European Journal of Pharmaceutics and Biopharmaceutics 141 (2019) 58–69

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European Journal of Pharmaceutics and Biopharmaceutics

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Review article Niclosamide repositioning for treating cancer: Challenges and nano-based T drug delivery opportunities ⁎ Eduardo José Barbosaa, Raimar Löbenbergb, Gabriel Lima Barros de Araujoa, , Nádia Araci Bou-Chacraa a Department of Pharmacy, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil b Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada

ARTICLE INFO ABSTRACT

Keywords: Drug repositioning may be defined as a process when new biological effects for known drugs are identified, Niclosamide leading to recommendations for new therapeutic applications. Niclosamide, present in the Model List of Essential Repositioning Medicines, from the World Health Organization, has been used since the 1960s for tapeworm infection. Several Physicochemical properties preclinical studies have been shown its impressive anticancer effects, which led to clinical trials for colon and Cancer prostate cancer. Despite high expectations, proof of efficacy and safety are still required, which are associated Clinical trials with diverse biopharmaceutical challenges, such as the physicochemical properties of the drug and its oral Nanostructured systems absorption, and their relationship with clinical outcomes. Nanostructured systems are innovative drug delivery strategies, which may provide interesting pharmaceutical advantages for this candidate. The aim of this review is to discuss challenges involving niclosamide repositioning for cancer diseases, and the opportunities of ther- apeutic benefits from nanosctrutured system formulations containing this compound.

1. Introduction particularly important for problematic drugs with low solubility and absorption, allowing dose and toxicity reduction [4]. Hence, as dif- The discovery of a new drug and its way to the market can be a risky ferent alternatives of creation of the formulation are associated with the process that may cost billions to pharmaceutical companies. The com- development of nanostructured systems [4], it also brings the per- pound may fail due to its toxicity or lack of efficacy in clinical trials, spective of patent protection for innovative products [3]. besides the long time involved in the process until reaching the market, In this context, it is noteworthy to mention that there are different which is estimated between 10 and 17 years [1]. In this context, to find terms to express the concept of “drug repositioning”. In the study of new uses for existing drugs, generally referred to as drug repositioning, Langedijk and colleagues, the authors analysed “repositioning” and is a strategy that has been gaining attention. It can provide advantages other words used in the literature until 2013 [5]. In this study, a great compared to the traditional approach, for instance, the reduced risk of increase was observed in the number of publications using terms related failure due to toxicity. In this case safety data and the pharmacokinetic to drug repositioning, after 2010. In addition, equivalent terms have profile of an approved drug are already known. It is also associated with been adopted, including “reprofiling” or “redirecting”, but “re- lower costs and reduced timeline, as the drug has already been eval- purposing” has been the most common and interchangeable with “re- uated in early stages of clinical trials [1,2]. This process has an esti- positioning”. However, a common definition was not identified. Among mated cost of around $300 million and may last for about 6.5 years [2]. them, terms referring to new therapeutic applications, or new uses for This strategy also provides an opportunity to develop innovative “old” or approved drugs were adopted. Hence, examples of successfully formulations, which may provide clinical benefits compared with those repositioned drugs include sildenafil, previously designed to treat cor- of older marketed drugs [3]. In this case, nanostructured systems are onary artery disease in the 1980s, but then during Phase I clinical trials, especially interesting. The reduction in particle size to the nanoscale it was discovered that it could treat erectile dysfunction. After its failure promotes changes in physicochemical and pharmacokinetic properties, in Phase II studies for treating angina, it was approved in 1998 for among them increasing dissolution rate and bioavailability. This is erectile dysfunction [6]. Another example is thalidomide, initially de-

⁎ Corresponding author. E-mail address: [email protected] (G.L.B. de Araujo). https://doi.org/10.1016/j.ejpb.2019.05.004 Received 3 March 2019; Received in revised form 23 April 2019; Accepted 8 May 2019 Available online 09 May 2019 0939-6411/ © 2019 Elsevier B.V. All rights reserved. E.J. Barbosa, et al. European Journal of Pharmaceutics and Biopharmaceutics 141 (2019) 58–69 signed to be used as a sedative, and then shown to treat morning 2. Mechanistic insights in the physicochemical properties of sickness for pregnant women in the 1960s, until its withdrawal from the niclosamide market due to birth defects. Then, several studies in the subsequent decades reported its anticancer effects, which eventually led to its ap- The development of innovative formulations aiming at drug re- proval for multiple myeloma in 2006 [6]. positioning requires knowledge of the physicochemical properties of Niclosamide is a drug present in the Model List of Essential the drug substance, since they are related to the complex drug ab- Medicines from the World Health Organization [7], used since the sorption process, and its distribution in the tissues [14]. These prop- 1960s for tapeworm infection [8]. For adults, the dose administered is erties greatly influence decisions about the design and development of 2 g orally, presenting efficacy and safety due to its local action inthe pharmaceutical products aiming at efficient drug delivery. Their me- gastrointestinal tract [9]. Its mechanism of action is attributed to un- chanistic understanding provides a scientific basis in drug product coupling of oxidative phosphorylation in the mitochondria, which in- performance [14,15]. terferes with the metabolism of these organisms [10,11]. Recently, new biological activities related to cancer diseases have been attributed to 2.1. Crystal stability and relationship with poor aqueous solubility this molecule, which perhaps make this drug one of the most promising candidates for repositioning [12,13]. Niclosamide is composed of yellow pale crystals, and it is practically Nonetheless, such expectations also must be accompanied by ra- insoluble in water, and moderately soluble in ethanol, chloroform and tional and realistic evaluation of its potential, since the repositioning ether [16]. This compound presents 327.12 g/mol molecular weight process still requires proof of efficacy and safety of the compound. This (M.W.) (Fig. 1), and three crystal forms are described in the literature: a implies different challenges to be dealt with, such as the physico- hygroscopic anhydrous form, and two monohydrates, HA and HB, with chemical properties of the drug, its oral absorption process and clinical melting points in the range 228–230 °C (Table 1) [17,18]. The existence outcomes. Therefore, this review aims to discuss challenges involving of different hydrated forms implies differences in physicochemical niclosamide repositioning for cancer diseases, in addition the prospect properties, such as water solubility and intrinsic dissolution [17]. In of therapeutic benefits that nano-based formulations may provide. aqueous medium, the anhydrous form rapidly tends to convert to HA, which in turn converts to the most stable, and the least water-soluble,

HB [17,18]. Conventional analytical techniques, such as thermal ana- lysis, infrared spectroscopy and X-ray diffraction, may be used to characterize these forms [17,18]. According to the Chemicalize.com prediction, the molecule is a weak acid, with higher proportion of anionic species in blood pH (7.4) (Fig. 2). According to the Biopharmaceutical Classification System (BCS), solubility and permeability are two important factors associated with drug absorption by oral route: since the drug is in a solid state, it has to be dissolved in gastrointestinal fluids to be absorbed by intestinal Fig. 1. Molecular structure of niclosamide (M.W. 327.12 g/mol). tract into the blood stream [21,22]. Considering the dose of 2 g used for tapeworm infection, niclosamide is classified as a low solubility drug by Table 1 BCS. In this case, its oral absorption is not an issue when the objective is Physicochemical properties of niclosamide. the local action in the gastrointestinal tract, but obviously becomes a pKa LogP Crystal form properties challenge when it is aimed at efficient systemic drug exposure. Neutral molecules are able to permeate biological membranes, a d Crystal form Dehydration (°C) Melting Water phenomenon energetically unfavourable to charged species [23]. Then, pointd solubilitye oral absorption of niclosamide becomes a “double-edged sword”: the (°C) (µg/mL) compound is a weak acid; therefore it is neutral at low pHs (Fig. 2), 6.89a 3.91a Anhydrous – 229 13.32 which restricts its solubility in the gastric region. On the other hand, as b c 7.25 4.45 HA 100 228 0.95 the molecule enters the small intestine, although an increase of pH HB 173 230 0.61 theoretically favours its solubilization, permeation through enterocyte a : In silico prediction by Chemicalize.com. membranes becomes less favoured with higher proportion of charged b : Determined at 25 °C by capillary electrophoresis [19]. species. In addition, no significant difference in solubility was observed c : Determined at 22 °C in octanol/water partition [20]. in acid medium (0.1 N HCl) and in buffered medium (6.8 pH) [24], d : Determined by differential scanning calorimetry (DSC) [17,18]. which further increases the challenge for the absorption. In this context, e : Measured at 25 °C [17]. based on its molecular and crystalline structure, and also considering

Fig. 2. pKa and logD in silico predictions by Chemicalize.com. At pH 6.89, distribution is 50% for neutral and anionic species, and at this pH LogD is 3.6.

59 E.J. Barbosa, et al. European Journal of Pharmaceutics and Biopharmaceutics 141 (2019) 58–69 the work of Bergström and colleagues [25], a hypothesis may be de- colleagues, when lipolysis-triggered supersaturation and precipitation rived to explain the fundamentals involved in niclosamide’s poor so- of eight compounds were evaluated, and correlated with their physi- lubility in water. cochemical properties [31]. Three types of formulations were prepared: In their study, the relationship between physicochemical properties one containing long-chain lipids (soybean oil and Maisine® 35-1), a and aqueous solubility were evaluated using statistical tools for a set of second containing medium-chain lipids (Capmul® MCM EP and Captex® 15 poor soluble drug substances [25]. According to the authors, “brick 355), and a third containing only surfactant and cosolvent (Cremophor® dust” and “grease balls” are terms generally used to refer to poor so- EL and Carbitol™). After in vitro lipolysis tests, using intestinal simu- luble compounds: the former refers to those composed of a stable lated fluid as medium, solubility and solid characterization were per- crystalline structure, marked by strong intermolecular bonds that pre- formed. In brief, niclosamide significantly precipitated as a crystalline vent interaction with water, whereas the latter refers to high lipophilic hydrate after addition of pancreatic extract and, despite the decrease in compounds that do not interact with this solvent [25]. In brief, solid- solubility due to the precipitation, the compound remained in a su- state properties (such as melting point, enthalpy and entropy of persaturated state during the tests [31]. melting) showed low correlation, while properties such as lipophilicity As a comparison, according to the authors, the compound showed and nonpolar surface area showed a correlation to aqueous solubility, poor saturation solubility in the medium with enzymes (30 µg/ml). indicating that these drugs would be “grease balls”, with solvation However, during lipolysis, the values related to the aqueous phase limited solubility [25]. dropped from 250 to around 80 µg/ml after 60 min of test, using the For niclosamide, the existence of NeH⋯O intramolecular interac- medium-chain rich lipid-based formulation as drug carrier [31]. Ob- tion favours a near planar conformation for the structure, whereas viously that solubility values may vary according to conditions of the OeH⋯O (from hydroxyl to carbonyl group) align the molecules along test, but this range is clearly higher than a reported value for the most an axis, being connected and stabilized by interactions between water-soluble anhydrate form (13.32 µg/ml) [17]. Therefore, although Cl⋯NO2 groups and between aromatic rings, promoting the formation the possibility of solid formation and the subsequent dissolution-rate of layered molecular chains [26,27]. The presence of hydrogen-bonding dependence for absorption, lipid-based formulations might improve sites (NO2, OH, carbonyl groups), and two Cl atoms contribute to sol- niclosamide bioavailability due to the ability to maintain some degree vation by water, and the anhydrous crystal lattice allows the entry of of supersaturation in the gastrointestinal fluids [32]. This could be water or other molecules in the cavities of the structure, forming hy- optimized if maintained as long as possible during the passage of the drates or solvates [26,27]. drug throughout the intestine [32]. Using multivariate analysis, Alskär Solvation and disruption of the crystal packing are among the pro- and colleagues observed that compounds with low molecular weight cesses related to solubilization of crystalline compounds, which re- (< 350 g/mol) and high melting point (> 200 °C), among them niclo- quires energy to occur [28]. Besides, the presence of water molecules samide and tolfenamic acid, tended to precipitate in crystalline forms, may provide stability to a crystal lattice, comparing to the anhydrous also indicating some similarity between these two molecules related to form [28]. Thus, a niclosamide crystalline structure might restrict in- the solid state [31]. teractions with water and breaking of the crystal, which is reflected in During the digestion of a formulation, the lipids can be absorbed or the low solubility of the anhydrous form (13.32 µg/mL). In addition, partitioned into micelles or mixed micelles, composed of bile salts and/ formation of stabilized hydrates might limit even more niclosamide or lipidic excipients [33,34]. Then, the drug released is also subjected to interactions with water, reflecting the lower aqueous solubility forHA partition into the micellar species formed [33,34]. Thus, super- and HB monohydrates (0.95 and 0.61 µg/mL, respectively). Therefore, saturation might not be the only phenomenon related to a possible niclosamide water solubility could also be associated with a solvation enhancement of niclosamide oral absorption. For instance, lipid parti- limited phenomenon. However, since it presents a stable crystalline cles can adhere to the membrane of the enterocytes, releasing the drug structure (M.P. 228–230 °C), its profile could be more similar to tolfe- within the cells, which can be optimized by reducing the particle size, namic acid. As referred to by Bergström and colleagues, specifically for providing a higher surface area for adhesion [34]. Additionally, in- this also small molecule (261.1 g/mol), the combination of lipophilicity hibition of drug efflux transporters by surfactants and other excipients, and stability of the crystal (M.P. 212 °C) was likely related to its low such as Tween® 80 and Cremophor® EL, were already proposed as a solubility [25]. possible mechanism, increasing the permeability and oral absorption of drugs [35–37]. Therefore, these examples reflect the need for a better 2.2. Lipophilicity: lipid-associated oral absorption and drug-likeness understanding of the phenomena that may be associated with oral ab- considerations sorption of niclosamide and other drugs. Regarding the lipophilicity of drugs, increasing logP leads to an If niclosamide has poor aqueous solubility, on the other hand, a high increase in the volume of distribution of the compound, and also in lipophilicity profile is indicated by the in silico and experimental logP central nervous system (CNS) penetration [29]. Nonetheless, a high values (3.91 and 4.5, respectively). These values show that niclosamide logP value (> 5) is not desirable for drug candidates because it may be solubility is thousands of times higher in the organic phase than in reflected in poor aqueous solubility and absorption, and likelihood of in aqueous phase, reflecting a tendency to dissolve in lipids, in crossing vivo toxicity, which could compromise approval in clinical trials [29]. biological membranes, and binding to proteins [29,30]. The LogD Thus, because niclosamide is a drug candidate, the possibility of sys- parameter also refers to lipophilicity, but the pH variable is included in temic action raises the question about its drug-like features, that is, if its the calculation. Hence, the value of 3.6 at pH 6.9 (Fig. 2) indicates a physicochemical properties are related to likelihood of successful ap- decreasing trend in its partition in the lipid phase due to a higher proval [38]. proportion of ionized species at this pH. The high lipophilicity also Based on molecular descriptors and physicochemical properties, might favour the development of lipid-based formulations [29]. How- different rules of thumb or guidelines have been proposed to guidethe ever, not all poor water-soluble compounds may present good solubility design and development of drug candidates [38,39]. One of the first, in lipids, since the high stability may also be an obstacle to solubili- and most known, was the work of Lipinski, which proposed a cLogP zation in these components [30]. Therefore, care should be taken with (calculated logP) ≤ 5 for good oral candidates, based on a statistic the development of the formulation, such as a rational approach for the evaluation of compounds that reached, at least, Phase II clinical trials selection of the excipients, and use of design of experiments (DOE) [38,39]. Another example was the work of Gleeson, who used a set of [30]. compounds from GlaxoSmithKline (GSK) to evaluate the influence of A possible mechanism by which lipid formulations might be asso- molecular descriptors in ADMET properties (absorption, distribution, ciated with niclosamide oral absorption was studied by Alskär and metabolism, excretion, toxicity). The author suggested that molecules

60 E.J. Barbosa, et al. European Journal of Pharmaceutics and Biopharmaceutics 141 (2019) 58–69 with both M.W. < 400 and clogP < 4 have a better chance of pre- senting desirable ADMET properties [40]. Lastly, Waring proposed an optimum range between 1 and 3 for lipophilicity, since high hydro- philic compounds also present undesirable properties, such as high 2022 2021 2021 2020 Estimated conclusion renal clearance and low permeability and tissue distribution [41]. 2017 (concluded) Hence, considering cLogP value (3.91), niclosamide would not be so distant from a desirable value for lipophilicity, further regarding LogD 3.6 at pH 6.9. However, obviously the experimental value (4.45), and its water solubility place this candidate in a “not ideal” drug-likeness region, compared to other compounds. This means that high doses might be required to achieve therapeutic blood levels. Hence, it justifies diverse pharmaceutical strategies, such as nanostructured systems, to improve its aqueous solubility and oral absorption. blood levels adverse events. treatment failure Outcome measures response, overall response 3. The discovery of niclosamide anticancer activities blood levels, PSA response. OS, PFS, PSA response, time to Dose limiting toxicity, niclosamide Dose limiting toxicity, niclosamide Dose limiting toxicity, OS, PFS, PSA A study by MacDonald and colleagues, in 2006, perhaps may be the Dose limiting toxicity, adverse events, first that indicated a possible repositioning process of niclosamide for cancer diseases, when unexpected results were revealed for this com- pound [42]. In this study, protein-protein interactions in HEK 293 cells were evaluated by protein-fragment complementation assays (PCA). The objective was to observe hidden or not expected biological activ- ities from 107 compounds, of different therapeutic classes. Using this website. approach, drug substances that could present unexpected anti- proliferative activities were identified. The action of niclosamide was confirmed in five different cancer cell lines (PC3, A549, MiaPaCa,

LOVO and U87MG), with a mean IC50 of 0.6 µM [42]. Treatment Then, several preclinical studies associated its anticancer activity primary tumour. ClinicalTrials.gov with the inhibition of the signaling pathways Wnt/β-catenin [43–49], STAT3 [50–60], Notch [61–64], NFκB [65–68] and mTOR [69–72],

which are cellular mechanisms related not only to different cell func- progression or unacceptable toxicity.

tions, but also to pathological conditions when deregulated. The effect disease progression or unacceptable toxicity. in multiple signaling pathways contributes to its action against different of disease progression or unacceptable toxicity. Treatment repeats every 4 weeks in the absence of disease types of cancer cells, among them colon, prostate, ovarian, breast and Niclosamide (PO QD) until disease progression or toxicity. OS, PFS, TP, disease control rate, lung [12,13]. acetate (PO QD). Treatment repeats every 4 weeks in the absence

4. Repositioning clinical studies for colon and prostate cancer 5 Niclosamide (PO TID) and enzalutamide (PO) for 28 days, until 18 Niclosamide (PO QD), during 7 days, prior to surgical resection40 of 37 Niclosamide (PO BID), prednisone (PO BID), and abiraterone 12 Niclosamide (PO BID) and enzalutamide (PO QD) on weeks 1–4. patients

From 2009 to subsequent years, preclinical studies that evaluated Estimated niclosamide against colon and prostate cancer cells provided encoura- ging results. For colon cancer, its activity was related to inhibition of Wnt signaling, which is an important mechanism for stem cells, asso- Colon Colon ciated with self-renew and homeostasis of tissues in adults, but also cancer Prostate Prostate Prostate related to several diseases when aberrantly active [73,74]. Niclosamide then showed its potential for use against colon cancer, including me- tastatic conditions [43,44,46]. For prostate cancer, promising results were reported by studies that evaluated niclosamide combined with drugs approved by the Food and Drug Administration (FDA) agency. Abiraterone (Zytiga®) is an an- Niclosamide Niclosamide Niclosamide, Niclosamide, enzalutamide drogen synthesis inhibitor approved in 2011, while enzalutamide enzalutamide Drug substances Type of

(Xtandi®) is an androgen receptor (AR) antagonist and inhibitor ap- abiraterone acetate proved in 2012 [75–77]. The main strategy for treating metastatic Niclosamide, prednisone, prostate cancer is castration by surgical or chemical intervention, which reduces androgen levels and, consequently, tumour growth [78,79]. However, in most cases, cellular adaptations involving AR signaling still promote disease progression [78,79]. The use of abiraterone or en- zalutamide provides clinical benefits, but therapy resistance may arise and can be associated with AR variants [78–80]. Hence, inhibition of the STAT3 pathway by niclosamide not only provided anticancer ef- (NCT02687009) (NCT02519582) (NCT02532114) fects, but also helped overcome resistance to these drugs [55–57]. USA (NCT03123978) USA (NCT02807805) Taken together, these results supported and prompted a series of clinical trials that began in 2015 (Table 2), with the first results being published in 2018 (Table 3). The first study completed was from the University of Washington (NCT02532114), in which relevant findings were published by Start Phase Sponsor (Identifier) 2017 I2017 I2016 Duke University University USA of California, Davis II2015 University of California, Davis II2015 Charité I University Germany University of Washington USA

Schweizer and colleagues [81]. In this study, niclosamide was Table 2 Niclosamide repositioning clinical studies for cancer diseases. For this search, the keyword “niclosamide” was used at the PO: by mouth, QD: once a day, BID: twice a day, TID: three-times-daily, OS: overall survival, PFS: progression-free survival, PSA: prostate-specific antigen, TP: time to progression.

61 E.J. Barbosa, et al. European Journal of Pharmaceutics and Biopharmaceutics 141 (2019) 58–69

Table 3 Treatments and first results from clinical trials for niclosamide repositioning. The study from the University of Washington was completed in 2017 andpublishedin 2018 [81]. University of California, Davis [82] and Charité University [83] published preliminary results in 2018.

Study (niclosamide treatment) Drug substances Patients Niclosamide Results (ages) Blood levels (ng/mL) Adverse effects

Target Achieved

University of Washington Niclosamide, enzalutamide 5 (60–84) > 163.5 35.7–182* Nausea, diarrhea, No PSA reduction (500–1000 mg/PO TD) colitis University of California, Davis Niclosamide, abiraterone, 6 (74–83) > 32.71 100–162** Nausea, diarrhea PSA reduction for at least four (1600 mg/PO TD) prednisone patients Charité University (2000 mg/PO QD) Niclosamide 5 – 429–1777* No drug related toxicity Possibility of longer time until reported disease progression

* Corresponds to Cmax values. ** Corresponds to trough levels. PO: by mouth, QD: once a day, BID: twice a day, TID: three-times-daily; PSA: prostate-specific antigen. administered in soft gelatin capsules of 500 mg, for patients with cas- higher concentrations can be obtained, that would be necessary for tration-resistant prostate cancer (CRPC), previously treated with abir- some anticancer effect. An indication for this is that, in this study, PSA aterone. Two dose levels were assessed: 500 and 1000 mg, three-times- reduction was reported for two patients (< 0.01 ng/mL). In addition, daily (TID) each. According to the authors, due to the poor oral bioa- two other patients presented reduction of ≥50% in PSA response [82]. vailability typically reported for niclosamide, the objective was to ad- In summary, the authors considered that the combination of niclosa- minister higher doses of the compound than those used for tapeworm mide with abiraterone and prednisone presented promising preliminary infection (2 g/day). safety and efficacy results [82]. Briefly, combined treatment was well tolerated with the 500mg For colon cancer, initial results from the Charité University trial dose regimen, but the two patients treated with 1000 mg dose level (NCT02519582) (Table 3) were described in the study of Burock and presented adverse events, among them nausea, colitis and diarrhea, colleagues (2018) [83]. Patients with metastasized colorectal cancer which were to some extent already predicted, as this dose level was received 2 g of niclosamide in tablets (Yomesan®), once a day, until clearly higher than those normally used for helminthic infection [81]. observation of disease progression or toxicity. In brief, no toxicity was

As previously mentioned, for adults the treatment is 2 g in a single dose, reported. In addition, Cmax values were clearly higher than values from and in the case of Hymenolepis nana infection, it is followed by 1 g daily the University of Washington trial (Table 3), which further indicates for 6 days [9]. Particularly, for one of the patients, symptoms began on that higher niclosamide blood levels might be achieved. A patient with day 26, suffering from diarrhea lasting > 72 h, whereas for the other, the highest median plasma level (598 ng/mL) showed stable disease at abdominal pain and diarrhea started on day 8, leading to hospitaliza- 4 months and, according to the authors, preliminary results indicated tion and medical care. Thus, the 500 mg dose was considered the that those with higher plasma concentrations might present longer time maximum tolerated dose [81]. Complementary studies showed that until disease progression, justifying more investigation [83]. even after administrating high doses of niclosamide no clinical evidence In summary, niclosamide showed consistent anticancer activity in of activity is observable [81]. This fact can be assigned to the inability preclinical studies, which justified initiating clinical trials for its re- to overcome the poor bioavailability and consistently achieve the positioning. Initial clinical results might show the importance of the plasma concentrations necessary to inhibit tumour growth, based on blood levels to present some effect against colon and prostate cancer. CRPC models [55–57]. However, despite the clinical failure of the However, high doses were administered to achieve target concentra- compound, the authors recognized some limitations of the study, such tions, as predicted by its physicochemical properties. This raises the as the small number of patients and non-evaluated drug-drug interac- question, in the case of approval, as to which doses will be ther- tion and the need for the development of alternatives with improved apeutically applied. Although it is well tolerated when used for tape- oral bioavailability [81]. In this context, as long as niclosamide shows worm infection, generally a single dose is administered in these cases. striking anticancer activity, a better understanding of the potential for Thus, the impact of its administration during longer periods for patients repositioning can be obtained from the ongoing clinical trials. with cancer diseases has still not been clarified. In this case, nausea, What reinforces this argument is that different findings were de- diarrhea and gastrointestinal irritation will likely be commonly ob- scribed in preliminary results from the University of California, Davis served adverse effects. Therefore, taken together, these considerations (NCT02807805) (Table 3), when niclosamide was evaluated in com- further justify the need for nano-based drug delivery systems. bination with abiraterone and prednisone [82]. In a preliminary phase, CRPC patients were evaluated, with dose escalation of niclosamide 5. Nanostructured systems: fundamentals, types, and methods of from 400 mg PO BID to 1600 mg PO TID (Table 3). According to results, production. the 1600 mg PO TID regimen was well tolerated in five patients, con- sidering the recommended dose for Phase II trial, which is, intriguingly, According to FDA guidelines for the industry, materials in the na- even higher than those assessed in the previous study from the Uni- noscale range can exhibit different physical or chemical properties, or versity of Washington, with nausea and diarrhea being reported. biological effects, differing from their larger counterparts [85]. This Although the niclosamide blood levels are in a range comparable to guidance defines these systems as (1) those that have, at least, oneof the failed study from the University of Washington (Table 3), the trial their dimensions in the range from 1 to 100 nm; or (2) if the dimensions from the University of California used “trough level” as a pharmaco- are in the range from 100 to 1000 nm, different properties or effects are kinetic parameter, which refers to the lowest blood concentrations attributed to their size [85]. Another feature commonly described in the during therapeutic drug monitoring, obtained at the end of a dose in- literature is that they have a higher surface to volume ratio [86]. This terval [84]. Thus, these Universities adopted different strategies to means that the reduction of size provides a higher surface area for the address the relationship between the anticancer activity and niclosa- system, compared to the bulk material, which also means that a higher mide blood levels. The University of California trial emphasized that proportion of atoms or molecules are present on the surface of the therapeutic blood levels are achievable. Hence, this might indicate that particle [86]. Hence, at the nanoscale, altered properties, such as

62 E.J. Barbosa, et al. European Journal of Pharmaceutics and Biopharmaceutics 141 (2019) 58–69 optical, electrical, magnetic and thermal, can emerge, which can be mixed micelles) which self-assemble into nanocarriers [101]. Still explored for pharmaceutical or biomedical applications, for instance as considering polymers, the application of a strong electric field is used to therapeutic or diagnostic purposes [86–88]. produce nanofibers, which can also act as drug carriers [102]. Examples One example is related to drug substances in their pure solid state, of carbon derived nanomaterials include nanotubes, graphene and when the reduction of particle size provides physicochemical im- fullerenes, which can also be functionalized with ligands [103]. Mag- provements supported by the Noyes-Whitney Eq. (1) [89]: netic nanostructures are composed of metal or metal oxides, which are dC DS directed to the targeted tissue by an external magnetic field [104]. = ()Cs C Two types of methods of production are used to prepare nanos- dt Vh (1) tructured systems: bottom-up and top-down. A third method is a com- According to this model, dC/dt is the dissolution rate, S is the sur- bination of these two [105,106]. In bottom-up techniques, the drug are face area, D is the diffusion coefficient, h is the diffusion layer thickness, dissolved in a solvent, and the production of nanoparticles are carried V is the volume of dissolution, Cs is the saturation solubility, and C is out by its precipitation, by adding an antisolvent. The advantages in- the concentration at time t [89]. Then, an increase of the surface area clude the low cost and use of low energy for preparation. On the other enhances dissolution rate. Another commonly reported effect is that hand, drawbacks include the use of toxic solvents, and the challenge of particle size also affects the diffusion layer thickness and saturation large-scale production [105,106]. In contrast, top-down techniques solubility [89,90]. Thus, increased saturation solubility and dissolution start from the micronized drug, and application of mechanical forces rate are two important features of solid particles at the nanoscale, reduces the particle size to the nanoscale. Therefore, these are high which is a valuable strategy to improve absorption and bioavailability energy techniques [105,106]. The disadvantages include the use of high of poor water-soluble drugs. energy equipment, and possibility of alteration of the crystal form of the Other advantages are usually attributed to nanomaterials. One ex- starting compound. Among the advantages, established techniques ample is that a higher surface area contributes to adhesiveness of the (media milling and high pressure homogenization) have good re- particles to biological membranes, which can improve their uptake by producibility, with the prospect of industrial production. The combi- cells [91]. In addition, incorporation of drugs into nanocarriers can nation of these techniques is generally done by preparing nanoparticles reduce degradation and toxicity [92]. Drug targeting, in turn, consists through a bottom-up technique, and then reducing the size by a top- of coating the surface of nanocarriers with ligands that bind to specific down method [105,106]. receptors, generally overexpressed by target cells, which is useful for cancer diseases [93]. This strategy, therefore, can provide specificity for 6. Niclosamide nano-based drug delivery formulations and treatment, with the prospect of reducing damage to normal tissues [93]. prospective therapeutic benefits However, if nanoparticles provide interesting advantages for phar- maceutical products, on the other hand one of the biggest challenges Table 4 presents research findings on nano-based formulations related to their development is the stability of the system. The higher containing niclosamide. From a total of 17 studies, distribution is: 3 surface to volume ratio also means that the higher proportion of che- included nanocrystals [116,122,123]; 6 included polymeric nano- mical species on the surface are not surrounded by their counterparts particles [108,110,117,119,120,123]; 3, lipid nanoparticles (comparing to the inner species of the particle), being in contact with [107,111,121]; 3, nanofibers [112,114,118]; 2, micelles [109,115]; and the external medium [86]. Therefore, these systems present high total 1, carbon nanoparticles [113]. In these studies, niclosamide also pre- surface energy, being thermodynamically unstable, and phenomena sented in vitro and in vivo anticancer activity when present in for- like agglomeration and Ostwald ripening reflect the tendency of the mulations. However, promising improvements in pharmacokinetic particles to aggregate, increasing the particle size [94]. Thus, the use of parameters were described in some of these studies. In addition, con- polymers (to promote steric hindrance) and/or surfactants (to provide sidering niclosamide’s diverse anticancer activity, the possibility of electrostatic repulsion or reduction of interfacial tension) are strategies synergy with other compounds and drug targeting were also explored. usually adopted to preserve the stability of the system [94]. Examples of pharmacokinetic improvements were reported by Different strategies have been adopted to develop nanostructured Zhang and colleagues when nanoemulsions were designed for evalu- systems. Using nanocrystals is the simplest approach. As previously ating niclosamide pharmacokinetic properties in rats [121]. In this mentioned, this consists of reducing the particle size of the micronized study, two types of nanoemulsions were prepared: with and without compound to the nanoscale, being stabilized in water by the presence of poly(ethylene glycol) monooleate (PEGM), in order to assess the ability polymers or surfactants [95]. Among the advantages related to them is of this polymer to provide “stealth” properties, that is, to avoid lipolysis the simplicity of the formulation, and the fact that established techni- by digestive enzymes. Among the results, no significant differences ques for large-scale production are used for their preparation, which were observed in the lyposis test, considering the presence of PEG, supports their approval for clinical use [90,96]. In fact, different pro- which the authors associated with the insufficient molecular mass of the ducts are already on the market [89]. One marked feature is related to polymer (∼1200) in to promote “stealth” properties. nanocrystals: the formulation does not require incorporation of the Regarding pharmacokinetic evaluation, niclosamide was adminis- drug into a matrix system, providing then 100% drug loading to the tered by oral gavage in Sprague-Dawley rats (250 ± 20 g), at the dose nanoparticle [95]. of 20 mg/kg. Then, Cmax values for nanoemulsions were 0.726 and The other types may be defined as matrix systems or nanostructured 0.432 µg/mL, for with and without PEGM, respectively, whereas carriers. These structures are classified differently, such as lipid-based, 0.195 µg/mL for the suspension [121]. Despite differences in particle polymeric-based, carbon or magnetic based [96]. Briefly, lipid-based size between the nanoformulations, similar bioavailability values were formulations include nanoemulsions, composed of mixture of two im- observed: ∼2.5 (AUC0-t µg h/mL) for nanoemulsions, whereas ∼0.5 for miscible liquids, with submicron droplets of a liquid dispersed in an- the suspension, resulting in a 5-fold increase. Hence, considering the other liquid, stabilized by the presence of surfactants [97]. The two possible mechanisms related to niclosamide oral absorption, these re- types of nanoparticles that are solid at ambient temperature are: solid- sults support the prospect of increasing bioavailability and reducing the lipid nanoparticles, composed of a solid lipid matrix containing the dose by lipid-based matrix systems. drug; and the nanostructured lipid carriers, composed of a mixture of The authors still note that while blood levels in rats are not directly solid and liquid lipids [98,99]. Polymeric nanoparticles include the use related to humans, a low dose (20 mg/kg) was administered to the of natural or synthetic polymers to encapsulate drugs in vesicular re- animals, which for them could be an indication that target blood levels servoirs or in solid polymeric matrix [100], whereas polymeric micelles in humans could be easily achieved. In this context, the pharmacoki- are composed of amphiphilic polymers (two or more in the case of netic of niclosamide had also been evaluated in a preclinical study from

63 ..Broa tal. et Barbosa, E.J. Table 4 Nanostructured systems containing niclosamide.

Year Type of nanoparticle (method of Components Nanoparticle characterization Performance Ref. preparation) Size (nm) PDI Z.P. (mV) E.E. (%) In vitro IC50 (µM) (cell) In vivo

2018 Solid Lipid (Solvent evaporation) Stearylamine, polysorbate 80, 112.18 ± 1.73 0.417 ± 0.026 +23.8 ± 2.7 82.21 ± 0.62 ∼18* (MDA-MB231) – [107] pluronic F-68 2018 Polymeric (solvent evaporation) PEGCE, PS-b-PAA, anti-CD44- 100 ± 25 – – – ∼2 (MCF-7, MDA-MB231) Regression in tumour growth [108] peptide from MCF-7 cells in mice 2017 Self-assembly polypeptidic micelles Elastin-like polypeptide 30–81 – – – 0.94 (HCT116) Reduced tumour volume from [109] (conjugation synthesis) HCT116 cells in mice 2017 Polymeric (desolvation) Chitosan, polysorbate 80, 100–120 – +24 > 90 7.5 (MCF-7), 8.75 (A549) – [110] glutaraldehyde, sodium sulfate, sodium metabisulfite 2017 Solid lipid (micro-emulsion) Stearic acid, polysorbate 80, 204.2 ± 3.2 0.328 ± 0.02 –33.16 ± 2 89.1 ± 0.03 – 11.08-fold increase in [111] PEG400 bioavailability in rabbits, compared to marketed drug 2016 Nanofiber (electrostatic spinning) PEO, Ag poly(e-caprolactone) 632 – – – 1.24 (A549), 1.21 (MCF-7) – [112] 2016 Pristine carbon nanoparticles Agave nectar, cucurbit[6]uril 88 ± 5 – –18 ± 5 – 21 ± 2 (MCF-7) Reduction of 50% in tumour [113] (Facile hydrothermal) size in mice, comparing to control 2016 Nanofibers crosslinked with bPEI, PEO, Fe3O4, folic acid, 655 ± 76 – – – – ** – [114] magnetic nanoparticle glutaraldehyde (electrostatic spinning) 2016 Mixed micelles (thin-film Pluronic®, biotin 31.8 ± 1.7 0.131 –3.37 ± 1.08 91.9 ± 1.9 < 0.9 (A549) – [115] hydration) 2016 Nanocrystals (electrospray) PVA 105 ± 21 – – – 3.59 (CP70), 3.38 (SKOV-3) Reduced tumour growth from [116]

64 CP70 and SKOV-3 cells in mice 2015 Polymeric (desolvation) Albumin, glutaraldehyde 199.9 – –34.2 92.36 5 (A549), 2.6 (MCF-7) – [117] 2015 Nanofiber (electrostatic spinning) bPEI, PEO, glutaraldehyde 430–576 – +6 to + 12 – 3.129 (A549), 2.147 (U87MG) – [118] 2015 Polymeric (solvent evaporation) Hyperstar polymers, amonafide 90 ± 10 – – – 1 ± 0.5 (MDA-MB231), – [119] 30 ± 5 (MCF-7), 20 ± 5 (SKBR-3), 5 ± 1 (BT549) 2015 Polymeric (solvent evaporation) PEGCE, PS-b-PAA ∼ 69 0.2 –12 86.9 12 ± 2 (MDA-MB231), – [120] European JournalofPharmaceuticsandBiopharmaceutics141(2019)58– 5 ± 1 (MCF-7), 1 ± 0.5 (C32) 2015 Nanoemulsions – a: PEG; b: PEGM, oleic acid, MCT, egg a: 162.2 ± 3.8 b: < 0.30 a: −21.8 ± 0.24 b: a: 9.12*** b: – ∼5-fold increase in [121] without PEG (melt dispersion/ lecithin, soybean lecithin, 307.8 ± 5.2 −20.8 ± 0.45 9.06*** bioavailability, compared to HPH) polysorbate 80, glycerin. drug solution, in rats 2015 Nanocrystals (wet milling) Polysorbate 80, poloxamer 188, 235.6 < 0.30 > +25 – 4.62 (EC9076) Delayed tissue distribution, [122] PVP, SDS, TPGS compared to drug solution, in rats 2013 a: nanocrystals b: polymeric a: PVA a: 105 ± 21 b1: – – – a: 2.44 (CP70), 5.52 (SKOV- – [123] (electrospray) b: PLGA**** 662 ± 121 b2: 3); b1: 1.37 (CP70), 7.81 584 ± 110 (SKOV-3); b2: 5.55 (CP70), 7.45 (SKOV-3)

PDI: Polydispersity index; Z.P.: zeta potential; E.E.: encapsulation efficiency; PEGCE: Polyethylene glycol cetyl ether; PS-b-PAA: poly(styrene)-block-poly(acrylic) acid; bPEI: branched poly(ethylenimine); MCF-7,MDA- MB231, SKBR-3 and BT549: breast cancer cells; HCT116: colon cancer cells; A549: lung cancer cells; PEG400: Polyethylene glycol 400; AUC: area under the curve; PEO: poly(ethylene oxide); PBS: phosphate-buffered saline; PVA: poly(vinyl alcohol); CP70 and SKOV-3: ovarian cancer cells; U87MG: brain cancer cells (glioma); C32: skin cancer cells (melanoma); HPH: High Pressure Homogenisation; PEGM: Polyethylene glycol monooleate; MCT: medium-chain triglyceride; PVP: polyvinylpyrrolidone; SDS: sodium dodecyl sulfate; TPGS: Tocopheryl polyethylene glycol succinate; EC9076: esophageal cancer cells (carcinoma); PLGA: poly lactic- co-glycolic acid. * : Measured as cytotoxic concentration (CTC50). ** : Percentage of L132 and KB cell viability were compared with formulations, not as a function of the concentrations. *** : Refers to drug loading. **** : b1 refers to single and b2 refers to dual-capillary electrospray system. 69 E.J. Barbosa, et al. European Journal of Pharmaceutics and Biopharmaceutics 141 (2019) 58–69

Duke University [44]. In this study, niclosamide was mixed into a Lin and co-workers, when niclosamide nanoparticles were prepared by polyethylene glycol system (90% polyethylene glycol-300, 10% 1-me- electrospray technique to evaluate its anticancer activity against thyl-2-pyrrolidone) for oral administration of 200 mg/kg, using NOD/ ovarian cancer cells [116]. The nanosuspension contained 1% polyvinyl SCID mice (23–25 g) as in in vivo model. Thus, Cmax was 0.893 µg/mL alcohol (PVA) in a phosphate-buffered saline solution, and formulation (tmax = 15 min), whereas blood levels ranged from ∼0.04 to ∼0.08 µg/ test presented an average particle diameter of 105 nm. Among the re- mL (0.5 to 12 h). According to the authors, high doses were adminis- sults, the nanosuspension was able to suppress the metabolism and the tered to the animals, which for them could be associated with complex in vitro growth of CP70 and SKOV3 cells, with IC50 (µM) of 3.59 and absorption behavior of the compound in different regions of the gas- 3.38, respectively. Besides, using NOD/SCID mice, and after adminis- trointestinal tract [44]. tration of 100 mg/kg of nanoniclosamide by oral gavage, reduced tu- Lipid-based nanoformulations were also designed and prepared in mour growth was observed in the animals [116]. Rehman et al. [111]. In this study, solid lipid nanoparticles were pre- In this study, they also evaluated the pharmacokinetic profile of pared by micro-emulsion technique, using stearic acid (SA), polysorbate niclosamide. Using Sprague-Dawley rats, nanocrystals dispersed in PBS 80 and polyethylene glycol (PEG). Four variables were evaluated: solution (0.46 mg/mL) were administered by oral gavage (5 mg/kg) or concentrations of SA, polysorbate-80 and PEG, and stirring time. Then, intravenous injection (2 mg/kg). In brief, the estimated bioavailability optimized formulations were prepared varying concentrations of the by oral route was 25%. According to the authors, despite the better drug. The objective was to evaluate the phamacokinetic profile of the results compared to a reported 10% value, they emphasized the need compound by oral route in rabbits (2 ± 0.3 kg), comparing with for improvement, considering its potential for ovarian cancer treatment marketed drug (Mesan®). In brief, Cmax values for the nanoparticles and [116]. Nonetheless, if this difference was maintained in humans, target marketed drug were 3.97 and 1.84 µg/mL, respectively, whereas re- blood levels could be more feasible to achieve, allowing the prospect of ported bioavailability (AUC0-t µg h/mL) were 16.74 for nanoformula- reducing the dose. Thus, strategies based on nanocrystals might work as tion, and 1.51 for Mesan® [111]. if they “brought” the compound closer to a desirable drug-likeness re- One of the justifications for this study was that the solid lipidna- gion. noparticles could improve absorption of niclosamide to the lymphatic Still considering their study, a second peak was observed in the system, reducing the first-pass effect by the liver. This possibility is plasma concentration profile by oral and IV route, which for them could attributed to lipid formulations in general, and it is based on the way be associated with an enterohepatic circulation phenomenon [116]. In that lipids from the diet are digested, and then absorbed in the intestine the study of Duke University, the authors associated the oral absorption [124]. This phenomenon is mediated by the synthesis and secretion of of the compound in rats with complex behavior in different regions of chylomicrons by enterocytes, which secrete the lipoproteins to the the gastrointestinal tract, but which was less likely to be related to lymphatic capillaries. The hypothesis for the absorption and transport enterohepatic circulation [44]. Lastly, in the study of Zhang and col- of drugs by lymphatics is that, after the digestion of the lipid compo- leagues, a similar pattern, with a second peak, is also observed in the nents of the formulations (a combination of lipids, surfactants and or pharmacokinetic profiles of the nanoemulsions, although a hypothesis co-solvents), drug and lipids (fatty acids and monoglycerides) are up- for this phenomenon was not discussed [121]. taken by the enterocytes [124–126]. Then, long-chain triglycerides are Drugs that reach the bloodstream are eliminated mainly by two synthetized in the cell and usually incorporated into chylomicrons, pathways: by the kidneys or the liver. In the latter case, compounds along with the drug and proteins. Thus, after secretion by the en- may be removed in an unchanged form or as metabolites, and some of terocytes, the size of these large lipoproteins limits their entry into them may be excreted via bile [127]. Hence, enterohepatic circulation blood vessels, which favours the entry into lymphatic capillaries. is the elimination of drugs or other substances from the bile to the small Hence, it is believed that drug and chylomicrons follow the unidirec- intestine, which are available for reabsorption, being subject to new tional flow of the lymphatic vessels, reaching the systemic blood cir- elimination by the liver or reaching systemic circulation [127,128]. culation by subclavian vein [124–126]. Therefore, despite a possible Examples of cases already observed in humans include the drugs dil- crystalline solid precipitation, lipid-based formulations might enhance tiazem, irinotecan, nevirapine, meloxicam and piroxican [127,128]. niclosamide oral absorption not only due to phenomena such as su- Molecules that undergo this phenomenon usually show multiple peaks persaturation in intestinal fluids, but also due its partition in the lipid in plasma concentration versus time profiles. However, obviously drug phase. In this case, the possibility of solubilization in triglycerides and metabolism in animals is not directly related to humans, and it is dif- incorporation into chylomicrons could favour its absorption toward the ficult to attribute a single cause for multiple peaking in pharmacoki- lymphatic system. This could be a strategy to achieve therapeutic netic profiles [128]. benefits, as increasing bioavailability, reduction of the first pass-effect Therefore, it is still not clarified if niclosamide is subject toen- by the liver and drug-drug interaction. terohepatic circulation. If it is another challenge for its oral absorption, Still considering lipid-based formulations, a pharmaceutical ad- bioavailability improvements provided by nanostructured systems vantage that might be achieved could be related to the crystal trans- might be achieved by two different strategies. Nanocrystals may pro- formations of niclosamide. This phenomenon might be critical to na- mote a higher amount of dissolved drug that reaches the portal circu- nocrystals, since the lack of a matrix allowed the nanosized crystalline lation, that might provide therapeutic concentrations more easily. The structure to be in contact with water, being subject to hydration. As other strategy is the drug delivery based on lipid formulations. This previously discussed, this implies changes in physicochemical proper- could contribute to niclosamide absorption not only due to super- ties, such as saturation solubility and intrinsic dissolution. Considering saturation, but also due to the transport by the lymphatic system, re- the in silico and experimental logP values (3.91 and 4.45, respectively), ducing the first-pass effect by the liver. These considerations aresum- niclosamide has a much higher tendency to dissolve in lipid phase than marized in Fig. 3. in aqueous phase. Hence, incorporation and solubilization of this drug A particular feature of niclosamide that may be explored, and which substance in a lipidic matrix system could diminish the influence of does not involve oral absorption necessarily, is its diversity for antic- niclosamide crystal transformations (anhydrous to monohydrate HA, ancer activity. As previously shown, this compound presents actions in and then to HB) in stabilizing the nanoparticles. On the other hand, different signaling pathways. Hence, this might provide synergistic ef- possible disadvantages include lower drug loading, compared with fects with other compounds, which was observed not only with abir- nanocrystals, and the required compatibility among the excipients. In aterone and enzalutamide, but also with erlotinib, a tyrosine kinase the case of solid lipid nanoparticles, polymorphic changes of the solid inhibitor used for lung cancer [51,52]. The therapeutic benefits include lipid may be a critical factor in stabilizing the system [99]. overcoming of drug resistance and reduction of dose [129,130]. Be- Considering nanocrystals, this strategy was presented in the work of sides, this may reinforce the argument for the repositioning process,

65 E.J. Barbosa, et al. European Journal of Pharmaceutics and Biopharmaceutics 141 (2019) 58–69

Fig. 3. Summary of nanocrystals and lipid-based strategies for niclosamide. Nanocrystals may improve the oral absorption due to an increase in dissolution rate and saturation solubility, and the enhanced surface area that favours adhesion to cell membranes. It does not prevent metabolization by the liver, but provides a higher amount of drug that reaches systemic circulation. Reduction of particle size of lipid-based formulations may improve adhesion to cell membranes and, after digestion, supersaturation provides a higher amount of solubilized drug available for absorption. This strategy is also associated with the hypothesis of targeting the lymphatic system, which is based on the incorporation of drugs into chylomicrons that, after secretion by enterocytes, enter lymphatic capillaries and follow the unidirectional flow of the vessels, reaching systemic circulation by the subclavian vein. M.W.: molecular weight; FA: fatty acids; MG: monoglycerides; TG: triglycerides. M.W.,pKa and logP values obtained by Chemicalize.com. further considering that a combination therapy is a common approach marker genes. Mechanistically, the authors attributed the anticancer for serious diseases like cancer [130,131]. effects to inhibition of STAT3 phosphorylation by niclosamide [108]. This idea was evaluated in the study of Misra and colleagues, when Different strategies against CSCs include inhibition of cellular me- polymeric nanoparticles were prepared to encapsulate niclosamide, a chanisms (Wnt, Hedgehog, Notch, NFκB) [134]. Niclosamide may block STAT3 blocker, and amonafide, a topoisomerase-II inhibitor, using three of these pathways (Wnt, Notch, NFκB) [12,13]. Besides, Misra and them against triple negative breast cancer (TNBC) cells [119]. The colleagues attributed its action by inhibition of STAT3 signaling. Hence, strategy was to prepare “hyperstar polymers” (HSP) as nanocarriers, since this compound may present activity against CSCs, which can be when hyperbranched macro-initiators were polymerized to produce a optimized with nanostructured systems, treatments including the core-shell structure to contain the two compounds. The spherical na- combination of this molecule with conventional drugs could provide nostructure was composed of protonable tertiary amine groups in the better results considering recurrence, therapy resistance and metastasis. shell (for water dispersion), and acid-degradable acetal groups in the core (for drug release under acid conditions). Then, the nanoparticles 7. Conclusion were compared with the compounds alone, and with a conventional formulation containing the two drugs substances, in MTT assays. The challenges involving niclosamide repositioning for cancer dis- Among the results, the combination were able to produce synergistic eases begin with its physicochemical properties, since its stable crys- effect against TNBC cells50 (IC ) (∼5 µM for BT549, and ∼1 µM, for talline structure and its lipophilicity restricts solubility in water, which MDA-MB231) compared to MCF-7 and SKBR-3 cells (∼30 and ∼20 µM, implies undesirable drug-like features that affect oral absorption. In respectively) [119]. fact, this was reflected by the high daily doses administered inpre- Later, Misra and colleagues also adopted the strategy of drug tar- liminary clinical studies, sometimes much higher than the usually used geting for breast cancer stem cells, which could be also associated with for tapeworm infection. This raises concerns of whether the dose will be synergy [108]. The concept of cancer stem cells (CSC), briefly, is based different if applied therapeutically, and what would be the impactof on evidence that, from a population of cancer cells, a small percentage administering it for long periods. Initial results indicated that the blood present stemness features, which can self-renew or differentiate into levels might be critical for some effect against prostate and colon rapidly proliferating ones [132,133]. Thus, conventional chemotherapy cancer. Thus, it also raises the question of whether the activity will be would have an effect against the majority of the cells, but the remaining confirmed in ongoing clinical trials. An unsuccessful performance does unaffected CSCs would be responsible for repopulation, therapy re- not exclude this candidate, since niclosamide already showed effects sistance and metastasis formation [133,134]. In the study, polyethylene against different types of cancer cells, but further highlights the chal- glycol cetyl ether (PEGCE) and poly(styrene)-block-poly(acrylic) acid lenges involving its repositioning. The drawbacks of the physicochem- (PS-b-PAA) were used to prepare polymeric nanoparticles containing ical properties bring the opportunity for the development of nano-based niclosamide, being marked with a CD44-targeting peptide. Among the + formulations, which were not evaluated in the clinical studies. results, from a population around 10% of CSC MCF-7 cells (CD44 ), Nanocrystals are the simplest strategy, as the increase in saturation maximum in vitro reduction was observed with targeted nanoparticles: solubility and dissolution rate might improve oral absorption and pro- around 60%, comparing with 20–30% using niclosamide alone or en- vide therapeutic blood levels. Despite the possibility of precipitation, capsulated in non-targeted structures. In addition the reduction of tu- drug supersaturation by lipid-based formulations might also improve mour growth in xenograft mice, the targeted nanostructures reduced + − absorption, and are associated with the hypothesis of targeting the the CD44 /CD24 CSC population in vivo and downregulated stemness lymphatic system, preventing metabolization by the liver as well.

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