Differential Analysis of Cereblon Neosubstrates in Rabbit Embryos Using Targeted Proteomics.

Targeted protein degradation is the selective removal of a protein of interest through hijacking intracellular protein cleanup machinery. This rapidly growing field currently relies heavily on the use of the E3 ligase cereblon (CRBN) to target proteins for degradation, including the immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide which work through a molecular glue mechanism of action with CRBN. While CRBN recruitment can result in degradation of a specific protein of interest (e.g., efficacy), degradation of other proteins (called CRBN neosubstrates) also occurs. Degradation of one or more of these CRBN neosubstrates is believed to play an important role in thalidomide-related developmental toxicity observed in rabbits and primates. We identified a set of 25 proteins of interest associated with CRBN-related protein homeostasis and/or embryo/fetal development. We developed a targeted assay for these proteins combining peptide immunoaffinity enrichment and high-resolution mass spectrometry and successfully applied this assay to rabbit embryo samples from pregnant rabbits dosed with three IMiDs. We confirmed previously reported in vivo decreases in neosubstrates like SALL4, as well as provided evidence of neosubstrate changes for proteins only examined in vitro previously. While there were many proteins that were similarly decreased by all three IMiDs, no compound had the exact same neosubstrate degradation profile as another. We compared our data to previous literature reports of IMiD-induced degradation and known developmental biology associations. Based on our observations, we recommend monitoring at least a major subset of these neosubstrates in a developmental test system to improve CRBN-binding compound-specific risk assessment. A strength of our assay is that it is configurable, and the target list can be readily adapted to focus on only a subset of proteins of interest or expanded to incorporate new findings as additional information about CRBN biology is discovered.

Reversible effects on female rat fertility with abrocitinib, a Janus kinase 1 inhibitor.

BACKGROUND: Abrocitinib is a Janus kinase (JAK) 1 selective inhibitor approved for the treatment of atopic dermatitis. Female reproductive tissues were unaffected in general toxicity studies, but an initial female rat fertility study resulted in adverse effects at all doses evaluated. A second rat fertility study was conducted to evaluate lower doses and potential for recovery. METHODS: This second study had 4 groups of 20 females each administered abrocitinib (0, 3, 10, or 70 mg/kg/day) 2 weeks prior to cohabitation through gestation day (GD) 7. In addition, 2 groups of 20 rats (0 or 70 mg/kg/day) were dosed for 3 weeks followed by a 4-week recovery period before mating. All mated females were evaluated on GD 14. RESULTS: No effects were observed at ≤10 mg/kg/day. At 70 mg/kg/day (29x human exposure), decreased pregnancy rate, implantation sites, and viable embryos were observed. All these effects reversed 4 weeks after the last dose. CONCLUSIONS: Based on these data and literature on the potential role of JAK signaling in implantation, we hypothesize that these effects may be related to JAK1 inhibition and, generally, that peri-implantation effects such as these, in the absence of cycling or microscopic changes in nonpregnant female reproductive tissues, are anticipated to be reversible.

The Postnatal Resolution of Developmental Toxicity Induced by Pharmacological Diacylglycerol Acyltransferase 2 (DGAT2) Inhibition During Gestation in Rats.

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.

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.

Knockout mouse models are predictive of malformations or embryo-fetal death in drug safety evaluations.

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.

Knockout mouse models are predictive of malformations or embryo-fetal death in drug safety evaluations.

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.

A multi-institutional study benchmarking the zebrafish developmental assay for prediction of embryotoxic plasma concentrations from rat embryo-fetal development studies.

Predicting embryotoxicity of pharmaceutical compounds or industrial chemicals is crucial for public safety. Conventional studies which monitor embryo-fetal development in rats and rabbits are costly and time consuming. Alternative assays which are simpler and less costly are being pursued. The purpose of this research was to assess the capacity for the zebrafish development assay to predict mammalian plasma levels that are embryotoxic. Previously published data on rat plasma levels associated with embryotoxicity were used to guide concentration ranges for each of 25 chemicals dissolved in the media bathing developing zebrafish embryos. Embryotoxic media concentrations were compared to embryotoxic rat plasma concentrations. Assays were conducted in parallel at multiple sites as a consortium effort through the Health and Environmental Sciences Institute (HESI). Considering results from all sites, the zebrafish embryo development assay predicted (within 1-log) the rat maternal exposure levels associated with embryotoxicity 75% of the time.