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Myocardial infarction (MI) is the leading cause of death worldwide. The most effective way to treat myocardial infarction is to rescue ischemic cardiomyocytes. After an ischemic event, the overproduction of reactive oxygen species (ROS) is a key driver of myocardial injury. The produced ROS affects mitochondrial function and induces apoptosis in cardiomyocytes. This was accomplished by constructing platelet-membrane-encapsulated ROS-responsive drug-releasing nanoparticles (PMN@NIC-MalNPs) to deliver malonate and niclosamide (NIC). The results revealed that PMN@NIC-MalNPs degraded and released malonate and niclosamide in a high-level ROS microenvironment, effectively reducing the oxidative stress and apoptosis rate. By enhancing basal mitochondrial oxygen consumption rate (OCR), adenosine triphosphate (ATP) production, and spare respiratory capacity (SRC) in vitro , reduced the oxidative stress levels and restored mitochondrial function. In vivo studies revealed that the PMN@NIC-MalNPs improved cardiac dysfunction, inhibited succinate dehydrogenase (SDH) activity, increased ATP production, and reduced the myocardial infarct size in myocardial infarction model mice. Further, transcriptome analysis and Western blot revealed that PMN@NIC-MalNPs prevented apoptosis by activating the expressions of the signal transducer and activator of transcription 3 (STAT3) and Bcl-2, and inhibiting the expression of Bax. Thus, this study provides a novel therapeutic solution for treating myocardial infarction and predicting the viability of an antioxidant and antiapoptotic therapeutic solution in the treatment of myocardial injury.

Niclosamide (NIC) is a commonly used molluscicide that reportedly disrupts the endocrine system and may lead to lipid metabolism disorders. However, few studies have investigated the mechanism by which NIC affects the endocrine system from the perspective of lipid metabolism. Adult female zebrafish were fed either a normal-fat diet (NFD) or a high-fat diet (HFD) and then exposed for 28 days to environmentally relevant concentrations of NIC. NIC accumulated most in the liver followed by the brain and then the gonads in both feeding conditions. Somatic index changes confirmed that HFD promotes fish growth, and NIC administration inhibits it. Lipid metabolites were decreased by NIC, as were levels of pregnenolone, androstenedione, estrogen, testosterone, and estradiol, suggesting that NIC impacted steroidogenesis. In addition, gene transcription changes related to the hypothalamic-pituitary-gonad-liver (HPGL) axis and altered ovarian histology strongly suggest that environmental relevant concentrations of NIC exposure may disrupt endocrine function. These findings highlighted that NIC exposure at environmentally relevant concentrations elicited endocrine-disruption effects may through impairing of steroid hormone synthesis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Sea lamprey (Petromyzon marinus) control in the Laurentian Great Lakes of North America makes use of two pesticides: 3-trifluoromethyl-4-nitrophenol (TFM) and niclosamide, which are often co-applied. Sea lamprey appear to be vulnerable to these agents resulting from a lack of detoxification responses with evidence suggesting that lampricide mixtures produce a synergistic effect. However, there is a lack of information pertaining to the physiological responses of sea lamprey to niclosamide and TFM:niclosamide mixtures. Here, we characterized the transcriptomic responses of the sea lamprey to TFM, niclosamide, and a TFM:niclosamide (1.5 %) mixture in the gill. Along with a control, larval sea lamprey were exposed to each treatment for 6 h, after which gill tissues were extracted for measuring whole-transcriptome responses using RNA sequencing. Differential gene expression patterns were summarized, which included identifying the broad roles of genes and common expression patterns among the treatments. While niclosamide treatment resulted in no differentially expressed genes, TFM- and mixture-treated fish had several differentially expressed genes that were associated with the cell cycle, DNA damage, metabolism, immune function, and detoxification. However, there was no common differential expression among treatments. For the first time, we characterized the transcriptomic response of sea lamprey to niclosamide and a TFM:niclosamide mixture and identified that these agents impact mRNA transcript abundance of genes associated with the cell cycle and cellular death, and immune function, which are likely mediated through mitochondrial dysregulation. These results may help to inform the production of more targeted and effective lampricides in sea lamprey control efforts., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Introduction: Idiopathic pulmonary fibrosis (IPF), a life-threatening interstitial lung disease, is characterized by excessive activation and proliferation of fibroblasts and epithelial-mesenchymal transition (EMT) of alveolar epithelial cells (AEC) accompanied by a large amount of extracellular matrix aggregation. There are no therapies to reverse pulmonary fibrosis, and nintedanib and pirfenidone could only slow down the decline of lung function of IPF patients and delay their survival time. Niclosamide (Ncl) is an antihelminthic drug approved by FDA, which has been reported to have pleiotropic pharmacological activities in recent years, but it's almost complete insolubility in water limits its clinical application., Objectives: To improve the water solubility of Ncl, explore its ability to reverse BLM-induced pulmonary fibrosis and its specific mechanism of action., Methods: The Niclosamide-loaded nanoparticles (Ncl-NPs) were formed by emulsification solvent evaporation method. A mouse model induced by bleomycin (BLM) was established to evaluate its effects and mechanisms of inhibiting and reversing fibrosis in vivo. The cell models treated by transforming growth factor-β1 (TGF-β1) were used to examine the mechanism of Ncl-NPs inhibiting fibrosis in vitro. Flow cytometry, IHC, IL-4-induced macrophage model and co-culture system were used to assess the effect of Ncl-NPs on M2 polarization of macrophages., Results: The Ncl-NPs improved the poor water solubility of Ncl. The lower dose of Ncl-NPs (2.5 mg/kg) showed the same effect of reversing established pulmonary fibrosis as free Ncl (5 mg/kg). Mechanistic studies revealed that Ncl-NPs blocked TGF-β/Smad and signaling transducer and activator of transcription 3 (Stat3) signaling pathways and inhibited the M2 polarization of macrophages. Additionally, H&E staining of the tissues initially showed the safety of Ncl-NPs., Conclusion: These results indicate Ncl-NPs may serve as a new idea for the treatment of pulmonary fibrosis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Production and hosting by Elsevier B.V.)

Niclosamide (NIC) is the only commercially available molluscicide for controlling schistosomiasis, and its negative effects on aquatic animals had been frequently reported in recent years. However, the toxicity mechanism of NIC on the Chinese soft-shelled turtle (Pelodiscus sinensis) have not yet been investigated. Therefore, juvenile turtles were exposed to 0 (control group), 10 (low NIC, L), and 50 (high NIC, H) μg/L NIC for 120 h and our results demonstrated that NIC exposure induced severe pathological changes in the liver of P. sinensis. And the typical symptom included edema, nuclear migration and deformation, and vacuolization. Compared with the liver, the NIC exposure did not cause significant damage in the gut tissue. In addition, the DHE staining demonstrated that the ROS production of liver and gut increased with the increase in concentration of NIC. The activities of antioxidant enzymes including superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) was inhibited with increased malondialdehyde (MDA) content, indicating that the antioxidant defense was significantly perturbed. Further, the transcriptome sequencing and was applied to evaluate the underlying toxicity mechanisms of NIC exposure in liver and gut of P. sinensis. Pathway enrichment showed that the disorder of lipid metabolism and innate immune regulation, including Toll-like receptors (TLRs), tumor necrosis factor (TNF), lectins, and complement and coagulation cascades, were toxicological properties of NIC on P. sinensis. Overall, the current study provides valuable information to understand the toxic effect of NIC on Chinese soft-shelled turtle., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Sea lamprey (Petromyzon marinus) control in the Laurentian Great Lakes of North America often relies on the application of 3-trifluoromethyl-4-nitrophenol (TFM) and niclosamide mixtures to kill larval sea lamprey. Selectivity of TFM against lampreys appears to be due to differential detoxification ability in these jawless fishes compared to bony fishes, particularly teleosts. However, the proximate mechanisms of tolerance to the TFM and niclosamide mixture and the mechanisms of niclosamide toxicity on its own are poorly understood, especially among non-target fishes. Here, we used RNA sequencing to identify specific mRNA transcripts and functional processes that responded to niclosamide or a TFM:niclosamide mixture in bluegill (Lepomis macrochirus). Bluegill were exposed to niclosamide or TFM:niclosamide mixture, along with a time-matched control group, and gill and liver tissues were sampled at 6, 12, and 24 h. We summarized the whole-transcriptome patterns through gene ontology (GO) term enrichment and through differential expression of detoxification genes. The niclosamide treatment resulted in an upregulation of several transcripts associated with detoxification (cyp, ugt, sult, gst), which may help explain the relatively high detoxification capacity in bluegill. Conversely, the TFM:niclosamide mixture resulted in an enrichment of processes related to arrested cell cycle and growth, and cell death alongside a diverse detoxification gene response. Detoxification of both lampricides likely involves the use of phase I and II biotransformation genes. Our findings strongly suggest that the unusually high tolerance of bluegill to lampricides is due to these animals having an inherently high capacity and flexible detoxification response to such compounds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Chemotherapy typically destroys the tumor mass but rarely eradicates the cancer stem cells (CSCs) that can drive metastatic recurrence. A key current challenge is finding ways to eradicate CSCs and suppress their characteristics. Here, we report a prodrug, Nic-A , created by combining a carbonic anhydrase IX (CAIX) inhibitor, acetazolamide, with a signal transducer and transcriptional activator 3 (STAT3) inhibitor, niclosamide. Nic-A was designed to target triple-negative breast cancer (TNBC) CSCs and was found to inhibit both proliferating TNBC cells and CSCs via STAT3 dysregulation and suppression of CSC-like properties. Its use leads to a decrease in aldehyde dehydrogenase 1 activity, CD44 high /CD24 low stem-like subpopulations, and tumor spheroid-forming ability. TNBC xenograft tumors treated with Nic-A exhibited decreased angiogenesis and tumor growth, as well as decreased Ki-67 expression and increased apoptosis. In addition, distant metastases were suppressed in TNBC allografts derived from a CSC-enriched population. This study thus highlights a potential strategy for addressing CSC-based cancer recurrence.

Niclosamide has been proposed as a possible candidate for a Covid-19 drug. However, the metabolites of niclosamide are difficult to investigate because they are usually not available commercially or they are quite expensive in the commercial market. In this study, the major metabolite of niclosamide in human liver microsomes (HLMs) was confirmed to be 3-OH niclosamide. Because the production of 3-OH niclosamide using HLMs has a slow turnover rate, a new method of producing niclosamide metabolite with an easier and highly cost-efficient method was thus conducted. Bacterial CYP102A1 (BM3) is one of the bacterial cytochrome P450s (CYPs) from Bacillus megaterium that structurally show similar activities to human CYPs. Here, the BM3 mutants were used to produce niclosamide metabolites and the metabolites were analyzed using high-performance liquid chromatography and LC-mass spectrometry. Among a set of mutants tested here, BM3 M14 mutant was the most active in producing 3-OH niclosamide, the major metabolite of niclosamide. Comparing BM3 M14 and HLMs, BM3 M14 production of 3-OH niclosamide was 34-fold higher than that of HLMs. Hence, the engineering of BM3 can be a cost-efficient method to produce 3-OH niclosamide., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

In the current study, adult male zebrafish fed a normal diet (ND) or high-fat diet (HFD) were exposed to niclosamide (NIC) at environmentally relevant concentrations to reveal the accumulation and distribution in different tissues and evaluate the effects on liver-gut axis. Chemical analysis indicated that the liver bore a greater burden of NIC compared with the brain and gonads in adult zebrafish, and the HFD-fed fish bore greater burden in their liver and brain than those ND-fed fish. The indications from body weight, growth rate, body mass index, micro-CT images, biochemical and pathological changes confirmed that NIC can efficaciously curb weight gain and improve overloads of in plasma insulin and glucose in HFD-fed zebrafish. However, the potential effects on liver-gut axis in ND-fed zebrafish were also elucidated: NIC disturbed mitochondrial energy production, inhibited the glycemic and triacylglycerol biosynthesis but promoted triacylglycerol and free fatty acid catabolism, therefore reduced lipid accumulation in hepatocytes; NIC also impaired the physical barrier, evoked inflammatory and oxidative stress and led to microbiota dysbiosis in the intestine. There findings highlighted the necessity for evaluating its potential impacts on the health of wild animals as well as human beings upon long-term exposure.

Tumor recurrence is often attributed to cancer stem cells (CSCs). We previously demonstrated that down-regulation of Pregnane X Receptor (PXR) decreases the chemoresistance of CSCs and prevents colorectal cancer recurrence. Currently, no PXR inhibitor is usable in clinic. Here, we identify miR-148a as a targetable element upstream of PXR signaling in CSCs, which when over-expressed decreases PXR expression and impairs tumor relapse after chemotherapy in mouse tumor xenografts. We then develop a fluorescent reporter screen for miR-148a activators and identify the anti-helminthic drug niclosamide as an inducer of miR-148a expression. Consequently, niclosamide decreased PXR expression and CSC numbers in colorectal cancer patient-derived cell lines and synergized with chemotherapeutic agents to prevent CSC chemoresistance and tumor recurrence in vivo. Our study suggests that endogenous miRNA inducers is a viable strategy to down-regulate PXR and illuminates niclosamide as a neoadjuvant repurposing strategy to prevent tumor relapse in colon cancer., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Aim/purpose: Niclosamide (NCL) is an anthelminthic drug, which is widely used to treat various diseases due to its pleiotropic anti-inflammatory and antiviral activities. NCL modulates of uncoupling oxidative phosphorylation and different signaling pathways in human biological processes. The wide-spectrum antiviral effect of NCL makes it a possible candidate for recent pandemic SARS-CoV-2 infection and may reduce Covid-19 severity. Therefore, the aim of the present study was to review and clarify the potential role of NCL in Covid-19., Methods: This study reviewed and highlighted the protective role of NCL therapy in Covid-19. A related literature search in PubMed, Scopus, Web of Science, Google Scholar, and Science Direct was done., Results: NCL has noteworthy anti-inflammatory and antiviral effects. The primary antiviral mechanism of NCL is through neutralization of endosomal PH and inhibition of viral protein maturation. NCL acts as a proton carrier, inhibits homeostasis of endosomal PH, which limiting of viral proliferation and release. The anti-inflammatory effects of NCL are mediated by suppression of inflammatory signaling pathways and release of pro-inflammatory cytokines. However, the major limitation in using NCL is low aqueous solubility, which reduces oral bioavailability and therapeutic serum concentration that reducing the in vivo effect of NCL against SARS-CoV-2., Conclusions: NCL has anti-inflammatory and immune regulatory effects by modulating the release of pro-inflammatory cytokines, inhibition of NF-κB /NLRP3 inflammasome and mTOR signaling pathway. NCL has an anti-SARS-CoV-2 effect via interruption of viral life-cycle and/or induction of cytopathic effect. Prospective clinical studies and clinical trials are mandatory to confirm the potential role of NCL in patients with Covid-19 concerning the severity and clinical outcomes., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Niclosamide (NIC), a helminthic drug used widely for controlling schistosomiasis, can reportedly disrupt the endocrine system. However, its underlying mechanisms are still unclear. In this study, we revealed the potential endocrine disruption mechanism of NIC by activating estrogen receptors (ERs) and estrogen-related receptors (ERRs). The binding potency of NIC with ERα, ERβ and ERRγ were determined by fluorescence competitive binding assays, which shows an IC 50 (the concentration of NIC needed to displace 50 % of the probe from the receptor) of 90 ± 4.1, 10 ± 1.7 nM and 0.59 ± 0.07 nM respectively. The IC 50 for ERRγ is the lowest one among the three detected receptors, which is three orders of magnitude lower than the known agonist GSK4716.The transcriptional activities of NIC on ERs and ERRs were detected by MVLN cells (stably transfected with ERs reporter gene) and HeLa cells (transiently transfected with ERRs reporter gene)-based luciferase reporter gene assay. The lowest observable effective concentration (LOEC) ranked as follows: ERRγ (0.5 nM) < ERRα (10 nM) < ERs (100 nM). The maximum observed induction rate for ERRγ (294 %) was higher than that for ERRα (191 %). The maximum observed induction rate of NIC for ERs was 30 % relative to 17β-estradiol. In addition, we simulated the interactions of NIC with ERs and ERRs by molecular docking. NIC could dock into the ligand binding pockets of ERs and ERRs and form hydrogen bonds with different amino acids. The binding energy ranked as follows: ERRγ (-8.90 kcal/mol) < ERβ (-7.57 kcal/mol) < ERRα (-7.15 kcal/mol) < ERα (-6.53 kcal/mol), which implied that NIC bound to ERRγ with higher binding affinity than the other receptors. Overall, we clarify that ERRγ might be the dominant target for NIC in cells rather than ERRα and ERs. We reveal potential novel mechanisms for the endocrine disruption effects of NIC by activating both ERRs and ERs at environmentally-related nanomolar levels., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Zika virus has emerged as a potential threat to human health globally. A previous drug repurposing screen identified the approved anthelminthic drug niclosamide as a small molecule inhibitor of Zika virus infection. However, as antihelminthic drugs are generally designed to have low absorption when dosed orally, the very limited bioavailability of niclosamide will likely hinder its potential direct repurposing as an antiviral medication. Here, we conducted SAR studies focusing on the anilide and salicylic acid regions of niclosamide to improve physicochemical properties such as microsomal metabolic stability, permeability and solubility. We found that the 5-bromo substitution in the salicylic acid region retains potency while providing better drug-like properties. Other modifications in the anilide region with 2'-OMe and 2'-H substitutions were also advantageous. We found that the 4'-NO 2 substituent can be replaced with a 4'-CN or 4'-CF 3 substituents. Together, these modifications provide a basis for optimizing the structure of niclosamide to improve systemic exposure for application of niclosamide analogs as drug lead candidates for treating Zika and other viral infections. Indeed, key analogs were also able to rescue cells from the cytopathic effect of SARS-CoV-2 infection, indicating relevance for therapeutic strategies targeting the COVID-19 pandemic., (Published by Elsevier Ltd.)

One avenue to combat multidrug-resistant Gram-negative bacteria is the coadministration of multiple drugs (combination therapy), which can be particularly promising if drugs synergize. The identification of synergistic drug combinations, however, is challenging. Detailed understanding of antibiotic mechanisms can address this issue by facilitating the rational design of improved combination therapies. Here, using diverse biochemical and genetic assays, we examine the molecular mechanisms of niclosamide, a clinically approved salicylanilide compound, and demonstrate its potential for Gram-negative combination therapies. We discovered that Gram-negative bacteria possess two innate resistance mechanisms that reduce their niclosamide susceptibility: a primary mechanism mediated by multidrug efflux pumps and a secondary mechanism of nitroreduction. When efflux was compromised, niclosamide became a potent antibiotic, dissipating the proton motive force (PMF), increasing oxidative stress, and reducing ATP production to cause cell death. These insights guided the identification of diverse compounds that synergized with salicylanilides when coadministered (efflux inhibitors, membrane permeabilizers, and antibiotics that are expelled by PMF-dependent efflux), thus suggesting that salicylanilide compounds may have broad utility in combination therapies. We validate these findings in vivo using a murine abscess model, where we show that niclosamide synergizes with the membrane permeabilizing antibiotic colistin against high-density infections of multidrug-resistant Gram-negative clinical isolates. We further demonstrate that enhanced nitroreductase activity is a potential route to adaptive niclosamide resistance but show that this causes collateral susceptibility to clinical nitro-prodrug antibiotics. Thus, we highlight how mechanistic understanding of mode of action, innate/adaptive resistance, and synergy can rationally guide the discovery, development, and stewardship of novel combination therapies. IMPORTANCE There is a critical need for more-effective treatments to combat multidrug-resistant Gram-negative infections. Combination therapies are a promising strategy, especially when these enable existing clinical drugs to be repurposed as antibiotics. We examined the mechanisms of action and basis of innate Gram-negative resistance for the anthelmintic drug niclosamide and subsequently exploited this information to demonstrate that niclosamide and analogs kill Gram-negative bacteria when combined with antibiotics that inhibit drug efflux or permeabilize membranes. We confirm the synergistic potential of niclosamide in vitro against a diverse range of recalcitrant Gram-negative clinical isolates and in vivo in a mouse abscess model. We also demonstrate that nitroreductases can confer resistance to niclosamide but show that evolution of these enzymes for enhanced niclosamide resistance confers a collateral sensitivity to other clinical antibiotics. Our results highlight how detailed mechanistic understanding can accelerate the evaluation and implementation of new combination therapies., (Copyright © 2020 Copp et al.)

Niclosamide, previously used as an anthelmintic drug is currently being repurposed for its anticancer activity. Niclosamide is a brick like biopharmaceutical classification system (BCS) class II drug with poor aqueous solubility and dissolution consequently leading to low bioavailability. By considering the physicochemical properties and geometry of niclosamide, inclusion complex with cyclodextrin was prepared by freeze drying method and characterized using FT-IR, DSC, PXRD, and 1 HNMR. In silico molecular modeling study was performed to study the possible interactions between niclosamide and cyclodextrin. The anticancer activity of niclosamide formulation was evaluated through in vitro cell cytotoxicity study using various cancer cell lines. The potential of niclosamide complex for improvement of the bioavailability was evaluated in male BALB/c mice. In vitro cytotoxicity studies indicated significantly higher cytotoxicity at lower concentrations and the pharmacokinetic studies showed significant improvement in C max and T max of niclosamide from cyclodextrin complex in comparison to pure niclosamide alone., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The release of aromatic amines from drugs and other xenobiotics resulting from the hydrolysis of metabolically labile amide bonds presents a safety risk through several mechanisms, including geno-, hepato- and nephrotoxicity. Whilst multiple in vitro systems used for studying metabolic stability display serine hydrolase activity, responsible for the hydrolysis of amide bonds, they vary in their efficiency and selectivity. Using a range of amide-containing probe compounds (0.5-10 µM), we have investigated the hydrolytic activity of several rat, minipig and human-derived in vitro systems - including Supersomes, microsomes, S9 fractions and hepatocytes - with respect to their previously observed human in vivo metabolism. In our hands, human carboxylesterase Supersomes and rat S9 fractions systems showed relatively poor prediction of human in vivo metabolism. Rat S9 fractions, which are commonly utilised in the Ames test to assess mutagenicity, may be limited in the detection of genotoxic metabolites from aromatic amides due to their poor concordance with human in vivo amide hydrolysis. In this study, human liver microsomes and minipig subcellular fractions provided more representative models of human in vivo hydrolytic metabolism of the aromatic amide compounds tested.

Niclosamide is a promising antitumor agent, but its low aqueous solubility places limit on its antitumor efficacy. The aim of this study was to improve the solubility and dissolution of niclosamide through using octenylsuccinate hydroxypropyl phytoglycogen (OHPP), an amphiphilic dendrimer-like biopolymer. The niclosamide-OHPP solid dispersion (niclo-OHPP SD) was prepared and characterized in terms of crystallinity, molecular interactions, solubility and dissolution profile, in-vitro antitumor efficacy, and in-vitro transdermal amount. X-ray powder diffraction analysis showed the amorphous state of niclosamide in niclo-OHPP SD. FTIR showed the formation of hydrogen bonding between niclosamide and OHPP. Solubility of niclosamide with niclo-OHPP SD was about 11,914 times that of niclosamide alone, and 61% of niclosamide with niclo-OHPP SD dissolved in 3 h upon dissolution. Against three cancer cell lines, cytotoxicity assays indicated greater inhibition by using niclo-OHPP SD than by using DMSO-assisted niclosamide solution. The cumulative transdermal amount of niclosamide with niclo-OHPP SD was 5.3 times that with niclosamide alone. This study showed that the use of OHPP could provide strong support for the development of niclosamide-based drug formulations., (Copyright © 2017 Elsevier B.V. All rights reserved.)

With the aim to develop alternative therapeutic tools for the treatment of resistant cancers, here we propose targeted Pluronic(®) P123/F127 mixed micelles (PMM) delivering niclosamide (NCL) as a repositioning strategy to treat multidrug resistant non-small lung cancer cell lines. To build multifunctional PMM for targeting and imaging, Pluronic(®) F127 was conjugated with biotin, while Pluronic(®) P123 was fluorescently tagged with rhodamine B, in both cases at one of the two hydroxyl end groups. This design intended to avoid any interference of rhodamine B on biotin exposition on PMM surface, which is a key fundamental for cell trafficking studies. Biotin-decorated PMM were internalized more efficiently than non-targeted PMM in A549 lung cancer cells, while very low internalization was found in NHI3T3 normal fibroblasts. Biotin-decorated PMM entrapped NCL with good efficiency, displayed sustained drug release in protein-rich media and improved cytotoxicity in A549 cells as compared to free NCL (P<0.01). To go in depth into the actual therapeutic potential of NCL-loaded PMM, a cisplatin-resistant A549 lung cancer cell line (CPr-A549) was developed and its multidrug resistance tested against common chemotherapeutics. Free NCL was able to overcome chemoresistance showing cytotoxic effects in this cell line ascribable to nucleolar stress, which was associated to a significant increase of the ribosomal protein rpL3 and consequent up-regulation of p21. It is noteworthy that biotin-decorated PMM carrying NCL at low doses demonstrated a significantly higher cytotoxicity than free NCL in CPr-A549. These results point at NCL-based regimen with targeted PMM as a possible second-line chemotherapy for lung cancer showing cisplatin or multidrug resistance., (Copyright © 2016 Elsevier B.V. All rights reserved.)

1. Niclosamide is an old anthelmintic drug that shows potential in fighting against cancers. Here, we characterized the metabolism of niclosamide by cytochrome P450 enzymes (CYPs) and UDP-glucuronosyltransferases (UGTs) using human liver microsomes (HLM) and expressed enzymes. 2. NADPH-supplemented HLM (and liver microsomes from various animal species) generated one hydroxylated metabolite (M1) from niclosamide; and UDPGA-supplemented liver microsomes generated one mono-O-glucuronide (M2). The chemical structures of M1 (3-hydroxy niclosamide) and M2 (niclosamide-2-O-glucuronide) were determined through LC-MS/MS and/or NMR analyses. 3. Reaction phenotyping revealed that CYP1A2 was the main enzyme responsible for M1 formation. The important role of CYP1A2 in niclosamide metabolism was further confirmed by activity correlation analyses as well as inhibition experiments using specific inhibitors. 4. Although seven UGT enzymes were able to catalyze glucuronidation of niclosamide, UGT1A1 and 1A3 were the enzymes showed the highest metabolic activities. Activity correlation analyses demonstrated that UGT1A1 played a predominant role in hepatic glucuronidation of niclosamide, whereas the role of UGT1A3 was negligible. 5. In conclusion, niclosamide was subjected to efficient metabolic reactions hydroxylation and glucuronidation, wherein CYP1A2 and UGT1A1 were the main contributing enzymes, respectively.

The interaction between niclosamide (NIC) and pepsin was investigated using multispectroscopic and molecular docking methods. Binding constant, number of binding sites, and thermodynamic parameters at different temperatures were measured. Results of fluorescence quenching and synchronous fluorescence spectroscopy in combination with three-dimensional fluorescence spectroscopy showed that changes occurred in the microenvironment of tryptophan residues and the molecular conformation of pepsin. Molecular interaction distance and energy-transfer efficiency between pepsin and NIC were determined based on Förster nonradiative energy-transfer mechanism. Furthermore, the binding of NIC inhibited pepsin activity in vitro. All these results indicated that NIC bound to pepsin mainly through hydrophobic interactions and hydrogen bonds at a single binding site. In conclusion, this study provided substantial molecular-level evidence that NIC could induce changes in pepsin structure and conformation.

Despite standard cancer therapies such as chemotherapy and targeted therapy have shown some efficacies, the cancer in many cases eventually relapses and metastasizes upon stopping the treatment. There is a small subpopulation of cancer cells within tumor, with specific characters similar to those found in stem cells. This group of cancer cells is known as tumor-initiating or cancer stem cells (CSCs), which have an ability to self-renew and give rise to cancer cell progeny. CSCs are related with drug resistance, metastasis and relapse of cancer, hence emerging as a crucial drug target for eliminating cancer. Rapid advancement of CSC biology has enabled researchers to isolate and culture CSCs in vitro, making the cells amenable to high-throughput drug screening. Recently, drug repositioning, which utilizes existing drugs to develop potential new indications, has been gaining popularity as an alternative approach for the drug discovery. As existing drugs have favorable bioavailability and safety profiles, drug repositioning is now actively exploited for prompt development of therapeutics for many serious diseases, such as cancer. In this review, we will introduce latest examples of attempted drug repositioning targeting CSCs and discuss potential use of the repositioned drugs for cancer therapy.

Electrochemistry combined with (liquid chromatography) high resolution mass spectrometry was used to simulate the general reductive metabolism of three biologically important nitro aromatic molecules: 3-trifluoromethyl-4-nitrophenol (TFM), niclosamide, and nilutamide. TFM is a pesticide used in the Laurential Great Lakes while niclosamide and nilutamide are used in cancer therapy. At first, a flow-through electrochemical cell was directly connected to a high resolution mass spectrometer to evaluate the ability of electrochemistry to produce the main reduction metabolites of nitro aromatic, nitroso, hydroxylamine, and amine functional groups. Electrochemical experiments were then carried out at a constant potential of -2.5 V before analysis of the reduction products by LC-HRMS, which confirmed the presence of the nitroso, hydroxylamine, and amine species as well as dimers. Dimer identification illustrates the reactivity of the nitroso species with amine and hydroxylamine species. To investigate xenobiotic metabolism, the reactivity of nitroso species to biomolecules was also examined. Binding of the nitroso metabolite to glutathione was demonstrated by the observation of adducts by LC-ESI(+)-HRMS and the characteristics of their MSMS fragmentation. In conclusion, electrochemistry produces the main reductive metabolites of nitro aromatics and supports the observation of nitroso reactivity through dimer or glutathione adduct formation.

Niclosamide produces genotoxic effects, such as point mutations in Salmonella sp., sperm-head abnormalities in mice and clastogenic effects in human lymphocytes in vitro and in vivo. As cytochrome P450 could be involved in the bioactivation of niclosamide, we investigated which subfamily was involved. We used liver microsomal fractions from rats treated with phenobarbital/β-naphthoflavone (PB/β-NF), benzo[a]pyrene (BaP) or cyclohexanol, which are known to induce different cytochrome P450 subfamilies, such as CYP2B, CYP1A1, CYP1A2 and CYP2E1. We also inhibited CYP1A and CYP2E using α-NF and diethyldithiocarbamate to identify the cytochrome P450 involved. Liver-S9 fractions obtained from PB/β-NF- and BaP-treated rats significantly increased the number of revertants induced by niclosamide, while the CYP1A1 inhibitor α-NF decreased the number of revertants. The incubation of niclosamide with CYP1A1 Supersomes™ increased the number of revertants, suggesting that CYP1A1 is responsible for the bioactivation of niclosamide. Nitroreduction is also involved in niclosamide bioactivation, as the nitroreductase-deficient strain YG7132 did not respond to the niclosamide treatment. Our findings indicated that a metabolite, derived from the action of CYP1A1 and a nitroreduction-reaction process, has a key role in the bioactivation of niclosamide.

The transformation of organic halogenated pesticides by laccase-mediator system has been investigated. Twelve pesticides were assayed in the presence of nine different mediators. Acetosyringone and syringaldehyde showed to be the best mediators. The halogenated pesticides bromoxynil, niclosamide, bromofenoxim and dichlorophen were transformed by the laccase-syringaldehyde system showing catalytic activities of 48.8, 142.0, 166.2 and 1257.6nmolmin(-1)U(-1), respectively. The highest pesticide transformation rates were obtained with a mediator-substrate proportion of 5:1, one of the lowest reported so far for the laccase-mediator systems. The analysis of the main product from the dichlorophen transformation showed that an oxidative dehalogenation is involved in the catalytic mechanism. Adduct formation between the mediator syringaldehyde and the pesticides dichlorophen or bromoxynil was also found after enzymatic oxidation. The main goal of this work is to evaluate environmental-friendly mediators for the pesticide transformation, and the potential of laccase-mediator system to efficiently reduce the environmental impact of organic halogenated pesticides is discussed.

Niclosamide is an anti-helminthic drug susceptible to being metabolized into a bacterial mutagen by the action of enzymes present in the S9 activation mixture. Additional results from genotoxic studies in rodents and humans suggest that the drug is absorbed from the gastrointestinal tract, and mutagenic metabolites are excreted both in the free form and as conjugated glucuronides. As in the case of other secondary amides, phase I metabolism of niclosamide may result in a hydrolytic cleavage of the amide bond, giving rise to 5-chlorosalicylic acid and 2-chloro-4-nitroaniline as the main metabolites. In this study, the mutagenicity of these compounds was tested using the Salmonella typhimurium assay. Bacterial mutagenicity tests with these 2 compounds reveal a non-mutagenic response with 5-chlorosalicylic acid and a mutagenic one with 2-chloro-4-nitroaniline. However, the mutagenic potency observed with this compound is lower than that of niclosamide. The role of nitroreduction in the activation of niclosamide and 2-chloro-4-nitroaniline was also investigated with the help of S. typhimurium strains TA98NR, YG1020, YG1021 and YG1024. The results show a pattern of response which is qualitatively similar for both compounds and this indicates that its mutagenicity depends on both nitroreduction and transacetylation.

A 2-5-month treatment with niclosamide, a widely used drug in developing countries, has been reported to induce lymphosarcomas in toad liver and kidney. The genotoxic effects of this drug have also been evaluated in Salmonella typhimurium, in somatic and germinal cells of mice and in human lymphocytes exposed in vitro and in vivo. The present study shows that niclosamide is also capable of inducing mitotic crossing-over and non-disjunction in Aspergillus nidulans, which points to the wide potential of this drug as a genotoxic agent.

1. Residues of the lampricides 14C-Bayer 73 (2',5-dichloro-4'-nitrosalicylanilide) and 14C-TFM (3-trifluoromethyl-4-nitrophenol) were absorbed from aqueous sublethal concn. by aquatic larvae of the midge Chironomus tentans. 2. Toxicant residues were widely distributed during early hours of exposure, but later and during a 24-h post-exposure period, they became concentrated in the trunk and intestinal tract; the posterior movement of Bayer 73 was more rapid than that of TFM. 3. During 4-h tests, Malpighian tubules of the chironomid actively transported Bayer 73 and TFM in vitro, and on a per tubule basis, more TFM was transported than Bayer 73. 4. Potassium cyanide inhibited tubule transport; serotonin had no effect.

Experiments on mice and rats were made to study a new medicinal form, fenasal granules given per os and to compare it with ground tablets and powder of fenasal as regards the intestinal content, absorption capacity, anthelminthic activity and toxicity. The intestinal content and absorption capacity of fenasal were medicinal form-dependent. The highest anthelminthic activity was exhibited by fenasal granules. Acute toxicity of powder, ground tablets and granules of fenasal administered to mice and rats per os remained the same.

1. Niclosamide and related nitro compounds were reduced to the corresponding amines by Moniezia expansa, Ascaris lumbricoides var suum and by enzymes prepared from these species and by mouse and sheep liver homogenates. The reduction of niclosamide by the helminths required as cofactors NADH2 and glutathione, but was inhibited 50% by 2 X 10(-7) M allopurinol. 2. Unlike benzanilide, niclosamide was not hydrolysed by either the helminths, or the mammalian liver preparations. Rates of hydrolysis of compounds related to niclosamide indicate that niclosamide was not hydrolysed because substituents in both benzene rings ortho to the amide bond sterically hinder the hydrolase. 3. Hydrolysis of benzanilide and related compounds was inhibited by anthelmintic organophosphates.