis or artifact. Birth Defects Res B 68:421–427, 2003. © 2003 Wiley-Liss, Inc.

BACKGROUND: A review of the scientific literature suggested the occurrence of low-level incidences of ventricular septal defect (VSD) and midline defect (MD) in rat fetuses and diaphragmatic hernia (DH), VSD, and MD in rabbit fetuses after maternal exposure to nonsteroidal anti-inflammatory drugs (NSAIDs). Aspirin, an NSAID that irreversibly inhibits cyclooxygenase 1 (COX-1) and COX-2, induces DH, VSD, and MD when administered as one dose during the sensitive periods of development in rats. Unlike aspirin, other NSAIDs, including selective COX-2 inhibitors, reversibly inhibit COX activity. To evaluate whether the dysmorphogenesis observed after maternal NSAID exposure correlates with COX-1 or COX-2 inhibition, a series of compounds with different capacities to inhibit COX-1 and COX-2 were administered to pregnant rats and rabbits during the sensitive period for heart development and midline closure. METHODS: The compounds selected, ranked from the most COX-2 selective to the most COX-1 selective based on COX inhibition in a human whole blood assay, were CJ-19,209, meloxicam, diclofenac, diflunisal, ibuprofen, and ketorolac. Rat dams were treated on gestation days (GDs) 9 and 10, and rabbit does were treated on GDs 9, 10, and 11. The doses selected for evaluation represented the maximum tolerable dose for the compound, with the exception of CJ-19,209, which was dosed at 1000 mg/kg. Fetuses were collected by cesarean section on GDs 21 and 29 for rats and rabbits, respectively, and all fetuses were examined for external and visceral developmental anomalies. RESULTS: In rabbits, diflunisal induced DH, VSD, and MD (omphalocele) and single incidences of VSD and MD (gastroschisis) were noted in the ibuprofen group; no other developmental findings were associated with treatment. In rats, ibuprofen, diflunisal, and ketorolac induced increases in the incidence of VSD. In general, the induction of developmental defects was associated with compounds that selectively inhibit COX-1 or have a high ratio of COX-1 to COX-2 inhibition. CONCLUSIONS: Inhibition of COX-1 may be involved in the disruption of heart development, whereas the selective inhibition of COX-2 (as assessed with CJ-19,209) appears to have no effect on heart development and midline closure in rats and rabbits. Birth Defects Research (Part B) 68: 47–56, 2003. r 2003 Wiley-Liss, Inc.

BACKGROUND: A review of the nonsteroidal anti-inflammatory drug (NSAID) literature suggested occurrences of low-level incidences of cardiovascular and midline defects in rabbit fetuses exposed in utero. Aspirin (acetylsalicylic acid, ASA) is a widely used NSAID that irreversibly inhibits cyclooxygenases (COXs) 1 and 2. ASA has been studied extensively in rats and has consistently increased low-incidence cardiovascular malformations and defects in midline closure. The objectives of the current study were to comprehensively define the developmental toxicology profile of ASA in rabbits by using a dosing paradigm encompassing the period of organogenesis and to test the hypothesis that maternal gastrointestinal toxicity after repeated dose administrations hampers the detection of low-incidence malformations with ASA in rabbits by limiting ASA administration to sensitive windows for cardiovascular development and midline closure. METHODS: ASA was administered to pregnant New Zealand White rabbits from gestation days (GDs) 7 to 19 at dose levels of 125, 250, and 350 mg/kg per day and as single doses of 500, 750, or 1000 mg/kg on GD 9, 10, or 11. Cesarean sections were performed on GD 29, and the fetuses were examined for external, visceral, and skeletal development. RESULTS: In the repeated dose study, maternal toxicity was exhibited in the 250- and 350-mg/kg per day groups by mortality and decreased food consumption and body weight gain. In the single dose studies, maternal toxicity was exhibited at all doses by reductions in body weight gain and food consumption for 3 days after treatment. Fetal body weight was significantly reduced in the repeated dose study at 350 mg/kg per day. Fetal weights were not affected by single doses of ASA on GD 9, 10, or 11. There were no treatment-related external, visceral, or skeletal malformations associated with ASA administration throughout organogenesis or with single doses administered during critical developmental windows. CONCLUSION: These findings supported previous work demonstrating that ASA is not teratogenic in rabbits, as opposed to rats, even when large doses are administered on single days during specific windows of development. Birth Defects Research (Part B) 68:38–46, 2003. © 2003 Wiley-Liss, Inc.

Peroxisome proliferators are a class of nongenotoxic rodent hepatocarcinogens that cause peroxisome proliferation and liver tumors when administered to rats and mice; but other species, including guinea pigs, dogs, and primates are less sensitive or refractory to the induction of peroxisome proliferation. Therefore, rodent peroxisome proliferators are not believed to pose a hepatocarcinogenic hazard to humans. Some peroxisome proliferators produce developmental toxicity in rats that is expressed as suppressed postnatal growth. To evaluate the relevance of the rat developmental effect to primates, groups of 4 lactating female Rhesus monkeys and their infants were exposed for 6 h/day, 7 days/week for 3 weeks to air or 1000 ppm HCFC-123. Animals were evaluated for clinical signs, body weights, clinical pathology parameters, and biochemical and pathological evaluations of liver biopsy samples. The effect of HCFC-123 exposure on milk quality (protein and fat concentration) was evaluated. The concentrations of HCFC-123 and the major metabolite, trifluoroacetic acid (TFA), were measured in the blood of the mothers and infants and in the milk. Exposure of monkeys to 1000 ppm HCFC-123 did not result in exposure-related clinical observations, or changes in body weight, appetence and behavior. There were no exposure-related effects on serum triglycerides, cholesterol, or glucose levels. HCFC-123 and TFA were present in milk, although maternal HCFC-123 exposure did not affect milk protein and fat content. In general, HCFC-123 was not detected in maternal or infant blood. TFA was detected in the majority of the mothers and TFA levels in infants ranged from 2 to 6 times higher than levels in the corresponding maternal blood. A pharmacokinetic analysis in a maternal animal indicated a peak concentration of TFA at approximately 1 h post-exposure, with a half-life of approximately 20 h. Liver microsomal P450 and peroxisome oxidase activities showed exposure-related decreases in CYP4A1 and CYP2E1 and acyl-CoA oxidase for animals exposed to HCFC-123. Microscopic evaluation of maternal liver from HCFC-123 exposed animals revealed mild to moderate centrilobular hepatocyte vacuolation, trace to mild centrilobular necrosis, and trace to mild subacute inflammation. The histopathological damage and altered hepatic biochemical activities produced by HCFC-123 in monkeys are not consistent with the HCFC-123 peroxisome proliferation response observed in rat livers. These findings demonstrate that HCFC-123 is not a peroxisome proliferator in adult Rhesus monkeys and postnatal exposure to HCFC-123 does not affect body weight of nursing infant monkeys.

Prenatal cocaine treatment produces equivocal effects on spatial learning and memory; however, no data are available on neonatal treatment as a model of human third-trimester exposure. Sprague-Dawley rats were treated on postnatal days (P) 1-10 or 11-20 with cocaine (15 mg/kg x 4 per day at 2-h intervals) or saline (P1-P20) and evaluated as adults in the Morris water maze and on tests of activity, startle, scent marking, swimming immobility, and sequential learning. Neonatal cocaine had no effect on mortality; however, early treatment reduced body weight, whereas later treatment did not. Neonatal cocaine had no effects on exploratory activity, swimming ability, sequential learning, multiday activity rhythms, scent marking, or swimming immobility, but augmented acoustic startle amplitude in the early-treated group. Neonatal cocaine also produced an interaction on spatial learning in which the cocaine early-treated males performed slightly more efficiently than controls. Plasma cocaine concentrations were significantly higher in the early-treated group than the later-treated group despite receiving the same weight-adjusted doses. It was concluded that neonatal cocaine, when administered during a stage of brain development analogous to human third trimester, induces few behavioral effects based on the assessments used in this study.

The neurotoxic potential of cocaine when administered under conditions conducive to the initiation of hyperthermia was investigated. Rats were administered cocaine at ambient temperatures of 22°C or 30°C. To determine the thermal response, body temperatures were measured every 30 min and the total thermal response (TTR), representing the area under the temperature vs. time curve, was calculated. Saline administered at 22°C or 30°C resulted in a normal thermal response (TTR = 9.8 ± 0.9 and 11.2 ± 0.9, respectively). Cocaine administration resulted in ambient temperature-dependent hyperthermia. Cocaine (4 × 25 mg/kg) administered at 22°C resulted in a TTR of 15.1 ± 0.9 whereas cocaine (4 × 15 or 25 mg/kg) administered at 30°C resulted in TTRs of 22.2 ± 0.9 and 21.9 ± 0.8, respectively. Regardless of the dose or thermal response, cocaine administration did not result in depletion of dopamine (DA) or serotonin (5-HT) in the caudate-putamen. Cocaine administration also failed to induce an increase in the concentration of glial fibrillary acidic protein (GFAP), a marker for neurotoxicity. These results demonstrate that hyperthermia does not promote cocaine-induced neurotoxicity in the rat caudate-putamen.

Methamphetamine (MA)-induced monoamine depletions in male and female Sprague–Dawley CD rats were studied under conditions in which the magnitude of MA-induced hyperthermia was comparable between the sexes. MA (5 or 10 mg/kg) or saline (3 ml/kg) was administered SC four times at 2-h intervals. Animals were sacrificed 3 days posttreatment for the determination of dopamine (DA), serotonin (5-HT), and metabolites. MA induced significant monoamine reductions but the magnitude of these reductions was not significantly different between males and females. In the MA 5 mg/kg groups, neostriatal DA content was reduced by 51.2% and 44.8%, whereas 5-HT content was reduced by 30.6% and 23.9% of controls for males and females, respectively. In the MA 10 mg/kg groups, neostriatal DA content was reduced by 72.9% and 65.8%, whereas striatal 5-HT content was reduced by 77.4% and 73.6% of controls for males and females, respectively. No significant differences in thermal responses to MA were observed between genders. Unlike reports in mice, gender does not play a role in MA-induced monoamine reductions in rat neostriatum when MA-induced hyperthermia is comparable across sexes. The data also showed a threshold effect in which dopamine depletions were not correlated with hyperthermia at core body temperatures above approximately 41°C.

Methamphetamine (MA) administration to adult rats (4 × 10 mg/kg s.c.) induces neurotoxicity predominately characterized by a persistent reduction of neostriatal dopamine (DA) content. Hyperthermia following MA administration potentiates the resulting DA depletion. DA-derived free radicals are postulated to be a mechanism through which MA-induced neurotoxicity is produced. The spin trapping agent PBN reacts with free radicals to form nitroxyl adducts, thereby preventing damaging free radical reactions with cellular substrates. MA with saline pretreatment (Sal-MA) reduced neostriatal DA by 55% (P < 0.01 vs. Sal-Sal). MA with PBN pretreatment (PBN-MA) at 36 or 60 mg/kg reduced neostriatal DA by 36 and 22%, respectively (P < 0.05 and P < 0.01 vs. Sal-MA) indicating partial protection. PBN pretreatment did not alter MA-induced hyperthermia. Thus, PBN does not attenuate MA-induced neurotoxicity by reducing MA-induced hyperthermia. These results support a role for free radicals in the generation of MA-induced dopaminergic neurotoxicity. © 1996 Wiley-Liss, Inc.

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.

Sunitinib (SUTENT, Pfizer Inc., New York, NY) is a multitargeted inhibitor of selected receptor tyrosine kinases, which produces an antiproliferative and antiangiogenic effect by blocking pathways fundamental to tumor growth and survival. We investigated the effects of sunitinib on male and female fertility and early embryonic development in the rat.In the female fertility and early embryonic development phase, untreated males were paired with treated females dosed at 0 (control), 0.5, 1.5, and 5 mg/kg/day from 14 days premating, through mating, to gestation day 7. In the male fertility phase, the same males were then treated 58 days at doses of 0 (control), 1, 3, and 10 mg/kg/day, mated with untreated females, with continued daily dosing for a total of 74 days.There was no systemic toxicity- or treatment-related effects on fertility in female rats. Females exposed at 5 mg/kg/day had an increase in the number of early resorptions with associated decrease in viable embryos. In the males, body weight and food consumption were decreased at 10 mg/kg/day compared to the controls. Male reproductive capacity, as assessed by copulation, fertility, and conception indices, was not impacted at any dose level. Sperm morphology, concentration, and motility were also unaffected by treatment.There were no effects on male reproduction. An increase in corpora lutea and an increase in early resorptions with associated reduction in viable embryos was noted in the females dosed 5 mg/kg/day. Sunitinib at doses up to 1.5 and 10 mg/kg/day had no effects on female and male reproduction, respectively.

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.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.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.The results of these studies indicate that lersivirine did not exhibit any evidence of teratogenicity in rabbits.

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.

Administration of methamphetamine (MA) induces degeneration of dopaminergic nerve terminals and astrogliosis, such as hypertrophy and an increase in apparent number, in the neostriatum. In this experiment adult rats were treated with MA (10 mg/kg, i.p.) 4 times in one day at 2 h intervals. Amfonelic acid (AFA), a dopamine reuptake inhibitor, was administered (20 mg/kg, i.p.) at the same time the last MA dose was given. Three days later, dopaminergic terminals and astrocytes were examined immunohistochemically and the contents of striatal dopamine and its metabolites were analyzed by HPLC. The results showed that MA-induced the typical depletion of dopaminergic terminals, reduction of dopamine content and astrogliosis in the neostriatum. AFA treatment completely prevented the effects of MA on the dopaminergic system, both morphologically and biochemically. However, the reaction of astrocytes remained in the region where the most severe depletion of dopaminergic terminals was seen in MA treated animals (ventral-lateral portion of neostriatum). The results support the concept that the dopamine transporter is involved in MA-induced dopaminergic nerve terminal degeneration. The results also indicate that blocking the dopamine transporter cannot completely prevent the reaction of astrocytes in the neostriatum, which indicates that the astrocytic reaction can be induced by factors other than degeneration of dopaminergic terminals in this region. Based on these and other data, it is hypothesized that MA may cause degeneration of corticostriatal glutamate pathways and this effect may be responsible for the astrogliosis in MA-AFA treated animals.

Animal and care use practices are constantly evolving. These can have unexpected consequences on the data collected from such procedures. One example is the recent change in our animal facility, based on recommendations from the Newcastle Consensus Meeting on Carbon Dioxide Euthanasia of Laboratory Animals, from CO(2) to isoflurane for anesthesia. The current study was conducted to determine the effects of isoflurane on sperm motility, as compared to two different CO(2) euthanasia procedures. Sperm motility was evaluated after euthanasia by a standard 5-minute CO(2) euthanasia procedure, an extended 10-minute CO(2) euthanasia procedure, or by isoflurane anesthesia followed by exsanguination (iso/exsanguination). The 5-minute CO(2) procedure produced sperm motility of 94.3 ± 1.7% motile sperm with 65.6 ± 16.8 sperm/field. By comparison, iso/exsanguination reduced that count to 3.3 ± 2.3 sperm/field and only 60.7 ± 32.0% motile sperm. The reduction in sperm motility after iso/exsanguination appeared to have been due primarily to the reduction in the number of sperm expelled from the vas deferens (3.3), compared to that after 5-minute CO(2) (65.6). This reduction in number of sperm available for evaluation, in the presence of a constant level of background debris, which was counted by the computer optics system as nonmotile sperm, resulted in an apparent reduction in motility. Using the extended 10-minute CO(2) procedure produced sperm data in between the other two extremes: 77.6 ± 36.1% motile sperm with 34.6 ± 28.3 sperm/field. The results of this study support the hypothesis that isoflurane inhibits contraction of the smooth muscle of the vas deferens, resulting in a decreased number of expelled sperm. Given these findings, it is important that careful consideration be taken to select an appropriate anesthesia/euthanasia method.

BACKGROUND: CP-601927 is a selective α4β2 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. Birth Defects Res (Part B) 92:323–332, 2011. © 2011 Wiley-Liss, Inc.