Several new approach methodologies (NAMs) for developmental toxicity (Dev Tox) testing are being used by pharmaceutical companies for derisking or for exploring Dev Tox mechanisms. Regulatory adoption of these NAMs-based approaches as being adequate for Dev Tox risk assessment has been more challenging, due, in part, to dynamic changes in the conceptus and placenta throughout development and the impact of the pharmaceutical on the mother's physiology, which may also have an embryo-fetal impact. Still, there is currently a recognition by Health Authorities that there are certain contexts-of-use under which Dev Tox NAMs can provide information that is adequate to inform risk. This has been adopted in the 3rd revision of the ICH S5 guideline, which provides a path to qualify Dev Tox NAMs for regulatory decision making. Despite this opportunity, pharmaceutical companies rarely submit Dev Tox NAMs to Health Authorities for qualification or with intent to support regulatory decision making. This may be in part due to the need for a greater understanding of the biological relationship between currently available Dev Tox NAMs and in vivo outcome, applicability domain, translatability, predictivity, and that these NAMs do not cover the complete scope of embryo-fetal development. Furthermore, there is a lack of Dev Tox NAMs data visibility to Health Authorities. To use Dev Tox NAMs for regulatory decision making, more data sharing with Health Authorities and further understanding the applicability domain of these methodologies are needed.

Per-and polyfluoro alkyl substances (PFAS), also known as "forever chemicals", are deemed as highly toxic with similar toxicological mode-of-action (MoA) and potency. However, varying carbon chain length and functional head-group of PFAS can affect their physicochemical properties, resulting in different toxicological properties. To assess PFAS toxicological MoA and to distinguish between high toxic PFAS and the low-toxic analogs, we tested a set of eight PFAS with varying carbon chain length (C2-C10) in the ToxProfiler assay. ToxProfiler is a human in vitro assay containing seven fluorescent reporters to visualize and quantify activation of the major cellular stress pathways: oxidative stress, cell cycle stress, endoplasmic reticulum (ER) stress, autophagy, ion stress, protein stress and inflammation. In addition, we evaluated teratogenicity potential of long-chain PFAS perfluorooctanoic acid (PFOA; C8), and the ultrashort-chain PFAS trifluoroacetic acid (TFA; C2) in ReproTracker, a human induced pluripotent stem cell (hiPSCs)-based assay in which differentiation into cardiomyocytes, hepatocytes, and neural rosettes is followed to identify developmental toxicity hazards of new drugs and chemicals. In this study, we identified long-chain PFAS (C8-C10), such as PFOA (C8) to be more cytotoxic than ultrashort-chain PFAS and to predominantly induce ER and oxidative stress at 130 mu M. PFAS with a carbon chain length of C4-C7 primarily induced autophagy (300 mu M) in ToxProfiler. Ultrashort-chain PFAS trifluoroacetic acid (TFA; C2) and perfluoropropionic acid (PFPrA; C3) did not activate any of the ToxProfiler stress response reporters and were not cytotoxic at their maximum tested concentrations (10 mM). In concordance, exposure of differentiating cells to PFOA in ReproTracker led to a concentration-dependent decrease in the hepatocyte-specific and neuroectodermal biomarker genes and disrupted their morphology at 30 and 60 mu M, respectively. TFA had no significant effect on biomarker expression, nor on the morphology/functionality of the three differentiated cells. Altogether, we demonstrated that the carbon chain length of PFAS can determine their in vitro toxicity and ultrashort-chain PFAS (TFA) were found to be less toxic when compared to long-chain PFAS.

IntroductionExposure to teratogenic compounds during pregnancy can lead to significant birth defects. Given the considerable variation in drug responses across species, along with the financial and ethical challenges associated with animal testing, the development of advanced human-based in vitro assays is imperative for effectively identifying potential human teratogens. Previously, we developed a human induced pluripotent stem cells (hiPSCs)-based biomarker assay, ReproTracker, that follows the differentiation of hiPSCs into hepatocytes and cardiomyocytes. The assay combines morphological profiling with the assessment of time-dependent expression patterns of cell-specific biomarkers to detect developmental toxicity responses.MethodsTo further increase the predictability of the assay in identifying potential teratogens, we added differentiation of hiPSCs towards neural rosette-like cells. We evaluated the performance of the extended assay with a set of 51 well-known in vivo teratogens and non-teratogens, including the compounds listed in the ICH S5 reference list.ResultsThe optimized assay correctly identified (neuro)developmental toxicants that were not detected in the hepatocyte and cardiomyocyte differentiation assays. These compounds selectively downregulated gene and protein expression of the neuroectodermal marker PAX6 and/or neural rosette marker NESTIN in a concentration-dependent manner and disrupted the differentiation of hiPSCs towards neural rosette-like cells. Overall, based on the current dataset, the addition of neural commitment improved the assay accuracy (from 72.55% to 86.27%) and sensitivity (from 67.50% to 87.50%), when compared to the previously described assay.DiscussionIn summary, trilineage differentiation expanded the spectrum of teratogenic agents detectable by ReproTracker, making the assay an invaluable tool for early in vitro teratogenicity screening.

In vitro chemical safety assessment often relies on simple and general cytotoxicity endpoint measurements and fails to adequately predict human toxicity. To improve the in vitro chemical safety assessment, it is important to understand the underlying mechanisms of toxicity. Here we introduce ToxProfiler, a novel human-based reporter assay that quantifies the chemical-induced stress responses at a single-cell level and reveals the toxicological mode-of-action (MoA) of novel drugs and chemicals. The assay accurately measures the activation of seven major cellular stress response pathways (oxidative stress, cell cycle stress, endoplasmic reticulum stress, ion stress, protein stress, autophagy and inflammation) that play a role in the adaptive responses prior to cellular toxicity. To assess the applicability of the assay in predicting the toxicity MoA of chemicals, we tested a set of 100 chemicals with well-known in vitro and in vivo toxicological profiles. Concentration response modeling and point-of-departure estimation for each reporter protein allowed for chemical potency ranking and revealed the primary toxicological MoA of chemicals. Furthermore, the assay could effectively group chemicals based on their shared toxicity signatures and link them to specific toxicological targets, e.g. mitochondrial toxicity and genotoxicity, and different human pathologies, including liver toxicity and cardiotoxicity. Overall, ToxProfiler is a quantitative in vitro reporter assay that can accurately provide insight into the toxicological MoA of compounds, thereby assisting in the future mechanism-based safety assessment of chemicals.

Nephropathic cystinosis is a rare monogenetic kidney disease caused by mutations in the lysosomal transporter cystinosin (encoded by CTNS ) that, to date, has no cure. The hallmark of this disease is lysosomal accumulation of cystine and decline in proximal tubular function leading to kidney failure early in life. In this project, we developed a novel gene repair strategy using CRISPR/Cas9 Homology-Independent Targeted Integration (HITI) to restore CTNS . A novel, non-viral peptide-mediated approach was used to deliver the Cas9-guideRNA ribonucleoprotein (RNP) complex and repair templates to conditionally immortalized proximal tubule epithelial cell (ciPTEC) lines. The repair constructs contained either mCherry (1.7 kb), the CTNS Superexon (1.7 Kb) or both (3.2 Kb). The results demonstrated that the smaller mCherry construct achieved a higher repair efficiency (63%) compared to the CTNS -mCherry construct (16%). Clonal expansion of repaired cells showed restoration of lysosomal cystine levels in 70-75% of the clones, which was accompanied by improved mitochondrial bioenergetics. In conclusion, CRISPR/Cas9 HITI can be used to precisely insert repair templates into the genome, resulting in a functional cystinosin restoration, and a reversal of the cystinotic disease phenotype.

Malaria in pregnancy remains a major public health issue capable of causing significant adverse perinatal outcomes. Due to Plasmodium sequestration in the placenta (placental malaria), and the immunosuppression during pregnancy, malaria represents a substantial risk for both the mother and fetus. Placental malaria occurs in 16%–63% of pregnant women (Darmstadt et al., 2011) with malaria and is the leading global cause of maternal anemia, low birth weight, preterm delivery, stillbirths, and miscarriages (Goldenberg et al., 2010). Each year, approximately 30–35 million women become pregnant in malaria-endemic areas of Africa, and similar numbers of women can be exposed to malaria in Asia, Oceania, and South America (Chico & Cano, 2019; World Health Organization (WHO), 2021). Unfortunately, due to a shortage of safety data on pregnant women for most of the currently recommended antimalarial drugs, there are limited treatment options for this vulnerable population, particularly in the first trimester. This highlights the need for continued improvement and development of novel antimalarial medicines with no developmental effects (teratogenicity). Over the past decades, novel antimalarials have been developed. Unfortunately, a number of these have shown to be teratogenic in preclinical animal models. Therefore, the key focus for Medicines for Malaria Venture (MMV) and partners is to incorporate embryo-fetal developmental (EFD) toxicity testing early into drug development, allowing for earlier prioritization of candidate compounds, and increasing the importance of having safe medicines for pregnancy by front-loading developmental toxicity evaluations (El Gaaloul et al., 2022). This de-risking process could act as the first step to include pregnant women and women of childbearing potential earlier in clinical trials. This population is currently excluded even though they represent a target population. In our recent study, we assessed the EFD toxicity of MMV390048, an antimalarial agent, in both rats and rabbits (Demarta-Gatsi et al., 2022). We observed adverse effects of MMV390048 on the rat fetus, specifically diaphragmatic hernia and ventricular septum defect, whereas no developmental effects were observed in rabbits. Based on these observations, MMV390048 was classified as teratogenic. In the current study, to verify the teratogenicity potential of MMV390048 and address the interspecies difference outcomes, in accordance with the 3R's (replace, reduce, refine) principles, we compared traditional EFD studies in mammals with the well-characterized new approach methodologies (NAMs; Green et al., 2016). In this study, we compared the outcomes obtained in mammalian EFDs with those observed in zebrafish (Cassar et al., 2019) and ReproTracker® (Jamalpoor et al., 2022) for the drug MMV390048. Zebrafish model has become a popular model for predicting embryotoxicity/malformations and has shown to be a good model for generating relevant information on mammalian developmental risks (Truong et al., 2014). Briefly, drug effects on zebrafish embryo development have been assessed after exposure to different concentrations (up to 100 μM) of MMV390048. However, the maximum concentration tested was limited by the compound's solubility in water. Consequently, the highest concentration achieved in fish (fish-uptake) was 8 μM at the highest concentration tested (100 μM). The second in vitro model used, ReproTracker, is a human-induced pluripotent stem cell (hiPSCs)-based biomarker assay that follows the cells differentiation during early embryonic development. Initially, cytotoxicity and solubility of MMV390048 were tested in nondifferentiated hiPSCs at different concentrations up to 1 mM. In this case, concentrations of MMV390048 were limited by cytotoxicity or solubility, and therefore the maximum noncytotoxic and/or soluble concentration (15.6 μM) was used. Next, hiPSCs were directed to differentiate toward three germ layer-specific cell types, hepatocytes, cardiomyocytes, and neural rosettes. Proper stem cell differentiation was investigated by morphological profiling and assessment of time-dependent expression patterns of cell-specific biomarkers. In this system, a decrease in the expression of specific biomarker genes and morphology disruption of the differentiated cells following compound treatment indicated teratogenicity. To allow for direct comparison between the previously published preclinical safety evaluation data for MMV390048 (Demarta-Gatsi et al., 2022) and the concentrations tested in alternative assays, we calculated and compared the maternal MMV390048 free concentrations (Cmax) in preclinical animal models with the concentrations of MMV390048 used in ReproTracker and zebrafish models (Table 1). Studies have shown that in vivo Cmax values can be compared with the in vitro concentrations and can be directly correlated to the in vitro potency values (Danielsson et al., 2000; Smith et al., 2010). In the main EFD study, MMV390048 induced diaphragmatic hernia only in one of the rat fetuses at a concentration of 13 μM (Table 1). However, in the investigative EFD study at concentrations equal to ≈20 μM, MMV390048 induced a notable developmental effect. In rabbits, MMV390048 (up to ≈10 μM, maximum tested concentration) showed no developmental effects. In the ReproTracker assay, MMV390048 up to 15.6 μM (maximum testable concentration) had no significant effect on the expression pattern of the biomarker genes nor on the functionality/morphology of all three cell types. Similarly, MMV390048 up to the highest testable concentration (8 μM) had no effect on larvae development in zebrafish (Table 1). The results of these studies suggest that MMV390048 may not be a teratogen at concentrations below 20 μM. In concordance, based on exposure data from EFD in vivo studies, IC50s ≥20 μM for individual kinases that are inhibited by MMV390048 were considered not to be relevant (Demarta-Gatsi et al., 2022). Moreover, PK/PD modeling-based simulations obtained from the Phase 1a human study predicted that a single 120 mg dose of MMV390048, resulting in a Cmax of no more than ≈3 μM in malaria naïve male individuals, would likely achieve cure in patients with Plasmodium falciparum malaria with 92% certainty (Table 2; McCarthy et al., 2020). Of note, Cmax achieved in malaria naïve male individuals can be significantly different (higher or lower) compared with individuals from endemic countries or pregnant women. In this study, we have combined traditional EFD studies in mammals with NAMs and revealed that the observed malformations of MMV390048 in rats could be related to the concentration differences or species-specific effect. Based on rat developmental studies, we expected MMV390048 to disrupt some developmental pathways in relation to diaphragmatic hernia and/or cardiovascular malformations (e.g., ventricular septum defect; Demarta-Gatsi et al., 2022) in other models, as well. However, the compound did not exhibit a teratogenic potential in any of the in vitro alternative models, nor in the rabbit study. Whether the rat or rabbit data are an outlier and their relevance to man remains an open question. The constant challenge of drug resistance and the difficulty of diagnosing malaria associated with pregnancy makes this population at risk. Therefore, MMV is working with its partners to establish in vitro screening methodologies to test antimalarials' potential teratogenicity and be able to front-load fetal/developmental toxicity studies early into drug development. In order to achieve this, a preliminary development dose range finding study will be conducted for each compound that results positive in NAMs. This study will include external fetal evaluation and visceral or skeletal examination depending on the target and expected toxicities. In this retrospective study, however, the use of NAMs would have been of limited value, as the compound had no teratogenic effect in any of the NAMs and still an EFD study at later stage of drug development would have been required. Nevertheless, this comparative approach has demonstrated that the use of NAMs can be helpful to better understand the outcome differences in animal testing and can be used for de-risking antimalarials early in drug development for teratogenicity hazards. This study further promoted MMV and partners to continue and develop a publicly supported value framework in which the degree of uncertainty is weighed up against the acceptance of the animal-free approaches. Moreover, this will allow to develop a broader normative framework for the responsible implementation of preclinical models in development and reproductive toxicology studies (DART) and make explicit possible normative presuppositions behind the current practice of (the lack of) using animal-free models in DART studies. The datasets analysed during the current study are available from the corresponding author on request and with permission of Medicines for Malaria Venture.

in vitro screening platforms to assess teratogenic potential of compounds are emerging rapidly. ReproTracker is a human induced pluripotent stem cells (hiPSCs)-based biomarker assay that is shown to identify the teratogenicity potential of new pharmaceuticals and chemicals reliably. In its current state, the assay is limited to identifying the potential teratogenic effects and does not immediately quantify a clinical dose relevant to the exposure of chemicals or drugs observable in mothers or fetuses. The goal of this study was to evaluate whether the ReproTracker assay can be extrapolated in vivo and quantitatively predict developmental toxicity exposure levels of two known human teratogens, thalidomide, and carbamazepine. Here, we utilized Physiologically Based Pharmacokinetic (PBPK) modeling to describe the pharmacokinetic behavior of these compounds and conducted an in vitro to in vivo extrapolation (IVIVE) approach to predict human equivalent effect doses (HEDs) that correspond with in vitro concentrations potentially associated with adverse outcomes in ReproTracker. The HEDs derived from the ReproTracker concentration predicted to cause developmental toxicity were close to the reported teratogenic human clinical doses and the HED derived from the rat or rabbit developmental toxicity study. The ReproTracker derived-HED revealed to be sensitive and protective of humans. Overall, this pilot study demonstrated the importance of integrating PBPK model in extrapolating and assessing developmental toxicity in vitro. The combination of these tools demonstrated that they could improve the safety assessment of drugs and chemicals without animal testing.

Testing for developmental toxicity according to the current regulatory guidelines requires large numbers of animals, making these tests very resource intensive, time-consuming, and ethically debatable. Over the past decades, several alternative in vitro assays have been developed, but these often suffered from low predictability and the inability to provide a mechanistic understanding of developmental toxicity. To identify embryotoxic compounds, we developed a human induced pluripotent stem cells (hiPSCs)-based biomarker assay. The assay is based on the differentiation of hiPSCs into functional cardiomyocytes and hepatocytes. Proper stem cell differentiation is investigated by morphological profiling and assessment of time-dependent expression patterns of cell-specific biomarkers. In this system, a decrease in the expression of the biomarker genes and morphology disruption of the differentiated cells following compound treatment indicated teratogenicity. The hiPSCs-based biomarker assay was validated with 21 well-established in vivo animal teratogenic and non-teratogenic compounds during cardiomyocyte and hepatocyte differentiation. The in vivo teratogenic compounds (e.g., thalidomide and valproic acid) markedly disrupted morphology, functionality, and the expression pattern of the biomarker genes in either one or both cell types. Non-teratogenic chemicals generally had no effect on the morphology of differentiated cells, nor on the expression of the biomarker genes. Compared to the in vivo classification, the assay achieved high accuracy (91%), sensitivity (91%), and specificity (90%). The assay, which we named ReproTracker®, is a state-of-the-art in vitro method that can identify the teratogenicity potential of new pharmaceuticals and chemicals and signify the outcome of in vivo test systems.

Testing for developmental toxicity according to the current regulatory guidelines requires large numbers of animals, making these tests very resource intensive, time‐consuming, and ethically debatable. Over the past decades, several alternative in vitro assays have been developed, but these often suffered from low predictability and the inability to provide a mechanistic understanding of developmental toxicity.

Identified as being the primary mechanism involved in the induction of torsades de pointes (TdP), early afterdepolarisation (EAD) formation is an important parameter in cardiac safety pharmacology. Easily observed experimentally at the cellular or tissue level, EAD can also be simulated by computer algorithms using animal or human models. During the last decade, confidence in these algorithms has greatly increased. We investigated the putative usefulness of EAD simulation for cardiac safety pharmacology.EAD simulations were performed in non-failing human ventricular myocytes using the O'Hara-Rudy dynamic model. The role of each cardiac current was investigated by modifying the amplitude of its activity in the model. Prediction of EAD induction by drugs was based on the ratio of their 50% inhibitory concentration values for various cardiac ionic currents to their maximal effective free therapeutic plasma concentration (EFTPCmax).In the ventricular endocardial myocytes, EAD was only induced by at least 85% inhibition of the rapid delayed rectifier K+ current (IKr). The other currents can either induce or prevent EAD under sub- (80% IKr inhibition) or up-threshold conditions (87% IKr inhibition) of EAD. The study of the ability of drugs to induce EAD resulted in a classification which was in agreement with the Tdp risk classification.Based on EAD computer simulation within the human situation, the present study identified the role of various cardiac currents in the EAD formation and suggested that prediction of EAD formation can be useful for early cardiac safety pharmacology.

Nephropathic cystinosis is a severe, monogenic systemic disorder that presents early in life and leads to progressive organ damage, particularly affecting the kidneys. It is caused by mutations in the CTNS gene, which encodes the lysosomal transporter cystinosin, resulting in intralysosomal accumulation of cystine. Recent studies demonstrated that the loss of cystinosin is associated with disrupted autophagy dynamics, accumulation of distorted mitochondria, and increased oxidative stress, leading to abnormal proliferation and dysfunction of kidney cells. We discuss these molecular mechanisms driving nephropathic cystinosis. Further, we consider how unravelling molecular mechanisms supports the identification and development of new strategies for cystinosis by the use of small molecules, biologicals, and genetic rescue of the disease in vitro and in vivo.

Nephropathic cystinosis is a rare disease caused by mutations of the CTNS gene that encodes for cystinosin, a lysosomal cystine/H+ symporter. The disease is characterized by early-onset chronic kidney failure and progressive development of extra-renal complications related to cystine accumulation in all tissues. At the cellular level, several alterations have been demonstrated, including enhanced apoptosis, altered autophagy, defective intracellular trafficking, and cell oxidation, among others. Current therapy with cysteamine only partially reverts some of these changes, highlighting the need to develop additional treatments. Among compounds that were identified in a previous drug-repositioning study, disulfiram (DSF) was selected for in vivo studies. The cystine depleting and anti-apoptotic properties of DSF were confirmed by secondary in vitro assays and after treating Ctns(-/-) mice with 200 mg/kg/day of DSF for 3 months. However, at this dosage, growth impairment was observed. Long-term treatment with a lower dose (100 mg/kg/day) did not inhibit growth, but failed to reduce cystine accumulation, caused premature death, and did not prevent the development of renal lesions. In addition, DSF also caused adverse effects in cystinotic zebrafish larvae. DSF toxicity was significantly more pronounced in Ctns(-/-) mice and zebrafish compared to wild-type animals, suggesting higher cell toxicity of DSF in cystinotic cells.