A dual-action niclosamide-based prodrug that targets cancer stem cells and inhibits TNBC metastasis.

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, CD44high/CD24low 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-loaded nanoparticles disrupt Candida biofilms and protect mice from mucosal candidiasis.

Candida albicans biofilms are a complex multilayer community of cells that are resistant to almost all classes of antifungal drugs. The bottommost layers of biofilms experience nutrient limitation where C. albicans cells are required to respire. We previously reported that a protein Ndu1 is essential for Candida mitochondrial respiration; loss of NDU1 causes inability of C. albicans to grow on alternative carbon sources and triggers early biofilm detachment. Here, we screened a repurposed library of FDA-approved small molecule inhibitors to identify those that prevent NDU1-associated functions. We identified an antihelminthic drug, Niclosamide (NCL), which not only prevented growth on acetate, C. albicans hyphenation and early biofilm growth, but also completely disengaged fully grown biofilms of drug-resistant C. albicans and Candida auris from their growth surface. To overcome the suboptimal solubility and permeability of NCL that is well known to affect its in vivo efficacy, we developed NCL-encapsulated Eudragit EPO (an FDA-approved polymer) nanoparticles (NCL-EPO-NPs) with high niclosamide loading, which also provided long-term stability. The developed NCL-EPO-NPs completely penetrated mature biofilms and attained anti-biofilm activity at low microgram concentrations. NCL-EPO-NPs induced ROS activity in C. albicans and drastically reduced oxygen consumption rate in the fungus, similar to that seen in an NDU1 mutant. NCL-EPO-NPs also significantly abrogated mucocutaneous candidiasis by fluconazole-resistant strains of C. albicans, in mice models of oropharyngeal and vulvovaginal candidiasis. To our knowledge, this is the first study that targets biofilm detachment as a target to get rid of drug-resistant Candida biofilms and uses NPs of an FDA-approved nontoxic drug to improve biofilm penetrability and microbial killing.

Niclosamide, but not ivermectin, inhibits anoctamin 1 and 6 and attenuates inflammation of the respiratory tract.

Inflammatory airway diseases like cystic fibrosis, asthma and COVID-19 are characterized by high levels of pulmonary cytokines. Two well-established antiparasitic drugs, niclosamide and ivermectin, are intensively discussed for the treatment of viral inflammatory airway infections. Here, we examined these repurposed drugs with respect to their anti-inflammatory effects in airways in vivo and in vitro. Niclosamide reduced mucus content, eosinophilic infiltration and cell death in asthmatic mouse lungs in vivo and inhibited release of interleukins in the two differentiated airway epithelial cell lines CFBE and BCi-NS1.1 in vitro. Cytokine release was also inhibited by the knockdown of the Ca2+-activated Cl- channel anoctamin 1 (ANO1, TMEM16A) and the phospholipid scramblase anoctamin 6 (ANO6, TMEM16F), which have previously been shown to affect intracellular Ca2+ levels near the plasma membrane and to facilitate exocytosis. At concentrations around 200 nM, niclosamide inhibited inflammation, lowered intracellular Ca2+, acidified cytosolic pH and blocked activation of ANO1 and ANO6. It is suggested that niclosamide brings about its anti-inflammatory effects at least in part by inhibiting ANO1 and ANO6, and by lowering intracellular Ca2+ levels. In contrast to niclosamide, 1 µM ivermectin did not exert any of the effects described for niclosamide. The present data suggest niclosamide as an effective anti-inflammatory treatment in CF, asthma, and COVID-19, in addition to its previously reported antiviral effects. It has an advantageous concentration-response relationship and is known to be well tolerated.

Chemical mixture that targets the epidermal growth factor pathway impairs human trophoblast cell functions.

Pregnant women are exposed to complex chemical mixtures, many of which reach the placenta. Some of these chemicals interfere with epidermal growth factor receptor (EGFR) activation, a receptor tyrosine kinase that modulates several placenta cell functions. We hypothesized that a mixture of chemicals (Chem-Mix) known to reduce EGFR activation (polychlorinated biphenyl (PCB)-126, PCB-153, atrazine, trans-nonachlor, niclosamide, and bisphenol S) would interfere with EGFR-mediated trophoblast cell functions. To test this, we determined the chemicals' EGFR binding ability, EGFR and downstream effectors activation, and trophoblast functions (proliferation, invasion, and endovascular differentiation) known to be regulated by EGFR in extravillous trophoblasts (EVTs). The Chem-Mix competed with EGF for EGFR binding, however only PCB-153, niclosamide, trans-nonachlor, and BPS competed for binding as single chemicals. The effects of the Chem-Mix on EGFR phosphorylation were tested by exposing the placental EVT cell line, HTR-8/SVneo to control (0.1% DMSO), Chem-Mix (1, 10, or 100 ng/ml), EGF (30 ng/ml), or Chem-Mix + EGF. The Chem-Mix - but not the individual chemicals - reduced EGF-mediated EGFR phosphorylation in a dose dependent manner, while no effect was observed in its downstream effectors (AKT and STAT3). None of the individual chemicals affected EVT cell invasion, but the Chem-Mix reduced EVT cell invasion independent of EGF. In support of previous studies that have explored chemicals targeting a specific pathway (estrogen/androgen receptor), current findings indicate that exposure to a chemical mixture that targets the EGFR pathway can result in a greater impact compared to individual chemicals in the context of placental cell functions.

Wnt Signaling Modulators Exhibit Neuroprotective Effects via Combating Astrogliosis and Balancing Synaptic Density at Early and Late Stage Temporal Lobe Epilepsy.

Temporal Lobe Epilepsy (TLE) is a severe neurological condition characterized by recurrent seizures that often do not respond well to available anti-seizure medications. TLE has been associated with epileptogenesis, a process that starts during the latent period following a neurologic insult and is followed by chronic phase. Recent research has linked canonical Wnt signaling to the pathophysiology of epileptogenesis and TLE. Our previous study demonstrated differential regulation of canonical Wnt signaling during early and late stage post status epilepticus (SE) induction. Building on these findings, our current study utilized Wnt modulators: GSK-3ß inhibitor 6-bromoindirubin-3'-oxime (6-Bio) and disheveled inhibitor niclosamide and investigated their impact on canonical Wnt signaling during the early (30 days) and later stages (60 days) following SE induction. We assessed several parameters, including seizure frequency, astrogliosis, synaptic density, and neuronal counts in hippocampal tissue. We used immunohistochemistry and Nissl staining to evaluate gliosis, synaptic density, and neuronal counts in micro-dissected hippocampi. Western blotting was used to examine the expression of proteins involved in canonical Wnt/ß-catenin signaling, and real-time PCR was conducted to analyze their relative mRNA expression. Wnt modulators, 6-Bio and Niclosamide were found to reduce seizure frequency and various other parameters including behavioral parameters, hippocampal morphology, astrogliosis and synaptic density at different stages of TLE.

In vitro synergistic antiviral activity of repurposed drugs against enterovirus 71.

Enteroviruses cause viral diseases that are harmful to children. Hand, foot, and mouth disease (HFMD) with neurological complications is mainly caused by enterovirus 71 (EV71). Despite its clinical importance, there is no effective antiviral drug against EV71. However, several repurposed drugs have been shown to have antiviral activity against related viruses. Treatments with single drugs and two-drug combinations were performed in vitro to assess anti-EV71 activity. Three repurposed drug candidates with broad-spectrum antiviral activity were found to demonstrate potent anti-EV71 activity: prochlorperazine, niclosamide, and itraconazole. To improve antiviral activity, combinations of two drugs were tested. Niclosamide and itraconazole showed synergistic antiviral activity in Vero cells, whereas combinations of niclosamide-prochlorperazine and itraconazole-prochlorperazine showed only additive effects. Furthermore, the combination of itraconazole and prochlorperazine showed an additive effect in neuroblastoma cells. Itraconazole and prochlorperazine exert their antiviral activities by inhibiting Akt phosphorylation. Repurposing of drugs can provide a treatment solution for HFMD, and our data suggest that combining these drugs can enhance that efficacy.

Repurposing Niclosamide as a plausible neurotherapeutic in autism spectrum disorders, targeting mitochondrial dysfunction: a strong hypothesis.

Autism Spectrum Disorders (ASD) are a complex set of neurodevelopmental manifestations which present in the form of social and communication deficits. Affecting a growing proportion of children worldwide, the exact pathogenesis of this disorder is not very well understood, and multiple signaling pathways have been implicated. Among them, the ERK/MAPK pathway is critical in a number of cellular processes, and the normal functioning of neuronal cells also depends on this cascade. As such, recent studies have increasingly focused on the impact this pathway has on the development of autistic symptoms. Improper ERK signaling is suspected to be involved in neurotoxicity, and the same might be implicated in autism spectrum disorders (ASD), through a variety of effects including mitochondrial dysfunction and oxidative stress. Niclosamide, an antihelminthic and anti-inflammatory agent, has shown potential in inhibiting this pathway, and countering the effects shown by its overactivity in inflammation. While it has previously been evaluated in other neurological disorders like Alzheimer's Disease and Parkinson's Disease, as well as various cancers by targeting ERK/MAPK, it's efficacy in autism has not yet been evaluated. In this article, we attempt to discuss the potential role of the ERK/MAPK pathway in the pathogenesis of ASD, specifically through mitochondrial damage, before moving to the therapeutic potential of niclosamide in the disorder, mediated by the inhibition of this pathway and its detrimental effects of neuronal development.

Repurposing Niclosamide to Modulate Renal RNA-Binding Protein HuR for the Treatment of Diabetic Nephropathy in db/db Mice.

Hu antigen R (HuR) plays a key role in regulating genes critical to the pathogenesis of diabetic nephropathy (DN). This study investigates the therapeutic potential of niclosamide (NCS) as an HuR inhibitor in DN. Uninephrectomized mice were assigned to four groups: normal control; untreated db/db mice terminated at 14 and 22 weeks, respectively; and db/db mice treated with NCS (20 mg/kg daily via i.p.) from weeks 18 to 22. Increased HuR expression was observed in diabetic kidneys from db/db mice, which was mitigated by NCS treatment. Untreated db/db mice exhibited obesity, progressive hyperglycemia, albuminuria, kidney hypertrophy and glomerular mesangial matrix expansion, increased renal production of fibronectin and a-smooth muscle actin, and decreased glomerular WT-1+-podocytes and nephrin expression. NCS treatment did not affect mouse body weight, but reduced blood glucose and HbA1c levels and halted the DN progression observed in untreated db/db mice. Renal production of inflammatory and oxidative stress markers (NF-κBp65, TNF-a, MCP-1) and urine MDA levels increased during disease progression in db/db mice but were halted by NCS treatment. Additionally, the Wnt1-signaling-pathway downstream factor, Wisp1, was identified as a key downstream mediator of HuR-dependent action and found to be markedly increased in db/db mouse kidneys, which was normalized by NCS treatment. These findings suggest that inhibition of HuR with NCS is therapeutic for DN by improving hyperglycemia, renal inflammation, and oxidative stress. The reduction in renal Wisp1 expression also contributes to its renoprotective effects. This study supports the potential of repurposing HuR inhibitors as a novel therapy for DN.

The Antifungal Potential of Niclosamide and Structurally Related Salicylanilides.

Human mycoses cover a diverse field of fungal diseases from skin disorders to systemic invasive infections and pose an increasing global health problem based on ineffective treatment options, the hampered development of new efficient drugs, and the emergence of resistant fungal strains. Niclosamide is currently applied for the treatment of worm infections. Its mechanisms of action, which include the suppression of mitochondrial oxidative phosphorylation (also known as mitochondrial uncoupling), among others, has led to a repurposing of this promising anthelmintic drug for the therapy of further human diseases such as cancer, diabetes, and microbial infections. Given the urgent need to develop new drugs against fungal infections, the considerable antifungal properties of niclosamide are highlighted in this review. Its chemical and pharmacological properties relevant for drug development are also briefly mentioned, and the described mitochondria-targeting mechanisms of action add to the current arsenal of approved antifungal drugs. In addition, the activities of further salicylanilide-based niclosamide analogs against fungal pathogens, including agents applied in veterinary medicine for many years, are described and discussed for their feasibility as new antifungals for humans. Preliminary structure-activity relationships are determined and discussed. Various salicylanilide derivatives with antifungal activities showed increased oral bioavailabilities when compared with niclosamide. The simple synthesis of salicylanilide-based drugs also vouchsafes a broad and cost-effective availability for poorer patient groups. Pertinent literature is covered until 2024.

Evaluation of concurrent use of Vitamin C and Niclosamide against methotrexate-induced liver injury in mice.

OBJECTIVE: To examine the effect of using vitamin C and niclosamide together on liver damage caused by methotrexate. METHODS: The study was conducted at the Pharmacology Department and the Iraqi Center for Cancer and Medical Genetics Research, College of Medicine, Mustansiriya University, Baghdad, Iraq, from November 2020 to July 2021, and comprised albino mice who were randomly assigned to 5 groups. Group 1 comprised controls, groups 2 to 5 was received methotrexate, niclosamide 70mg/kg/day, vitamin C 100mg/kg/day, and a combination of niclosamide and vitamin C, respectively. Mice in groups 3, 4 and 5 also received an intraperitoneal injection of methotrexate 20mg/kg to induce hepatotoxicity. After 48 hours of the injection, the mice were sacrificed under chloroform anaesthesia. Cardiac blood samples were drawn for biochemical examination. The liver, after being washed, was divided into two parts; one part was taken for histological examination, and the other was preserved in formalin 10% for histopathological analyses. Data was analysed using SPSS 16. RESULTS: Of the 35 mice, there were 7(20%) in each of the 5 groups. The overall age ranged between 9-12 weeks and weight between 18-38gm. The results show significant hepatoprotection ( p-value <0.05) produced by both niclosamide and Vitamin C separately, reflected by a decrease in ALP, ALT, and LDH, while the combination of (niclosamide and Vitamin C) showed no additive effect (p>0.05) on enhancement of liver function. CONCLUSIONS: Niclosamide alone was found to be superior than in combination with vitamin C for treating methotrexate-induced liver damage.

Targeting the epithelial-mesenchymal transition (EMT) pathway with combination of Wnt inhibitor and chalcone complexes in lung cancer cells.

Non-small cell lung cancer (NSCLC) is the most common type of the lung cancer. Despite development in treatment options in NSCLC, the overall survival ratios is still poor due to epithelial and mesenchymal transition (EMT) feature and associated metastasis event. Thereby there is a need to develop strategy to increase antitumor response against the NSCLC cells by targeting EMT pathway with combination drugs. Niclosamide and chalcone complexes are both affect cancer cell signaling pathways and therefore inhibit the EMT pathway. In this study, it was aimed to increase antitumor response and suppress EMT pathway in NSCLC cells by combining niclosamide and chalcone complexes. SRB cell viability assay was performed to investigate the anticancer activity of drugs. The drugs were tested on both NSCLC cells (A549 and H1299) and normal lung bronchial cells (BEAS-2B). Then the two drugs were combined and their effects on cancer cells were evaluated. Fluorescence imaging and enzyme-linked immunosorbent assay were performed on treated cells to observe the cell death manner. Wound healing assay, real-time quantitative polymerase chain reaction, and western blot analysis were performed to measure EMT pathway activity. Our results showed that niclosamide and chalcone complexes combination kill cancer cells more than normal lung bronchial cells. Compared to single drug administration, the combination of both drugs killed NSCLC cells more effectively by increasing apoptotic activity. In addition, the combination of niclosamide and chalcone complexes decreased multidrug resistance and EMT activity by lowering their gene expressions and protein levels. These results showed that niclosamide and chalcone complexes combination could be a new drug combination for the treatment of NSCLC.

Mitochondrial uncouplers impair human sperm motility without altering ATP content†.

In human spermatozoa, the electrochemical potentials across the mitochondrial and plasma membranes are related to sperm functionality and fertility, but the exact role of each potential has yet to be clarified. Impairing sperm mitochondrial function has been considered as an approach to creating male or unisex contraceptives, but it has yet to be shown whether this approach would ultimately block the ability of sperm to reach or fertilize an egg. To investigate whether the mitochondrial and plasma membrane potentials are necessary for sperm fertility, human sperm were treated with two small-molecule mitochondrial uncouplers (niclosamide ethanolamine and BAM15) that depolarize membranes by inducing passive proton flow, and evaluated the effects on a variety of sperm physiological processes. BAM15 specifically uncoupled human sperm mitochondria while niclosamide ethanolamine induced proton current in the plasma membrane in addition to depolarizing the mitochondria. In addition, both compounds significantly decreased sperm progressive motility with niclosamide ethanolamine having a more robust effect. However, these uncouplers did not reduce sperm adenosine triphosphate (ATP) content or impair other physiological processes, suggesting that human sperm can rely on glycolysis for ATP production if mitochondria are impaired. Thus, systemically delivered contraceptives that target sperm mitochondria to reduce their ATP production would likely need to be paired with sperm-specific glycolysis inhibitors. However, since niclosamide ethanolamine impairs sperm motility through an ATP-independent mechanism, and niclosamide is FDA approved and not absorbed through mucosal membranes, it could be a useful ingredient in on-demand, vaginally applied contraceptives.

Strategies to target SARS-CoV-2 entry and infection using dual mechanisms of inhibition by acidification inhibitors.

Many viruses utilize the host endo-lysosomal network for infection. Tracing the endocytic itinerary of SARS-CoV-2 can provide insights into viral trafficking and aid in designing new therapeutic strategies. Here, we demonstrate that the receptor binding domain (RBD) of SARS-CoV-2 spike protein is internalized via the pH-dependent CLIC/GEEC (CG) endocytic pathway in human gastric-adenocarcinoma (AGS) cells expressing undetectable levels of ACE2. Ectopic expression of ACE2 (AGS-ACE2) results in RBD traffic via both CG and clathrin-mediated endocytosis. Endosomal acidification inhibitors like BafilomycinA1 and NH4Cl, which inhibit the CG pathway, reduce the uptake of RBD and impede Spike-pseudoviral infection in both AGS and AGS-ACE2 cells. The inhibition by BafilomycinA1 was found to be distinct from Chloroquine which neither affects RBD uptake nor alters endosomal pH, yet attenuates Spike-pseudovirus entry. By screening a subset of FDA-approved inhibitors for functionality similar to BafilomycinA1, we identified Niclosamide as a SARS-CoV-2 entry inhibitor. Further validation using a clinical isolate of SARS-CoV-2 in AGS-ACE2 and Vero cells confirmed its antiviral effect. We propose that Niclosamide, and other drugs which neutralize endosomal pH as well as inhibit the endocytic uptake, could provide broader applicability in subverting infection of viruses entering host cells via a pH-dependent endocytic pathway.

Niclosamide inhibits TGF-ß1-induced fibrosis of human Tenon's fibroblasts by regulating the MAPK-ERK1/2 pathway.

Preventing postoperative bleb scar formation is an effective way of improving glaucoma filtration surgery (GFS) outcome. Use of more effective antifibrotic drugs with fewer adverse effects may be a good way to address the problem. In the present study, we use a primary cell model, consisting of Tenon's fibroblasts obtained from patients with glaucoma, which were stimulated with TGF-ß1 to induce the fibrotic phenotype. We explored the effects of niclosamide on TGF-ß1-induced fibrosis in these cells and examined its underlying mechanism of action. A transcriptome sequencing assay was used to explore possible signaling pathways involved. Niclosamide inhibited cell proliferation and migration, and decreased the levels of alpha-smooth muscle actin, type I and type III collagen in human Tenon's fibroblasts induced by TGF-ß1. Niclosamide also induced apoptosis and counteracted TGF-ß1-induced cytoskeletal changes and extracellular matrix accumulation. Moreover, niclosamide decreased TGF-ß1-induced phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) protein expression in human Tenon's fibroblasts. The results indicate that niclosamide inhibits TGF-ß1-induced fibrosis in human Tenon's fibroblasts by blocking the MAPK-ERK1/2 signaling pathway. Thus, niclosamide is a potentially promising antifibrotic drug that could improve glaucoma filtration surgery success rate.

Desolvation Processes in Channel Solvates of Niclosamide.

The antiparasitic drug niclosamide (NCL) is notable for its ability to crystallize in multiple 1:1 channel solvate forms, none of which are isostructural. Here, using a combination of time-resolved synchrotron powder X-ray diffraction and thermogravimetry, the process-induced desolvation mechanisms of methanol and acetonitrile solvates are investigated. Structural changes in both solvates follow a complicated molecular-level trajectory characterized by a sudden shift in lattice parameters several degrees below the temperature where the desolvated phase first appears. Model fitting of kinetic data obtained under isothermal heating conditions suggests that the desolvation is rate-limited by the nucleation of the solvent-free product. The desolvation pathways identified in these systems stand in contrast to previous investigations of the NCL channel hydrate, where water loss by diffusion initially yields an anhydrous isomorph that converts to the thermodynamic polymorph at significantly higher temperatures. Taking the view that each solvate lattice is a unique "pre-organized" precursor, a comparison of the pathways from different starting topologies to the same final product provides the opportunity to reevaluate assumptions of how various factors (e.g., solvent binding strength, density) influence solid-state desolvation processes.

Single-dose pharmacokinetics and lung function of nebulized niclosamide ethanolamine in sheep.

PURPOSE: Niclosamide is approved as an oral anthelminthic, but its low oral bioavailability hinders its medical use requiring high drug exposure outside the gastrointestinal tract. An optimized solution of niclosamide for nebulization and intranasal administration using the ethanolamine salt has been developed and tested in a Phase 1 trial. In this study we investigate the pulmonary exposure of niclosamide following administration via intravenous injection, oral administration or nebulization. METHODS: We characterized the plasma and pulmonary pharmacokinetics of three ascending doses of nebulized niclosamide in sheep, compare it to intravenous niclosamide for compartmental PK modelling, and to the human equivalent approved 2 g oral dose to investigate in the pulmonary exposure of different niclosamide delivery routes. Following a single-dose administration to five sheep, niclosamide concentrations were determined in plasma and epithelial lining fluid (ELF). Non-compartmental and compartmental modeling was used to characterize pharmacokinetic profiles. Lung function tests were performed in all dose groups. RESULTS: Administration of all niclosamide doses were well tolerated with no adverse changes in lung function tests. Plasma pharmacokinetics of nebulized niclosamide behaved dose-linear and was described by a 3-compartmental model estimating an absolute bioavailability of 86%. ELF peak concentration and area under the curve was 578 times and 71 times higher with nebulization of niclosamide relative to administration of oral niclosamide. CONCLUSIONS: Single local pulmonary administration of niclosamide via nebulization was well tolerated in sheep and resulted in substantially higher peak ELF concentration compared to the human equivalent oral 2 g dose.

SAR study of niclosamide derivatives for neuroprotective function in SH-SY5Y neuroblastoma.

Neurodegenerative disease is a debilitating and incurable condition that affects millions of people around the world. The loss of functions or malfunctions of neural cells are the causes of mortality. A proteosome inhibitor, MG132, is well known to cause neurodegeneration in vitro when model neuronal-derived cell lines are exposed to it. Niclosamide, an anthelmintic drug, which has been used to treat tapeworm infections for more than 50 years, has recently attracted renewed attention in drug repurposing because it has been found to be a good candidate in many drug development screenings. We recently found that all markers of MG132-induced neuronal cell toxicity, including the accumulation of ubiquitinated proteins, were prevented by the presence of niclosamide. In addition, niclosamide was shown to enhance autophagy induced by MG132. There results suggested that niclosamide could act as a neuroprotective agent. In the present study, niclosamide derivatives were synthesized, and the structure-activity relationship (SAR) were determined with respect to protein ubiquitination induced by MG132 and effect on cell survival signaling pathways for neuroprotective function. Our results indicate that phenol OH plays a significant role in neuroprotective activity while the niclosamide derivatives without Cl (5- or 2'-Cl) showed almost the same neuroprotective effect. 4'-NO2 can be replaced by N3 or CF3 whereas NH2 significantly decreased activity. These findings provide guidance for the development of new niclosamide analogues against neurodegenerative diseases including Parkinson's disease.

Sublethal effects of niclosamide on the aquatic snail Pomacea canaliculata.

Pomacea canaliculata is a malignant invasive aquatic snail found worldwide, and niclosamide (NS) is one of the primary agents used for its control. NS applied to water will exist in non-lethal concentrations for some time due to degradation or water exchange, thus resulting in sublethal effects on environmental organisms. To identify sublethal effects of NS on Pomacea canaliculata, we studied the aspects of histopathology, oxygen-nitrogen ratio (RO∶N), enzyme activity determination, and gene expression. After LC30 NS treatment (0.310 g/L), many muscle fibers of the feet degenerated and some acinar vesicles of the hepatopancreas collapsed and dissolved. The oxygen-nitrogen ratio (RO∶N) decreased significantly from 15.0494 to 11.5183, indicating that NS had changed the metabolic mode of Pomacea canaliculata and shifted it primarily to protein catabolism. Transcriptome analysis identified the sublethal effects of LC30 NS on the snails at the transcriptional level. 386, 322, and 583 differentially expressed genes (DEGs) were identified in the hepatopancreas, gills, and feet, respectively. GO (Gene Ontology) functional analysis and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway annotations showed that DEGs in the hepatopancreas were mainly enriched for sugar metabolism, protein biosynthesis, immune response, and amino acid metabolism functional categories; DEGs in the gills were mainly enriched for ion transport and amino acid metabolism; DEGs in the feet were mainly enriched for transmembrane transport and inositol biosynthesis. In the future, we will perform functional validation of key genes to further explain the molecular mechanism of sublethal effects.

Niclosamide exposure disrupts antioxidant defense, histology, and the liver and gut transcriptome of Chinese soft-shelled turtle (Pelodiscus sinensis).

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.

Development and validation of HPLC method for simultaneous estimation of erlotinib and niclosamide from liposomes optimized by screening design.

The emerging drug resistance to the approved first-line drug therapy leads to clinical failure in cancer. Drug repurposing studies lead to the identification of many old drugs to be used for cancer treatment. Combining the repurposed drugs (niclosamide) with first-line therapy agents like erlotinib HCl showed improved efficacy by inhibiting erlotinib HCl acquired resistance. But there is a need to develop a sensitive, accurate, and excellent analytical method and drug delivery system for successfully delivering drug combinations. In the current study, an HPLC method was developed and validated for the simultaneous estimation of niclosamide and erlotinib HCl. The retention time of niclosamide and erlotinib hydrochloride was 6.48 and 7.65 min at 333 nm. The developed method was rapid and sensitive to separating the two drugs with reasonable accuracy, precision, robustness, and ruggedness. A Plackett-Burman (PBD) screening design was used to identify the critical parameters affecting liposomal formulation development using particle size, size distribution, zeta potential, and entrapment efficiency as the response. Lipid concentration, drug concentration, hydration temperature, and media volume were critical parameters affecting the particle size, polydispersity index (PDI), ZP, and %EE of the liposomes. The optimized NCM-ERL liposomes showed the particle size (126.05 ± 2.1), PDI (0.498 ± 0.1), ZP (-16.2 ± 0.3), and %EE of NCM and ERL (50.04 ± 2.8 and 05.42 ± 1.3). In vitro release studies indicated the controlled release of the drugs loaded liposomes (87.06 ± 9.93% and 42.33 ± 0.89% in 24 h).