New approach methodologies to confirm developmental toxicity of pharmaceuticals based on weight of evidence.

The aim of embryo-fetal developmental toxicity assessments for pharmaceuticals is to inform potential risk of adverse pregnancy outcome, which has traditionally relied on studies in pregnant animals. Recent updates to international safety guidelines (ICH S5R3) have incorporated information on how to use weight of evidence and alternative assays to reduce animal use while still informing risk of fetal harm. Uptake of these alternative approaches has been slow due to limitations in understanding how alternative assays translate to in vivo effects and then relevance to human exposure. To understand the predictivity of new approach methodologies for developmental toxicity (DevTox NAMs), we used two pharmaceutical examples (glasdegib and lorlatinib) to illustrate the value of DevTox NAMs to complement weight of evidence (WoE) assessments while considering the relationship of concentration-effect levels in NAMs to in vivo studies. The in vitro results generated in a battery of assays (mEST, rWEC, zebrafish, and human based stem cells) confirmed the WoE based on literature and further confirmed by preliminary embryo-fetal development data. The data generated for these two compounds supports integrating DevTox NAMs into the developmental toxicity assessment for advanced cancer indications.

Juvenile toxicity study of PF-07256472/recifercept, a recombinant human soluble fibroblast growth factor receptor 3, in 2-3-month-old cynomolgus monkeys.

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.

Regulatory Experience Assessing the Carcinogenic Potential of a Monoclonal Antibody Inhibiting PCSK9, Bococizumab, Including a 2-Year Carcinogenicity Study in Rats.

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.

Inhibition of Acetyl-CoA Carboxylase Causes Malformations in Rats and Rabbits: Comparison of Mammalian Findings and Alternative Assays.

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.

Reproductive toxicity assessment of sunitinib, a multitargeted receptor tyrosine kinase inhibitor, in male and female rats.

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.

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

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

Histologic and cytologic detection of endocrine and reproductive tract effects of exemestane in female rats treated for up to twenty-eight days.

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.

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

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