Lipid-Based Self-Microemulsion of Niclosamide Achieved Enhanced Oral Delivery and Anti-Tumor Efficacy in Orthotopic Patient-Derived Xenograft of Hepatocellular Carcinoma in Mice.

Introduction: We previously identified niclosamide as a promising repurposed drug candidate for hepatocellular carcinoma (HCC) treatment. However, it is poorly water soluble, limiting its tissue bioavailability and clinical application. To overcome these challenges, we developed an orally bioavailable self-microemulsifying drug delivery system encapsulating niclosamide (Nic-SMEDDS). Methods: Nic-SMEDDS was synthesized and characterized for its physicochemical properties, in vivo pharmacokinetics and absorption mechanisms, and in vivo therapeutic efficacy in an orthotopic patient-derived xenograft (PDX)-HCC mouse model. Niclosamide ethanolamine salt (NEN), with superior water solubility, was used as a positive control. Results: Nic-SMEDDS (5.6% drug load) displayed favorable physicochemical properties and drug release profiles in vitro. In vivo, Nic-SMEDDS displayed prolonged retention time and plasma release profile compared to niclosamide or NEN. Oral administration of Nic-SMEDDS to non-tumor bearing mice improved niclosamide bioavailability and Cmax by 4.1- and 1.8-fold, respectively, compared to oral niclosamide. Cycloheximide pre-treatment blocked niclosamide absorption from orally administered Nic-SMEDDS, suggesting that its absorption was facilitated through the chylomicron pathway. Nic-SMEDDS (100 mg/kg, bid) showed greater anti-tumor efficacy compared to NEN (200 mg/kg, qd); this correlated with higher levels (p < 0.01) of niclosamide, increased caspase-3, and decreased Ki-67 in the harvested PDX tissues when Nic-SMEDDS was given. Biochemical analysis at the treatment end-point indicated that Nic-SMEDDS elevated lipid levels in treated mice. Conclusion: We successfully developed an orally bioavailable formulation of niclosamide, which significantly enhanced oral bioavailability and anti-tumor efficacy in an HCC PDX mouse model. Our data support its clinical translation for the treatment of solid tumors.

Pharmacokinetic considerations for enhancing drug repurposing opportunities of anthelmintics: Niclosamide as a case study.

Recently, anthelmintics have showcased versatile therapeutic potential in addressing various diseases, positioning them as promising candidates for drug repurposing. However, challenges such as low bioavailability and a lack of a solid pharmacokinetic basis impede successful repurposing. To overcome these flaws, we aimed to investigate the key pharmacokinetic factors of anthelmintics mainly focusing on the absorption, distribution, and metabolism profiles by employing niclosamide (NIC) as a model drug. The intestinal permeability of NIC is significantly influenced by solubility and doesn't function as a substrate for efflux transporters. It showed high plasma protein binding. Also, the metabolism study indicated that NIC would have low metabolic stability by extensively undergoing the intestinal glucuronidation. Additionally, we investigated the CYP-mediated drug-drug interaction potential of NIC in both direct and time-dependent ways. NIC showed strong inhibitory effects on CYP1A2 and CYP2C8 and is not likely to become a time-dependent inhibitor. Our findings could contribute to the identification of essential factors in the pharmacokinetics of anthelmintics, potentially facilitating their repositioning.

Computational drug repositioning identifies niclosamide and tribromsalan as inhibitors of Mycobacterium tuberculosis and Mycobacterium abscessus.

Tuberculosis (TB) is still a major global health challenge, killing over 1.5 million people each year, and hence, there is a need to identify and develop novel treatments for Mycobacterium tuberculosis (M. tuberculosis). The prevalence of infections caused by nontuberculous mycobacteria (NTM) is also increasing and has overtaken TB cases in the United States and much of the developed world. Mycobacterium abscessus (M. abscessus) is one of the most frequently encountered NTM and is difficult to treat. We describe the use of drug-disease association using a semantic knowledge graph approach combined with machine learning models that has enabled the identification of several molecules for testing anti-mycobacterial activity. We established that niclosamide (M. tuberculosis IC90 2.95 µM; M. abscessus IC90 59.1 µM) and tribromsalan (M. tuberculosis IC90 76.92 µM; M. abscessus IC90 147.4 µM) inhibit M. tuberculosis and M. abscessus in vitro. To investigate the mode of action, we determined the transcriptional response of M. tuberculosis and M. abscessus to both compounds in axenic log phase, demonstrating a broad effect on gene expression that differed from known M. tuberculosis inhibitors. Both compounds elicited transcriptional responses indicative of respiratory pathway stress and the dysregulation of fatty acid metabolism.

ROS-Suppression Nanoplatform Combined Activation of STAT3/Bcl-2 Pathway for Preventing Myocardial Infarction in Mice.

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.

Inhibition of mucus secretion by niclosamide and benzbromarone in airways and intestine.

The Ca2+ activated Cl- channel TMEM16A (anoctamin 1; ANO1) is expressed in secretory epithelial cells of airways and intestine. Previous studies provided evidence for a role of ANO1 in mucus secretion. In the present study we investigated the effects of the two ANO1-inhibitors niclosamide (Niclo) and benzbromarone (Benz) in vitro and in vivo in mouse models for cystic fibrosis (CF) and asthma. In human CF airway epithelial cells (CFBE), Ca2+ increase and activation of ANO1 by adenosine triphosphate (ATP) or ionomycin was strongly inhibited by 200 nM Niclo and 1 µM Benz. In asthmatic mice airway mucus secretion was inhibited by intratracheal instillation of Niclo or Benz. In homozygous F508del-cftr mice, intestinal mucus secretion and infiltration by CD45-positive cells was inhibited by intraperitoneal injection of Niclo (13 mg/kg/day for 7 days). In homozygous F508del-cftr rats intestinal mucus secretion was inhibited by oral application of Benz (5 mg/kg/day for 60 days). Taken together, well tolerated therapeutic concentrations of niclosamide and benzbromarone corresponding to plasma levels of treated patients, inhibit ANO1 and intracellular Ca2+ signals and may therefore be useful in inhibiting mucus hypersecretion and mucus obstruction in airways and intestine of patients suffering from asthma and CF, respectively.

Pharmacokinetic interactions of niclosamide in rats: Involvement of organic anion transporters 1 and 3 and organic cation transporter 2.

Niclosamide is an anthelmintic drug with a long history of use and is generally safe and well tolerated in humans. As the conventional dose of niclosamide results in a low but certain level in systemic circulation, drug interactions with concomitant drugs should be considered. We aimed to investigate the interaction between niclosamide and drug transporters, as such information is currently limited. Niclosamide inhibited the transport activity of OATP1B1, OATP1B3, OAT1, OAT3, and OCT2 in vitro. Among them, the inhibitory effects on OAT1, OAT3, and OCT2 were strong, with IC50 values of less than 1 µM. When 3 mg/kg of niclosamide was co-administered to rats, systemic exposure to furosemide (a substrate of OAT1/3) and metformin (a substrate of OCT2) increased, and the renal clearance (CLr) of the drugs significantly decreased. These results suggest that niclosamide inhibits renal transporters, OAT1/3 and OCT2, not only in vitro but also in vivo, resulting in increased systemic exposure to the substrates of the transporters by strongly blocking the urinary elimination pathway in rats. The findings of this study will support a meticulous understanding of the transporter-mediated drug interactions of niclosamide and consequently aid in effective and safe use of niclosamide.

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.

Colon-targeted delivery of niclosamide from solid dispersion employing a pH-dependent polymer via hotmelt extrusion for the treatment of ulcerative colitis in mice.

Niclosamide (NCL) is repurposed to treat inflammatory bowel disease due to its anti-inflammatory properties and potential to reduce oxidative stress. This therapeutic activity remains challenging if administered directly due to its low solubility and high recrystallization tendency in gastric pH. Solid dispersions using pH-dependent polymer will be a better idea to improve the solubility, dissolution and targeted delivery at the colon. Hot melt extrusion was used to formulate a solid dispersion with 30% NCL utilising hydroxypropyl methylcellulose acetate succinate as a pH-dependent polymer. In vitro drug release studies revealed formulation (F1) containing 10%w/w Tween 80 showed minimal release (2.06%) at the end of 2 h, followed by 47.87% and 82.15% drug release at 6 h and 14 h, respectively, indicating the maximum amount of drug release in the colon. The drug release from the formulations containing no plasticiser and 5%w/w plasticiser was comparable to the pure crystalline drug (approximately 25%). Solid-state analysis confirmed particle conversion of crystalline NCL to amorphous form, and the optimised formulation was stable for 6 months without significant changes in dissolution profile. In contrast to pure NCL, the F1 formulation substantially reduced the disease activity index, colonic inflammation, histological alterations and oxidative damage in colitis mice. These findings reveal that the prepared formulation can potentially deliver the drug locally at the colon, making it an effective tool in treating ulcerative colitis.

The Impact of Niclosamide Exposure on the Activity of Antioxidant Enzymes and the Expression of Glucose and Lipid Metabolism Genes in Black Carp (Mylopharyngodon piceus).

Niclosamide (NIC, 2',5-dichloro-4'-nitrosalicylanilide) is a salicylanilide molluscicide, and the extensive utilization and environmental pollution associated with NIC engender a potential hazard to both human health and the wellbeing of aquatic organisms. However, the mechanism of the chronic toxicity of NIC at environmentally relevant concentrations in terms of oxidative stress, metabolic disorder, and barrier functions in black carp (Mylopharyngodon piceus) is unknown. Therefore, healthy juvenile black carp (M. piceus) (average weight: 38.2 ± 2.5 g) were exposed to NIC at an environmentally realistic concentration (0, 10, and 50 µg/L) for 28 days. The findings of this study indicate that exposure to NIC resulted in reductions in weight gain, decreased activity of antioxidant enzymes, and increased expression of the Nrf2 gene. Furthermore, the liver demonstrated a greater accumulation of NIC than that in the gut and gills, as determined with a chemical analysis. Additionally, NIC exposure led to a significant reduction in ATP content and the activity of Na+/K+-ATPase and Ca2+/Mg2+-ATPase in the gut. Meanwhile, exposure to NIC resulted in a decrease in the liver glucose (Glu) level, gut cholesterol (CHO), and glycogen (Gln) and triglyceride (TG) content in all examined tissues. Conversely, it led to an increase in tissue lactic acid (LA) and acetyl-CoA levels, as well as LDH activity. Furthermore, NIC exposure at environmentally relevant concentrations demonstrated an upregulation in the expression of genes associated with glycolysis, such as PK and GK, while concurrently downregulating the gluconeogenesis gene G6Pase. Additionally, NIC exhibited an upregulation in the expression of genes related to ß-oxidation, such as CPT1 and ACOX, while downregulating genes involved in triglyceride synthesis, including SREBP1, GPAT, FAS, and ACC1. Moreover, NIC facilitated fatty acid transportation through the overexpression of FATP and Fat/cd36. These results suggest that chronic exposure to NIC is associated with oxidative stress, compromised barrier function, and metabolic disorder. Moreover, these results underscore the significance of assessing the potential consequences of NIC for black carp and aquatic environments for aquaculture.

[Molluscicidal effect of spraying 5% niclosamide ethanolamine salt granules with drones against Oncomelania hupensis in hilly regions].

OBJECTIVE: To establish a snail control approach for spraying chemicals with drones against Oncomelania hupensis in complex snail habitats in hilly regions, and to evaluate its molluscicidal effect. METHODS: The protocol for evaluating the activity of spraying chemical molluscicides with drones against O. hupensis snails was formulated based on expert consultation and literature review. In August 2022, a pretest was conducted in a hillside field environment (12 000 m2) north of Dafengji Village, Dacang Township, Weishan County, Yunnan Province, which was assigned into four groups, of no less than 3 000 m2 in each group. In Group A, environmental cleaning was not conducted and 5% niclosamide ethanolamine salt granules were sprayed with drones at a dose of 40 g/m2, and in Group B, environmental cleaning was performed, followed by 5% niclosamide ethanolamine salt granules sprayed with drones at a dose of 40 g/m2, while in Group C, environmental cleaning was not conducted and 5% niclosamide ethanolamine salt granules were sprayed with knapsack sprayers at a dose of 40 g/m2, and in Group D, environmental cleaning was performed, followed by 5% niclosamide ethanolamine salt granules sprayed with knapsack sprayers at a dose of 40 g/m2. Then, each group was equally divided into six sections according to land area, with Section 1 for baseline surveys and sections 2 to 6 for snail surveys after chemical treatment. Snail surveys were conducted prior to chemical treatment and 1, 3, 5, 7 days post-treatment, and the mortality and corrected mortality of snails, density of living snails and costs of molluscicidal treatment were calculated in each group. RESULTS: The mortality and corrected mortality of snails were 69.49%, 69.09%, 53.57% and 83.48%, and 68.58%, 68.17%, 52.19% and 82.99% in groups A, B, C and D 14 days post-treatment, and the density of living snails reduced by 58.40%, 63.94%, 68.91% and 83.25% 14 days post-treatment relative to pre-treatment in four groups, respectively. The median concentrations of chemical molluscicides were 37.08, 35.42, 42.50 g/m2 and 56.25 g/m2 in groups A, B, C and D, and the gross costs of chemical treatment were 0.93, 1.50, 0.46 Yuan per m2 and 1.03 Yuan per m2 in groups A, B, C and D, respectively. CONCLUSIONS: The molluscicidal effect of spraying 5% niclosamide ethanolamine salt granules with drones against O. hupensis snails is superior to manual chemical treatment without environmental cleaning, and chemical treatment with drones and manual chemical treatment show comparable molluscicidal effects following environmental cleaning in hilly regions. The cost of chemical treatment with drones is slightly higher than manual chemical treatment regardless of environmental cleaning. Spraying 5% niclosamide ethanolamine salt granules with drones is recommended in complex settings with difficulty in environmental cleaning to improve the molluscicidal activity and efficiency against O. hupensis snails.

Combination therapy of niclosamide with gemcitabine inhibited cell proliferation and apoptosis via Wnt/ß-catenin/c-Myc signaling pathway by inducing ß-catenin ubiquitination in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a type of cancer with high morbidity and mortality rates worldwide. Owing to a lack of therapeutic options, the overall survival rate of patients with pancreatic cancer is low. Gemcitabine has been mainly used to treat patients with pancreatic cancer, but its efficacy is limited by chemoresistance. Therefore, a novel therapeutic agent for PDAC therapy is urgently needed. An anthelminthic drug, niclosamide, has already been researched in breast, lung, colon, and pancreatic cancer as an anti-cancer purpose by re-positioning its original purpose. However, combination therapy of gemcitabine and niclosamide was not informed yet. Here, we found that niclosamide co-administered with gemcitabine significantly inhibited tumorigenesis of pancreatic cancer compared to gemcitabine alone. Further, combining niclosamide and gemcitabine inhibited cell proliferation and induced apoptosis. Niclosamide induced cell cycle arrest at the G1 phase, and the levels of CDK4/6 and cyclin D1 were lowered after gemcitabine treatment. In addition, the combination of these chemical compounds more effectively increased the binding level of activated ß-catenin destruction complex and ß-catenin to enable phosphorylation, compared to gemcitabine alone. After phosphorylation, niclosamide - gemcitabine upregulated the ubiquitin level, which caused phosphorylated ß-catenin to undergo proteasomal degradation; the combination was more potent than gemcitabine alone. Finally, the combination more effectively suppressed tumor growth in vivo, compared to gemcitabine alone. Altogether, our results indicate that niclosamide synergistically enhances the antitumor effect of gemcitabine in pancreatic cancer, by inducing the degradation of ß-catenin with ubiquitination. Therefore, this drug combination can potentially be used in PDAC therapy.

Metabolomic analyses uncover an inhibitory effect of niclosamide on mitochondrial membrane potential in cholangiocarcinoma cells.

Background: Niclosamide is an oral anthelminthic drug that has been used for treating tapeworm infections. Its mechanism involves the disturbance of mitochondrial membrane potential that in turn inhibits oxidative phosphorylation leading to ATP depletion. To date, niclosamide has been validated as the potent anti-cancer agent against several cancers. However, the molecular mechanisms underlying the effects of niclosamide on the liver fluke Opisthorchis viverrini (Ov)-associated cholangiocarcinoma (CCA) cell functions remain to be elucidated. The aims of this study were to investigate the effects of niclosamide on CCA cell proliferation and on metabolic phenoconversion through the alteration of metabolites associated with mitochondrial function in CCA cell lines. Materials and Methods: The inhibitory effect of niclosamide on CCA cells was determined using SRB assay. A mitochondrial membrane potential using tetramethylrhodamine, ethyl ester-mitochondrial membrane potential (TMRE-MMP) assay was conducted. Liquid chromatography-mass spectrometry-based metabolomics was employed to investigate the global metabolic changes upon niclosamide treatment. ATP levels were measured using CellTiter-Glo® luminescent cell viability assay. NAD metabolism was examined by the NAD+/NADH ratio. Results: Niclosamide strongly inhibited CCA cell growth and reduced the MMP of CCA cells. An orthogonal partial-least square regression analysis revealed that the effects of niclosamide on suppressing cell viability and MMP of CCA cells were significantly associated with an increase in niacinamide, a precursor in NAD synthesis that may disrupt the electron transport system leading to suppression of NAD+/NADH ratio and ATP depletion. Conclusion: Our findings unravel the mode of action of niclosamide in the energy depletion that could potentially serve as the promising therapeutic strategy for CCA treatment.

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.

Differences in the transcriptome response in the gills of sea lamprey acutely exposed to 3-trifluoromethyl-4-nitrophenol (TFM), niclosamide or a TFM:niclosamide mixture.

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.

Identification of a drug binding pocket in TMEM16F calcium-activated ion channel and lipid scramblase.

The dual functions of TMEM16F as Ca2+-activated ion channel and lipid scramblase raise intriguing questions regarding their molecular basis. Intrigued by the ability of the FDA-approved drug niclosamide to inhibit TMEM16F-dependent syncytia formation induced by SARS-CoV-2, we examined cryo-EM structures of TMEM16F with or without bound niclosamide or 1PBC, a known blocker of TMEM16A Ca2+-activated Cl- channel. Here, we report evidence for a lipid scrambling pathway along a groove harboring a lipid trail outside the ion permeation pore. This groove contains the binding pocket for niclosamide and 1PBC. Mutations of two residues in this groove specifically affect lipid scrambling. Whereas mutations of some residues in the binding pocket of niclosamide and 1PBC reduce their inhibition of TMEM16F-mediated Ca2+ influx and PS exposure, other mutations preferentially affect the ability of niclosamide and/or 1PBC to inhibit TMEM16F-mediated PS exposure, providing further support for separate pathways for ion permeation and lipid scrambling.

Praziquantel meets Niclosamide: A dual-drug Antiparasitic Cocrystal.

In this paper we report a successful example of combining drugs through cocrystallization. Specifically, the novel solid is formed by two anthelminthic drugs, namely praziquantel (PZQ) and niclosamide (NCM) in a 1:3 molar ratio, and it can be obtained through a sustainable one-step mechanochemical process in the presence of micromolar amounts of methanol. The novel solid phase crystallizes in the monoclinic space group of P21/c, showing one PZQ and three NCM molecules linked through homo- and heteromolecular hydrogen bonds in the asymmetric unit, as also attested by SSNMR and FT-IR results. A plate-like habitus is evident from scanning electron microscopy analysis with a melting point of 202.89 °C, which is intermediate to those of the parent compounds. The supramolecular interactions confer favorable properties to the cocrystal, preventing NCM transformation into the insoluble monohydrate both in the solid state and in aqueous solution. Remarkably, the PZQ - NCM cocrystal exhibits higher anthelmintic activity against in vitro S. mansoni models than corresponding physical mixture of the APIs. Finally, due to in vitro promising results, in vivo preliminary tests on mice were also performed through the administration of minicapsules size M.

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.

Establishment and application of unbiased in vitro drug screening assays for the identification of compounds against Echinococcus granulosus sensu stricto.

Echinococcus multilocularis and E. granulosus s.l. are the causative agents of alveolar and cystic echinococcosis, respectively. Drug treatment options for these severe and neglected diseases are limited to benzimidazoles, which are not always efficacious, and adverse side effects are reported. Thus, novel and improved treatments are needed. In this study, the previously established platform for E. multilocularis in vitro drug assessment was adapted to E. granulosus s.s. In a first step, in vitro culture protocols for E. granulosus s.s. were established. This resulted in the generation of large amounts of E. granulosus s.s. metacestode vesicles as well as germinal layer (GL) cells. In vitro culture of these cells formed metacestode vesicles displaying structural characteristics of metacestode cysts generated in vivo. Next, drug susceptibilities of E. multilocularis and E. granulosus s.s. protoscoleces, metacestode vesicles and GL cells were comparatively assessed employing established assays including (i) metacestode vesicle damage marker release assay, (ii) metacestode vesicle viability assay, (iii) GL cell viability assay, and (iv) protoscolex motility assay. The standard drugs albendazole, buparvaquone, mefloquine, MMV665807, monepantel, niclosamide and nitazoxanide were included. MMV665807, niclosamide and nitazoxanide were active against the parasite in all four assays against both species. MMV665807 and monepantel were significantly more active against E. multilocularis metacestode vesicles, while albendazole and nitazoxanide were significantly more active against E. multilocularis GL cells. Albendazole displayed activity against E. multilocularis GL cells, but no effects were seen in albendazole-treated E. granulosus s.s. GL cells within five days. Treatment of protoscoleces with albendazole and monepantel had no impact on motility. Similar results were observed for both species with praziquantel and its enantiomers against protoscoleces. In conclusion, in vitro culture techniques and drug screening methods previously established for E. multilocularis were successfully implemented for E. granulosus s.s., allowing comparisons of drug efficacy between the two species. This study provides in vitro culture techniques for the reliable generation of E. granulosus s.s. metacestode vesicles and GL cell cultures and describes the validation of standardized in vitro drug screening methods for E. granulosus s.s.

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.

Niclosamide inhibits Newcastle disease virus replication in chickens by perturbing the cellular glycolysis.

Newcastle disease virus (NDV), a member of Paramyxoviridae family, is one of the most important pathogens in poultry. To ensure optimal environments for their replication and spread, viruses rely largely on host cellular metabolism. In the present study, we evaluated the small drug molecule niclosamide for its anti-NDV activity. Our study has shown that a sublethal dose of 1 µM niclosamide could drastically reduce NDV replication. The results showed that niclosamide has antiviral activity against NDV infection during in vitro, in ovo and in vivo assays. Pharmacologically inhibiting the glycolytic pathway remarkably reduced NDV RNA synthesis and infectious virion production. Our results suggest that the effect of niclosamide on cellular glycolysis could be the possible reason for the specific anti-NDV effect. This study could help us understand antiviral strategies against similar pathogens and may lead to novel therapeutic approaches through targeted inhibition of specific cellular metabolic pathways.