Long-Acting Tumor-Activated Prodrug of a TGFβR Inhibitor.

Inhibition of TGFβ signaling in concert with a checkpoint blockade has been shown to provide improved and durable antitumor immune response in mouse models. However, on-target adverse cardiovascular effects have limited the clinical use of TGFβ receptor (TGFβR) inhibitors in cancer therapy. To restrict the activity of TGFβR inhibitors to tumor tissues and thereby widen the therapeutic index, a series of tumor-activated prodrugs of a selective small molecule TGFβR1 inhibitor were prepared by appending to a serine protease substrate and a half-life extension fatty acid carbon chain.

Concordance of 3 alternative teratogenicity assays with results from corresponding in vivo embryo-fetal development studies: Final report from the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) DruSafe working group 2.

An IQ DruSafe working group evaluated the concordance of 3 alternative teratogenicity assays (rat whole embryo culture, rWEC; zebrafish embryo culture, ZEC; and murine embryonic stem cells, mESC) with findings from rat or rabbit embryo-fetal development (EFD) studies. Data for 90 individual compounds from 9 companies were entered into a database. In vivo findings were deemed positive if malformations or embryo-fetal lethality were reported in either species.

TGFβ2 and TGFβ3 mediate appropriate context-dependent phenotype of rat valvular interstitial cells.

This study focused on characterizing the potential mechanism of valvular toxicity caused by TGFβ receptor inhibitors (TGFβRis) using rat valvular interstitial cells (VICs) to evaluate early biological responses to TGFβR inhibition. Three TGFβRis that achieved similar exposures in the rat were assessed. Two dual TGFβRI/-RII inhibitors caused valvulopathy, whereas a selective TGFβRI inhibitor did not, leading to a hypothesis that TGFβ receptor selectivity may influence the potency of valvular toxicity.

T4-mediated rescue of aortic malformations in hypothyroid rats indicates maternal thyroid status can affect great vessel development.

Pexacerfont is a corticotrophin-releasing factor subtype 1 receptor (CRF-1) antagonist developed for potential treatment of anxiety and stress-related disorders. In male rats, pexacerfont caused hepatic enzyme induction leading to increased thyroxine (T4) clearance. When administered to pregnant rats on gestation day 6 to 15, pexacerfont at 300 mg/kg/day (30× mean AUC in humans at 100 mg/day) produced similar effects on thyroid homeostasis with serum T4 and thyroid-stimulating hormone levels that were 0.

Discovery of BMS-986260, a Potent, Selective, and Orally Bioavailable TGFβR1 Inhibitor as an Immuno-oncology Agent.

Novel imidazole-based TGFβR1 inhibitors were identified and optimized for potency, selectivity, and pharmacokinetic and physicochemical characteristics. Herein, we report the discovery, optimization, and evaluation of a potent, selective, and orally bioavailable TGFβR1 inhibitor, (BMS-986260). This compound demonstrated functional activity in multiple TGFβ-dependent cellular assays, excellent kinome selectivity, favorable pharmacokinetic properties, and curative efficacy in combination with anti-PD-1 antibody in murine colorectal cancer (CRC) models.

Intermittent dosing of the transforming growth factor beta receptor 1 inhibitor, BMS-986260, mitigates class-based cardiovascular toxicity in dogs but not rats.

Small-molecule inhibitors of transforming growth factor beta receptor 1 (TGFβRI) have a history of significant class-based toxicities (eg, cardiac valvulopathy) in preclinical species that have limited their development as new medicines. Nevertheless, some TGFβRI inhibitors have entered into clinical trials using intermittent-dosing schedules and exposure limits in an attempt to avoid these toxicities. This report describes the toxicity profile of the small-molecule TGFβRI inhibitor, BMS-986260, in rats and dogs.

Discovery of 4-Azaindole Inhibitors of TGFβRI as Immuno-oncology Agents.

The multifunctional cytokine TGFβ plays a central role in regulating antitumor immunity. It has been postulated that inhibition of TGFβ signaling in concert with checkpoint blockade will provide improved and durable immune response against tumors. Herein, we describe a novel series of 4-azaindole TGFβ receptor kinase inhibitors with excellent selectivity for TGFβ receptor 1 kinase.

In Vitro Developmental Toxicology Screens: A Report on the Progress of the Methodology and Future Applications.

There has been increasing focus on generation and assessment of in vitro developmental toxicology models for assessing teratogenic liability of chemicals. The driver for this focus has been to find reliable in vitro assays that will reduce or replace the use of in vivo tests for assessing teratogenicity. Such efforts may be eventually applied in testing pharmaceutical agents where a developmental toxicology assay or battery of assays may be incorporated into regulatory testing to replace one of the two species currently used in teratogenic assessment.

A Developmental Toxicology Assay Platform for Screening Teratogenic Liability of Pharmaceutical Compounds.

Increasing need for proactive safety optimization of pharmaceutical compounds has led to generation and/or refinement of in vitro developmental toxicology assays. Our laboratory has developed three in vitro developmental toxicology assays to assess teratogenic liability of pharmaceutical compounds. These assays included a mouse molecular embryonic stem cell assay (MESCA), a dechorionated zebrafish embryo culture (ZEC) assay, and a streamlined rat whole embryo culture (rWEC) assay.

Optimization and Performance Assessment of the Chorion-Off [Dechorinated] Zebrafish Developmental Toxicity Assay.

The Dechorinated Zebrafish Embryo Developmental toxicity assay was originally developed from a training set of 31 compounds and reported to be 87% concordant with in vivo teratogenicity data (Brannen, K. C., Panzica-Kelly, J.

Fishing for teratogens: a consortium effort for a harmonized zebrafish developmental toxicology assay.

A consortium of biopharmaceutical companies previously developed an optimized Zebrafish developmental toxicity assay (ZEDTA) where chorionated embryos were exposed to non-proprietary test compounds from 5 to 6 h post fertilization and assessed for morphological integrity at 5 days post fertilization. With the original 20 test compounds, this achieved an overall predictive value for teratogenicity of 88% of mammalian in vivo outcome [Gustafson, A. L.

The rat whole embryo culture assay using the Dysmorphology Score system.

The rat whole embryo culture (WEC) system has been used extensively for characterizing teratogenic properties of test chemicals. In this chapter, we describe the methodology for culturing rat embryos as well as a new morphological score system, the Dysmorphology Score (DMS) system for assessing morphology of mid gestation (gestational day 11) rat embryos. In contrast to the developmental stage focused scoring associated with the Brown and Fabro score system, this new score system assesses the respective degree of severity of dysmorphology, which delineates normal from abnormal morphology of specific embryonic structures and organ systems.

Establishment of a molecular embryonic stem cell developmental toxicity assay.

The mouse embryonic stem cell test (EST) is a 10-day screen for teratogenic potential developed to reduce animal use for embryotoxicity testing of chemicals (Spielmann, 2005; Spielmann et al., 1997). In this study, we used the cytotoxicity IC(50) values and transcriptional expression changes as primary endpoints in a shorter 4-day version of the EST, the molecular embryonic stem cell assay.

Zebrafish embryo developmental toxicology assay.

A promising in vitro zebrafish developmental toxicology assay was generated to test compounds for their teratogenic potential. The assay's predictivity is approximately 87% in AB strain fish (Brannen KC et al., Birth Defects Res B Dev Reprod Toxicol 89:66-77, 2010).

Development of a streamlined rat whole embryo culture assay for classifying teratogenic potential of pharmaceutical compounds.

This study describes a novel rat whole embryo culture (rWEC) teratogenicity assay that applies a simplified experimental design and statistical prediction model, resulting in reduced animal requirements and increased throughput with low prediction error rate for classifying teratogenic potential of compounds. A total of 70 compounds (38 teratogens and 32 nonteratogens) were evaluated, and the prediction model was generated from a dataset of 59 compounds. The rWEC assay requires only one test concentration (1μM) and three structural endpoints (group average morphological scores of spinal cord, heart, and number of somite pairs), which are used in a recursive partition model for classifying teratogenic liability.

Inter-laboratory assessment of a harmonized zebrafish developmental toxicology assay - progress report on phase I.

This report provides a progress update of a consortium effort to develop a harmonized zebrafish developmental toxicity assay. Twenty non-proprietary compounds (10 animal teratogens and 10 animal non-teratogens) were evaluated blinded in 4 laboratories. Zebrafish embryos from pond-derived and cultivated strain wild types were exposed to the test compounds for 5 days and subsequently evaluated for lethality and morphological changes.

A dysmorphology score system for assessing embryo abnormalities in rat whole embryo culture.

Background: The rodent whole embryo culture (WEC) system is a well-established model for characterizing developmental toxicity of test compounds and conducting mechanistic studies. Laboratories have taken various approaches in describing type and severity of developmental findings of organogenesis-stage rodent embryos, but the Brown and Fabro morphological score system is commonly used as a quantitative approach. The associated score criteria is based upon developmental stage and growth parameters, where a series of embryonic structures are assessed and assigned respective scores relative to their gestational stage, with a Total Morphological Score (TMS) assigned to the embryo.

In vitro developmental toxicology assays: A review of the state of the science of rodent and zebrafish whole embryo culture and embryonic stem cell assays.

In vitro developmental model systems have been an important tool for advancing basic research in the embryology and teratology fields. The rat and zebrafish embryo models have had broad utility in both fields for many decades. Furthermore embryonic stem cells, applied as a basic research tool, have broad applications across the development fields and many other fields including cancer, regeneration and epigenetic research.

Development of a zebrafish embryo teratogenicity assay and quantitative prediction model.

Background: A zebrafish (Danio rerio) teratogenicity assay has been developed and evaluated for its ability to predict the teratogenic potential of chemicals.

Methods: Zebrafish embryos were dechorionated and then exposed to a test solution from 4-6 hours post-fertilization, and embryos or larvae were assessed up to 5 days post-fertilization (dpf) for viability and morphology. In preliminary experiments, the potential time points for assessment of compound-induced dysmorphology and general toxicity parameters were evaluated, and 5 dpf was found to be the optimum developmental stage for evaluation.

Predictive teratology: teratogenic risk-hazard identification partnered in the discovery process.

Unexpected teratogenicity is ranked as one of the most prevalent causes for toxicity-related attrition of drug candidates. Without proactive assessment, the liability tends to be identified relatively late in drug development, following significant investment in compound and engagement in pre clinical and clinical studies. When unexpected teratogenicity occurs in pre-clinical development, three principle questions arise: Can clinical trials that include women of child bearing populations be initiated? Will all compounds in this pharmacological class produce the same liability? Could this effect be related to the chemical structure resulting in undesirable off-target adverse effects? The first question is typically addressed at the time of the unexpected finding and involves considering the nature of the teratogenicity, whether or not maternal toxicity could have had a role in onset, human exposure margins and therapeutic indication.

State of the art in developmental toxicity screening methods and a way forward: a meeting report addressing embryonic stem cells, whole embryo culture, and zebrafish.

A meeting was convened so that users of three models for in vitro developmental toxicity (embryonic stem cells, whole embryo culture, and zebrafish) could share their experiences with each model, and explore the areas for improvement. We present a summary of this meeting and the recommendations of the group.

Alternative experimental approaches for interpreting skeletal findings in safety studies.

Standard evaluations for characterizing selective developmental toxicity are traditionally undertaken in vivo. These studies incur significant cost in animal use, labor and compound, ultimately limiting the selection of compounds that can be evaluated in vivo. Such limitations hinder the ability to address questions regarding whether teratogenic outcome was caused by intended pharmacology or attributed to off-target effects associated with the structure of the small molecule.

SB-236057: Critical window of sensitivity study and embryopathy of a potent musculoskeletal teratogen.

Background: SB-236057 is a potent skeletal teratogen in rodents and rabbits. The study objective was to identify the critical developmental window of compound sensitivity and to characterize the early onset of SB-236057 embryopathy.

Methods: SB-236057 was orally administered to Sprague Dawley dams at 100 mg/kg/day on days 6-7, 8-11, 12-14, or 15-17 postcoitus (pc).

Evidence for a molecular mechanism of teratogenicity of SB-236057, a 5-HT1B receptor inverse agonist that alters axial formation.

Background: SB-236057 is a potent skeletal teratogen in rodents and rabbits, producing axial and posterior somite malformations in cultured rat embryos. The compound shares some structural similarity to cyclopamine.

Methods: M13 phage display was used to identify amino acid motifs with binding affinity to SB-236057.