Niclosamide: Beyond an antihelminthic drug

Niclosamide was discovered in the Bayer chemotherapy research laboratories in 1953. It was originally developed as a molluscicide to kill snails, an intermediate host of schistosomiasis, and was marketed as Bayluscide in 1959 [1] . In 1960, scientists at Bayer found it to be effective against human tapeworm (cestoda) infection, and it was marketed as Yomesan for human use in 1962 [1] , [2] . Niclosamide was approved by the US FDA for use in humans to treat tapeworm infection in 1982 and is included in the World Health Organization's list of essential medicines [3] . It has been used to safely treat millions of patients. For such a widely-used drug, Niclosamide's mechanism of action has not been well-delineated, although it has been reported to involve uncoupling of oxidative phosphorylation [4] , [5] , [6] , [7] . In the past several years, mounting evidence has accumulated that niclosamide is a multifunctional drug that is able to inhibit or regulate multiple signaling pathways and biological processes, suggesting that it may be developed as a novel treatment for more than just helminthic disease.

Beyond its approved medical use for parasitic disease treatment, niclosamide has demonstrated preclinical activity in many disease models, ranging from cancer and metabolic diseases to multiple types of infections ( Table 2 ). Currently there are four clinical trials of niclosamide in colon cancer and prostate cancer in the ClinicalTrials.gov clinical trials registry. Others will surely follow as the beneficial effects of niclosamide are appreciated in specific diseases. Improvement of the pharmacological and pharmacokinetic properties of niclosamide through re-formulation or pro-drug strategies are approaches to make more widespread use of this drug. The development of novel niclosamide derivatives that are biased toward targeting specific signaling pathways or biological functions in specific systemic diseases is a second approach to make use of the remarkable power of niclosamide.

Systemic sclerosis is a connective tissue disorder characterized by fibrosis of the skin and internal organs, vascular alterations, and dysimmunity including the presence of autoantibodies to nuclear proteins, all without a defined pathological cause [93] . Morin et al. reported that niclosamide treatment led to an improvement of the disease in a mouse model of systemic sclerosis induced by hypochlorous acid. Niclosamide-induced inhibition of STAT3, AKT, and Wnt/β-catenin pathways were observed [94] .

Identifying unifying mechanisms underlying niclosamide's pleotropic biological activities is difficult due to gaps in our knowledge of targets that interact directly with niclosamide. For many of the activities reported within it is unclear if a specific interaction with a biological target molecule drives the observed result, if an indirect mechanism is operating, or if combinations of both occur. Most of the studies do not address this mechanistic issue. Thus no direct binding interaction between niclosamide and a distinct biological target molecule has been established to account for the reported impact on signaling pathways or biological observations cited within ( Table 1 , Table 2 ). Niclosamide's ability to act as a protonophore, uncouple oxidative phosphorylation, or affect pH balance in some cells has been proposed as underlying indirect mechanisms to account for Niclosamide's activity against helminths, activity against mTORC1, activity in mouse models of Type 2 diabetes and fatty liver disease, activity against bacteria and viruses, and activity in antihypertension models. Given niclosamide's ability to inhibit signal transduction pathways that drive the transcription of multiple gene products, it is likely that some of niclosamide's reported biological activities may result from cross-talk between signaling pathways [95] , [96] , [97] , [98] , [99] , [100] . Table 2 Summary of signaling pathways and biological processes in disease models cited within affected by niclosamide. Table 2 Pathway or process affected by Niclosamide Tape worm Cancer Bacteria Virus Metabolic syndrome Artery constriction Endo-metriosis Neuro-pathic pain Rheumatoid arthritis Graft-versus-host disease Systemic sclerosis Uncoupling of oxidative phosphorylation 4–7 9 79 82 Wnt 9, 11, 19, 23-25, 29, 35, 44, 45, 55 87 92 94 mTORC1 13, 29 STAT3 14, 15, 31, 37, 38, 50 84 92 94 NF-κB 29, 34, 35 84 89 Notch 26, 29, 33 92 AKT/ERK/Src 20 92 94 AR-V7 49 C-Fos, C-Jun, E2F1, c-Myc 41 mGluRs 86 Metabolic pathways 42 ROS 34 Mitochondria 52, 55 82 pH 78 13, 52 58 70 Summary of signaling pathways and biological processes in disease models cited within affected by niclosamide. The chemical structure of niclosamide contains structural features associated with pleotropic pharmacologic activity. Niclosamide is a member of the salicylanilide class of pharmacologic agents and is a derivative of salicylic acid. Imbedded within these classes and within niclosamide is an aryl β-hydroxy-carbonyl pharmacophore motif. This structural motif is resident in a large number of diverse biological natural products isolated from plants, fungus and bacteria, and it is resident in multiple approved medicines across a variety of therapeutic categories. Representative examples of pharmacologic agents containing this motif are salicylic acid, diflunisal, aminosalicylic acid, antimycin, balanol, mycophenolate, flavonoids, doxycycline, daunorubicin, and eticlopride. In many of these examples a direct binding target and mechanism has been identified. Given the presence of this structural motif, it is not surprising that niclosamide has pleotropic biological activities and has the potential to interact with multiple biological targets. More research is needed to define the structure-activity relationships of niclosamide and the biological targets to which it binds in order to identify more selective agents and define underlying mechanisms. Toward this end, recent structure-activity studies have demonstrated that niclosamide's effects on ATP homeostasis can be separated from its effect on Wnt signaling [101] .

Niclosamide is a monohydrate that dehydrates above 50 °C and melts at 232.2 ± 0.2 °C, with a heat of fusion of 40.7 ± 6.5 kJ/mol. Its LogD at pH = 7 is 4.48 and it is essentially insoluble in water [102] . Niclosamide has low oral toxicity in mammals, and an oral median lethal dose (LD 50 ) in rats of > 5000 mg/kg [103] , [104] . Chang et al. reported the pharmacokinetic parameters of niclosamide in the rat when administered orally at 5 mg/kg [105] . Niclosamide exhibits a short half-life (6.0 ± 0.8 h). Niclosamide was rapidly absorbed with a Tmax of less than 30 min. The Cmax is 354 ± 152 ng/mL. AUC and bioavailability were 429 ± 100 and 10%, respectively. Osada et al. reported the pharmacokinetic parameters in mice orally dosed at 200 mg/kg, and observed a similar kinetic profile [24] . They also demonstrated that niclosamide concentrations in tumor tissue (37 ng/g tissue) were similar to those in plasma (38 ng/mL) measured at 24 h after the final administration, a concentration well-below the IC 50 of niclosamide in vitro in Wnt signaling and in cell growth assays [24] . For treatment of systemic diseases, efforts to improve systemic exposure to drug have been focused on employing nanotechnology and pro-drug approaches. Ye et al. used a wet media milling technique to prepare niclosamide nanocrystals approximately 235 nm in size [106] . However, this nanocrystal formulation showed no significant improvement in plasma concentration vs. time profiles between nanocrystals and control niclosamide when administered intravenously (i.v.) to rats, though an increased tissue concentration was observed at 2 h. Lin et al. reported that by using single-capillary electrospray method, they developed a water-soluble form of nano-niclosamide. The plasma concentration of niclosamide in this nano-formulation, via both oral and IV administration, peaked right after the distribution phase at 4 h as previously reported [24] , [107] . Recently, our group reported that an acyl derivative of niclosamide, DK-520, significantly increased both the plasma concentration and the duration of exposure to niclosamide when dosed orally [108] . This is the first report to successfully increase the systemic drug exposure of niclosamide in plasma and to extend its duration of exposure. In order to make more effective use of niclosamide, additional work needs to be done to improve its solubility, absorption and systemic bioavailability.