Reproductive Toxicity Studies Research Articles

MON-010 Effects of Delta-9-Tetrahydrocannabinol (THC) on Oocyte Competence and Early Embryonic Development

Cannabis is the highest used recreational drug amongst individuals of reproductive age. Fertility clinics advise against cannabis use when undergoing fertility treatments, but the literature backing this statement is weak. This rise in cannabis use has occurred simultaneously with the increase in the percentage of the main psychoactive component of cannabis, delta-9 tetrahydrocannabinol (THC) (1). Current literature suggests that THC mimics the effects of endogenous cannabinoids, binding to cannabinoid receptor 1 (CB1), which has been identified in reproductive tissues (2).Our research aims to study the impact of THC on oocyte maturation and pre-implantation embryonic development. An in vitro bovine system was used as it is the most appropriate translational model to humans for in vitro reproductive toxicity studies. Bovine oocytes were collected and matured under five treatment groups: control, vehicle (1:1:18 ethanol: TWEEN: saline), low THC (0.032uM), mid THC (0.32uM) and high THC (3.2uM). These doses mimic plasma concentrations reached after therapeutic (0.032uM) or low/high recreational (0.32uM and 3.2uM) cannabis use (3). We hypothesise that THC affects oocyte competence and proper early embryonic development in vitro.A negative THC dose-dependent response in cleavage rate was observed, with the highest THC group cleaving at 70.2% rate compared to 86.8% and 85.5% of control and vehicle groups, respectively (p<0.0001, n=7). There was no significant difference in blastocyst rate, suggesting that oocyte THC exposure affects the numbers of oocytes capable of development, but those able to cleave will properly reach blastocyst stage. We analyzed changes in gene expression, i) by a full RNA transcriptome analysis (24,128 transcripts screened) and ii) by quantification of Connexin 37 (CX37) and 43 (CX43) mRNA levels. Connexin expression is correlated to oocyte competence (4). RNA transcriptome analysis showed 62 genes that were significantly downregulated only in the low THC group. CX mRNA levels were measured via droplet digital PCR in both cumulus-oocyte complexes (COCs) and blastocysts. No significant differences were detected in blastocysts, however, a significant decrease in both CX37 and CX43 levels was measured in the low THC group in COCs (p<0.05, n= 9). Differences seen exclusively at the low THC dose suggest a role of THC as partial agonist of CB1.This research aims to understand the effects of cannabis on fertility, as current knowledge during pre-implantation development is limited, making it difficult for physicians to properly advise patients undergoing IVF. Reference: (1) ElSohly et al., Biol Psychiatry. 2016 Apr 1;79(7):613–9. (2) El-Talatini et al., PLoS ONE. 2009 Feb 24;4(2):e4579. (3) Whan et al., Fertil Steril. 2006 Mar;85(3):653–60. (4) Wang et al., Am J Physiol Endocrinol Metab. 2012 Jun 15;302(12):1511–8

Evaluation of 2-methoxy-4-nitroaniline (MNA) in hypersensitivity, 14-day subacute, reproductive, and genotoxicity studies

2-Methoxy-4-nitroaniline (MNA), an intermediate in the synthesis of azo dyes used in textiles and paints, is structurally similar to carcinogenic anilines. Human exposure occurs primarily in the occupational setting through handling of dye dust, and through use and disposal of MNA-containing products. MNA has been reported to induce contact hypersensitivity in a human, myocardial necrosis in rats, and bacterial mutagenicity. This study assessed the subacute toxicity, genotoxicity, contact hypersensitivity, and reproductive toxicity of MNA in rodents in an effort to more fully characterize its toxicological profile. B6C3F1/N mice were exposed to 0, 650, 1250, 2500, 5000, or 10,000 ppm MNA by dosed feed for 14-days to evaluate subacute toxicity and histopathological endpoints. In female mice, decreased body weight (13.5 %) and absolute kidney weight (14.8 %), compared to control, were observed at 10,000 ppm MNA; increased relative liver weight (10–12 %), compared to control, occurred at 5,000–10,000 ppm MNA. In male mice, absolute (15 %) and relative liver weights (9–13 %) were increased at 2,500−5,000 ppm and 1250–10,000 ppm MNA, compared to control, respectively. In both sexes of mice, minimal elevations of hemosiderin pigmentation (a breakdown product of erythrocytes), relative to control, were observed in the liver (10,000 ppm); minimal to moderate elevations of hemosiderin pigmentation (5,000–10,000 ppm) and minimal increases in hematopoietic cell proliferation occurred in the spleen (≥ 1250 ppm). In a reproductive toxicity study, timed-mated female Harlan Sprague Dawley rats were exposed to 0–10,000 ppm MNA by dosed feed from gestation day 6 through postnatal day (PND) 21. Decreases in mean litter weights were observed at 5000 ppm MNA, compared to control, beginning at PND1. To evaluate potential contact hypersensitivity, MNA (2.5–50 %, in dimethylformamide) was applied to the dorsa of both ears of female Balb/c mice once daily for three days. The increase observed in lymph node cell proliferation (10–50 % increase in thymidine uptake compared to control) did not reproducibly achieve the Sensitization Index (SI) 3 level, and there was no ear swelling evident following sensitization with 10–50 % MNA and challenge with 25 % MNA in the mouse ear swelling test. In bacterial mutagenicity assays, MNA (250−1000 μg/plate) induced significant increases, compared to control, in mutant colonies with and without metabolic activation enzymes in Salmonella typhimurium strains TA100 and TA98. These data indicate that MNA is genotoxic, and may induce erythrocyte damage and reactive phagocytosis by macrophages in the liver and spleen.

Rethinking toxicity testing: Influence of aging on the outcome of long-term toxicity testing and possible remediation

Traditionally, toxicity testing is conducted at fixed dose rates (i.e., mg/kg/day) without considering life-changing events, e.g., stress, sickness, aging- and/or pregnancy-related changes in physical, physiological and biochemical parameters. In humans, life-changing events may cause systemic dose non-proportionality requiring modulation of drug dosage; similar changes occur in animals altering systemic dose during chronic/carcinogenic testing leading to “late-occurring” effects in some studies. For example, propylene monomethyl ether, an industrial chemical, initially induced sedation in rats and mice with recovery upon induction of hepatic CYPs after ~1 week. Sedation reappeared in rats but not in mice after ~12 months of exposure due to decreased CYP activity in rats, elderly mice were able to maintain slightly higher CYP activity avoiding recurrence of sedation. The systemic dose of two pharmaceuticals (doxazosin and brimonidine tartrate) increased up to 6-fold in ≥12-month old rats with no toxicity. In a rat reproductive toxicity study, systemic dose of 2,4-D, an herbicide, rapidly increased due to increased consumption of 2,4-D-fortified diet during pregnancy, lactation and neonatal growth, requiring adjustment to maintain the targeted systemic dose. Ideally, toxicological studies should be based on systemic dose with the option of modulating external dose rates to maintain the targeted systemic dose. Systemic dose can easily be monitored in selected core study animals at desired intervals considering recent developments in sampling and analysis at a fraction of the overall cost of a study.

Short-term toxicogenomics as an alternative approach to chronic in vivo studies for derivation of points of departure: A case study in the rat with a triazole fungicide

The derivation of an apical endpoint point of departure (POD) from animal-intensive testing programs has been the traditional cornerstone of human health risk assessment. Replacement of in vivo chronic studies with novel approaches, such as toxicogenomics, holds promise for future alternative testing paradigms that significantly reduce animal testing. We hypothesized that a toxicogenomic POD following a 14 day exposure in the rat would approximate the most sensitive apical endpoint POD derived from a battery of chronic, carcinogenicity, reproduction and endocrine guideline toxicity studies. To test this hypothesis, we utilized myclobutanil, a triazole fungicide, as a model compound. In the 14 day study, male rats were administered 0 (vehicle), 30, 150, or 400 mg/kg/day myclobutanil via oral gavage. Endpoints evaluated included traditional apical, hormone, and liver and testis transcriptomic (whole genome RNA sequencing) data. From the transcriptomic data, liver and testis biological effect POD (BEPOD) values were derived. Myclobutanil exposure for 14 days resulted in increased liver weight, altered serum hormones, liver histopathology, and differential gene expression in liver and testis. The liver and testis BEPODs from the short-term study were 22.2 and 25.4 mg/kg/day, respectively. These BEPODs were approximately an order of magnitude higher than the most sensitive apical POD identified from the two year cancer bioassay based on testis atrophy (1.4 mg/kg/day). This study demonstrates the promise of using a short-term study BEPOD to derive a POD for human health risk assessment while substantially reducing animal testing.

Effects of endocrine disruptor furan on reproductive physiology of Sprague Dawley rats: An F1 Extended One-Generation Reproductive Toxicity Study (EOGRTS).

The present study investigated the reproductive toxicity of furan in an Extended One-Generation Reproductive Toxicity Study in rats. Sprague Dawley F0 weaning rats (30 per sex per group) were exposed to furan orally at 0, 1, 2.5, 5, and 10 mg kg-1 for 10 weeks (males) and 2 weeks (females) and then mated. Results of F0 indicated that in the furan-treated groups (5 mg kg-1 and 10 mg kg-1), body weight (bw) gain decreased during prebreed and gestational period while increased during lactation periods. F0 animals prebreeding exposure resulted in head tilt and foot splay at 10 mg kg-1. Number of live pups at birth were decreased (p < 0.001) at 10 mg kg-1. At postnatal day (PND) 70, a significant (p = 0.03) decrease in testosterone levels of male rats and estrogen levels of female rats (p = 0.05) was observed in 10 mg kg-1 furan-treated group in F1 generation. Luteinizing hormone, follicle-stimulating hormone, and progesterone levels were also reduced, but their reduction was not statistically significant in all groups. In higher dose furan group (10 mg kg-1), testicular and ovarian weights were reduced in F1 generation at PND 70, with decreased daily sperm production (p = 0.01) and disturbed estrous cyclicity (p < 0.01). Some histopathological changes were also observed in testis and ovaries in groups whose parents were previously exposed to 10 mg kg-1 bw of furan group. Based on the above results, it is suggested that exposure to food-based contaminant furan induced remarkable changes in the F0 (parental stage) and F1 (offspring, pubertal, and adult stage) generations of Sprague Dawley rats.

Is there a rationale for direct dosing of chemicals to nursing pups in the EOGRTS (OECD 443)?

In OECD guideline 443 – Extended One Generation Reproductive Toxicity Study (EOGRTS) – to be used for testing industrial and agrochemicals, it has been indicated that careful consideration of benefits and disadvantages should be made prior to conducting direct-dosing studies in nursing pups. Nursing pups will not be directly dosed in dietary and drinking water studies whereas in oral gavage studies this possibility exists. Besides the risk of intubation trauma and overdosing due to direct exposure and exposure via the mother's milk, direct dosing could lead to a different hazard assessment of chemicals depending on the choice of the route of administration. In addition, in case of industrial and agrochemicals used in industrial or professional settings only, there will never be direct exposure of newborns. Moreover, direct dosing of nursing pups is an artificial, non-physiological, route of exposure and as such it would hamper risk assessment. It should therefore only be considered in exceptional cases and justified on a case-by-case approach.

Rat two-generation reproductive toxicity and teratogenicity studies of a novel coccidiostat – Ethanamizuril

In the current study, to support the safety assessment of ethanamizuril as a new potent anticoccidial agent of triazine compounds, a reproductive toxicity and teratogenic potential assay of ethanamizuril was investigated. Groups of 30 males and 30 females were administered 0, 0.02, 0.1 or 0.2 mg/ml ethanamizuril by gastric incubation through a 10-week prebreed period as well as during mating, gestation, parturition and lactation in any generation. Compared to the control group, no test compound-related changes in copulation index, fertility index, gestation length, litter size, pup weight, pup sex ratio, pup viability, epididymal sperm counts or motility or other functional reproductive measures were noted in any generation, except few significant changes in high dose group in the number of sperm motility at III level in F0 males and the body weights of GD14 and GD21 in F1 rats. There were no compound-related necropsy findings or effects on organ weight. Histopathologic examinations revealed no evidence of compound-related changes in any organs including the reproductive organs of both sexes. In conclusion, long-term administrated 0.2, 1.0 and 2.0 mg/kg of ethanamizuril by means of oral gavage did not affect the reproduction of Sprague-Dawley rats and the development ability of their offspring under the experimental conditions.

Absence of developmental and reproductive toxicity in animals exposed to dolutegravir

The success of new antiretroviral medicines for HIV resulted in a change to guidelines of standard therapy where continuation of antiretroviral therapy is recommended to maintain the low viral load during pregnancy, thereby preventing transmission of the virus to the fetus. As a result, pregnancy related exposure to HIV medicines has increased. Understanding the safety of these medicines during pregnancy is of paramount importance to ensure health of mothers and their offspring; well-designed animal studies that evaluate the reproductive life cycle play a key role in this effort. As part of the medicine development program for dolutegravir (DTG), a series of reproductive and developmental toxicity studies were conducted using rats and rabbits. In a fertility study, where exposure to DTG occurred in female rats before mating through conception and up to implantation of the embryo, no effects on reproductive cycles, ovulation, fertility) or preimplantation embryonic growth were observed. In rat and rabbit embryo-fetal development studies, where exposure to DTG occurred during organogenesis, no malformations or other developmental abnormalities were observed. In a rat pre- and post-natal development study, where DTG exposure to the pups occurred during pregnancy and postnatally via milk, no malformations or other developmental abnormalities were observed. In these studies, no DTG-related effects occurred on fertility, embryonic (pre- and post-implantation loss, resorptions, abortions, and malformations) or fetal development where the multiples of exposure at the maximum recommended human dose were up to 27 times higher in rats or below the human exposure in rabbits.

A critical Assessment of the Genotoxicity Profile of the Fungicide Tricyclazole.

Tricyclazole (8-methyl-[1,2,4]triazolo[3,4-b][1,3]benzothiazole) is a fungicide used globally on rice for treatment of the seasonal rice blast disease. Human exposure to this fungicide can occur via dietary and nondietary routes. In a battery of in vitro assays, tricyclazole did not induce gene mutations in bacteria (Ames test) or at the Hprt locus of CHO cells. It was also negative for the induction of micronuclei in human lymphocyte cultures and unscheduled DNA synthesis (UDS) in primary rat hepatocyte. Paradoxically, tricyclazole induced a mutagenic response at the Tk locus of the mouse lymphoma L5178Ycells (MLA), which occurred equally among small/large colony phenotypes. Selection of preexisting mutants leading to a false-positive response in the MLA was ruled out in follow-up experiments. In vivo, tricyclazole was negative in the rat liver UDS assay, mouse bone micronucleus test and a transgenic (MutaMouse) gene mutation assay in glandular stomach, liver, and kidney. Other supporting evidence for the lack of genotoxicity for tricyclazole comes from an in vivo study for sister chromatid exchanges in Chinese hamsters, and a dominant lethal test in the male germ cells of mice. The combined evidence from the genotoxicity studies together with the evidence from toxicokinetic, carcinogenicity, developmental, and reproductive toxicity studies confirm that mutagenicity does not occur in relevant in vivo systems. Data were also compared to potential animal and human exposure, mechanistic data on biological targets and data on analogues, confirming adequacy of the available data for hazard identification and risk assessment. Environ. Mol. Mutagen. 61:300-315, 2020. © 2019 Wiley Periodicals, Inc.

Developmental and reproductive toxicity studies of BIA 10-2474

A series of regulatory studies were carried out to investigate the effects of the FAAH inhibitor BIA 10–2474 on fertility, embryo-fetal toxicity and pre- and post-natal development in rats and rabbits. Despite some reductions in sperm count in rats from 50 mg/kg, there were no major changes in male fertility up to 100 mg/kg. In female rats administered up to GD6, there were increases in pre-implantation loss at 50 and 100 mg/kg but neither post-implantation loss nor early embryonic development was affected. In contrast, when administered to female rats during pregnancy (GD6-GD17), BIA 10–2474 at 75 mg/kg/day reduced food consumption resulting in weight loss, increased post-implantation loss and reduced mean fetal body weight. In rabbits, the same maternal toxicity was seen but there were no effects in this species on post-implantation loss or fetal body weights. There were no teratological effects clearly due to BIA 10–2474 and developmental milestones and behavior of offspring were not affected. When administered during pregnancy and lactation (GD6-PND20), some post-implantation loss was seen from 20 mg/kg/day, but developmental milestones and behavior of the offspring were not affected, although males tended to have lower body weight. Based on these data the NOAEL for parental fertility was established as 50 mg/kg/day, the maternal NOAEL during pregnancy was 25 mg/kg/day in rats and developmental NOAEL was 25 and 75 mg/kg/day in rats and rabbits, respectively. When administered during post-natal development to rats the maternal NOAEL was 6 mg/kg/day. The parental reproductive NOAEL, the NOAEL for viability and growth of the F1 offspring, the F1 parental NOAEL and the F1 reproductive NOAEL were all considered to be 20 mg/kg/day.

“Simulect” as a model compound for assessing placental transfer of monoclonal antibodies in minipigs

The aim of this study was to directly test and measure in vivo, if placental transfer of monoclonal antibodies takes place in pregnant Göttingen Minipigs to assess their suitability for reproductive assessment of therapeutic monoclonal antibodies.Simulect®, an approved anti CD25 (anti IL-2 receptor alpha) chimeric monoclonal IgG1 antibody, was used as a model monoclonal antibody. Maternal systemic exposure and potential placental transfer of Simulect® to fetuses were investigated following 4 weekly bolus intravenous administration of 5.0 mg/kg from gestation day (GD) 79 or 80 (e.g GD 79, 86, 93 and 100) and with terminal Caesarean section on GD 108 or GD 109 respectively. Results clearly showed exposure in maternal animals, detectable compound in the amniotic fluid from one out of 9 maternal animals, but no exposure in fetuses confirming absence of placental transfer of the selected model antibody Simulect® in minipigs. The absence of Simulect® in the fetuses further supports that the presence of Simulect® in the amniotic fluid in one maternal animal was likely due to contamination with maternal blood during sampling. The demonstrated absence of fetal exposure clearly indicates that, the minipig is not a suitable species for conduct of reproductive toxicity studies with monoclonal antibodies.

Evaluation of Resmethrin Toxicity to Neonatal Testes in Organ Culture.

Resmethrin is a widely used pyrethroid insecticide, which causes low toxicity in mammals. However, its toxicity in testes has not been fully investigated. Therefore, we evaluated the toxicity of resmethrin in mouse testes using an in vitro organ culture. Mouse testicular fragments (MTFs) derived from neonates were cultured in medium containing resmethrin for 30 days. Effects on spermatogenesis in the cultured testes were investigated as functions of both time and dose. Resmethrin significantly downregulated the transcription levels of marker genes for spermatogonia and the number of spermatogenic germ cells relative to those of the controls, according to quantitative PCR and immunostaining. In addition, spermatocyte was observed in the control, but not in 50 μM resmethrin-exposed cultures. Levels of the SYCP3 meiotic marker and phosphorylated H2AX decreased by resmethrin treatment, as observed by Western blotting. Toxic or apoptotic effects of resmethrin in Sertoli and Leydig cells from MTFs were not observed by immunostaining and Tunnel assay. No changes in the expression of steroidogenic enzymes were noted. Apoptosis was only detected in the germ cells of resmethrin-treated MTFs. Thus, the highest dose of resmethrin tested (50 μM) completely inhibited spermatogenesis, because of apoptosis of germ cells and spermatocytes. Although the in vivo toxicity of resmethrin has not yet been studied in detail, significant evidence for cytotoxicity was observed in our organ cultures. This methodological approach is useful for the study of reproductive toxicity before proceeding to animal models, as it greatly reduces the use of laboratory animals.

Developmental and reproductive toxicity studies in Sprague-Dawley rats and New Zealand white rabbits with palbociclib

Developmental and reproductive toxicity studies in Sprague-Dawley rats and New Zealand white rabbits with palbociclib

The Contribution of In Vivo Mammalian Studies to the Knowledge of Adverse Effects of Radiofrequency Radiation on Human Health.

The proliferation of cellular antennas and other radiofrequency radiation (RFR) generating devices of the last decades has led to more and more concerns about the potential health effects from RFR exposure. Since the 2011 classification as a possible carcinogen by the International Agency for Research on Cancer (IARC), more experimental studies have been published that support a causal association between RFR exposure and health hazards. As regard cancer risk, two long-term experimental studies have been recently published by the US National Toxicology Program (NTP) and the Italian Ramazzini Institute (RI). Despite important experimental differences, both studies found statistically significant increases in the development of the same type of very rare glial malignant tumors. In addition to carcinogenicity, reproductive organs might be particularly exposed, as well as sensitive to RFR. In this work, we reviewed the currently available evidence from in vivo studies on carcinogenicity and reproductive toxicity studies in order to summarize the contribution of experimental research to the prevention of the adverse effects of RFR on human health.

Determination of ethanamizuril, a novel triazine coccidiostat, in rat plasma by ultra-performance liquid chromatography system-tandem mass spectrometry and its application in a toxicological study.

Ethanamizuril is a new triazine compound that has the potential to be a novel anticoccidial drug. Toxicological studies in experimental rats were performed to understand the safety profile of ethanamizuril for drug product development. In this study, a novel, selective and accurate ultra-performance liquid chromatography tandem mass spectrometry method has been developed for the determination of ethanamizuril concentrations in rat plasma. With 4-nitro-o-cresol as an internal standard, sample pretreatment involved a one-step extraction with acetonitrile of 100 μL plasma. The detection was carried out by electrospray ionization mass spectrometry in negative ion mode with selected ion recording. The standard curves were linear (r2 ≥ 0.999) over the concentration range of 0.1-100 μg/mL. The relative standard deviations of intra- and inter-day precisions were less than 8.4 and 8.87%, respectively. The mean extraction recovery of ethanamizuril from rat plasma was 97.68-102.57%. The method was fully validated and successfully applied to monitor plasma concentrations of ethanamizuril in a short-term toxicity study and two-generation reproduction toxicity study. The result of the study confirmed that the elimination of ethanamizuril in rats is slow.

New toxicity testing of PQ grass allergy immunotherapy to support product development.

PQ Grass represents an allergen-specific immunotherapy for pre-seasonal treatment of patients with seasonal allergic rhinitis (or rhinoconjunctivitis) with or without mild-to-moderate bronchial asthma. It consists of a native pollen extract for 13 grass species, chemically modified with glutaraldehyde, and adsorbed to l-tyrosine in a microcrystalline form with addition of the adjuvant Monophosphoryl Lipid A (MPL® ). Previous non-clinical safety testing, including rat repeat dose toxicity in adult and juvenile animals, rat reproductive toxicity and rabbit local tolerance studies showed no safety findings of concern. A new Good Laboratory Practice compliant rat subcutaneous repeat dose toxicity study to evaluate a higher clinical dose and modified posology (once every 2weeks for 13weeks) showed no signs of toxicity. As seen in previous studies, relatively minor, immunostimulatory effects were seen such as reversible increased white cell count (notably neutrophils), increased globulin level (resulting in decreased A/G ratio) and increased fibrinogen as well as minor dose site reaction in the form of inflammatory cell infiltrate. These findings are likely due to the immunostimulatory nature of MPL and/or the presence of l-tyrosine within the adjuvanted vaccine. This new toxicity study with PQ Grass therefore supports longer posology with higher dose levels.

Exposure assessments in reproductive and developmental toxicity testing: An IQ-DruSafe industry survey on current practices and experiences in support of exposure-based high dose selection

The draft ICH S5(R3) guideline includes an exposure-based endpoint as an option for selecting the high dose in developmental and reproductive toxicity (DART) studies. In 2016, IQ DruSafe conducted an anonymous survey to identify industry practices and experiences related to pharmacokinetic assessments in DART studies in order to facilitate a pragmatic data-driven approach to development of an acceptable multiple of the clinical exposure to be proposed for dose selection in the guideline. Questions in the survey were designed to explore pharmacokinetic differences in pregnant versus non-pregnant animals, and to assess exposure levels attained in the absence of maternal toxicity as well as DART outcomes in animal studies associated with those exposures. Small molecule and therapeutic proteins were analyzed separately. The key findings for small molecules were: a) differences in exposures between pregnant and non-pregnant animals were generally ≤3-fold, b) Cmax or AUC exposures ≥25-fold the clinical exposure were achieved in the absence of maternal toxicity for 31% and 23% of rat and rabbit developmental toxicity studies, respectively, and c) only 3.3% (5/153) and 1.6% (2/128) of the developmental toxicity studies were positive for malformations or embryofetal lethality in rats and rabbits, respectively, that were not observed until exposure margins were ≥25-fold.

Bioanalytical assays in support of tanezumab developmental and reproductive toxicity studies: challenges and learnings.

Bioanalytical challenges were encountered during developmental and reproductive toxicity studies of tanezumab in cynomolgus monkeys. Possible changes in breast milk composition over the postpartum period potentially complicated assessment of tanezumab concentration in this matrix, requiring validation of the quantification assay across different time intervals. Immunogenicity assessment in maternal serum was complicated by apparent increases in the incidence of antidrug antibody-positive results in treatment-naive samples as pregnancy progressed that were due to changes in the concentration of nerve growth factor, tanezumab's target protein. This was overcome by employing gestational day-specific cut points throughout pregnancy.Researchers should recognize potential challenges associated with dynamic matrices/physiological conditions and anticipate that assays developed under normal conditions may require adaptation for specialized situations.

A new split-luciferase complementation assay identifies pentachlorophenol as an inhibitor of apoptosome formation.

The expense and time required for in vivo reproductive and developmental toxicity studies have driven the development of in vitro alternatives. Here, we used a new in vitro split luciferase‐based assay to screen a library of 177 toxicants for inhibitors of apoptosome formation. The apoptosome contains seven Apoptotic Protease‐Activating Factor‐1 (Apaf‐1) molecules and induces cell death by activating caspase‐9. Apaf‐1‐dependent caspase activation also plays an important role in CNS development and spermatogenesis. In the in vitro assay, Apaf‐1 fused to an N‐terminal fragment of luciferase binds to Apaf‐1 fused to a C‐terminal fragment of luciferase and reconstitutes luciferase activity. Our assay indicated that pentachlorophenol (PCP) inhibits apoptosome formation, and further investigation revealed that PCP binds to cytochrome c. PCP is a wood preservative that reduces male fertility by ill‐defined mechanisms. Although the data show that PCP inhibited apoptosome formation, the concentration required suggests that other mechanisms may be more important for PCP's effects on spermatogenesis. Nonetheless, the data demonstrate the utility of the new assay in identifying apoptosome inhibitors, and we suggest that the assay may be useful in screening for reproductive and developmental toxicants.

MON-102 Nonclinical Development of AZP-531 (Livoletide): A Peptide Analog of Unacylated Ghrelin for the Treatment of Hyperphagia in Prader-Willi Syndrome

Prader-Willi syndrome (PWS) is a rare and complex endocrine disease characterized by hyperphagia and abnormal food-related behaviors that contribute to severe morbidity and early mortality and to a significant burden on patients and caregivers. Hyperghrelinemia may be involved in the underlying mechanisms of hyperphagia. While ghrelin, the most potent appetite-stimulating hormone, is present at higher concentrations in the plasma of PWS patients compared to age-matched controls, detailed studies of ghrelin and unacylated ghrelin (UAG) reveal a relative deficit of UAG in hyperphagic individuals. UAG is a 28-amino-acid peptide that does not bind the growth hormone secretagogue receptor (GHSR), in contrast to acylated ghrelin. UAG has intrinsic activities that often counteract effects of ghrelin, and exerts its actions through a GHSR-independent mechanism. Livoletide is a cyclic 8-amino-acid analogue of UAG with improved plasma stability and pharmacokinetics. The objective of this nonclinical safety program was to support the clinical development of livoletide including a pivotal Phase 2b/3 clinical trial in patients with PWS. The program was designed to define the safety pharmacology and the chronic toxicologic and toxicokinetic profile, and identify parameters for clinical monitoring of potential adverse effects. Genotoxicity, safety pharmacology, reproductive toxicity, and repeat-dose 13-week toxicology studies were all completed. In the in vivo studies, livoletide was administered subcutaneously consistent with the clinical route of delivery. Livoletide was not cytotoxic or genotoxic. Safety pharmacology studies indicated no treatment-related effects on major physiological systems. Results from preliminary embryo-fetal developmental toxicity studies in rat and rabbit indicated that livoletide at high multiples of the anticipated human exposure is not associated with adverse maternal toxicity, embryo-fetal toxicity or teratogenic potential when administered throughout the period of organogenesis. Repeat-dose toxicity studies of up to 13 weeks’ duration in rats and dogs demonstrated that livoletide is very well-tolerated, with no evidence of systemic toxicity. Cumulative data indicated that livoletide has a wide safety margin relative to planned clinical exposures. The highest chronic doses tested were 75 mg/kg in rat and 30 mg/kg in dog; these were considered to be the NOAELs. These dose levels provided AUC values of ≥50-fold the intended clinical systemic exposure (~1200 ng·h/mL). No anti-livoletide antibodies were detected in any of the toxicology studies. These results confirm the favorable long-term safety profile of livoletide and support the subcutaneous administration of the highest anticipated human clinical dose in the Phase 2b/3 study.