Niclosamide-loaded nanoparticles (Ncl-NPs) reverse pulmonary fibrosis in vivo and in vitro.

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-ß1 (TGF-ß1) 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-ß/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.

Phase Ib trial of reformulated niclosamide with abiraterone/prednisone in men with castration-resistant prostate cancer.

Niclosamide has preclinical activity against a wide range of cancers. In prostate cancer, it inhibits androgen receptor variant 7 and synergizes with abiraterone. The approved niclosamide formulation has poor oral bioavailability. The primary objective of this phase Ib trial was to identify a maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) of a novel reformulated orally-bioavailable niclosamide/PDMX1001 in combination with abiraterone and prednisone in men with castration-resistant prostate cancer (CRPC). Eligible patients had progressing CRPC, adequate end-organ function, and no prior treatment with abiraterone or ketoconazole. Patients were treated with escalating doses of niclosamide/PDMX1001 and standard doses of abiraterone and prednisone. Peak and trough niclosamide plasma levels were measured. Common Terminology Criteria for Adverse Events (CTCAE) v4.0 and Prostate Cancer Working Group 2 criteria were used to evaluate toxicities and responses. Nine patients with metastatic CRPC were accrued, with no dose-limiting toxicities observed at all dose levels. The recommended Phase II dose of niclosamide/PDMX1001 was 1200 mg orally (PO) three times daily plus abiraterone 1000 mg PO once daily and prednisone 5 mg PO twice daily. Trough and peak niclosamide concentrations exceeded the therapeutic threshold of > 0.2 µM. The combination was well tolerated with most frequent adverse effects of diarrhea. Five out of eight evaluable patients achieved a PSA response; two achieved undetectable PSA and radiographic response. A novel niclosamide/PDMX1001 reformulation achieved targeted plasma levels when combined with abiraterone and prednisone, and was well tolerated. Further study of niclosamide/PDMX1001 with this combination is warranted.

A phase I study of niclosamide in combination with enzalutamide in men with castration-resistant prostate cancer.

BACKGROUND: Niclosamide, an FDA-approved anti-helminthic drug, has activity in preclinical models of castration-resistant prostate cancer (CRPC). Potential mechanisms of action include degrading constitutively active androgen receptor splice variants (AR-Vs) or inhibiting other drug-resistance pathways (e.g., Wnt-signaling). Published pharmacokinetics data suggests that niclosamide has poor oral bioavailability, potentially limiting its use as a cancer drug. Therefore, we launched a Phase I study testing oral niclosamide in combination with enzalutamide, for longer and at higher doses than those used to treat helminthic infections. METHODS: We conducted a Phase I dose-escalation study testing oral niclosamide plus standard-dose enzalutamide in men with metastatic CRPC previously treated with abiraterone. Niclosamide was given three-times-daily (TID) at the following dose-levels: 500, 1000 or 1500mg. The primary objective was to assess safety. Secondary objectives, included measuring AR-V expression from circulating tumor cells (CTCs) using the AdnaTest assay, evaluating PSA changes and determining niclosamide's pharmacokinetic profile. RESULTS: 20 patients screened and 5 enrolled after passing all screening procedures. 13(65%) patients had detectable CTCs, but only one was AR-V+. There were no dose-limiting toxicities (DLTs) in 3 patients on the 500mg TID cohort; however, both (N = 2) subjects on the 1000mg TID cohort experienced DLTs (prolonged grade 3 nausea, vomiting, diarrhea; and colitis). The maximum plasma concentration ranged from 35.7 to 182 ng/mL and was not consistently above the minimum effective concentration in preclinical studies. There were no PSA declines in any enrolled subject. Because plasma concentrations at the maximum tolerated dose (500mg TID) were not consistently above the expected therapeutic threshold, the Data Safety Monitoring Board closed the study for futility. CONCLUSIONS: Oral niclosamide could not be escalated above 500mg TID, and plasma concentrations were not consistently above the threshold shown to inhibit growth in CRPC models. Oral niclosamide is not a viable compound for repurposing as a CRPC treatment. CLINICAL TRIAL REGISTRY: Clinicaltrials.gov: NCT02532114.

Phase II trial to investigate the safety and efficacy of orally applied niclosamide in patients with metachronous or sychronous metastases of a colorectal cancer progressing after therapy: the NIKOLO trial.

BACKGROUND: Colorectal cancer (CRC) is the second most common cause of all cancer deaths in Europe and the Western world with a lifetime risk of approximately 5%. Despite several improvements in the treatment of patients with unresectable CRC prognosis is poor and there is the need of developing new treatment strategies for patients with metastatic chemorefractory disease. The S100 calcium binding protein A4 (S100A4) predicts metastasis formation and reduced CRC patient survival. S100A4 was previously identified as transcriptional target of the Wnt/ß-catenin signaling pathway. The Food and Drug Administration (FDA)-approved anti-helminthic drug niclosamide is known to intervene in the Wnt/ß-catenin pathway signaling, leading to reduced expression of S100A4 linked to restricted in vivo metastasis formation. Thus, we aim at translation of our findings on restricting S100A4-driven metastasis into clinical practice for treating metastasized CRC patients progressing after standard therapy. METHODS/DESIGN: NIKOLO is a phase II, single center, one-arm open-label clinical trial to investigate the safety and efficacy of niclosamide tablets in patients with metastasized CRC progressing under standard therapy. Eligible patients will receive 2 g of orally applied niclosamide once a day and will continue with the treatment once daily till disease progression or toxicity. Toxicities will be graded according to National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) v4.03. The primary objective of this trial is to assess the progression free survival after 4 months, secondary objectives are overall survival, time to progression, disease control rate (remission + partial remission + stable disease), and safety. Furthermore, pharmacokinetic analysis will be conducted to evaluate niclosamide plasma concentration. DISCUSSION: This study is expected to provide evidence of the feasibility, toxicity and efficacy of niclosamide in the treatment of patients with metastasized CRC and could help to establish a new treatment option. TRIAL REGISTRATION: The study is registered with ClinicalTrials.gov (NCT02519582) and the European Clinical Trials Database (EudraCT 2014-005151-20).

The Simultaneous Inhibitory Effect of Niclosamide on RANKL-Induced Osteoclast Formation and Osteoblast Differentiation.

The bone destruction disease including osteoporosis and rheumatoid arthritis are caused by the imbalance between osteoblastogenesis and osteoclastogenesis. Inhibition of the NF-κB pathway was responsible for decreased osteoclastogenesis. Recently many studies indicated that niclosamide, the FDA approved an antihelminth drug, inhibits prostate and breast cancer cells growth by targeting NF-κB signaling pathways. This study evaluated the effects of niclosamide on osteoclast and osteoblast differentiation and function in vitro. In RANKL-induced murine osteoclast precursor cell RAW264.7 and M-CSF/RANKL-stimulated primary murine bone marrow-derived macrophages (BMM), niclosamide dose-dependently inhibited the formation of TRAP-positive multinucleated osteoclasts and resorption pits formation between 0.5uM and 1uM. In addition, niclosamide suppressed the expression of nuclear factor of activated T cells c1 (NFATc1) and osteoclast differentiated-related genes in M-CSF/ RANKL-stimulated BMM by interference with TRAF-6, Erk1/2, JNK and NF-κB activation pathways. However, the cytotoxic effects of niclosamide obviously appeared at the effective concentrations for inhibiting osteoclastogenesis (0.5-1uM) with increase of apoptosis through caspase-3 activation in osteoblast precursor cell line, MC3T3-E1. Niclosamide also inhibited ALP activity, bone mineralization and osteoblast differentiation-related genes expression in MC3T3-E1. Therefore, our findings suggest the new standpoint that niclosamide's effects on bones must be considered before applying it in any therapeutic treatment.

The anthelmintic drug niclosamide induces apoptosis, impairs metastasis and reduces immunosuppressive cells in breast cancer model.

Breast carcinoma is the most common female cancer with considerable metastatic potential. Discovery of new therapeutic approaches for treatment of metastatic breast cancer is still needed. Here, we reported our finding with niclosamide, an FDA approved anthelmintic drug. The potency of niclosamide on breast cancer was assessed in vitro and in vivo. In this investigation, we found that niclosamide showed a dramatic growth inhibition against breast cancer cell lines and induced apoptosis of 4T1 cells in a dose-dependent manner. Further, Western blot analysis demonstrated the occurrence of its apoptosis was associated with activation of Cleaved caspases-3, down-regulation of Bcl-2, Mcl-1 and Survivin. Moreover, niclosamide blocked breast cancer cells migration and invasion, and the reduction of phosphorylated STAT3(Tyr705), phosphorylated FAK(Tyr925) and phosphorylated Src(Tyr416) were also observed. Furthermore, in our animal experiments, intraperitoneal administration of 20 mg/kg/d niclosamide suppressed 4T1 tumor growth without detectable toxicity. Histological and immunohistochemical analyses revealed a decrease in Ki67-positive cells, VEGF-positive cells and microvessel density (MVD) and an increase in Cleaved caspase-3-positive cells upon niclosamide. Notably, niclosamide reduced the number of myeloid-derived suppressor cells (MDSCs) in tumor tissues and blocked formation of pulmonary metastases. Taken together, these results demonstrated that niclosamide may be a promising candidate for breast cancer.

Bioensaio de atividade moluscicida adaptado para a avaliação de extratos de plantas medicinais / Bioensayo de actividad moluscicida adaptado para evaluación de extractos de plantas medicinales / Bioassay of molluscicidal activity adapted for the evaluation of medicinal plant extracts

Nesta nota é apresentada detalhadamente a metodologia (preparação dos extratos, adaptação dos caramujos, ensaio de atividade, destino dos caramujos) usada para a avaliação da atividade moluscicida de extratos de plantas frente a caramujos da espécie Biomphalaria glabrata. A adaptação desta metodologia tem o propósito de avaliar extratos naturais para a busca de produtos alternativos mais baratos, biodegradáveis, seguros e disponíveis localmente, para o controle das populações de caramujos.

The methodology (extract preparation, adaptation of the snails, activity test, destiny of the snails) used for the evaluation of the molluscicidal activity of plant extracts in relation to snails from the Biomphalaria glabrata species appears in detail in this note. The adaptation of this methodology has the purpose of evaluating natural extracts in order to find cheaper, biodegradable, safe and easily available alternative products for the control of the populations of snails.

En este apunte se presenta detalladamente la metodología (preparación de los extractos, adaptación de los caracoles, ensayo de actividad, destino de los caracoles) utilizada para la evaluación de la actividad moluscicida de extractos de plantas frente a caracoles de la especie Biomphalaria glabrata. La adaptación de esta metodología tiene el propósito de evaluar extractos naturales para la búsqueda de productos alternativos más baratos, biodegradables, seguros y disponibles localmente, para el control de las poblaciones de caracoles.

Impact of certain plants and synthetic molluscicides on some fresh water snails and fish.

The LC50 (78, 85 ppm) and LC90 (88, 135 ppm) of Anagalis arvensis and Calendula micrantha respectively against Biomphalaria alexandrina were higher than those of the non-target snails, Physa acuta, Planorbis planorbis, Helisoma duryi and Melanoides tuberculata. In contrast, the LC50 of Niclosamide (0.11 ppm) and Copper sulphate (CuSO4) (0.42 ppm) against B. alexandrina were lower than those of the non-target snails. The mortalities percentage among non-target snails ranged between 0.0 & 20% when sublethal concentrations of CuSO4 against B. alexandrina mixed with those of C. micrantha and between 0.0 & 40% when mixed with A. arvensis. Mortalities ranged between 0.0 & 50% when Niclosamide was mixed with each of A. arvensis and C. micrantha. A. arvensis induced 100% mortality on Oreochromis niloticus after 48 hrs exposure and after 24 hrs for Gambusia affinis. C. micrantha was non-toxic to the fish. The survival rate of O. niloticus and G. affinis after 48 hrs exposure to 0.11 ppm of Niclosamide were 83.3% & 100% respectively. These rates were 91.7% & 93.3% respectively when each of the two fish species was exposed to 0.42 ppm of CuSO4. Mixture of sub-lethal concentrations of A. arvensis against B. alexandrina and those of Niclosamide or CuSO4 at ratios 10:40 & 25:25 induced 66.6% mortalities on O. niloticus and 83.3% at 40:10. These mixtures caused 100% mortalities on G. affinis at all ratios. A. arvensis CuSO4 mixtures at 10:40 induced 83.3% & 40% mortalities on O. niloticus and G. affinis respectively and 100% mortalities on both fish species at ratios 25:25 & 40:10. A mixture of sub-lethal concentrations of C. micrantha against B. alexandrina and of Niclosamide or CuSO4 caused mortalities of O. niloticus between 0.0 & 33.3% and between 5% & 35% of G. affinis. The residue of Cu in O. niloticus were 4.69, 19.06 & 25.37 mg/1kgm fish after 24, 48 & 72 hrs exposure to LC0 of CuSO4 against B. alexandrina respectively.

Effect of niclosamide on the marketable fish Liza ramada (Risso, 1826) concerning accumulation in muscles and activities of three metabolic liver enzymes.

The accumulation in muscles and the effect of different concentrations of niclosamide (Bayluscide) on the metabolic enzymes; succinate dehydrogenase (SDH), pyruvate dehydrogenase (PDH) and lactate dehydrogenase (LDH) in the liver of the Thin-lip grey mullet after 1, 2 & 3 weeks were studied. The molluscicide residues were detected in the muscles. The residues increased with the increase of concentrations of niclosamide as well as the time of exposure. On the other hand, LDH showed significant increase. But, SDH and PDH showed significant decrease. No doubt, this disturbance in the three liver enzymes cause disturbance in the process of metabolism. The important point is what about man who consumes a fish with niclosamide accumulated in its muscles.