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bioRxiv2024

Cadmium Disrupts Blood-Testis Barrier (BTB) via An Oxidative Stress-Dependent Autophagy in Prepubertal Rats

Blood testis barrier (BTB) is an important target of cadmium (Cd) toxicology, but the mechanism underlying the Cd-induced impairment of BTB function remains fully elucidated. The mammalian target of rapamycin (mTOR) complex 2 (mTOR2) regulates BTB function via its effects on cellular junctions and the cytoskeleton of Sertoli cells. In this study, we investigated whether mTOR2 was involved in the effects of Cd exposure on BTB integrity. A Cd exposure model in vivo was established in prepubertal male rats using a single intraperitoneal injection of Cadmium Chloride (CdCl2). A Cd exposure model of Sertoli cell was established using a CdCl2-treated TM4 cell line. The Acute cadmium exposure decreased the activity of mTOR2 signaling and adhesin proteins which is linked to the induction of oxidative stress-induced autophagy. In the presence of CdCl2, mTOR, the catalytic subunit of the mTOR2 complex, exhibits a reduction in levels of phosphorylation, accompanied by decreased adhesin proteins and Rictor, the key component of the mTOR2 complex. CdCl2 treatment also drives a process of oxidative stress-induced autophagy, evidenced by alterations in cellular markers for oxidative stress and autophagy. Pharmaceutical inhibition of oxidative stress and/or autophagy alleviates the alternations in mTOR2 signaling and adhesin proteins upon CdCl2 treatment in TM4, a Sertoli cell line. This work is the first to examine the effects of cadmium exposure on rictor/mTOR2 signaling pathways. Our results suggest that Cadmium might exert testicular toxicology via the perturbation in mTOR2 signaling, which can be associated with the cellular stress-related protolysis in Sertoli cells.

pharmacology and toxicology↗

bioRxiv2025

A Precision Gene Engineered B Cell Medicine Producing Sustained Levels of Active Factor IX for Hemophilia B Therapy

Hemophilia B gene therapy treatments currently have not addressed the need for predictable, durable, active, and redosable factor IX (FIX). Unlike conventional gene therapy, engineered B Cell Medicines (BCMs) are durable, redosable, and titratable, and thus have the potential to address significant unmet needs in the Hemophilia B treatment paradigm. BE-101 is an autologous BCM comprised of expanded and differentiated B lymphocyte lineage cells genetically engineered ex vivo to secrete FIX-Padua. CRISPR/Cas9 mediated gene editing at the C-C chemokine receptor type 5 locus was used to facilitate transgene insertion of an AAV6-encoded DNA template via homology-directed repair. Transgene insertion did not alter B cell biology, viability, or differentiation into plasma cells. Appreciable levels of BE-101-derived FIX-Padua were detected within 1 day after IV administration in mouse and steady state was reached within 2 weeks and persisted for over 184 days. Redosing produced an increase in FIX-Padua production close to linear dose proportionality. Comprehensive genotoxicity analysis found no off-target issues of concern. No safety signals were observed in animal tolerability and GLP toxicology studies. In conclusion, BE-101 produces sustained levels of active FIX-Padua with the ability to engraft without host preconditioning and with the potential for redosing and titratability.

pharmacology and toxicology↗

bioRxiv2019

The flavor enhancer maltol increases pigment aggregation in dermal and neural melanophores in Xenopus laevis tadpoles

Melanophores are pigmented cells that change the distribution of pigmented melanosomes, enabling animals to appear lighter or darker for camouflage, thermoregulation, and UV-protection. A complex series of hormonal and neural mechanisms regulates melanophore pigment distribution, making these cells a valuable tool to screen toxicants as a dynamic cell type that responds rapidly to the environment. We found that maltol, a naturally occurring flavor enhancer and fragrance agent, induces melanophore pigment aggregation in a dose-dependent manner in Xenopus laevis tadpoles. To determine if maltol affects camouflage adaptation, we placed tadpoles into maltol baths situated over either white or black background. Maltol induced pigment aggregation in a similar dose-dependent pattern regardless of background color. We also tested how maltol treatment compares to melatonin treatment and found that the degree of pigment aggregation induced by maltol is similar to treatment with melatonin, but the time course differs significantly. Last, maltol had no effect on mRNA expression of pro-opiomelanocortin or melanin concentrating hormone receptor in the brain, both of which regulate camouflage-related pigment aggregation. Our results suggest that maltol does not exert its effects via the camouflage adaptation mechanism nor via melatonin-based mechanisms. These results are the first to identify a specific toxicological effect of maltol exposure and rules out several mechanisms by which maltol may exert its effects on pigment aggregation.

pharmacology and toxicology↗

bioRxiv2022

Toxicological study on methanol root bark extract of Acacia sieberiana (Fabaceae) in Wistar rats

BackgroundThe plant Acacia sieberiana belongs to the family Fabaceae. It has been used in ethnomedical practice to manage bleeding, rheumatism, pain, pyrexia, kidney diseases, gastrointestinal problems, parasitic and infectious diseases, hepatitis, cough, epilepsy, mouth ulcer and many more. Phytochemical compounds such as ellagic acid, quercetin, isoferulic acid, gallic acid, kaempferol, luteolin, apigenin, glucoside dihydroacacipetalin, acacipetalin and many others were isolated from Acacia sieberiana. Previous pharmacological investigations have reported that the plant has anticancer, antimicrobial, antidiarrhoeal and antitrypanosomal effects. Despite the therapeutic properties of this plant, no safety information is available in the literature. Hence, this work intends to investigate the sub-acute toxicity effects of Acacia sieberiana root bark extract (ASE). The phytochemical and oral median lethal dose (LD50) evaluations on the ASE were done in line with the standard protocols. The sub-acute toxic effects of the ASE (250, 750, and 1,500 mg/kg) were investigated following administration of the ASE daily for 28-consecutive days based on the Organization of Economic Cooperation and Development (OECD) 407 protocols in rats. The weekly body weights were monitored and the rats were euthanized on the 29th day. The blood samples from the animals were obtained for biochemical and haematological determinations. The liver, kidney, lung and heart were removed for histological investigations. ResultsThe ASE revealed triterpenes, tannins, saponins, cardiac glycosides, flavonoids, and alkaloids. The oral LD50 values was >5,000 mg/kg. The ASE remarkably (p<0.05) declined the body weight of the rats in consideration to the control categories. There was also a remarkable (p<0.05) elevation in ALP, urea and lymphocytes. The cardiac histology revealed no abnormalities. However, the liver produced dose-dependent hepatocellular necrosis and vacuolations. Besides, lymphocyte hyperplasia and glomerular necrosis were observed in the kidneys and alveolar congestion in the lungs. ConclusionsThe ASE is relatively non-toxic on acute administration. In contrast, it could pose slight hepatic and renal toxicity on sub-acute administration.

pharmacology and toxicology↗

bioRxiv2025

Chronic Cadmium Exposures and Hyperglycemia Additively Drive Mitochondrial Dysfunction in Hepatic Cells: Key Implications for MASLD Etiopathogenesis

Effects of chronic heavy metal stress on hepatocellular pathophysiology remains ill-understood. Human livers are a long-term accumulative site for many toxic heavy metals (e.g., cadmium and arsenic) whose effects are unknown. In the current study, we studied effects of chronic, low-dose exposures of cadmium (CLEC) modulated by normoglycemic (5.6 mM) and hyperglycemic (15 mM) exposures, focusing on hepatocellular mitochondrial function. HepG2 and HUH7 cell lines were exposed to CLEC and glucose for 24 weeks, mimicking a chronic heavy metal exposure paradigm seen in normal and type II diabetic individuals. We observe that CLEC exposures significantly affect the long-term health of mitochondria, including decreased mitochondrial mass, increased superoxide production, and loss of mitochondrial membrane potential (MMP) in a CLEC and glucose-dependent manner. Furthermore, the Seahorse MitoStress assay revealed CLEC induced significant chronic oxidative stress. In particular, CLEC cells showed altered levels of basal and non-mitochondrial respiration, causing dysregulation in mitochondrial oxygen consumption rates (OCRs). Lastly, we identified significant impacts of CLEC and glucose exposures on the mitochondrial dynamics (fission/fusion) of the CLEC cells, which showed enhanced mitochondrial fragmentation and turnover rates. We also identified novel cell compensatory mechanisms that may mask the true extent of chronic Cd exposure induced damage in liver cells. CLEC and glucose work additively to damage hepatocellular mitochondrial function. New approach methodologies (NAMs), such as the current vitro toxicology study, establish the insidious effects of chronic heavy metal pollutant exposures on human hepatocellular function.

pharmacology and toxicology↗

bioRxiv2024

Network toxicology focused investigation on the impacts of inorganic arsenic and cadmium on human and ecosystem health

Heavy metals like arsenic and cadmium are persistent environmental pollutants that pose serious health risks to humans and ecosystems due to their toxicity, bioaccumulation potential, and frequent presence in consumer products. Network toxicology offers a holistic in silico framework to elucidate the complex biological mechanisms of toxicity, thereby supporting New Approach Methodologies (NAMs) for toxicity assessment. In this study, network toxicological tools were utilized to investigate arsenic- and cadmium-induced toxicities. Toxicity endpoints associated with inorganic arsenic and cadmium compounds were curated from six exposome-relevant databases and mapped to key events (KEs) across adverse outcome pathways (AOPs) cataloged in AOP-Wiki. This led to construction of stressor-AOP networks, revealing 51 AOPs associated with arsenic and 78 with cadmium, and facilitated mechanistic case studies of pathways relevant to human and ecological health. Toxicity concentrations and bioconcentration factors from the ECOTOX database were then used to construct stressor-species networks, that helped identify species that are vulnerable and potentially bioaccumalate these chemicals. Further, the construction of species sensitivity distributions (SSDs) and toxicity-normalized SSDs (SSDn), provided a comparative framework for prioritizing these compounds in risk assessments. Further, integrating SSD data with stressor-species networks identified species groups particularly sensitive to arsenic and cadmium exposure, enhancing these networks utility for ecological risk assessment. The networks and related data generated in this study are freely available for further research at https://cb.imsc.res.in/heavymetaltox/. Overall, this study offers a comprehensive perspective on the toxicological impact of inorganic arsenic and cadmium compounds, supporting a One Health approach to their regulatory and mitigation strategies.

pharmacology and toxicology↗

bioRxiv2026

Single-cell transcriptomics reveals a differential response of human bronchial epithelial cell-types to cadmium chloride

Exposure of cells or tissues to chemical compounds can be analyzed through transcriptomic signatures, which can be used to classify chemical agents. This information can also enrich Adverse Outcome Pathways (AOP). Transcriptional signatures have generally been obtained using "bulk" analysis, by which the global gene expression pattern of an entire tissue is determined. Although this approach has been useful in toxicology, some information is lost, especially when tissues containing multiple cell types are considered. With the advent of single-cell transcriptomics (scRNA-seq), it is now possible to obtain higher resolution, cell type-specific responses in complex tissues. The aim of the present study was to evaluate the added value of scRNA-seq in analysis of the acute response of human bronchial epithelial cells grown at the air/liquid interface (ALI) to a known toxic compound, CdCl2, with well described transcriptional signatures of exposure. Fully differentiated mucocilliary epithelia obtained from three independent donors were exposed to 10 {micro}M CdCl2 and scRNA-seq analysis was performed on a total of 18255 cells to obtain cell type-specific signatures. Our results show that the contribution of each cell type to the overall transcriptomic bulk response varies. For example, the classical heavy metal detoxification response was only detected in multiciliated and secreting cells, while absent in basal cells. The data demonstrate that scRNA-seq provides high-resolution transcriptional signatures with unexpected features. This added information is likely to have implications for the refinement of AOPs and could serve as a basis for a new generation of tests in predictive toxicology.

pharmacology and toxicology↗

bioRxiv2026

Unveiling the Toxicological and Allelopathic Effects of Pteridium aquilinum: Chemical Profiling and Biological Assays

The fern Pteridium aquilinum (bracken) is among the world most widespread plants and poses serious ecological and health risks due to the presence of illudane glycosides (IGs), carcinogenic compounds that particularly affect grazing livestock. This study determined the presence of IGs in plant extracts and in water samples from the Asturias region (northern Spain), an area characterized by intensive cattle farming, and assessed the genotoxic potential, and allelopathic effects of these samples. The glycosides ptaquiloside (PTA) and ptesculentoside (PTE), together with the degradation products pterosin A and pterosin B, were quantified in in vitro-cultured gametophytes and young sporophytes, spring-collected croziers, and autumn water samples from several bracken-infested sites. All sampling locations contained IGs in both plant and water samples, with particularly high concentrations of PTA detected in croziers from Brana Vieja (Somiedo), where cattle deaths had been reported. In vivo genotoxicity assays, using Drosophila melanogaster, revealed induction of somatic mutations and recombination events by aqueous plant extracts and of somatic mutations by water samples. The genotoxic activity of plant extracts, but not that of water samples, was associated with pterosins A and B. Moreover, comet assays in cultured human cells confirmed the genotoxic activity of plant extracts. Unexpectedly, allelopathic bioassays indicated a possible phytostimulatory rather than inhibitory effect of bracken extracts on the germination of meadow species. These findings underscore the widespread presence and biological activity of bracken in northern Spain and highlight the urgent need for management strategies to mitigate the ecological and toxicological threats posed by bracken proliferation.

pharmacology and toxicology↗

bioRxiv2025

Hepatic cellular stress response pathways exhibit species differences in basal and inducible activity

Cellular stress response pathways such as the NRF2 oxidative stress response, endoplasmic reticulum (ER) stress response and macroautophagy afford protection against many forms of drug toxicity, including the liver toxicity associated with the formation of reactive drug metabolites. To maximise the translatability of preclinical toxicology studies, an understanding of the relative hepatic stress response capacities of humans and widely-used preclinical animal species is vital. In control liver tissue, the basal gene and protein expression of stress response pathway components was found to be greater in rodents than non-rodent preclinical species and humans. In addition, following in vitro exposure to pharmacological modulators of the NRF2 and ER stress responses, rodent hepatocytes generally displayed a greater capacity, relative to those of non-rodent preclinical species and humans, for adaptation to cellular stress. Consistent with the reported lower concordance of drug toxicity between humans and rats, the latter displayed a greater level of Torin1-induced autophagic flux than all other species, while the robust transcriptional responses to thapsigargin-induced endoplasmic reticulum stress and Bardoxolone- or Ki696-mediated NRF2 activation were comparable between mouse and rat hepatocytes. In all, our results indicate that rodent preclinical species possess a greater basal and adaptive hepatic capacity for mitigation of chemical insult than non- rodent preclinical species and humans. This study represents the first to provide a comprehensive comparison of stress response pathway capacity of humans and the animal species most commonly used for preclinical drug safety assessment. Our findings can be used to inform the selection of species for safety testing of drugs with a liability for reactive metabolite-mediated liver toxicity.

pharmacology and toxicology↗

bioRxiv2024

A network toxicology approach for mechanistic modelling of nanomaterial hazard and adverse outcomes

Hazard assessment is the first step in evaluating the potential adverse effects of chemicals. Traditionally, toxicological assessment has focused on the exposure, overlooking the impact of the exposed system on the observed toxicity. However, systems toxicology emphasises how system properties significantly contribute to the observed response. Hence, systems theory states that interactions store more information than individual elements, leading to the adoption of network based models to represent complex systems in many fields of life sciences. Here, we developed a network-based approach to characterise toxicological responses in the context of a biological system, inferring biological system specific networks. We directly linked molecular alterations to the adverse outcome pathway (AOP) framework, establishing connections with toxicologically relevant phenotypic events. We applied this framework on a dataset including 31 engineered nanomaterials with different physicochemical properties in two different in vitro and one in vivo models and demonstrated how the biological system is the driving force of the observed response. This work highlights the potential of network-based methods to significantly improve our understanding of toxicological mechanisms from a systems biology perspective, guiding the hazard assessment of nanomaterials and other advanced materials.

pharmacology and toxicology↗

bioRxiv2026

Systematic toxicological study of PFOS/PFOA co-exposure driving prostate cancer: Core target identification, TME immune remodeling, and combination drug prediction

BackgroundPer- and polyfluoroalkyl substances (PFAS), particularly perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), are persistent organic pollutants ubiquitous in the environment. Epidemiological evidence has closely linked them to an elevated risk of prostate cancer (PCa). However, the precise molecular mechanisms by which combined PFOS/PFOA exposure promotes prostate cancer and their dynamic effects on the tumor microenvironment remain unclear. MethodsThis study constructed a multi-module analytical framework integrating network pharmacology and computational biology: (1) Through ADMET toxicity prediction, multi-database target collection (three-way Venn analysis), panoramic GO/KEGG enrichment, focused androgen receptor (AR) axis analysis, GWAS genetic association validation, protein-protein interaction (PPI) network construction, machine learning-based independent screening, and a relaxed intersection strategy, we systematically identified PFOS/PFOA-prostate cancer core targets. (2) Subsequently, a PFAS-PTS score weighted purely by Cox coefficients was employed to drive gene set variation analysis (GSVA)-based pathway enrichment, tumor microenvironment (TME) deconvolution, ordinary differential equation (ODE)-based kinetic modeling, and drug intervention prediction. ResultsTarget collection identified 100 shared PFOS/PFOA-prostate cancer targets, from which 18 core targets were determined after multi-module screening. These targets were significantly enriched in the AR signaling axis, the PI3K-AKT pathway, and cell cycle regulation. Molecular docking confirmed strong binding affinities of PFOS/PFOA with AR (-9.49/-8.56 kcal/mol), AKT1 (-7.56/-6.93 kcal/mol), and PTEN (-6.36/-6.08 kcal/mol). GSVA revealed that the G2M checkpoint and E2F target gene pathways were significantly upregulated in the high-risk group (padj < 0.001), whereas the androgen response pathway was downregulated (padj = 4.8e-4). TME deconvolution (GSE141445, NNLS) revealed a significantly increased proportion of tumor cells (PCa) (p = 2.4e-4) and markedly reduced CD8+ T cell infiltration (p = 5.7e-4) in the high-risk group, indicating immunosuppressive microenvironment remodeling. ODE-based kinetic modeling confirmed that PFAS promoted tumor cell proliferation and suppressed immune surveillance in a dose-dependent manner. Drug intervention simulation demonstrated that the combination of enzalutamide and Alpelisib achieved optimal tumor cell inhibition (33.9% predicted by the ODE model). ConclusionPFOS/PFOA promote prostate cancer progression primarily through multi-target synergy involving AR axis disruption, PI3K-AKT pathway activation, and cell cycle dysregulation, while reshaping an immunosuppressive tumor microenvironment. The integrative computational framework established in this study provides systematic computational evidence for risk assessment and therapeutic intervention in PFAS-associated prostate cancer.

pharmacology and toxicology↗

bioRxiv2026

Antimicrobial cetylpyridinium chloride disrupts the mitochondrial electron transport chain as potently as cyanide, via cardiolipin interference at cytochrome C

People are widely exposed to the antimicrobial cetylpyridinium chloride (CPC) via consumer products, but CPC is a mitochondrial toxicant with potency comparable to that of canonical mitotoxicants. CPC is largely unregulated despite growing usage, bioavailability, and ability to cross the blood-brain barrier. Previously, we showed, in several cell types at non-cytotoxic and exposure-relevant doses, CPC inhibits ATP and OCR, endpoints of the electron transport chain (ETC). Mitochondrial toxicity is linked to multiple diseases (e.g., diabetes, Parkinsons, myalgic encephalomyelitis), but CPC has not been studied epidemiologically, and little mechanistic information is available. To determine why OCR and ATP are hampered by CPC, we hypothesized that CPC inhibits individual ETC components, cardiolipin, or TCA enzymes. Here, we show that, in primary human skin cells, an immune mast cell model, and isolated mitochondria, CPC apparently inhibits multiple ETC Complexes. Detailed investigation pinpointed the mechanism to the distal end of ETC: Complex III-cytochrome C-Complex IV. Using multiple approaches, we show that CPC does not directly inhibit any of the Complexes (not even Complex I as earlier reported), nor TCA enzymes, nor coenzyme Q. Yet, we found that CPC exhibits mitotoxicity as potent as cyanide. Anionic lipid cardiolipin attracts cytochrome C to the inner mitochondrial membrane so that it may shuttle electrons from Complex III to IV. Despite not altering levels of cardiolipin, CPC hinders cytochrome C by electrostatically interfering with cardiolipin. To aid epidemiology, risk analysis, and predictive toxicology, we have determined the precise biochemical mechanism of action of this ubiquitous compound.

pharmacology and toxicology↗

bioRxiv2023

SQ3370, the first clinical click chemistry-activated cancer therapeutic, shows safety in humans and translatability across species

BackgroundSQ3370 is the first demonstration of the Click Activated Protodrugs Against Cancer (CAPAC) platform that uses click chemistry to activate drugs directly at tumor sites, maximizing therapeutic exposure. SQ3370 consists of a tumor-localizing biopolymer (SQL70) and a chemically-attenuated doxorubicin (Dox) protodrug SQP33; the protodrug is activated upon clicking with the biopolymer at tumor sites. Here, we present data from preclinical studies and a Phase 1 dose-escalation clinical trial in adult patients with advanced solid tumors (NCT04106492) demonstrating SQ3370s activation at tumor sites, safety, systemic pharmacokinetics (PK), and immunological activity. MethodsTreatment cycles consisting of an intratumoral or subcutaneous injection of SQL70 biopolymer followed by 5 daily intravenous doses of SQP33 protodrug were evaluated in tumor-bearing mice, healthy dogs, and adult patients with solid tumors. ResultsSQL70 effectively activated SQP33 at tumor sites, resulting in high Dox concentrations that were well tolerated and unachievable by conventional treatment. SQ3370 was safely administered at 8.9x the veterinary Dox dose in dogs and 12x the conventional Dox dose in patients, with no dose-limiting toxicity reported to date. SQ3370s safety, toxicology, and PK profiles were highly translatable across species. SQ3370 increased cytotoxic CD3+ and CD8+ T-cells in patient tumors indicating T-cell-dependent immune activation in the tumor microenvironment. ConclusionsSQ3370, the initial demonstration of click chemistry in humans, enhances the safety of Dox at unprecedented doses and has the potential to increase therapeutic index. Consistent safety, toxicology, PK, and immune activation results observed with SQ3370 across species highlight the translatability of the click chemistry approach in drug development. Trial registrationNCT04106492; 7 September 2019

pharmacology and toxicology↗

bioRxiv2026

Visual - Acoustic Thigmotaxis in Zebrafish Larvae: A High throughput NAM for Neurotoxicity Assessment

The nervous system is highly vulnerable to chemical disruption, yet current regulatory guidelines do not include behavioral endpoints that capture changes in stress-related responses. Zebrafish larvae, up to 5 days old, have emerged as a promising model to bridge this gap, offering genetic and neurochemical similarity to humans together with high throughput potential. In this work, we have developed and evaluated a larval thigmotaxis assay as a new approach methodology (NAM) to detect behavioral alterations caused by neuroactive substances. Thigmotaxis, or edge-preference behavior, was studied in zebrafish larvae exposed to a range of model compounds and challenged with both visual (light/dark) and acoustic (sound/silence) stimuli. We compared 24 round well plates, commonly used in behavioral assays, with 96 square well plates to increase throughput. The two formats showed equivalent results, supporting the use of the higher-capacity system. Classical controls confirmed assay performance with caffeine increasing thigmotaxis, while diazepam decreased it. Additional neuroactive substances with diverse modes of action (chlorpyrifos, nicotine, dexamethasone, ethylenethiourea) produced stimulus-dependent responses, whereas negative controls (saccharin, amoxicillin) had little or no effect. Benchmark dose modeling showed that thigmotaxis was generally more sensitive than traditional locomotor activity endpoints. Overall, this multiplexed visual-acoustic thigmotaxis assay proved reproducible, scalable, and sensitive. In neurotoxicity testing this method could be used both as a stand-alone assay or as part of a broader behavioral NAM battery to assess potential effects on the vertebrate nervous system. This method provides a practical and ethical tool to improve chemical safety assessment both in ecotoxicology and human toxicology. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=62 SRC="FIGDIR/small/703464v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1ceea53org.highwire.dtl.DTLVardef@17a2a8borg.highwire.dtl.DTLVardef@17f16b1org.highwire.dtl.DTLVardef@aac24f_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2021

Niclosamide Inhalation Powder Made by Thin-Film Freezing: Pharmacokinetic and Toxicology Studies in Rats and Hamsters

In this work, we have developed and tested in vivo a dry powder form of niclosamide made by thin-film freezing (TFF) and administered it by inhalation to rats and hamsters. The niclosamide dry powder, suitable for inhalation, is being developed as a therapeutic agent against COVID-19 infection. Niclosamide, a poorly water-soluble drug, is an interesting drug candidate because it was approved over 60 years ago for use as an anthelmintic medication, but recent studies demonstrated its potential as a broad-spectrum antiviral with a specific pharmacological effect against SARS-CoV-2 infection. In the past, clinical trials for other indications were terminated prior to completion due to low and highly variable oral bioavailability. In order to quickly address the current pandemic, targeting niclosamide directly to the lungs is rational to address the COVID-19 main clinical complications. Thin-film freezing technology was used to develop a niclosamide inhalation powder composition that exhibited acceptable aerosol performance with a fine particle fraction (FPF) of 86.0% and a mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of 1.11 m and 2.84, respectively. This formulation not only proved to be safe after an acute three-day, multi-dose pharmacokinetic study in rats as evidenced by histopathology analysis, but also was able to achieve lung concentrations above the required IC50 and IC90 levels for at least 24 h after a single administration in a Syrian hamster model. To conclude, we successfully developed a niclosamide dry powder inhalation formulation by thin-film freezing for further scale-up and clinical testing against the COVID-19 infection. This approach overcomes niclosamides limitation of poor oral bioavailability by targeting the drug directly to the primary site of infection, the lungs.

pharmacology and toxicology↗

bioRxiv2024

Predicting uptake and elimination kinetics of chemicals in invertebrates: a technicalnote on residual variance modeling

Toxicokinetic models for predicting contents of nanomaterials and other toxic chemicals are often fitted without evaluation of the residual variance structure. The aim of the present study was to evaluate various residual variance structures, assuming either homoscedasticity or heteroscedasticity, when fitting non-linear toxicokinetic one-compartment models for predicting uptake, bioaccumulation and elimination of chemicals in invertebrate organisms. Data describing the exposure of several aquatic and terrestrial invertebrates to specific metal nanomaterials and other chemicals were available from real experiments for evaluating the residual variance functions for toxicokinetic models. As proof of concept, datasets of truly homoscedastic and heteroscedastic nature were simulated. Depending the dataset, applying models with different residuals variance assumption largely affected the residual plots and the error margins of parameters or the predicted content of a chemical. Consequently, selecting the most accurate residual variance functions for toxicokinetic modeling, either homoscedastic or heteroscedastic, improves the prediction of chemical contents in invertebrate organisms and the estimation of the associated uptake and elimination rates. HighlightsO_LIResidual plots indicate if an accurate model was fitted to the toxicological data C_LIO_LIChoice of residual variance function affects the error margins of predicted chemicals C_LIO_LISelecting proper residual variance models may prevent false positives and negatives C_LI

pharmacology and toxicology↗

bioRxiv2019

Assessment of chemotherapy-induced organ damage with 68Ga-labeled duramycin

Compared to standard toxicological techniques in preclinical toxicity studies, non-invasive imaging of organ toxicity enables fast and longitudinal investigation of the whole animal. Therefore, we set out to evaluate [68Ga]Ga-NODAGA-duramycin as a positron emission tomography (PET)-tracer of cell death for detecting chemotherapy-induced organ toxicity.\n\nMethodsNODAGA-duramycin was radiolabeled with 68Ga, and quality control was done by thin layer chromatography and high performance liquid chromatography. Tracer specificity was determined in vitro by performing competitive binding experiments on ethanol treated cells. To optimize the timing of the PET/CT-based tracer evaluation, kinetic studies were performed in untreated and cisplatin-treated (20 mg/kg BW, intraperitoneal (i.p.)) BALB/cAnNRj mice. Organ uptake was analyzed in doxorubicin (4 mg/kg BW, i.p.)-, busulfan (18.8 mg/kg KG, i.p.)-, and cisplatin-treated (20 mg/kg BW, i.p.) mice, and in untreated control mice 2 hours after intravenous injection of 5-10 MBq [68Ga]Ga-NODAGA-duramycin. For immunofluorescence validation, tissue sections were stained with anti-active caspase-3 antibody. Blood and serum samples were collected to determine platelet count, aspartate transaminase, alanine transaminase, urea, creatinine, and creatine kinase values.\n\nResultsIn vitro experiments confirmed specific binding of [68Ga]Ga-NODAGA-duramycin to dying cells. The biodistribution analysis revealed a blood half-life of 10-17 minutes and a predominantly urinary excretion of the radiotracer. Doxorubicin-, busulfan-, and cisplatin-induced organ toxicities were detected successfully using [68Ga]Ga-NODAGA-duramycin PET/CT and confirmed by immunohistochemistry as well as blood parameter analysis. Busulfan-related spleno-, cardio-, and pneumotoxicity as well as cisplatin-induced cardio- and pneumotoxicity were detected even earlier by [68Ga]Ga-NODAGA-duramycin PET/CT than by blood parameters and histological stainings. In livers and kidneys, differences between treated and untreated animals tended to occur in PET/CT at later time points than in histology due to the relatively high background in these organs. However, trends over time were comparable.\n\nConclusion[68Ga]Ga-NODAGA-duramycin PET/CT was successfully applied to non-invasively detect chemotherapy-induced organ toxicity with high sensitivity in preclinical studies. It even depicted some toxic effects prior to immunohistochemistry and blood parameter analysis and represents a promising alternative or complementary method to standard toxicological analyses. Furthermore, the tracer has a high translational potential and may provide a valuable link between preclinical and clinical research.

pharmacology and toxicology↗

bioRxiv2024

Identification of non-charged 7.44 analogs interacting with the NHR2 domain of RUNX1-ETO and exhibiting an improved, selective antiproliferative effect in RUNX-ETO positive cells

The RUNX1/ETO fusion protein is a chimeric transcription factor in acute myeloid leukemia (AML) created by chromosomal translocation t(8;21)(q22;q22). t(8;21) abnormality is associated with 12% of de novo AML cases and up to 40% in the AML subtype M2. Previously, we identified the small-molecule inhibitor 7.44, which specifically interferes with NHR2 domain tetramerization of RUNX1/ETO, restores gene expression down-regulated by RUNX1/ETO, inhibits proliferation, and reduces RUNX1/ETO-related tumor growth in a mouse model. However, despite generally favorable physicochemical, pharmacokinetic, and toxicological properties, 7.44 is negatively charged at physiological pH and was predicted to have low to medium membrane permeability. Here, we identified M23, M27, and M10 as non-charged analogs of 7.44 using ligand-based virtual screening, in vivo hit identification, biophysical and in vivo hit validation, and integrative modeling and ADMET predictions. All three compounds interact with the NHR2 domain and show KD,app values of 39-114 {micro}M in Microscale Thermophoresis experiments as well as IC50 values of 33-77 M as to cell viability in RUNX1/ETO-positive KASUMI cells, i.e., are [~]5 to 10-fold more potent than 7.44. M23 is [~]10-fold more potent than 7.44 in inhibiting cell proliferation of RUNX1/ETO-positive cells. M23 and M27 are negligibly protonated or in a [~]1:1 ratio at physiological pH, while M10 has no (de-)protonatable group. The non-protonated species are predicted to be highly membrane-permeable, along with other favorable pharmacokinetic and toxicological properties. These compounds might serve as lead structures for the optimization of binding affinity, bioavailability, and anti-leukemic effects of compounds inhibiting RUNX1/ETO oncogenic function in t(8;21) AML.

pharmacology and toxicology↗