Chronic liver diseases, including metabolic dysfunction-associated steatosic liver disease (MASLD) and hepatocellular carcinoma (HCC), are on the rise, potentially due to daily exposure to complex mixtures of chemical compounds forming part of the exposome. Understanding the mechanisms involved in hepatotoxicity of these mixtures is essential to identify common molecular targets that may highlight potential interactions at the molecular level. We illustrated this issue with two families of environmental contaminants, per- and polyfluoroalkyl substances (PFAS) and heterocyclic aromatic amines (HAAs), both of which could be involved in the progression of chronic liver diseases, and whose toxicity may be potentiated by interactions at the level of xenobiotic metabolism. In the study of exposome effects on chronic liver disease, New Approach Methodologies (NAMs) including omics analyses and data from various in vitro, in vivo and in silico approaches, are crucial for improving predictivity of toxicological studies in humans while reducing animal experimentation. Additionally, the development of complex in vitro human liver cell models, such as organoids, is essential to avoid interspecies differences that minimize the risk for humans. All together, these approaches will contribute to construct Adverse Outcome Pathways (AOPs) and could be applied not only to PFAS mixtures but also to other chemical families, providing valuable insights into mixture hepatotoxicity prediction in the study of the exposome. A better understanding of toxicological mechanisms will clarify the role of environmental contaminant mixtures in the development of MASLD and HCC, supporting risk assessment for better treatment, monitoring and prevention strategies.

To reduce costs and delays related to developing new and effective drugs, there is a critical need for improved human liver tissue models. Here we describe an approach for 3D bioprinting functional human liver tissue models, in which we fabricate disc-shaped structures (discoids) 200 μm in thickness and 1-3 mm in diameter from mixtures of cells and collagen-1, embedded in a highly permeable support medium made from packed polyethylene glycol (PEG) microgels. We demonstrate that the method is precise, accurate, and scalable; up to 100 tissues/h can be manufactured with a variability and error in diameter of about 4 %. Histologic and immunohistochemical evaluation of printed discs reveal self-organization, cell cohesion, and key liver marker expression. Over the course of three weeks in culture, the tissues stably synthesize albumin and urea at high levels, outperforming spheroid tissue models. We find the tissues express >100 genes associated with molecular absorption, distribution, metabolism, and excretion (ADME) at levels within the range of human liver. The liver tissue models exhibit enzymatic formation of metabolites after exposure to multiple test compounds. Together, these results demonstrate the promise of 3D printed discoids for pharmacological and toxicological applications.

Gastrointestinal toxicities (GITs) in clinical trials often lead to dose-limitations that reduce drug efficacy and delay treatment optimization. Preclinical animal models do not accurately replicate human physiology, leaving few options for early detection of GITs, such as diarrhea, before human studies. Chemotherapeutic agents, known to cause clinical diarrhea, frequently target mitotic cells. Therefore, we hypothesized a model utilizing proliferative cell populations derived from human intestinal crypts would predict clinical diarrhea occurrence with high accuracy. Here, we describe the development of a diarrhea prediction assay utilizing RepliGut® Planar, a primary intestinal stem cell-derived platform. To evaluate the ability of this model to predict clinical diarrhea risk, we assessed toxicity of 30 marketed drugs by measuring cell proliferation (EdU incorporation), cell abundance (nuclei quantification), and barrier formation (TEER) in cells derived from three human donors. Dose response curves were generated for each drug, and the IC15 to Cmax ratio was used to identify a threshold for assay positivity. This model accurately predicted diarrhea potential, achieving an accuracy of 91 % for proliferation, 90 % for abundance, and 88 % for barrier formation. In vitro toxicity screening using primary proliferative cells may reduce clinical diarrhea and ultimately lead to safer and more effective treatments for patients.

In this study, two novel anti-inflammatory peptides were identified from the hydrolysates of crocodile heads using computer-assisted techniques and in vitro cellular experiments. Their interaction mechanisms were elucidated through molecular docking and molecular dynamics simulations. A total of 67 peptides were identified via LC-MS/MS, with AKLDLEEVIK and DFLDLPSIER emerging as promising candidates for anti-inflammatory activity. Results of in vitro cellular experiments demonstrated that these peptides significantly inhibited LPS-stimulated macrophage hyperactivation, resulting in reduced release of nitric oxide (NO) and pro-inflammatory cytokines, i.e., TNF-α, IL-1β, and IL-6. Molecular dynamics (MD) simulations and docking analyses revealed that AKLDLEEVIK and DFLDLPSIER displayed high affinity for the TLR4-MD2 complex, with stable and tight interactions. Key residues Arg90, Ser118, Cys133, and Arg264 were identified as critical for the recognition and interaction between the peptides and TLR4-MD2. This study provides a theoretical foundation for the development of anti-inflammatory functional foods derived from crocodile heads.

Nutmeg (Myristica fragrans Houtt.) is widely cultivated in tropical regions and valued for its culinary and medicinal uses. However, its safety profile remains underexplored. This study aimed to evaluate the safety of nutmeg powder through in-silico toxicity predictions, acute oral toxicity, and a 13-week repeated-dose toxicity assessment. In the in-silico study, bioactive compounds identified via High-Performance Liquid Chromatography (HPLC) were analyzed for toxicity parameters using the ProTox II server. For acute toxicity, a single dose of nutmeg powder was administered to mice, with no mortality observed over 14 days, and an LD50 greater than 5000 mg/kg body weight was determined. In the sub-chronic toxicity study, rats received nutmeg powder at 1400 mg/kg and 3500 mg/kg (35 and 87.5 times the human therapeutic dosage, respectively) for 90 days. A 28-day recovery phase was included to assess delayed or reversible effects. The results indicate that a dose of 1400 mg/kg leads to liver damage and hemosiderin deposition in the spleen, while a dose of 3500 mg/kg causes liver and kidney damage, as well as hemosiderin deposition in the spleen. These findings highlight the potential toxicity of long-term nutmeg consumption and underscore the need for further research to ensure its safety in preclinical.

The application of human enteroid systems presents a significant opportunity within the drug development pipeline, highlighting considerable potential for advancements in the characterization and evaluation of new molecular entities. Derived from LGR5+ crypt-based columnar cells, enteroid systems more accurately recapitulate the microanatomy and physiological processes of the human intestinal mucosa compared to traditionally used systems. They contain the complement of major mucosal epithelial cell types, maintain the genetic identity of the donor and intestinal segment they were derived from, and exhibit biological functions and specific activities that are seen in vivo. In this review, we examine the applications of human enteroid systems in nonclinical drug development and compare findings to existing and emerging in vitro models of the small intestine. Specifically, we explore enteroid systems in the context of predicting oral drug disposition, focusing on apparent permeability, intestinal first-pass metabolism, and drug interactions, as well as their utility in assessing drug-induced gastrointestinal toxicity and screening therapeutic efficacy against enteric diseases. Additionally, we highlight aspects of enteroid systems that warrant further study.

Machine learning (ML) is increasingly valuable for predicting molecular properties and toxicity in drug discovery. However, toxicity-related end points have always been challenging to evaluate experimentally with respect to in vivo translation due to the required resources for human and animal studies; this has impacted data availability in the field. ML can augment or even potentially replace traditional experimental processes depending on the project phase and specific goals of the prediction. For instance, models can be used to select promising compounds for on-target effects or to deselect those with undesirable characteristics (e.g., off-target or ineffective due to unfavorable pharmacokinetics). However, reliance on ML is not without risks, due to biases stemming from nonrepresentative training data, incompatible choice of algorithm to represent the underlying data, or poor model building and validation approaches. This might lead to inaccurate predictions, misinterpretation of the confidence in ML predictions, and ultimately suboptimal decision-making. Hence, understanding the predictive validity of ML models is of utmost importance to enable faster drug development timelines while improving the quality of decisions. This perspective emphasizes the need to enhance the understanding and application of machine learning models in drug discovery, focusing on well-defined data sets for toxicity prediction based on small molecule structures. We focus on five crucial pillars for success with ML-driven molecular property and toxicity prediction: (1) data set selection, (2) structural representations, (3) model algorithm, (4) model validation, and (5) translation of predictions to decision-making. Understanding these key pillars will foster collaboration and coordination between ML researchers and toxicologists, which will help to advance drug discovery and development.

Drug-induced liver injury (DILI) is a leading cause of acute liver failure. Hepatotoxicity typically occurs only in a subset of individuals after prolonged exposure and constitutes a major risk factor for the termination of drug development projects.

We provide an overview of available human liver models for DILI research and discuss how they have been used to aid in early risk assessments and to mitigate the risk of project closures due to DILI in clinical stages. We summarize the different data that can be provided by such models and illustrate how these diverse data types can be interfaced with machine learning strategies to improve predictions of liver safety liabilities.

Advanced human liver models closely mimic human liver phenotypes and functions for many weeks, allowing for the recapitulation of hepatotoxicity events in vitro. Integration of the biochemical, histological, and toxicogenomic output data from these models with physicochemical compound properties using different machine learning architectures holds promise to enhance preclinical DILI predictions. However, to realize this aim, it is important to benchmark the available liver models on test sets of DILI positive and negative compounds and to carefully annotate and share the resulting data.

To investigate the toxicity of N-n-butyl haloperidol iodide (F2), a quaternary ammonium salt derivative of haloperidol, in mice for potential therapeutic purposes.

The acute median lethal dose (LD50) of F2 was determined using the Bliss method following intravenous administration in mice. Routine surface electrocardiograms (ECGs) and arterial blood pressures (aBPs) were recorded under general anesthesia in untreated and pharmacologically vagotomized mice injected with F2. Sublethal doses of F2 were tested for their effects on aBP, heart rate, and biochemical parameters such as lactate dehydrogenase (LDH), blood urea nitrogen (BUN), and serum lactate levels. Histopathological changes in the heart, lungs, liver, and kidneys were evaluated after F2 administration.

The acute LD50 of F2 was determined to be 5.11 mg/kg. A 10 mg/kg dose of F2 caused severe hypotension, second-degree atrioventricular block, progressive prolongation of Pmurr intervals, and death due to cardiac asystole. Similar ECG and aBP changes were observed in atropine-pretreated mice, indicating that cholinergic effects do not play a major role in F2-induced toxicity. Sublethal doses of F2 (1.2 and 2.4 mg/kg) caused dose-dependent decreases in aBP and increases in heart rate. F2 induced significant, dose-dependent increases in LDH, BUN, and serum lactate levels. Histopathological analysis revealed acute lung lesions at 10 mg/kg, with no significant changes observed in the heart, liver, or kidneys.

Acute intravenous injection of F2 exhibits dose-dependent cardiopulmonary toxicity, characterized by severe hypotension, arrhythmias, and biochemical changes. These findings highlight the potential risks of F2 and the need for further evaluation of its safety profile for therapeutic use.

Two observational studies were conducted to support an initiative to qualify translational kidney safety biomarkers as clinical drug development tools that identify tubular injury prior to changes in estimated glomerular filtration rate (eGFR). Normal healthy volunteers provided three morning spot urine collections over 4 weeks. Patients undergoing surgical resection and intrathoracic cisplatin for malignant pleural mesothelioma provided urine samples pre- and postoperatively at 4, 8, and 12 hours and daily for 6 days. Using receiver-operating characteristics curves, "statistically significant thresholds" established peak longitudinal changes for 8 biomarkers to differentiate mesothelioma patients who developed acute kidney injury (AKI) from normal healthy volunteers. We also assessed "medically significant thresholds" to differentiate mesothelioma patients who did vs. did not develop AKI. Statistically and medically significant thresholds for a fold-change from baseline of urine creatinine (UCr)-normalized values were established for 6 biomarkers: clusterin (2.2, 5.1); osteopontin (3.1, 7.1); N-acetyl-ß-D-glucosaminidase (2.7, 8.1); kidney injury molecule-1 (4.3, 7.5); cystatin C (1.8, 4.5); neutrophil gelatinase-associated lipocalin (2.9, 7.8). For urine albumin and total protein, thresholds were established based on UCr-normalized absolute values: (> upper limit normal, > 10× upper limit normal). Statistically significant thresholds for all biomarkers outperformed eGFR at discriminating mesothelioma subjects exposed to cisplatin from healthy volunteers, demonstrating their utility for enhancing safe drug development. Medically significant thresholds provide perspective on when patients begin to exhibit AKI. These studies have established guideposts for confirmatory studies with additional cohorts and nephrotoxicants to formally qualify the selected biomarkers with worldwide regulatory authorities.

ZhenzhuXiaoji Decoction (ZZXJD) is a traditional Chinese medicine formulation composed of five herbs: Ligustrum lucidum, Curcuma zedoaria, Prunella vulgaris, Hedyotis diffusa, and Glycyrrhiza uralensis, developed for the treatment of hepatocellular carcinoma (HCC). Although early studies have demonstrated the therapeutic potential of ZZXJD, its safety profile, particularly regarding potential toxicity, remains underexplored. This study aims to evaluate both the pharmacological effects and toxicity of ZZXJD in preclinical models to determine its clinical applicability.

This study employed in vitro and in vivo experiments to assess the pharmacological effects and safety of ZZXJD. HHL-5 and HEK-293 cell lines were treated with ZZXJD at varying concentrations (125, 250, 500, and 1,000 μg/mL) for 24, 48, and 72 h to evaluate its effects on cell viability, apoptosis, and necrosis. Acute and subacute toxicity studies were conducted in male and female mice, including assessments of behavioral changes, body weight, organ weight, and liver/kidney functions. Additionally, routine blood tests were performed to identify potential immunostimulatory effects.

In vitro experiments demonstrated that ZZXJD inhibited the proliferation of HHL-5 and HEK-293 cells in a dose-dependent manner and induced apoptosis and necrosis. In subacute toxicity studies, mice in the low and mid-dose groups exhibited no significant behavioral changes, whereas the high-dose group showed transient alterations in liver and kidney function markers, particularly in female mice. These changes were reversible following treatment cessation. Blood tests indicated increased lymphocyte and monocyte counts in treated male mice; however, these increases were not statistically significant. Organ weight and histopathological analyses revealed no significant signs of toxicity at therapeutic doses.

Treatment with ZZXJD at standard therapeutic dosage did not produce acute or subacute toxic effects on liver or kidney functions in vivo, suggesting its safety for continued use in cancer treatment. However, reversible abnormalities in liver and kidney function markers were observed at higher doses. Thus, regular monitoring of liver and kidney functions is recommended during clinical use, especially when higher doses are employed.

Liver toxicity poses a critical challenge in drug development due to the liver's pivotal role in drug metabolism and detoxification. Accurately predicting liver toxicity is crucial but is hindered by scattered information sources, a lack of curation standards, and the heterogeneity of data perspectives. To address these challenges, we developed the HepatoToxicity Portal (HTP), which integrates an expert-curated knowledgebase (HTP-KB) and a state-of-the-art machine learning model for toxicity prediction (HTP-Pred). The HTP-KB consolidates hepatotoxicity data from nine major databases, carefully reviewed by hepatotoxicity experts and categorized into three levels: in vitro, in vivo, and clinical, using the Medical Dictionary for Regulatory Activities (MedDRA) terminology. The knowledgebase includes information on 8,306 chemicals. This curated dataset was used to build a hepatotoxicity prediction module by fine-tuning a GNN-based foundation model, which was pre-trained with approximately 10 million chemicals in the PubChem database. Our model demonstrated excellent performance, achieving an area under the ROC curve (AUROC) of 0.761, surpassing existing methods for hepatotoxicity prediction. The HTP is publicly accessible at https://kobic.re.kr/htp/ , offering both curated data and prediction services through an intuitive interface, thus effectively supporting drug development efforts.Scientific contributionsHTP-KB consolidates comprehensive curated information on liver toxicity gathered from nine sources. HTP-Pred utilizes advanced deep learning techniques, significantly enhancing predictive accuracy. Together, these tools provide valuable resources for researchers and practitioners in drug development, accessible through a user-friendly interface.

Tumoroids are cultures of patient-derived tumor cells, which are grown in 3D in the presence of an extracellular matrix extract and specific growth factors. Tumoroids can be generated from adult as well as pediatric cancers, including epithelial cancers, sarcomas, and brain cancers. Tumoroids retain multi-omic characteristics of their corresponding tumor and recapitulate interpatient and intratumor heterogeneity. Retrospective and prospective studies have demonstrated that tumoroids predict patient responses to anticancer therapies, making them a promising tool for precision oncology. However, several challenges remain before tumoroids can be fully integrated into clinical decision-making, including success rates of tumoroid establishment and turnaround times. This review discusses the current advances, challenges, and future directions of tumoroid-based models in cancer research and precision therapy.

Essential information about the effects of a pollutant on an ecosystem can be obtained by observing how it influences a bioindicator organism. Hexavalent chromium (Cr VI+) naturally occurs in Irish agricultural soils at levels of up to 250 mg/kg, which raises concerns about potential negative impacts on human health and the surrounding areas. This research aimed to assess the sublethal effect concentrations (up to 300 ppm) of Cr VI + on the entomopathogenic nematode (EPN) Steinernema feltiae focusing on endpoints such as nematode movement and host finding ability in contaminated sand and pathogenicity, percentage penetration, sex ratio and reproduction in Galleria mellonella. To achieve that, an Irish isolate of S. feltiae [strain SB 12 (1)], was used in all experiments. The attraction of nematodes to the insect host was tested using PVC tubes of various lengths, containing sand with various concentrations of Cr VI + (50-300 ppm in increments of 50). The replication was tenfold and the insect mortality was recorded at the end of the experiment. Results showed that there was a significant effect of Cr VI + on the pathogenicity, movement and host finding ability of the nematodes in contaminated sand, and on the percentage of penetration in an insect host. However, no significant effects among the studied Cr VI + concentrations were observed in S. feltiae reproduction in G. mellonella. Similarly, the presence of the toxicant (at low concentration of 12ppm) did not affect the growth of the nematode symbiotic bacteria in liquid and solid media (TSA and NBTA). Reproduction, unlike the other sublethal parameters tested, appeared not to be an optimal endpoint for assessing soil Cr VI + risk contamination. Overall, this study confirms the excellent potential of S. feltiae to be used as a suitable sentinel organism in assessing the risk of Cr VI + soil contamination especially in the contexts of agriculture and soil health.

Chimeric mice with humanized liver are considered a useful tool to predict drug pharmacokinetics and in vivo toxicity in humans. The PXB-mouse is one of such chimeric (humanized) mouse models with more than 70% of human hepatocytes in their liver, which can produce human albumin with human-type bile secretion and express human xenobiotic metabolizing enzymes. However, data are limited regarding the properties of such humanized mice in hepatotoxicity studies. This study aimed to explore the distinctive characteristics of chimeric PXB-mice with humanized liver that can influence susceptibility to hepatotoxicity. Morphologically, the PXB-mice have a diffuse hepatic macrovesicular and microvesicular steatosis in the transplanted human hepatocytes, which can be suppressed after human growth hormone treatment. The humanized liver of the PXB-mice has a metabolic zonation of glutamine synthetase, cytochrome P450 2E1, and argininosuccinate synthase 1, similar to normal liver in rodents and humans. The transplanted human hepatocytes in the PXB liver have a markedly decreased N-cadherin expression compared with normal human liver. Scanning electron microscopy revealed formation of septum-like structures encircling the transplanted human hepatocytes in the PXB liver, which consists of an accumulation of fibers in the space of Disse under transmission electron microscopy and is immunolabeled for laminin. Overall, the present report demonstrated the morphological and immunohistochemical characteristics of the PXB-mice with humanized liver along with some abnormalities in the cell adhesion of the transplanted human hepatocytes. These findings would be useful for hepatotoxicity studies using humanized animal models.

Model organisms (MO) are widely used in neuroscience to study brain processes, behavior, and the biological foundation of human diseases. However, the use of MO has also been criticized for low reliability and insufficient success rate in the development of therapeutic approaches, because the success of MO use also led to overoptimistic and simplistic applications, which sometimes resulted in wrong conclusions. Here, we develop a conceptual framework of MO to support scientists in their practical work and to foster discussions about their power and limitations. For this purpose, we take advantage of concepts developed in the philosophy of science and adjust them for practical application by neuroscientists. We suggest that MO can be best understood as tools that are used to gain information about a group of species or a phenomenon in a species of interest. These learning processes are made possible by some properties of MO, which facilitate the process of acquisition of understanding or provide practical advantages, and the possibility to transfer information between species. However, residual uncertainty in the reliability of information transfer remains, and incorrect generalizations can be side-effects of epistemic benefits, which we consider as representational and epistemic risks. This suggests that to use MO most effectively, scientists should analyze the similarity relation between the involved species, weigh advantages and risks of certain epistemic benefits, and invest in carefully designed validation experiments. Altogether, our analysis illustrates how scientists can benefit from philosophical concepts for their research practice.

Therapeutic drugs can sometimes cause adverse effects in a nonclinical species that do not translate to other species, including human. Species-specific (rat, dog, and human) in vitro liver spheroids were employed to understand the human relevance of cholestatic liver injury observed with a selective estrogen receptor degrader (amcenestrant) in dog, but not in rat, during preclinical development. Amcenestrant showed comparable cytotoxicity in liver spheroids from all 3 species; however, its M5 metabolite (RA15400562) showed dog preferential cytotoxicity after 7 days of treatment. Whole genome transcript profiles generated from liver spheroids revealed downregulation of genes related to bile acid synthesis and transport indicative of strong farnesoid X receptor (FXR) antagonism following treatment with both amcenestrant and its M5 metabolite in the dog but not in rat or human. In human spheroids, upregulation of genes for detoxification enzymes indicative of pregnane X receptor (PXR) agonism was observed following amcenestrant treatment, whereas in the dog these genes were downregulated. The M5 metabolite showed gene dysregulation indicating PXR agonism in both rat and human, and antagonism in dog. Analysis of liver samples from a 3-mo dog toxicity study conducted with amcenestrant showed downregulation of several genes associated with PXR and FXR, corroborating the in vitro results. These results support the hypothesis that dogs are uniquely susceptible to cholestatic hepatotoxicity following administration of amcenestrant due to species-specific antagonism of FXR and highlight the value of in vitro liver spheroids to investigating mechanisms of toxicity and possible species differences.

Cardiovascular disease remains the leading cause of death worldwide, yet despite massive investment in drug discovery, the progress of cardiovascular drugs from lab to clinic remains slow. It is a complex, costly pathway from drug discovery to the clinic and failure becomes more expensive as a drug progresses along this pathway. The focus has begun to shift to optimisation of in vitro culture methodologies, not only because these must be undertaken are earlier on in the drug discovery pathway, but also because the principles of the 3Rs have become embedded in national and international legislation and regulation. Numerous studies have shown myocyte cell behaviour to be much more physiologically relevant in 3D culture compared to 2D culture, highlighting the advantages of using 3D-based models, whether microfluidic or otherwise, for preclinical drug screening. This review aims to provide an overview of the challenges in cardiovascular drug discovery, the limitations of traditional routes, and the successes in the field of preclinical models for cardiovascular drug discovery. It focuses on the particular role biomimicry can play, but also the challenges around implementation within commercial drug discovery.

Drug-induced liver injury (DILI) presents a major challenge not only in new drug development but also in post-marketing withdrawals and the safety of food, cosmetics, and chemicals. Experimental model organisms such as the rodents have been widely used for preclinical toxicological testing. However, the tension exists associated with the ethical and sustainable use of animals in part because animals do not necessarily inform the human-specific ADME (adsorption, dynamics, metabolism and elimination) profiling. To establish alternative models in humans, in vitro hepatic tissue models have been proposed, ranging from primary hepatocytes, immortal hepatocytes, to the development of new cell resources such as stem cell-derived hepatocytes. Given the evolving number of novel alternative methods, understanding possible combinations of cell sources and culture methods will be crucial to develop the context-of-use assays. This review primarily focuses on 3D liver organoid models for conducting. We will review the relevant cell sources, bioengineering methods, selection of training compounds, and biomarkers towards the rationale design of in vitro toxicology testing.

This study explores the anticancer potential of methanolic extract from Ganoderma applanatum, focusing on its cytotoxicity across various cancer cell lines and its safety and efficacy in an in vivo hepatocellular carcinoma (HCC) model, along with molecular docking analysis of its bioactive compounds targeting B-cell lymphoma 2 (Bcl-2) protein. The MTT assay revealed significant cytotoxicity of the extract against epidermoid carcinoma (A431), human alveolar carcinoma (A549), and hepatocellular carcinoma (HepG2) cell lines, with the extract exhibiting the highest potency (IC50 of 95.65 µg/ml) against HepG2 cells. Apoptosis induction and DNA degradation in HepG2 cells were confirmed through mitochondrial membrane potential analysis, ethidium bromide/acridine orange staining, and DNA fragmentation assays. In vivo studies on Wistar albino rats showed that administration of the extract up to 1000 mg/ml did not significantly affect body weight or hematological parameters, suggesting a favorable safety profile. Histopathological examination revealed normal liver architecture at most doses, with mild inflammation observed at the highest dose (1000 mg/ml). The G. applanatum extract were showed reducing liver weight and improving body weight in a Diethylnitrosamine (DEN)-induced HCC model was comparable to cyclophosphamide, indicating its potential as a less toxic alternative or adjunct to conventional chemotherapy. Additionally, the extract reduced elevated serum liver enzymes, demonstrating hepatoprotective effects. Molecular docking of nine bioactive compounds from G. applanatum identified 2h-3,11c-(epoxymethano)phenanthro[10,1-bc]pyran as a promising candidate for further investigation. These findings suggest G. applanatum as a novel anticancer agent with the potential for natural, effective cancer therapy.

The SLC22A2 gene encodes organic cation transporter 2 (OCT2), which is predominantly expressed in renal proximal tubule cells. OCT2 is critical for the active renal excretion of various cationic drugs and endogenous metabolites. OCT2 expression varies across species, with higher levels in mice and monkeys compared with humans and rats. The human OCT2 protein consists of 555 amino acids and contains 12 transmembrane domains. OCT2 functions as a uniporter, facilitating the bidirectional transport of organic cations into renal tubular cells, driven by the inside-negative membrane potential. Its expression is regulated by sex hormones, contributing to potential sex differences in Oct2 activity in rodents. OCT2 has been linked to tissue toxicity, such as cisplatin-induced nephrotoxicity. Factors such as genetic variants, age, disease states, and the coadministration of drugs, including tyrosine kinase inhibitors, contribute to interindividual variability in OCT2 activity. This, in turn, impacts the systemic exposure and elimination of drugs and endogenous substances. Regulatory agencies recommend evaluating the potential of a drug to inhibit OCT2 through in vitro and clinical drug-drug interaction (DDI) studies, often using metformin as a probe substrate. Emerging tools like transporter biomarkers and physiologically based pharmacokinetic modeling hold promise in predicting OCT2-mediated DDIs. While several OCT2 biomarkers, such as N1-methylnicotinamide, have been proposed, their reliability in predicting renal DDIs remains uncertain and requires further study. Ultimately, a better understanding of the factors influencing OCT2 activity is essential for achieving precision medicine and minimizing renal and systemic toxicity. SIGNIFICANCE STATEMENT: Organic cation transporter 2 (OCT2) is essential for the active tubular secretion of xenobiotics and endogenous cationic substances in the kidneys. This article offers a comprehensive overview of the tissue distribution, interspecies differences, and factors affecting its activity-critical for evaluating tissue toxicity and systemic exposure to cationic substances. Using OCT2 biomarkers and integrating OCT2 activity and expression data into physiologically based pharmacokinetic models are valuable tools for predicting OCT2 function and its clinical implications.

Drug-induced hepatotoxicity is the leading cause of attrition of drug candidates and withdrawal of marketed drugs owing to safety concerns. In most hepatotoxicity cases, the parent drugs are metabolized by cytochrome P450 (CYP) enzymes, generating reactive metabolites that bind to intracellular organelles and proteins, ultimately causing hepatocellular damage. A major limitation of animal models, which are widely used for toxicity assessment, is the discrepancy in CYP-mediated drug metabolism and toxicological outcomes owing to species differences between humans and animals. Two-dimensional (2D) hepatocytes were first developed as a promising alternative model using human pluripotent stem cells (hPSCs). However, their CYP expression was similar to that of the fetal liver, and they lacked CYP-mediated hepatic metabolism. CYP expression in hPSC-derived hepatic models is closely correlated with liver maturity. Therefore, liver organoids that are more mature than hPSC-derived hepatic models and mimic the structure and physiological functions of the human liver have emerged as new alternatives. In this review, we explored the role and essentiality of CYPs in human hepatotoxicity, their expression, and epigenetic regulation in hPSC-derived hepatocytes and liver organoids, as well as the current state of liver organoid technology in terms of CYP expression and activity, drug metabolism, and toxicity. We also discussed the current challenges and future directions for the practical use of liver organoids. In conclusion, we highlight the importance of methods and metrics for accurately assessing CYP expression and activity in liver organoids to enable the development of feasible models that reproduce hepatotoxicity in humans.

Background/Objectives: The kidneys are essential for eliminating drugs and chemicals from the human body and renal epithelial cells are particularly vulnerable to damage caused by xenobiotics and their metabolites. Drug-induced kidney toxicity is a major cause of drug attrition during preclinical and clinical development and the ability to predict renal toxicity remains a pressing challenge, necessitating more predictive in vitro models. However, the abundance of commercially available renal proximal tubule epithelial cell (RPTEC) sources complicates the selection of the most predictive cell types. Methods: This study compared a wide range of RPTEC sources, including primary cells (Lonza) and various RPTEC lines from different vendors, such as ciPTECs (Cell4Pharma), TERT1/RPTECs (ATCC), and HEK293 (GenoMembrane), including OAT1-overexpressing variants. HepG2 cells were included for a comparison of organ specificity. The different cells were cultured in 96- or 384-well plates and exposed to 12 drugs for 72 h at a concentration yielding a response (0.3-300 µM) to evaluate their ability to predict clinical outcomes. The CellTiterGlo® assay was used to measure cell viability, and transcriptome data from unexposed cells was analyzed using the TempO-seq® S1500+ platform. Results: Gene expression data showed that the primary kidney cells most closely matched the transcriptome of the human kidney medulla, followed by the TERT1 and ciPTEC lines, with the HEK lines showing the lowest similarity. The RPTEC sources showed clustering by cell type, with OAT1 overexpression driving changes in metabolic, detoxification, and immune pathways, especially in TERT1 cells. Cell viability data were used to determine points of departure (PODs) which were compared to human serum Cmax values to assess safety margins. The TERT1 and ciPTEC RPTEC lines demonstrated the highest predictive performance for nephrotoxicity, with OAT1 overexpression significantly enhancing sensitivity, accuracy, and overall predictive power (MCC scores: 0.764 and 0.667, respectively). In contrast, HepG2 cells showed the lowest performance across all metrics, highlighting the critical role of cell type and transporter expression in nephrotoxicity prediction. Conclusions: This study highlights important differences among RPTEC sources and their utility in drug safety studies of the renal proximal tubule. We show that while improved cell options for renal proximal tubule are needed, OAT1-overexpressing RPTECs are a superior model to the background cell type.

Assessments of a chemical agent's carcinogenicity based on rodent bioassays rely on their appropriate interpretation. This involves attention to study details, including reliable histopathologic diagnoses, and proper statistical analyses, including consideration of multiple comparisons, concurrent and historical controls. A major factor is evaluation of their likely mode of action and the human relevance of any identified tumors. We present a critical evaluation of the assessment of the 2-year inhalation bioassays of n-butyl methacrylate (n-BMA) in rats and mice performed by the Japan Bioassay Research Center (JBRC) and an assessment of the International Agency for Research on Cancer (IARC) review and classification as Group 2B, possible human carcinogen. The tumors of concern for assessment of its carcinogenicity included mononuclear cell leukemia (MCL) in male rats, thyroid C-cell tumors in female rats, liver tumors and histiocytic sarcomas in male mice, and hemangiosarcomas in female mice. Our review of these studies raises concerns regarding the accuracy of histopathology diagnoses and human relevance of MCL. Most critically, the statistical evaluation/interpretation of all tumor types indicates no carcinogenic effects, since the frequency of increases (at p < 0.05) in tumor incidences in the study is totally consistent with chance expectation (i.e. not treatment related). Furthermore, the plausibility of n-BMA being carcinogenic is questionable since it is non-genotoxic, and the weight of evidence including read-across to the close structural analog methyl methacrylate indicates no concern for cancer. After thorough review of these bioassays, we conclude that there is no convincing evidence of carcinogenicity for n-BMA, contrary to the conclusion of the JBRC and the decision by the IARC.

Ferula communis has demonstrated an abundance of pharmacological and antioxidative qualities.

This study investigates the antioxidant activity of F. communis leaf aqueous extract, total polyphenol and flavonoid concentrations, and ultra-high-performance liquid chromatography (UHPLC) composition and then evaluates the toxicity of the plant's leaves in vitro and in silico. The major compound of the studied extract, namely, p-hydroxybenzoic acid, was chosen for a molecular docking technique to discover the inhibition mechanism toward antioxidant proteins. In addition, a detailed molecular dynamics simulation was carried out to examine the thermodynamic stability of the produced intermolecular interactions. The antioxidant capacity of the extracts of F. communis was evaluated using 2,2-diphenylpicryl hydroxyl (DPPH) radical and ferric reducing antioxidant power (FRAP) procedures. Acute toxicity was tested on albino mice at doses of 200, 300, and 400 mg/kg.

The results show that the polyphenol and flavonoid contents of the extract are significant (0.257 ± 0.003 mg Eq AG/mg and 0.32 ± 0.04 mg Eq Q/mg, respectively). The antioxidant activity illustrates that the extracts have notable activity in DPPH and FRAP assays. The toxicity study revealed that the mice's behavior, body weight, and organ weights (liver and kidneys) were unaffected by Ferula communis leaf extract administration compared to controls. UHPLC-tandem mass spectrometry (MS/MS) analysis of the extract highlights the presence of 11 compounds, the most abundant of which is p-hydroxybenzoic acid, representing 53.65%. The predicted pharmacokinetic characteristics of absorption, distribution, metabolism, excretion, and toxicity (ADMET) attest to the well-absorbed nature of the isolated compounds, with human intestinal absorption (HIA) varying from 42% for arbutin (M3) to 100% for ursolic acid (M4).

In conclusion, the leaves of Ferula communis are a good source of natural antioxidants and phenolic compounds. Thus, this study demonstrates that this plant has a wide range of applications, including natural food preservatives, pharmaceuticals, and cosmetics, as evidenced by ongoing research.

Drug-induced intestinal toxicity (GIT) is a frequent dose-limiting adverse event that can impact patient compliance and treatment outcomes. In vivo, there are proliferative and differentiated cell types critical to maintaining intestinal homeostasis. Traditional in vitro models using transformed cell lines do not capture this cellular complexity, and often fail to predict intestinal toxicity. Primary tissue-derived intestinal organoids, on the other hand, are a scalable Complex in vitro Model (CIVM) that recapitulates major intestinal cell lineages and function. Intestinal organoid toxicity assays have been shown to correlate with clinical incidence of drug-induced diarrhea, however existing studies do not consider how differentiation state of the organoids impacts assay readouts and predictivity. We employed distinct proliferative and differentiated organoid models of the small intestine to assess whether differentiation state alone can alter toxicity responses to small molecule compounds in cell viability assays. In doing so, we identified several examples of small molecules which elicit differential toxicity in proliferative and differentiated organoid models. This proof of concept highlights the need to consider which cell types are present in CIVMs, their differentiation state, and how this alters interpretation of toxicity assays.

Spheroids and other 3D cellular models more accurately recapitulate physiological responses when compared to 2D models and represent potential alternatives to animal testing. The cryopreservation of spheroids remains challenging, limiting their wider use. Standard DMSO-only cryopreservation results in supercooling to low subzero temperatures, reducing viability, shedding surface cells, and perforating spheroid interiors. Here, cocultured spheroids with differentially labeled outer cell layers allow spatial evaluation of the protective effect of macromolecular ice nucleators by microscopy and histology. Extracellular nucleation is shown to reduce damage to both interior and exterior regions of the spheroids, which will support the development of "off-the-shelf" 3D models.

Skin wound treatments require efficient and targeted delivery of therapeutic agents to promote fast tissue regeneration and prevent infections. Hydrogels are one of the most popular products in the wound care market, although their use as medicated wound dressings remains a massive challenge when hydrophobic drugs are needed due to the hydrophilic nature of these soft materials. In this study, we developed innovative, dynamic hydrogels based on polyvinyl alcohol (PVA), pyrogallol as a hydrogen bond crosslinker, and casein micelles as hydrophobic reservoirs of silver sulfadiazine (SSD) for enzyme-activated smart delivery at wound sites. The hydrogel formulation was optimized for mechanical strength, viscoelastic behavior, water absorption capacity, and drug-loading efficiency. In vitro drug delivery studies revealed a sustainable release profile of SSD for over 24 h from the micelles within the hydrogel network. Furthermore, biocompatibility evaluation using mouse fibroblast L929 cells demonstrated that the hydrogel did not inhibit cell viability, while in vivo experiments on Caenorhabditis elegans (C. elegans) proved its safety in complex organisms. This versatile hydrogel also has anti-inflammatory and antibacterial effects stemming from the therapeutic polyphenol, which could benefit the healing process. The combination of PVA, pyrogallol, and casein-based nanocarriers could offer an approach to wound healing, providing a new platform for hosting hydrophobic therapeutic substances. Overall, this hydrogel system shows great promise in wound care and could broaden the applications of this family of soft materials for treating various skin injuries.

Pharmaceutical developers are encouraged to adopt the best practices of being purposefully thoughtful about the use of animals, seeking alternatives wherever possible. They should engage with health authorities to increase their familiarity with the methods, study designs, data outputs, and the context of use for new approach methodologies (NAMs). Although current state of technology does not yet provide adequate models to fully replace in vivo studies, many models are sufficiently good for an augmented approach that will enhance our understanding of in vitro to in vivo correlations and advance the long-term goal of reducing animal use through innovative NAMs. The goal of future nonclinical safety packages is to advance the utilization of such enabling technologies toward appropriate human risk characterization. Establishing confidence in NAMs is a critical first step. For example, sponsors may include both "traditional" and NAM-based nonclinical safety data in regulatory submissions to establish confidence with health authorities. In addition, regulators should create a "safe harbor" for hybrid nonclinical data packages to facilitate iterative learning, refinement, and implementation of NAM-based safety assessment strategies. Sponsors are urged to contribute to NAMs evolution through consortia participation, peer-reviewed publications, and documenting animal reduction in studies/programs, accelerating the eventual elimination of animal use in pharmaceutical development, as envisioned in the FDA Modernization Act 3.0.

Abutilon indicum, a shrub of the Malvaceae family, is found abundantly in tropical countries like India. A. indicum is widely used for its high medicinal properties. Traditionally, A. indicum seed powder is consumed to treat piles, constipation, chronic cystitis, gonorrhea, gleet, and pregnancy-related problems. Despite having numerous medicinal properties and widespread traditional use of A. indicum seeds, scientific validation, and toxicity studies have yet to be documented.

The primary objective of this study is to conduct a comprehensive study on phytochemical profiling, in-vitro cytotoxicity, mutagenicity, and in-vivo acute and sub-acute toxicity, and genotoxicity on animal models of methanolic extract of A. indicum seed (MAS).

The qualitative analysis of MAS was explored through FTIR and HR LC-MS. For in-vitro cytotoxicity, the HEK-293 cell line was used, and the TA100 (Staphylococcus typhimurium) bacterial strain was used for the Ames mutagenicity test. A single oral dose of 250, 500, 1000, or 2000 mg/kg body weight of MAS was given to each male and female rat for acute toxicity study and observed for 14 days for any toxicity signs. In the sub-acute toxicity study, 250, 500, or 1000 mg/kg body weight of MAS was administered orally to each rat for 28 days. The experimental animals were weighed weekly, and general behavior was monitored regularly. After 28 days of the experiment, the rats were sacrificed, and different serum biochemical, hematological, and histological analyses were performed. The blood samples of different doses of MAS were used for genotoxicity study through comet assay.

FTIR analysis found different functional groups, which indicated the presence of phenolics, flavonoids, and alkaloids. HR LC-MS analysis depicts several components with different biological functions. The cell cytotoxicity and Ames mutagenicity results showed minimal toxicity and mutagenicity up to a certain dose. The acute toxicity study conducted in Wistar albino rats demonstrated zero mortality among the animals, and the LD50 value for seed extract was determined to be 2000 mg/kg body weight. Sub-acute toxicity assessments indicated that the administration of seed extract resulted in no adverse effects at dosages of 250 and 500 mg/kg body weight. However, at higher doses, specifically 1000 mg/kg body weight, the liver of the experimental rats exhibited some toxic effects. In the genotoxicity study, minimal DNA damage was found in 250 and 500 mg/kg doses, respectively, but slightly greater DNA damage was found in 1000 mg/kg doses in both male and female rats.

The consumption of A. indicum seed powder is deemed safe; however, doses exceeding 500 mg/kg body weight may raise concerns regarding use. These findings pave the path for the creation of innovative medicines with improved efficacy and safety profiles.

Salvia blancoana subsp. mesatlantica (Maire) Figuerola (SBm) is a plant endemic to Morocco and is one of the less studied species of Salvia. Herbal therapy is becoming more and more popular, especially in underdeveloped nations where access to medicinal herbs is affordable. However, some plants demonstrated toxic effects in animals and humans.

Our study aimed to evaluate the SBm-extract for both acute and sub-chronic toxicity.

Aqueous extracts were obtained from the aerial parts of SBm collected from Immouzer Kander commune (Middle Atlas, Morocco). Total Phenolic Content (TPC) and Flavones and flavonols Content (FFC), Antioxidant activity (2,2-diphenyl-1-picrylhydrazyl (DPPH) and reducing power) was determined, and chemical composition was determined by High-Performance Liquid Chromatography with Diode Array Detection (HPLC-DAD). Toxicity tests were conducted on mice and rats.

In acute toxicity, Swiss albino mice (mass of 25-35 g) received SBm-extract orally and intraperitoneally at doses (0.5-11 g/kg, bm). The sub-chronic toxicity was tested in Wistar albino rats (mass of 200-240 g) for 90 days at doses of 0, 250, 500, or 1000 mg/kg, bm. Values of TPC and FFC were estimated to be 157.56 ± 0.32 mg GAE/g DW and 7.89 ± 0.05 mg QE/g DW, respectively. DPPH scavenging (IC50) was estimated to be 26.9 ± 0.08 µg/mL while reducing power was 12.41 ± 0.03 µg/mL. No toxicity or deaths were observed in acute tests after oral exposure, while intraperitoneal administration resulted in dose-dependent acute toxicity, with an LD50 value of 6.82 g/kg. In sub-chronic tests, most hematological and biochemical parameters remained unchanged, except for transient fluctuations in specific blood constituents and a transitory reduction in serum glucose levels observed at elevated dosages. Histopathological investigation revealed no organ abnormalities. The SBm-extract exhibited minimal toxicity, supporting its safe use.

Despite the relevant results of this study, future studies need to confirm these findings and expand our understanding of the safety characteristics of Salvia. Further investigations are needed to explore the effects of other solvents on the extraction of bioactive compounds from the underground and aerial parts of this endemic species. Evaluation of other biological properties such as anti-microbial, anti-cancer, and anti-inflammatory activities are needed.

Addressing drug-induced liver injury is crucial in drug development, often causing Phase III trial failures and market withdrawals. Traditional animal models fail to predict human liver toxicity accurately. Virtual twins of human organs present a promising solution. We introduce the Virtual Hepatic Lobule, a foundational element of the Living Liver, a multi-scale liver virtual twin. This model integrates blood flow dynamics and an acetaminophen-induced injury model to predict hepatocyte injury patterns specific to patients. By incorporating metabolic zonation, our predictions align with clinical zonal hepatotoxicity observations. This methodology advances the development of a human liver virtual twin, aiding in the prediction and validation of drug-induced liver injuries.

The screening of plant-derived compounds with anti-cancer properties is a promising strategy to meet the growing need for new, safe and effective anti-cancer drugs. Justicidin B is a plants secondary metabolite that displays anti-cancer properties in several tumor cells. Therefore, it represents a good candidate. We used the 3R-compliant organism Caenorhabditis elegans to evaluate the safety of justicidin B produced by in vitro-grown adventitious roots of Linum lewisii. We showed that a dose of 100 µg/mL justicidin B does not affect worm vitality in either short-term or chronic administration; in contrast, the 200 µg/mL dose induces a lifespan reduction, but only in short-term daily treatment. We attributed this effect to its accumulation in lipofuscin granules in the pharynx as observed through confocal analysis. HPLC analysis confirmed the higher accumulation justicidin B with a 200 µg/mL dose but also revealed the presence of metabolic derivatives that could be responsible for the toxicity. We also demonstrated that the 100 µg/mL dose does not affect worm fertility or development. Our results highlight the safety of justicidin B, supporting its employment in cancer therapy, and encourage the use of a C. elegans model as an appropriate tool to assess compounds' toxicity before moving to more complex organisms.

The IQ Consortium's DruSafe Leadership Group previously reported results of a nonclinical to clinical translational database for First-In-Human (FIH)-enabling animal toxicology studies. We have completed an additional translational database populated with longer duration (>1 month) animal toxicology studies and longer duration (Phase 2 and beyond) clinical trials. The blinded database was composed of 127 molecules. Animal and clinical data were categorized by organ system and animal model (e.g. rodent, dog). The 2 × 2 contingency table (true positive, false positive, true negative, false negative) was used for statistical analysis and both the positive predictive value (PPV) and negative predictive value (NPV) were determined. As also reported in the FIH database, the NPV was the strongest predictive performance measure at 96 %. The PPV was lower than the FIH database with the rodent at 29 %, dog at 21 % and NHP at 20 %. No new additional target organs were observed in 62 % of the entries. A new target organ was identified in 38 % of the entries, with the majority in a rodent (26 %) and fewer in the dog (8 %) or NHP (12 %). However, new target organ data resulted in only a PPV of 13 %, suggesting that current ICH requirements for longer duration animal general toxicology studies should be re-evaluated and better aligned with the 3Rs. A newer paradigm could include an appropriately justified single animal model for longer duration studies, in addition to utilizing New Approach Methods (NAMs) that would provide translational safety data, but additional research is needed.

The Bhasmas, herbo-minerals, are reported to produce adverse effects when used clinically. Therefore, the present study aimed to assess the safety of Krishna Vajra Abhraka Bhasma (KVB), through acute and subacute toxicity testing.

After ethics committee approval, total 66 Wistar albino rats weighing 180-200 gms were used. Experiments were conducted as per CCSEA guidelines. In acute toxicity study, a single limit dose of 2000 mg/kg of KVB with honey was administered to six female rats. Subacute toxicity studies involved 60 Wistar albino rats of either sex, randomly divided into six groups and received KVB in low (highest clinical dose extrapolated to rats), medium (double the low dose) and high (double the medium dose) dose administered orally for 28 days.

No mortality or adverse effects on rat's general behaviour or internal organs was observed in acute toxicity study The results of subacute study indicated no significant differences in body and organ weights, hematological or renal function between control and treated groups. However, mid and high doses of KVB revealed reversible, dose-dependent hepatotoxicity through biochemical and histopathological evaluation, while the satellite high dose group did not exhibit this effect, confirming it as drug-induced hepatotoxicity.

The acute toxicity results positioned KVB in class V (safe) of the GHS classification. Subacute toxicity findings suggested that while low therapeutic doses were non-toxic, mid and high doses induced reversible hepatotoxicity, emphasizing the importance of careful dosage considerations and patient monitoring in Ayurvedic practice. The study contributes valuable insights into the safety profile of KVB, addressing concerns for both users and practitioners of Ayurvedic medicine.

This review presents a comprehensive analysis of current research on biological treatment processes for removing pharmaceutical compounds (PhCs) from wastewater. Unlike previous studies on this topic, our study specifically delves into the effectiveness and drawbacks of various treatment approaches such as traditional wastewater treatment facilities (WWTP), membrane bioreactors (MBRs), constructed wetlands (CW), and moving bed biofilm reactors (MBBR). Through the examination and synthesis of information gathered from more than 200 research studies, we have created a comprehensive database that delves into the effectiveness of eliminating 19 particular PhCs, including commonly studied compounds such as acetaminophen, ibuprofen, diclofenac, naproxen, ketoprofen, indomethacin, salicylic acid, codeine, and fenoprofen, amoxicillin, azithromycin, ciprofloxacin, ofloxacin, tetracycline, atenolol, propranolol, and metoprolol. This resource provides a depth and scope of information that was previously lacking in this area of study. Notably, among these pharmaceuticals, azithromycin demonstrated the highest removal rates across all examined treatment systems, with the exception of WWTPs, while carbamazepine consistently exhibited the lowest removal efficiencies across various systems. The analysis showcases the diverse results in removal efficiency impacted by factors such as system configuration, operation specifics, and environmental circumstances. The findings emphasize the critical need for continued innovation and research, specifically recommending the integration of advanced oxidation processes (AOPs) with existing biological treatment methods to improve the breakdown of recalcitrant compounds like carbamazepine. PRACTITIONER POINTS: Persistent pharmaceuticals harm aquatic ecosystems and human health. Biological systems show varying pharmaceutical removal efficiencies. Enhancing HRT and SRT improves removal but adds complexity and costs. Tailored treatment approaches needed based on contaminants and conditions.

Regulatory studies have revolutionised over time. Today, the focus has shifted from animal toxicity testing to non-animal for regulatory safety testing. This move is in line with the international 3Rs (Replacement, Reduction, and Refinement) principle and has also changed the regulator's perspective. The 3R principle has stimulated changes in policy, regulations, and new approaches to safety assessment in drug development in many countries. The 3Rs approach has led to the discovery and application of new technologies and more human-relevant in vitro approaches that minimise the use of animals including non-human primates, in research and improve animal welfare. In 2016, the European Medicines Agency published the Guidelines on the principles of regulatory acceptance of 3Rs testing approaches, followed by a conceptual paper in 2023 to align with current 3R standards. Additionally, the United States Food and Drug Administration passed new legislation in 2023 that no longer requires all new human drugs to be tested on animals, which will change the current testing paradigm. This review paper provides the adoption of the 3Rs and the current regulatory perspective regarding their implementation.

Research within the hepato-biliary system and hepatic function is currently experiencing heightened interest, this is due to the high frequency of relapse rates observed in chronic conditions, as well as the imperative for the development of innovative therapeutic strategies to address both inherited and acquired diseases within this domain. The most commonly used sources for studying hepatocytes include primary human hepatocytes, human hepatic cancer cell lines, and hepatic-like cells derived from induced pluripotent stem cells. However, a significant challenge in primary hepatic cell culture is the rapid decline in their phenotypic characteristics, dedifferentiation and short cultivation time. This limitation creates various problems, including the inability to maintain long-term cell cultures, which can lead to failed experiments in drug development and the creation of relevant disease models for researchers' purposes. To address these issues, the creation of a powerful 3D cell model could play a pivotal role as a personalized disease model and help reduce the use of animal models during certain stages of research. Such a cell model could be used for disease modelling, genome editing, and drug discovery purposes. This review provides an overview of the main methods of 3D-culturing liver cells, including a discussion of their characteristics, advantages, and disadvantages.

The proliferation of multidrug-resistant and biofilm-forming pathogenic bacteria poses a serious threat to public health. The limited effectiveness of current antibiotics motivates the search for new antibacterial compounds. In this study, a novel strain, RG-5, was isolated from desert soil. This strain exhibited potent antibacterial and antibiofilm properties against multidrug-resistant and biofilm-forming pathogenic bacteria. Through phenotypical characterizations, 16S rRNA gene sequence and phylogenetic analysis, the strain was identified as Streptomyces pratensis with 99.8% similarity. The active compound, RG5-1, was extracted, purified by reverse phase silica column HPLC, identified by ESI-MS spectrometry, and confirmed by 1H and 13C NMR analysis as 2,5-Piperazinedione, 3,6-bis(2-methylpropyl), belonging to cyclic peptides. This compound showed interesting minimum inhibitory concentrations (MICs) of 04 to 15 µg/mL and minimum biofilm inhibitory concentrations (MBICs 50%) of ½ MIC against the tested bacteria. Its molecular mechanism of action was elucidated through a molecular docking study against five drug-protein targets. The results demonstrated that the compound RG5-1 has a strong affinity and interaction patterns with glucosamine-6-phosphate synthase at - 6.0 kcal/mol compared to reference inhibitor (- 5.4 kcal/mol), medium with penicillin-binding protein 1a (- 6.1 kcal/mol), and LasR regulator protein of quorum sensing (- 5.4 kcal/mol), confirming its antibacterial and antibiofilm activities. The compound exhibited minimal toxicity and favorable physicochemical and pharmacological properties. This is the first report that describes its production from Streptomyces, its activities against biofilm-forming and multidrug-resistant bacteria, and its mechanism of action. These findings indicate that 2,5-piperazinedione, 3,6-bis(2-methylpropyl) has the potential to be a promising lead compound in the treatment of antibiotic-resistant and biofilm-forming pathogens.

Hepatic bile acid regulation is a multifaceted process modulated by several hepatic transporters and enzymes. Drug-induced cholestasis (DIC), a main type of drug-induced liver injury (DILI), denotes any drug-mediated condition in which hepatic bile flow is impaired. Our ability in translating preclinical toxicological findings to human DIC risk is currently very limited, mainly due to important interspecies differences. Accordingly, the anticipation of clinical DIC with available in vitro or in silico models is also challenging, due to the complexity of the bile acid homeostasis. Herein, we assessed the in vitro inhibition potential of 47 marketed drugs with various degrees of reported DILI severity towards all metabolic and transport mechanisms currently known to be involved in the hepatic regulation of bile acids. The reported DILI concern and/or cholestatic annotation correlated with the number of investigated processes being inhibited. Furthermore, we employed univariate and multivariate statistical methods to determine the important processes for DILI discrimination. We identified time-dependent inhibition (TDI) of cytochrome P450 (CYP) 3A4 and reversible inhibition of the organic anion transporting polypeptide (OATP) 1B1 as the major risk factors for DIC among the tested mechanisms related to bile acid transport and metabolism. These results were consistent across multiple statistical methods and DILI classification systems applied in our dataset. We anticipate that our assessment of the two most important processes in the development of cholestasis will enable a risk assessment for DIC to be efficiently integrated into the preclinical development process.

The pre-clinical animal models often fail to predict intrinsic and idiosyncratic drug induced liver injury (DILI), thus contributing to drug failures in clinical trials, black box warnings and withdrawal of marketed drugs. This suggests a critical need for human-relevant in vitro models to predict diverse DILI phenotypes. In this study, a porcine liver extracellular matrix (ECM) based biomaterial ink with high printing fidelity, biocompatibility and tunable rheological and mechanical properties is formulated for supporting both parenchymal and non-parenchymal cells. Further, we applied 3D printing and microfluidic technology to bioengineer a human physiomimetic liver acinus model (HPLAM), recapitulating the radial hepatic cord-like structure with functional sinusoidal microvasculature network, biochemical and biophysical properties of native liver acinus. Intriguingly, the human derived hepatic cells incorporated HPLAM cultured under physiologically relevant microenvironment, acts as metabolic biofactories manifesting enhanced hepatic functionality, secretome levels and biomarkers expression over several weeks. We also report that the matured HPLAM reproduces dose- and time-dependent hepatotoxic response of human clinical relevance to drugs typically recognized for inducing diverse DILI phenotypes as compared to conventional static culture. Overall, the developed HPLAM emulates in vivo like functions and may provide a useful platform for DILI risk assessment to better determine safety and human risk.