Silver nanoparticles (AgNPs) have emerged as promising agents in cancer diagnostics and/or therapy, demonstrating a lot of possible pharmacological actions. However, understanding the pharmacokinetics and safety profiles of nanoparticles, which is crucial for their clinical application, still raises many questions. Studies indicate that AgNPs can accumulate in tumour tissues, improving drug delivery and specificity. However, their interaction with biological systems necessitates thorough safety evaluations. Classical methods for assessing AgNPs' safety include cytotoxicity assays, genotoxicity tests, and histopathological examinations. However, novel techniques are emerging, such as advanced imaging and biomarker analysis, offering more precise toxicity assessments. Prediction models, including computational simulations and in silico analyses, are being developed to forecast AgNPs' toxicity profiles. These models aim to reduce reliance on animal testing and expedite the evaluation process. To mitigate potential risks associated with nanoparticle-based therapies, strategies such as surface modification, controlled release systems, and targeted delivery are being explored. These methods aim to enhance therapeutic efficacy while minimizing adverse effects. The main aim of this review article is to describe AgNPs from the point of view of their pharmacokinetic/toxicokinetic profile in the light of modern knowledge. Special attention will be given to novel methods for assessing the safety and toxicity profiles of AgNPs, providing insights into their interactions with cancer therapies and their potential clinical applications.

Basal cortisol concentration is used to screen for hypoadrenocorticism (HAD) in dogs with chronic gastrointestinal disease, but low values often lead to unnecessary ACTH stimulation tests. This study evaluated treadmill exercise as a potential alternative method for stimulating cortisol release. Nineteen healthy pet dogs (mean age: 5.2 years, median weight: 23.4 kg) were included. After a baseline blood draw (T0), dogs were acclimated to a treadmill with treats to minimize stress. They then walked and trotted for 10 min, followed by a 30 min rest before a second blood draw (T1). Pre- and post-treadmill exercise cortisol levels were compared using a randomization (permutation) test for paired data. Median pre-exercise serum cortisol was 1.36 μg/dL (IQR: 0.9-2.095), and post-exercise was 1.1 μg/dL (IQR: 0.895-1.585), with no significant difference (p = 0.0915). After exercise cortisol increased in 9 of 19 and decreased in 10 of 19 dogs. These findings suggest that treadmill exercise for 10 min does not reliably stimulate cortisol release, assessed 30 min post-exercise. Basal cortisol concentrations in this cohort of clinically healthy dogs were lower than previously described.

Ethnopharmacology, a discipline focused on studying the medicinal use of natural materials by humans, plays a crucial role in addressing challenges in modern drug development. However, the traditional 3R principle-Replacement, Reduction, and Refinement-have limitations in guiding the ethical management of animal experimentation, conducting animal studies, and utilizing animal-derived materials in ethnopharmacological research. To address these gaps, the field has introduced the 4R principles, which expand the original framework by adding "Responsibility." The Responsibility principle highlights the ethical obligation of researchers to consider the welfare of experimental animals during all procedures. It calls for researchers to take accountability for their actions and decisions, ensuring that they actively protect animal welfare and exhibit empathy across species. This principle reinforces the ethical foundation of ethnopharmacological research. To implement the 4R principles effectively, this article explores the dimensions of Reduction, Refinement, Replacement, and Responsibility in detail. For Reduction, strategies include minimizing animal use by developing optimized, efficient experimental designs, creating tissue banks to recycle animal samples, and improving success rates in animal modeling. These efforts collectively aim to enhance ethical standards while advancing scientific outcomes. In terms of Refinement, the goal is to minimize animal distress and pain by improving the experimental environment, refining operational procedures, ensuring strict control of experiments under anesthesia, and prioritizing non-invasive or minimally invasive techniques for data collection. For Replacement, the aim is to reduce the need for experimental animals by exploring alternative solutions. This includes substituting in vitro experiments for in vivo ones, using 3D organoids to replace animal organs, and applying deep learning technologies in ways that decrease animal use. The Responsibility principle focuses on enhancing researchers' ethical obligations toward animal welfare. This can be achieved by improving regulations and policies governing animal experimentation, providing ethical training for technical personnel, and promoting awareness of animal welfare and ethical practices. The introduction and implementation of the 4R principles provide valuable guidance for the ethical conduct of animal experimentation in ethnopharmacological research, offering new insights and methodologies that support the responsible use of animals in scientific studies.

Shunt systems for hydrocephalus therapy are commonly based on passive mechanical pressure valves that are driven by the intracranial, intra-abdominal, and hydrostatic pressure. The differential pressure acting on the valve determines the drainage rate of cerebrospinal fluid (CSF) but is not a gauge of the physiological condition of the patient. Internal and external influences can cause over- or underdrainage and lead to pathological levels of intracranial pressure (ICP).

The first prototype of a ventricular intelligent and electromechanical shunt (VIEshunt) is developed, tested, and compared with previous efforts towards the development of a smart shunt. Its key components are a micro pump, a flow meter, a pressure sensor, an inertial measurement unit, a wireless communication interface, and a microcontroller. The VIEshunt prototype was tested in vitro using a hardware-in-the-loop (HiL) test bench that runs real-time patient simulations involving changes in intracranial and intra-abdominal pressure, as well as changes in posture ranging between supine and upright position. The prototype was subsequently tested in an in vivo pilot study based on an acute ovine animal model (n=1) with infusions of artificial CSF.

During 24 h in vitro testing, the prototype detected the simulated posture changes of the patient and automatically adapted the controller reference. The posture-specific ICP references of 12 mmHg for supine and -3 mmHg for upright position were tracked without offset, thus preventing adverse over- and underdrainage during the investigated HiL test scenario. During acute in vivo testing, the prototype first regulated the mean ICP of a sheep from 22 mmHg down to 20 mmHg. Each of the three subsequent intraventricular bolus infusions of 1 mL saline solution increased mean ICP by approximately 11 mmHg. While natural absorption alone decreased ICP by only 5 mmHg within 9 min, the prototype was able to regulate ICP back to the pre-bolus pressure value within 5 min.

The developed VIEshunt prototype is capable of posture-dependent ICP regulation and CSF drainage control. Smart shunt systems based on VIEshunt could improve patient monitoring and enable optimal physiologic therapy by adapting to the individual patient. To derive statistically relevant conclusions for the performance of VIEshunt, future work will focus on the development of a next generation prototype for use in pre-clinical studies.

The androgen receptor (AR) activation by androgens is vital for tissue development, sexual differentiation, and reproductive attributes in zebrafish (Danio rerio). However, our understanding of the molecular mechanisms behind their activation remains limited. In this study, we employed both ab initio (AlphaFold) and homology (SWISS-MODEL) structure models of zebrafish androgen receptor ligand-binding domain (zAR-LBD) to explore the binding specificity, binding affinity, and molecular interactions of endogenous hormones (testosterone (T), 11-ketotestosterone (11-KT), and dihydrotestosterone (DHT)) in a computational simulation. Molecular docking analysis showed that both structures formed the same interactions and similar patterns of binding energy with androgens. Molecular Dynamics (MD) simulation analysis revealed that hydrogen bond occupancy aligned with in vitro findings related to androgenic effect. When comparing complexes modeled by SWISS-MODEL and AlphaFold, significant differences were observed in root mean square deviation (RMSD) and root mean square fluctuations (RMSF). The AlphaFold structures also exhibited a clear separation between ligands in principal component analysis. Further correlation analysis between in silico features and in vitro EC50 values identified MMPBSA energies as the most significant contributors to ligand-specific variance in the in silico complexes (p < 0.05). Overall, this integrative approach offers significant insights into the molecular mechanisms underlying zebrafish AR activity.

(1) Background: Osteoarthritis is a degenerative disease of the whole joint marked by cartilage-bone interface (CBI) remodeling, but methods to monitor subtle changes in mineralization are lacking. We optimized a non-destructive ultrasound imaging method to monitor incremental shifts in mineralization, using brief decalcification as a mimetic of CBI remodeling. (2) Methods: We used a 35-MHz transducer to scan 3 mm diameter bovine osteochondral explants wrapped with parafilm to produce surface-directed decalcification and dedicated 3D-printed holders to maintain sample orientation. Customized MATLAB codes and a matched pair design were used for quantitative hypothesis testing. (3) Results: Optimal scan precision was obtained when the High-Frequency Ultrasound (HFUS) focal distance was trained at the CBI. HFUS cartilage thickness increased by 53 ± 21 µm or 97 ± 28 µm after three or seven hours of ethylene diamine tetra-acetic acid (EDTA) (but not PBS), respectively, and was highly correlated with histological cartilage thickness (R = 0.98). The en face CBI backscatter pattern was irregular and shifted after the EDTA-displacement of the mineral front. Collective data suggested that the -10 dB echogenic CBI signal originated from the mineral front and varied topographically with undulating mineral thickness. (4) Conclusions: This imaging approach could be used to monitor tidemark remodeling in live explant cultures, toward identifying new treatments that inhibit tidemark advancement and slow osteoarthritis progression.

Epilepsy is a prevalent neurological condition marked by seizures that lead to neurobiological and behavioral impairments. Caffeine (CAF), the world's most consumed stimulant, reportedly affects both epileptic seizures and the efficacy of antiepileptic drugs, particularly topiramate (TPM). This study aimed to investigate the effects of CAF on TPM in a pentylenetetrazol (PTZ)-induced seizure model using zebrafish larvae. Four days post-fertilization Danio rerio larvae were incubated for 18 hours with CAF, TPM, or CAF+TPM, followed by an assessment of locomotor activity. Seizures were induced by adding PTZ to achieve a final concentration of 20 mM. In the PTZ-induced seizure model, the application of CAF in doses over 50 mg/L resulted in a decrease in the average movement. TPM ( > 50 μM) significantly protected larvae against the PTZ. The addition of 15 mg/L CAF to TPM did not affect larval activity at any TPM concentration tested; however, higher doses of CAF significantly reduced larval activity. CAF doses above 25 mg/L altered the activity of larvae treated with TPM in the PTZ-induced seizure model. Larvae exhibited differential heart rate (HR) responses to CAF exposure across doses. CAF at 75 mg/L significantly increased HR, while doses of 175 mg/L and higher induced bradycardia. TPM, across all tested doses, did not independently influence HR. The study provides valuable insights into the interactions between CAF and TPM, which may inform future research on human epilepsy. However, the extrapolation of these results to other species should be approached cautiously due to physiological differences.

Retinal models play a pivotal role in translational drug development, bridging preclinical research and therapeutic applications for both ocular and systemic diseases. This review highlights the retina as an ideal organ for studying advanced therapies, thanks to its immune privilege, vascular and neuronal networks, accessibility, and advanced imaging capabilities. Preclinical retinal disease models offer unparalleled insights into inflammation, angiogenesis, fibrosis, and hypoxia, utilizing clinically translatable bioimaging tools like fundoscopy, optical coherence tomography, confocal scanning laser ophthalmoscopy, fluorescein angiography, optokinetic tracking, and electroretinography. These models have driven innovations in anti-inflammatory, anti-angiogenic, and neuroprotective strategies, with broader implications for systemic diseases such as rheumatoid arthritis, Alzheimer's, and fibrosis-related conditions. By emphasizing the integration of the 3Rs principles and novel imaging modalities, this review highlights how retinal research not only enhances therapeutic precision but also minimizes ethical concerns, paving the way for more predictive and human-relevant approaches in drug development.

The development of anticancer therapies has increasingly relied on advanced 3D in vitro models, which more accurately mimic the tumor microenvironment compared to traditional 2D cultures. This review describes the evolution of these 3D models, highlighting significant advancements and their impact on cancer research. We discuss the integration of machine learning (ML) and artificial intelligence (AI) in enhancing the predictive power and efficiency of these models, potentially reducing the dependence on animal testing. ML and AI offer innovative approaches for analyzing complex data, optimizing experimental conditions, and predicting therapeutic outcomes with higher accuracy. By leveraging these technologies, the next generation of 3D in vitro models could revolutionize anticancer drug development, offering effective alternatives to animal experiments.

Human precision-cut lung slices (hPCLS) are a unique platform for functional, mechanistic, and drug discovery studies in the field of respiratory research. However, tissue availability, generation, and cultivation time represent important challenges for their usage. Therefore, the present study evaluated the efficacy of a specifically designed tissue preservation solution, TiProtec, complete or in absence (-) of iron chelators, for long-term cold storage of hPCLS.

hPCLS were generated from peritumor control tissues and stored in DMEM/F-12, TiProtec, or TiProtec (-) for up to 28 days. Viability, metabolic activity, and tissue structure were determined. Moreover, bulk-RNA sequencing was used to study transcriptional changes, regulated signaling pathways, and cellular composition after cold storage. Induction of cold storage-associated senescence was determined by transcriptomics and immunofluorescence (IF). Finally, cold-stored hPCLS were exposed to a fibrotic cocktail and early fibrotic changes were assessed by RT-qPCR and IF.

Here, we found that TiProtec preserves the viability, metabolic activity, transcriptional profile, as well as cellular composition of hPCLS for up to 14 days. Cold storage did not significantly induce cellular senescence in hPCLS. Moreover, TiProtec downregulated pathways associated with cell death, inflammation, and hypoxia while activating pathways protective against oxidative stress. Cold-stored hPCLS remained responsive to fibrotic stimuli and upregulated extracellular matrix-related genes such as fibronectin and collagen 1 as well as alpha-smooth muscle actin, a marker for myofibroblasts.

Optimized long-term cold storage of hPCLS preserves their viability, metabolic activity, transcriptional profile, and cellular composition for up to 14 days, specifically in TiProtec. Finally, our study demonstrated that cold-stored hPCLS can be used for on-demand mechanistic studies relevant for respiratory research.

Background/Objectives MC4R expression and its role in colorectal and anaplastic thyroid cancers, where resistance to therapy and lack of standard treatments remain significant challenges, are poorly understood. This study aimed to investigate MC4R as a potential therapeutic target in these cancers using the selective antagonist ML00253764 (ML), alone and in combination with vinorelbine (VNR) and irinotecan (or its active metabolite SN-38). Methods: Human colorectal adenocarcinoma HT-29, Caco-2, and anaplastic thyroid carcinoma 8305C cell lines were used. MC4R expression was assessed by Real-Time PCR with validated primers (Assay ID Hs00271877_s1), immunofluorescence, and Western blotting. Proliferation and apoptosis assays were conducted with ML, and synergy with VNR and SN-38 was evaluated by Combination Index and Loewe methods. ERK1/2 phosphorylation was measured using an ELISA assay. In vivo studies were conducted by injecting tumor cells into Athymic Nude-Foxn1nu mice, treated with ML, VNR, irinotecan, or their combinations. Results: MC4R expression was confirmed in all cell lines. ML treatment inhibited MC4R, producing antiproliferative and pro-apoptotic effects, with IC50 values of 7667 ± 2144.6 nM (8305C), 806.4 ± 321.8 nM (HT-29), and 2993 ± 1135.2 nM (Caco-2). In combination with VNR and SN-38, ML exhibited significant synergy in vitro and reduced tumor volume in vivo without causing weight loss or adverse effects in mice. Conclusions This study identifies ML as a promising therapeutic agent that, when combined with chemotherapy, may offer a novel strategy for treating colorectal and anaplastic thyroid cancers.

Rat pharmacokinetic studies are commonly utilized in early discovery to support absorption, distribution, metabolism, and excretion optimization of active pharmaceutical ingredients (APIs). The aim of this work was to compare exposures from fit-for-purpose oral suspension and solution formulations in rats to guidance provided by the refined Developability Classification System (rDCS) with respect to identifying potential limits to oral absorption, formulation strategy selection, and to optimize oral bioavailability (BA). This investigation utilized six diverse APIs covering a large range of biorelevant solubility, metabolic stability, and oral BA in rats. While results for our model compounds acetaminophen, voriconazole, fedratinib, lemborexant, and istradefylline indicated oral BA in rats was limited by first-pass metabolism, only the results for voxelotor indicated an oral absorption limitation by intestinal dissolution/solubility. The in vivo studies highlighted challenges and limitations often encountered in early discovery. The rDCS analysis provided a more differentiated developability risk assessment associated with oral solid dosage form development by incorporating compound-specific physicochemical attributes and human physiology without the need of preclinical data. The rDCS results were shown to align well with the clinical/marketed formulation strategies for the investigated APIs.

The Thymus vulgaris and Origanum vulgare essential oils (contained thymol and carvacrol in a range of 35-80%) are used in various products in the fields of medicine, cosmetics, and foods. Molecular hybridization between benzothiazole (BT) and phenolic monoterpenoids is a promising method for the development of biologically active compounds. New benzothiazole-monoterpenoid hybrids were synthesized through a regioselective α-amidoalkylation reaction of thymol and carvacrol with high yields (70-96%). This approach is both simple and cost-effective, employing easily accessible and inexpensive reagents to produce target molecules. The structure of the synthesized compounds was characterized spectrally using 1H-, 13C-NMR, FT-IR, and HRMS data. The newly obtained compounds are structural analogues of the UVB filter PBSA, which is used in cosmetics. The spectral properties of the aromatic products thymol hybrid (2-(4-hydroxy-5-isopropyl-2-methylphenyl)benzo[d]thiazole) and carvacrol hybrid (2-(4-hydroxy-2-isopropyl-5-methylphenyl)benzo[d]thiazole) were successfully examined, using a validated spectrophotometric method. SPF values varied from 31 to 36, compared to the PBSA (30), and were observed at concentrations of 1-0.25 mM. 2-Hydroxyphenylbenzothiazoles are known antimicrobial and antioxidant agents that have potential applications in the food industry and cosmetics as preservatives and antioxidants. In this context, antimicrobial activity of the hybrid compounds was evaluated using the agar diffusion method against E. coli, S. aureus, P. aeruginosa, and C. albicans. Compounds of methyl-2-(4-hydroxy-2-isopropyl-5-methylphenyl)benzo[d]thiazole-3(2H)-carboxylate containing carvacrol fragments showed high activity against Staphylococcus aureus ATCC 25923 (with 0.044 μmol content). The radical scavenging activity was determined using ABTS and DPPH assays, the highest activity was exhibited by the thymol hybrids ethyl-2-(4-hydroxy-5-isopropyl-2-methylphenyl)benzo[d]thiazole-3(2H)-carboxylate (IC50-133.70 ± 10 µM) and methyl-2-(4-hydroxy-5-isopropyl-2-methylphenyl)benzo[d]thiazole-3(2H)-carboxylate (IC50-157.50 ± 10 µM), defined by ABTS. The aromatic benzothiazole-monoterpenoid hybrids are classified using in silico analyses as non-mutagenic, with low toxicity, and they are non-irritating to the skin. These compounds were identified as new hit scaffolds for multifunctional molecules in cosmetics.

In experimental research, animal welfare should always be of the highest priority. Currently, physical in-person observations are the standard. This is time-consuming, and results are subjective. Video-based machine learning models for monitoring experimental pigs provide a continuous and objective observation method for animal misthriving detection. The aim of this study was to develop and validate a pig tracking technology, using video-based data in a machine learning model to analyze the posture and activity level of experimental pigs living in single-pig pens. A research prototype was created using a microcomputer and a ceiling-mounted camera for live recording based on the obtained images from the experimental facility, and a combined model was created based on the Ultralytics YOLOv8n for object detection trained on the obtained images. As a second step, the Lucas-Kanade sparse optical flow technique for movement detection was applied. The resulting model successfully classified whether individual pigs were lying, standing, or walking. The validation test showed an accuracy of 90.66%, precision of 90.91%, recall of 90.66%, and correlation coefficient of 84.53% compared with observed ground truth. In conclusion, the model demonstrates how machine learning can be used to monitor experimental animals to potentially improve animal welfare.

The international gold standard for avian influenza virus (AIV) diagnosis is virus isolation (VI) in specific pathogen-free embryonated chicken eggs (ECEs). AIV isolation typically involves a 6-day turnaround, during which premises under suspicion for notifiable AIV infection are held under restriction regardless of molecular diagnoses, often with significant welfare implications.

A reduction in time for negation by VI was investigated following experimental inoculation of AIV from known-positive original clinical material into ECEs. VI data derived from more than 600 case investigations from epizootics of high-pathogenicity AIV (HPAIV) in Great Britain since 2016 and from low-pathogenicity AIV (LPAIV) cases in Great Britain since 2014 were examined to support a reduction in test timing using alternative regimens.

HPAIVs were isolated during the first passage, and for LPAIV VI, the second passage could be reduced to 2 days. Power analysis showed that the benefit of reducing the number of days outweighed the risk of missing a positive isolate.

Limited data were available from experimental inoculations.

This truncated methodology, which enables an earlier release of restrictions, may substantially ease the economic implications of restriction. It could also reduce bird welfare implications and improve international standards without loss of test performance.

Immortalised cell lines that mimic their primary cell counterparts are fundamental to research, particularly when large cell numbers are required. Here, we report that immortalisation of bone marrow-derived macrophages (iBMDMs) using the J2 virus resulted in the loss of a protein of interest, MSR1, in WT cells by an unknown mechanism. This led us to perform an in-depth mass spectrometry-based proteomic characterisation of common murine macrophage cell lines (J774A.1, RAW264.7, and BMA3.1A7), in comparison with the iBMDMs, as well as primary BMDMs from both C57BL/6 and BALB/c mice. This analysis revealed striking differences in protein profiles associated with macrophage polarisation, phagocytosis, pathogen recognition, and interferon signalling. Among the cell lines, J774A.1 cells were the most similar to the gold standard primary BMDM model, whereas BMA3.1A7 cells were the least similar because of the reduction in abundance of several key proteins related closely to macrophage function. This comprehensive proteomic dataset offers valuable insights into the use and suitability of macrophage cell lines for cell signalling and inflammation research.

A transgenic protein is frequently expressed as different homologous variants in genetically modified crops due to differential processing of targeting peptides or optimization of activity and specificity. The aim of this study was to develop a science-based approach for risk assessment of homologous protein variants using dicamba mono-oxygenase (DMO) as a case study. In this study, DMO expressed in the next-generation dicamba-tolerant maize, sugar beet and soybean crops exhibited up to 27 amino acid sequence differences in the N-terminus. Structure modeling using AlphaFold, ESMFold and OpenFold demonstrates that these small N-terminal extensions lack an ordered secondary structure and do not disrupt the DMO functional structure. Three DMO variants were demonstrated to have equivalent immunoreactivity and functional activity ranging from 214 to 331 nmol/min/mg. Repeated toxicity studies using each DMO variant found no test substance-related adverse effects. These results support that homologous protein variants, which have demonstrated physicochemical and functional equivalence, can leverage existing safety data from one variant without requiring additional de novo safety assessments.

Cell-based assays are widely exploited for drug screening and biosensing, providing useful information about bioactivity of target analytes and complex biological samples. It is well recognized that 3D cell models are required to achieve highly valuable information, also from the perspective of replacing animal models. However, bioassays relying on 3D cell models are generally highly demanding in terms of facilities, equipment, and skilled personnel requirements. To reduce cost, increase sustainability, and provide a flexible 3D cell-based platform for bioassays, we here report a novel approach based on a 3D-printed microtissue device. To assess the suitability of this strategy for reporter gene technology, we selected to monitor two molecular pathways which were of interest in several applications, hypoxia signaling and the p53 pathway. The investigation of such pathways is highly relevant in fields spanning from drug screening to bioactivity monitoring for industrial by-product valorization. Microtissues of human hepatocarcinoma (HepG2) and human embryonic kidney (Hek293T) cell lines were obtained with a low-cost and sustainable chip platform and bioassays were developed to monitor the hypoxia-inducible factors (HIFs) and the p53 tumor suppressor pathway. HepG2 and Hek293T 3D cell models were genetically engineered to express the Luc2P from Photinus pyralis firefly either under the regulation of p53 or HIF response elements. The bioassays allowed quantitative assessment of hypoxia and tumoral activity with 1,10-phenanthroline for HIF and with doxorubicin for p53 pathway activation, respectively, showing good potential for applications of this sustainable and low-cost 3D-printed microfluidic platform for bioactivity analyses, drug screening, and precision medicine.

The refined Developability Classification System (rDCS) provides a comprehensive animal-free approach for assessing biopharmaceutical risks associated with developing oral formulations. This work demonstrates practical application of a recently advanced rDCS framework guiding formulation design for six diverse active pharmaceutical ingredients (APIs) and compares rDCS classifications with those of the Biopharmaceutics Classification System (BCS). While the BCS assigns five of the APIs to class II/IV, indicating potentially unfavorable biopharmaceutical attributes, the rDCS provides a more nuanced risk assessment. Both BCS and rDCS assign acetaminophen to class I at therapeutic doses. Voriconazole and lemborexant (both BCS II) are classified in rDCS class I at therapeutic doses, indicating suitability for development as conventional oral formulations. Fedratinib is classified as BCS IV but the rDCS indicates a stratified risk (class I, IIa or IIb), depending on the relevance of supersaturation/precipitation in vivo. Voxelotor and istradefylline (both BCS II) belong to rDCS class IIb, requiring solubility enhancement to achieve adequate oral bioavailability. Comparing the rDCS analysis with literature on development and pharmacokinetics demonstrates that the rDCS reliably supports oral formulation design over a wide range of API characteristics, thus providing a strong foundation for guiding development.

The technique of using naturally occurring light-emitting reactants (photoproteins and luciferases] that have been extracted from a wide range of animals is known as bioluminescence imaging, or BLI. This imaging offers important details on the location and functional state of regenerative cells inserted into various disease-modeling animals. Reports on gene expression patterns, cell motions, and even the actions of individual biomolecules in whole tissues and live animals have all been made possible by bioluminescence. Generally speaking, bioluminescent light in animals may be found down to a few centimetres, while the precise limit depends on the signal's brightness and the detector's sensitivity. We can now spatiotemporally visualize cell behaviors in any body region of a living animal in a time frame process, including proliferation, apoptosis, migration, and immunological responses, thanks to BLI. The biological applications of in vivo BLI in nondestructively monitoring biological processes in intact small animal models are reviewed in this work, along with some of the advancements that will make BLI a more versatile molecular imaging tool.

Man-made vitreous fibers (MMVF) comprise diverse materials for thermal and acoustic insulation, including stone wool. Depending on dimension, durability, and dose, MMVF might induce adverse health effects. Therefore, early predictive in vitro (geno)toxicity screening of new MMVF is highly desired to ensure safety for exposed workers and consumers. Here, we investigated, as a starting point, critical in vitro screening determinants and pitfalls using primary rat alveolar macrophages (AM) and normal rat mesothelial cells (NRM2). A stone wool fiber (RIF56008) served as an exemplary MMVF (fibrous vs. ground to estimate impact of fiber shape) and long amosite (asbestos) as insoluble fiber reference. Materials were comprehensively characterized, and in vivo-relevant in vitro concentrations defined, based on different approaches (low to supposed overload: 0.5, 5 and 50 µg/cm2). After 4-48 h of incubation, certain readouts were analyzed and material uptake was investigated by light and fluorescence-coupled darkfield microscopy. DNA-strand break induction was not morphology-dependent and nearly absent in both cell types. However, NRM2 demonstrated material-, morphology- and concentration-dependent membrane damage, CINC-1 release, reduction in cell count, and induction of binucleated cells (asbestos > RIF56008 > RIF56008 ground). In contrast to NRM2, asbestos was nearly inactive in AM, with CINC-1 release solely induced by RIF56008. In conclusion, to define an MMVF-adapted, predictive in vitro (geno)toxicity screening tool, references, endpoints, and concentrations should be carefully chosen, based on in vivo relevance, and sensitivity and specificity of the chosen cell model. Next, further endpoints should be evaluated, ideally with validation by in vivo data regarding their predictivity.

Ecological risk assessments of agrochemicals have traditionally depended on in vivo guideline tests using northern bobwhite and mallard to provide relevant endpoints for avian species. However, these studies have limitations, including animal welfare concerns, the time and cost involved, limited potential for extrapolation to more realistic exposure conditions, and the lack of mechanistic understanding. The proof-of-concept work presented a case study for thiamethoxam in three avian species, demonstrating the potential of physiologically based kinetic (PBK) modeling to enable dosimetry extrapolations that inform hazard characterization in risk assessment, and reduce the use of avian testing. The model structure for northern bobwhite and mallard contained ten compartments, while an additional ovulation model was included for chicken in the physiological state of egg-laying. The model was first parameterized and evaluated for chicken and northern bobwhite using in vitro kinetic measurements and in vivo toxicokinetic (TK) data. The chicken model was then extrapolated to mallard based on allometric scaling. The models were then used to map the TK profiles across species by simulating internal dose metrics in different avian toxicology studies. These metrics, including peak blood concentrations (Cmax) and area under the curve (AUC) for blood concentration, were determined for acute, subacute, or chronic toxicity endpoints for mallard and northern bobwhite, enabling a quantitative cross-species and cross-route comparison of dosimetry. The results suggested that the chronic toxicological response of birds exposed to thiamethoxam is highly dependent on internal exposure, while mallard appeared to be more dynamically sensitive to thiamethoxam on an acute oral exposure basis. The case study increases the confidence in using new approach methodologies (NAMs) for interpreting avian toxicity studies and facilitating in vitro-in silico-based ecological risk assessments of agrochemicals.

Biomaterial-associated infections pose severe challenges in modern medicine. Previously, we reported that polyanionic DNA surface coatings repel bacterial adhesion and support osteoblast-like cell attachment in monoculture experiments, candidate for orthopaedic implant coatings. However, monocultures lack the influence of bacteria or bacterial toxins on osteoblast-like cell adhesion to biomaterial surfaces. In this study, co-culture of staphylococcus (S. epidermidis and S. aureus) and SaOS-2 osteosarcoma cells was studied on chitosan-DNA polyelectrolyte multilayer coated glass based on the concept of `the race for the surface`. Staphylococcus was first deposited onto the surface in a microfluidic chamber to mimic peri-operative contamination, and subsequently, SaOS-2 cells were seeded. Both staphylococcus and SaOS-2 cells were cultured together on the surfaces for 24 h under flow. The presence of S. epidermidis decreased SaOS-2 cell number on all surfaces after 24 h. However, the cells that adhered spread equally well in the presence of low virulent S. epidermidis. However, highly virulent S. aureus induced cell death of all adherent SaOS-2 cells on chitosan-DNA multilayer coated glass, a worse outcome than on uncoated glass. The outcome of our co-culture study highlights the limitations of monoculture models. It demonstrates the need for in vitro co-culture assays to meaningfully bridge the gap in lab testing of biomaterials and their clinical evaluations where bacterial infection can occur. The relative failure of cell-adhesive and bacteria-repelling DNA coatings in co-cultures also suggests the need to incorporate bactericidal in addition to non-adhesive functions to protect competitive cell spreading over a long period.

The goal of the current study was to develop a pig model to investigate oxidative stress with a low negative impact on piglet welfare. Four independent trials (A, B, C, and D) were performed using a single intraperitoneal shot of lipopolysaccharide (LPS) as an immune challenge, aiming to assess the minimal LPS dose for piglets of different age to trigger a measurable acute oxidative stress response in healthy animals. In trial A, piglets received an LPS dose of 25 µg/KgBW at 41 days post-weaning (p.w.). In trial B, piglets received 25 µg/KgBW of LPS at 28 days p.w., in trials C And D, piglets were injected with 50 µg/KgBW of LPS at 21 days p.w., respectively. Piglets were randomly allocated either to the T1) Control group with saline solution (Ctrl), or T2) LPS challenge (LPS). The oxidative stress response was measured through the enzymatic activity of glutathione peroxidase (GPx), glutathione-S-transferase (GST), superoxide dismutase (SOD), and catalase (CAT), in both plasma and intestinal tissues. Intestinal gene expression of oxidative stress and inflammatory markers was assessed. Discomfort behaviors (panting, prostration, trembling, and vomits) were also recorded. Plasmatic and intestinal oxidative stress response was inconsistent across the four trials even when the dose and pig age were similar, possibly due to individual variability. Relative gene expression differences of anti-inflammatory cytokines (IL10), oxidation precursor (iNOS), and antioxidant markers (GPx4, MnSOD, and CAT) were detected between Ctrl and LPS treatment (P < 0.05) when assessed. Behavioral observations were sensitive to the LPS dose relative to Ctrl (P < 0.05) in all four trials. These results suggest that behavioral observations can be used as a non-invasive methodology to detect the presence of oxidative stress in pigs in challenging conditions. Behavioral observations were more sensitive than other indicators (i.e., biomarkers and gene expression) in the current study. However, a sensitivity scale system needs to be developed to qualify and rank the impact of oxidative stress in pigs.

The application of nanomaterials in industry and consumer products is growing exponentially, which has pressed the development and use of predictive human in vitro models in pre-clinical analysis to closely extrapolate potential toxic effects in vivo. The conventional cytotoxicity investigation of nanomaterials using cell lines from cancer origin and culturing them two-dimensionally in a monolayer without mimicking the proper pathophysiological microenvironment may affect a precise prediction of in vitro effects at in vivo level. In recent years, complex in vitro models (also belonging to the new approach methodologies, NAMs) have been established in unicellular to multicellular cultures either by using cell lines, primary cells or induced pluripotent stem cells (iPSCs), and reconstituted into relevant biological dimensions mimicking in vivo conditions. These advanced in vitro models retain physiologically reliant exposure scenarios particularly appropriate for oral, dermal, respiratory, and intravenous administration of nanomaterials, which have the potential to improve the in vivo predictability and lead to reliable outcomes. In this perspective, we discuss recent developments and breakthroughs in using advanced human in vitro models for hazard assessment of nanomaterials. We identified fit-for-purpose requirements and remaining challenges for the successful implementation of in vitro data into nanomaterials Safe and Sustainable by Design (SSbD), Risk Assessment (RA), and Life Cycle Assessment (LCA). By addressing the gap between in vitro data generation and the utility of in vitro data for nanomaterial safety assessments, a prerequisite for SSbD approaches, we outlined potential key areas for future development.

Patient-derived tumour organoids (PDOs) are highly advanced in vitro models for disease modelling, yet they lack vascularisation. To overcome this shortcoming, organoids can be inoculated onto the chorioallantoic membrane (CAM); the highly vascularised, not innervated extraembryonic membrane of fertilised chicken eggs. Therefore, we aimed to (1) establish a CAM patient-derived xenograft (PDX) model based on PDOs generated from the liver metastasis of a colorectal cancer (CRC) patient and (2) to evaluate the translational pipeline (patient - in vitro PDOs - in vivo CAM-PDX) regarding morphology, histopathology, expression of C-X-C chemokine receptor type 4 (CXCR4), and radiotracer uptake patterns.

The main liver metastasis of the CRC patient exhibited high 2-[18F]FDG uptake and moderate and focal [68Ga]Ga-Pentixafor accumulation in the peripheral part of the metastasis. Inoculation of PDOs derived from this region onto the CAM resulted in large, highly viable, and extensively vascularised xenografts, as demonstrated immunohistochemically and confirmed by high 2-[18F]FDG uptake. The xenografts showed striking histomorphological similarity to the patient's liver metastasis. The moderate expression of CXCR4 was maintained in ovo and was concordant with the expression levels of the patient's sample and in vitro PDOs. Following in vitro re-culturing of CAM-PDXs, growth, and [68Ga]Ga-Pentixafor uptake were unaltered compared to PDOs before transplantation onto the CAM. Although [68Ga]Ga-Pentixafor was taken up into CAM-PDXs, the uptake in the baseline and blocking group were comparable and there was only a trend towards blocking.

We successfully established an in vivo CAM-PDX model based on CRC PDOs. The histomorphological features and target protein expression of the original patient's tissue were mirrored in the in vitro PDOs, and particularly in the in vivo CAM-PDXs. The [68Ga]Ga-Pentixafor uptake patterns were comparable between in vitro, in ovo and clinical data and 2-[18F]FDG was avidly taken up in the patient's liver metastasis and CAM-PDXs. We thus propose the CAM-PDX model as an alternative in vivo model with promising translational value for CRC patients.

Holistic phenotyping of rodent models is increasing, with a growing awareness of the 3Rs and the fact that specialized experimental setups can also impose artificial restrictions. Activity is an important parameter for almost all basic and applied research areas involving laboratory animals. Locomotor activity, the main form of energy expenditure, influences metabolic rate, muscle mass, and body weight and is frequently investigated in metabolic disease research. Additionally, it serves as an indicator of animal welfare in therapeutic, pharmacological, and toxicological studies. Thus, accurate and effective measurement of activity is crucial. However, conventional monitoring systems often alter the housing environment and require handling, which can introduce artificial interference and lead to measurement inaccuracies.

Our study focused on evaluating circadian activity profiles derived from the DVC and comparing them with conventional activity measurements to validate them statistically and assess their reproducibility. We utilized data from metabolic studies, an Alzheimer's disease model known for increased activity, and included DVC monitoring in a project investigating treatment effects on activity in a type-1-like diabetes model.

The DVC data yielded robust, scientifically accurate, and consistent circadian profiles from group-housed mice, which is particularly advantageous for longitudinal experiments. The activity profiles from both systems were fully comparable, providing matching profiles. Using DVC monitoring, we confirmed the hyperactivity phenotype in an AD model and reproduced a decline in activity in type-1-like diabetes model.

In our work, we derived robust circadian activity profiles from the DVC data of group-housed mice, which were scientifically accurate, reproducible and comparable to another activity measurement. This approach can not only improve animal welfare according to the 3R principles but can also be implement in high-throughput longitudinal studies. Furthermore, we discuss the advantages and limitations of DVC activity measurements to highlight its potential and avoid confounders.

Neurological disorders have for a long time been a global challenge dismissed by drug companies, especially due to the low efficiency of most therapeutic compounds to cross the brain capillary wall, that forms the blood-brain barrier (BBB) and reach the brain. This has boosted an incessant search for novel carriers and methodologies to drive these compounds throughout the BBB. However, it remains a challenge to artificially mimic the physiology and function of the human BBB, allowing a reliable, reproducible and throughput screening of these rapidly growing technologies and nanoformulations (NFs). To surpass these challenges, brain-on-a-chip (BoC) - advanced microphysiological platforms that emulate key features of the brain composition and functionality, with the potential to emulate pathophysiological signatures of neurological disorders, are emerging as a microfluidic tool to screen new brain-targeting drugs, investigate neuropathogenesis and reach personalized medicine. In this review, the advance of BoC as a bioengineered screening tool of new brain-targeting drugs and NFs, enabling to decipher the intricate nanotechnology-biology interface is discussed. Firstly, the main challenges to model the brain are outlined, then, examples of BoC platforms to recapitulate the neurodegenerative diseases and screen NFs are summarized, emphasizing the current most promising nanotechnological-based drug delivery strategies and lastly, the integration of high-throughput screening biosensing systems as possible cutting-edge technologies for an end-use perspective is discussed as future perspective.

Uveitis is a group of inflammatory ocular pathologies. Endotoxin-Induced Uveitis (EIU) model represent a well-known model induced by administration of Lipopolysaccharide (LPS). The aim is to characterize two models of EIU through two routes of administration with novel noninvasive imaging techniques. 29 rats underwent Intraocular Pressure (IOP) measurements, Optical Coherence Tomography (OCT), proteomic analysis, and Positron Emission Tomography and Computed Tomography (PET/CT). Groups included healthy controls (C), BSS administered controls (Ci), systemically induced EIU with LPS (LPSs), and intravitreally induced EIU with LPS (LPSi) for IOP, OCT, and proteomic studies. For 18F-FDG PET/CT study, animals were divided into FDG-C, FDG-LPSs and FDG-LPSi groups and scanned using a preclinical PET/CT system. LPSi animals exhibited higher IOP post-induction compared to C and LPSs groups. LPSi showed increased cellular infiltrate, fibrotic membranes, and iris inflammation. Proinflammatory proteins were more expressed in EIU models, especially LPSi. PET/CT indicated higher eye uptake in induced models compared to FDG-C. FDG-LPSi showed higher eye uptake than FDG-LPSs but systemic uptake was higher in FDG-LPSs due to generalized inflammation. OCT is valuable for anterior segment assessment in experimental models. 18F-FDG PET/CT shows promise as a noninvasive biomarker for ocular inflammatory diseases. Intravitreal induction leads to higher ocular inflammation. These findings offer insights for future inflammatory disease research and drug studies.

Vitamin D deficiency is a global problem. Vitamin D, the vitamin D receptor, and its enzymes are found throughout neuronal, ependymal, and glial cells in the brain and are implicated in certain processes and mechanisms in the brain. To investigate the processes affected by vitamin D deficiency in adults, we studied vitamin D deficient, control, and supplemented diets over 6 weeks in male and female C57Bl/6 mice. The effect of the vitamin D diets on proliferation in the neurogenic niches, changes in glial cells, as well as on memory, locomotion, and anxiety-like behavior, was investigated. Six weeks on a deficient diet was adequate time to reach deficiency. However, vitamin D deficiency and supplementation did not affect proliferation, neurogenesis, or astrocyte changes, and this was reflected on behavioral measures. Supplementation only affected microglia in the dentate gyrus of female mice. Indicating that vitamin D deficiency and supplementation do not affect these processes over a 6-week period.

The animal product most used as a stimulatory additive for cell cultivation is still fetal bovine serum (FBS). Besides the ethical concerns regarding serum collection, the main problems of FBS are batch-to-batch variability and the resulting risk of lower reproducibility, the differences between species, the presence of undefined/unknown components, and the risk of contamination. In contrast, pig blood, which is a by-product of slaughter, is a sufficiently available and sustainable resource with a high degree of standardization in terms of donor age, weight, and genetics. The variations in preparations from pig slaughter blood seem to be comparatively low, and consequently, batch effects might be much smaller, suggesting that the reproducibility of the research data obtained may be increased. Our pilot study aimed to investigate, as a proof of concept, whether adult human and porcine stem cells of different tissue origins proliferate and differentiate adequately when FBS is completely or partially replaced by porcine serum (PS). We could show that the human and porcine stem cells were vital and proliferated under partial and full PS supplementation. Furthermore, using PS, the two cell types studied showed tissue-specific differentiation (i.e., lipid vacuoles as a sign of adipogenic or myotubes as a sign of myogenic differentiation). In conclusion, the pig slaughter blood-derived serum has promising potential to be a replacement for FBS in adult stem cell cultures. Therefore, it could serve as a basis for the development of new cell culture supplements.

Ablation of the immune-specific catalytic subunit Cβ2 of protein kinase A is associated with a proinflammatory phenotype and increased sensitivity to autoimmunity in mice. Here we show that tumour growth of the adenocarcinoma cell line EO771 in the breast and in the lung after injection into the mammary fat pad and tail vein, respectively, was significantly reduced in mice ablated for Cβ2 compared to wild-type mice. In both cases, the breast and lung tumours showed increased infiltration of immune cells in the mice lacking Cβ2 compared to wild-type mice. Despite this, it appeared that solid tissue- versus intravenously injected EO771 cells evoked different immune responses. This was reflected by significantly increased levels of splenic proinflammatory immune cells and circulating cytokines in Cβ2 ablated mice carrying breast- but not the lung tumours. Moreover, Cβ2 ablated mice injected with EO771 cells showed increased overall survival compared to wild-type mice. Taken together, our results suggest for a role for immune cell-specific Cβ2 in protecting against tumour growth induced by EO771 cells in mice that is reflected in improved overall survival.

Near-infrared fluorescence (NIRF) angiography with intraoperative administration of indocyanine green (ICG) has rapidly disseminated in clinical practice. Another clinically approved, and widely available dye, methylene blue (MB), has up to now not been used for this purpose. Recently, we demonstrated promising results for the real-time evaluation of intestinal perfusion using this dye. The primary aim of this study was to perform a quantitative analysis of bowel perfusion assessment for both ICG and MB.

Four mature female Landrace pigs underwent laparotomy under general anesthesia. An ischemic bowel loop with five regions of interest (ROIs) with varying levels of perfusion was created in each animal. An intravenous (IV) injection of 0.25 mg/kg-0.50 mg/kg MB was administered after 10 min, followed by NIRF imaging in MB mode and measurement of local lactate levels in all corresponding ROIs. This procedure was repeated in ICG mode (IV dose of 0.2 mg/kg) after 60 min. The quest spectrum fluorescence camera (Quest Medical Imaging, Middenmeer, The Netherlands) was used for NIRF imaging of both MB and ICG.

Intraoperative NIRF imaging of bowel perfusion assessment with MB and ICG was successful in all studied animals. Ingress (i/s) levels were calculated and correlated with local lactate levels. Both MB and ICG ingress values showed a significant negative correlation (r = - 0.7709; p =  < 0.001; r = - 0.5367, p = 0.015, respectively) with local lactate levels. This correlation was stronger for MB compared to ICG, although ICG analysis showed higher absolute ingress values.

Our fluorescence quantification analysis validates the potential to use MB for bowel perfusion assessment besides the well-known and widely used ICG. Further human studies are necessary to translate our findings to clinical applications.

The field of nasal drug delivery gained enormously on interest over the past decade. Performing nasal in vivo studies is expensive and time-consuming, but also unfeasible for an initial high-throughput compound and formulation screening. Therefore, the development of fast and high-throughput in vitro models to screen compounds for their permeability through the nasal epithelium and mucosa is constantly expanding. Yet, the protocols used for nasal in vitro permeability studies are varying, which limits the comparability and reproducibility of generated data. This project aimed to elucidate the influence of different culture and assay parameters of RPMI 2650 cells grown under air-liquid interface (ALI) conditions on the transepithelial electrical resistance (TEER) and apparent permeability (Papp) values of five selected reference compounds, covering the range of low to moderate to high permeability. The influence of the passage number, seeding density, and timepoint of airlift was minimal in our approach, while the substrate pore density had a significant influence on the Papp values of carbamazepine, propranolol, and metoprolol, classified as highly permeable compounds, but not on atenolol and aciclovir. Elevation of the experimental concentration of carbamazepine, propranolol, and metoprolol in the donor compartment had an increasing effect on the Papp values, while prolonging the assay time did not have a significant influence. Based on the results reported here, RPMI 2650 cells cultured under ALI conditions offer the possibility of a standardized high-throughput screening model for small molecules and their formulations for in vitro drug permeation studies to predict and select optimal conditions for their nasal delivery.

Chagas disease is spreading faster than expected in different countries, and little progress has been reported in the discovery of new drugs to combat Trypanosoma cruzi infection in humans. Recent clinical trials have ended with small hope. The pathophysiology of this neglected disease and the genetic diversity of parasites are exceptionally complex. The only two drugs available to treat patients are far from being safe, and their efficacy in the chronic phase is still unsatisfactory.

This review offers a comprehensive examination and critical review of data reported in the last 10 years, and it is focused on findings of clinical trials and data acquired in vivo in preclinical studies.

The in vivo investigations classically in mice and dog models are also challenging and time-consuming to attest cure for infection. Poorly standardized protocols, availability of diagnosis methods and disease progression markers, the use of different T. cruzi strains with variable benznidazole sensitivities, and animals in different acute and chronic phases of infection contribute to it. More synchronized efforts between research groups in this field are required to put in evidence new promising substances, drug combinations, repurposing strategies, and new pharmaceutical formulations to impact the therapy.

Toxicity profiling is an integral part of the drug discovery pipeline. The 3Rs principle-Replacement, Reduction, and Refinement, is considered a golden rule in determining the most appropriate approach for toxicity studies. The acute toxicity study with proper estimate of median lethal dose (LD50) is usually an initial procedure for the determination of most suitable test doses for preclinical toxicological and pharmacological profiling. Several methods, which have been devised to determine the LD50, are faced with the challenge of using a large number of animals and time constraints. Despite the inherent advantage of the newer OECD Test Guidelines, the increasing concerns among toxicologists, the regulatory authorities and the general public, on the need to adhere to 3Rs principle, necessitated the need for an improved approach. Such an approach should not only minimize the time and number of animals required, but also take into cognizance animal welfare, and give accurate, comparable, and reproducible results across laboratories. While taking advantage of the inherent merits of the existing methods, here is presented the mathematical basis and evaluation of an improved method for toxicity profiling of test substances and estimation of LD50. The method makes use of the generated Table of values for the selection of appropriate test doses. Our proposed method has capacities to optimize the time and number of animal use, ensure more reliable and reproducible results across laboratories, allow for easy selection of doses for subsequent toxicity profiling, and be adaptable to other biological screening beyond toxicity studies.

The irreproducibility in scientific research has become a critical issue. Despite the essential role of rigorous methodology in constructing a scientific article, more than half of publications, on average, are considered non-reproducible. The implications of this irreproducibility extend to reliability problems, hindering progress in technological production and resulting in substantial financial losses. In the context of laboratory animal research, this work emphasizes the importance of choosing an appropriate experimental model within the 3R's principle (Refine, Reduce, Replace). This study specifically addresses a deficiency in data specification in scientific articles, revealing inadequacies in the description of crucial details, such as environmental conditions, diet, and experimental procedures. For this purpose, 124 articles from journals with relevant impact factors were analyzed, conducting a survey of data considered important for the reproducibility of studies. Important flaws in the presentation of data were identified in most of the articles evaluated. The results of this study highlight the need to improve the description of essential information, standardizing studies, and ensuring the reproducibility of experiments in areas such as metabolism, immunity, hormones, stress, among others, to enhance the reliability and reproduction of experimental results, aligning with international guidelines such as ARRIVE and PREPARE.

Adult neurotoxicity (ANT) and developmental neurotoxicity (DNT) assessments aim to understand the adverse effects and underlying mechanisms of toxicants on the human nervous system. In recent years, there has been an increasing focus on the so-called new approach methodologies (NAMs). The Organization for Economic Co-operation and Development (OECD), together with European and American regulatory agencies, promote the use of validated alternative test systems, but to date, guidelines for regulatory DNT and ANT assessment rely primarily on classical animal testing. Alternative methods include both non-animal approaches and test systems on non-vertebrates (e.g., nematodes) or non-mammals (e.g., fish). Therefore, this review summarizes the recent advances of NAMs focusing on ANT and DNT and highlights the potential and current critical issues for the full implementation of these methods in the future. The status of the DNT in vitro battery (DNT IVB) is also reviewed as a first step of NAMs for the assessment of neurotoxicity in the regulatory context. Critical issues such as (i) the need for test batteries and method integration (from in silico and in vitro to in vivo alternatives, e.g., zebrafish, C. elegans) requiring interdisciplinarity to manage complexity, (ii) interlaboratory transferability, and (iii) the urgent need for method validation are discussed.

To meet the growing demand for developmental toxicity assessment of chemicals, New Approach Methodologies (NAMs) are needed. Previously, we developed two 3D in vitro assays based on human-induced pluripotent stem cells (hiPSC) and cardiomyocyte differentiation: the PluriBeat assay, based on assessment of beating differentiated embryoid bodies, and the PluriLum assay, a reporter gene assay based on the expression of the early cardiac marker NKX2.5; both promising assays for predicting embryotoxic effects of chemicals and drugs. In this work, we aimed to further describe the predictive power of the PluriLum assay and compare its sensitivity with PluriBeat and similar human stem cell-based assays developed by others. For this purpose, we assessed the toxicity of a panel of ten chemicals from different chemical classes, consisting of the known developmental toxicants 5-fluorouracil, all-trans retinoic acid and valproic acid, as well as the negative control compounds ascorbic acid and folic acid. In addition, the fungicides epoxiconazole and prochloraz, and three perfluoroalkyl substances (PFAS), PFOS, PFOA and GenX were tested. Generally, the PluriLum assay displayed higher sensitivity when compared to the PluriBeat assay. For several compounds the luminescence readout of the PluriLum assay showed effects not detected by the PluriBeat assay, including two PFAS compounds and the two fungicides. Overall, we find that the PluriLum assay has the potential to provide a fast and objective detection of developmental toxicants and has a level of sensitivity that is comparable to or higher than other in vitro assays also based on human stem cells and cardiomyocyte differentiation for assessment of developmental toxicity.

Currently, there are no FDA-approved medications for the treatment of psychostimulant use disorders (PSUD). We have previously discovered "atypical" dopamine transporter (DAT) inhibitors that do not display psychostimulant-like behaviors and may be useful as medications to treat PSUD. Lead candidates (e.g., JJC8-091, 1) have shown promising in vivo profiles in rodents; however, reducing hERG (human ether-à-go-go-related gene) activity, a predictor of cardiotoxicity, has remained a challenge. Herein, a series of 30 (([1,1'-biphenyl]-2-yl)methyl)sulfinylalkyl alicyclic amines was synthesized and evaluated for DAT and serotonin transporter (SERT) binding affinities. A subset of analogues was tested for hERG activity, and the IC50 values were compared to those predicted by our hERG QSAR models, which showed robust predictive power. Multiparameter optimization scores (MPO > 3) indicated central nervous system (CNS) penetrability. Finally, comparison of affinities in human DAT and its Y156F and Y335A mutants suggested that several compounds prefer an inward facing conformation indicating an atypical DAT inhibitor profile.