Jepretan Layar 2022-12-03 Pada 01.42.13
Jepretan Layar 2022-12-03 Pada 01.42.13
Jepretan Layar 2022-12-03 Pada 01.42.13
PII: S0939-6411(19)30240-1
DOI: https://doi.org/10.1016/j.ejpb.2019.05.004
Reference: EJPB 13045
Please cite this article as: E. José Barbosa, R. Löbenberg, G. Lima Barros de Araujo, N. Araci Bou Chacra,
Niclosamide repositioning for treating cancer: Challenges and nano-based drug delivery opportunities, European
Journal of Pharmaceutics and Biopharmaceutics (2019), doi: https://doi.org/10.1016/j.ejpb.2019.05.004
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Niclosamide repositioning for treating cancer: challenges and nano-based
drug delivery opportunities
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
* Corresponding author
Email address: [email protected]
Abstract
1
1. Introduction .....................………………………………….………………..……................ 2
2. Mechanistic insights in the physicochemical properties of niclosamide .…………….… 4
2.1 Crystal stability and relationship with poor aqueous solubility ……………….…… 4
2.2 Lipophilicity: lipid-associated oral absorption and drug-likeness considerations .. 7
3. The discovery of niclosamide anticancer activities ………………....……………….....… 10
4. Repositioning clinical studies for colon and prostate cancer ..………………………..…. 11
5. Nanostructured systems: fundamentals, types, methods of production ……………….. 16
6. Niclosamide nano-based drug delivery formulations and prospective therapeutic benefits
………………………………………………………………………………………... 18
7. Conclusion …………………...…………..………………..………….……………............... 28
Acknowledgments ……………..………………………….……………………………..…… 29
References .………………………………………………………..….……….……………… 29
1. Introduction
The discovery of a new drug and its way to the market can be a risky
process that may cost billions to pharmaceutical companies. The compound
may fail due to its toxicity or lack of efficacy in clinical trials, besides the long
time involved in the process until reaching the market, which is estimated
between 10 – 17 years [1]. In this context, to find new uses for existing drugs,
generally referred to as drug repositioning, is a strategy that has been gaining
attention. It can provide advantages compared to the traditional approach, for
instance, the reduced risk of failure due to toxicity. In this case safety data and
the pharmacokinetic profile of an approved drug are already known. It is also
associated with lower costs and reduced timeline, as the drug has already been
evaluated in early stages of clinical trials [1,2]. This process has an estimated
cost of around $300 million and may last for about 6.5 years [2].
This strategy also provides an opportunity to develop innovative
formulations, which may provide clinical benefits compared with those of older
marketed drugs [3]. In this case, nanostructured systems are especially
interesting. The reduction in particle size to the nanoscale promotes changes in
physicochemical and pharmacokinetic properties, among them increasing
dissolution rate and bioavailability. This is particularly important for problematic
2
drugs with low solubility and absorption, allowing dose and toxicity reduction [4].
Hence, as different alternatives of creation of the formulation are associated
with the development of nanostructured systems [4], it also brings the
perspective of patent protection for innovative products [3].
In this context, it is noteworthy to mention that there are different terms to
express the concept of “drug repositioning”. In the study of Langedijk and
colleagues, the authors analysed “repositioning” and other words used in the
literature until 2013 [5]. In this study, a great increase was observed in the
number of publications using terms related to drug repositioning, after 2010. In
addition, equivalent terms have been adopted, including “reprofiling” or
“redirecting”, but “repurposing” has been the most common and
interchangeable with “repositioning”. However, a common definition was not
identified. Among them, terms referring to new therapeutic applications, or new
uses for “old” or approved drugs were adopted. Hence, examples of
successfully repositioned drugs include sildenafil, previously designed to treat
coronary artery disease in the 1980s, but then during Phase I clinical trials, it
was discovered that it could treat erectile dysfunction. After its failure in Phase II
studies for treating angina, it was approved in 1998 for erectile dysfunction [6].
Another example is thalidomide, initially designed to be used as a sedative, and
then shown to treat morning sickness for pregnant women in the 1960s, until its
withdrawal from the market due to birth defects. Then, several studies in the
subsequent decades reported its anticancer effects, which eventually led to its
approval for multiple myeloma in 2006 [6].
Niclosamide is a drug present in the Model List of Essential Medicines
from the World Health Organization [7], used since the 1960s for tapeworm
infection [8]. For adults, the dose administered is 2 g orally, presenting efficacy
and safety due to its local action in the gastrointestinal tract [9]. Its mechanism
of action is attributed to uncoupling of oxidative phosphorylation in the
mitochondria, which interferes with the metabolism of these organisms [10,11].
Recently, new biological activities related to cancer diseases have been
attributed to this molecule, which perhaps make this drug one of the most
promising candidates for repositioning from new therapeutic indications [12,13].
Nonetheless, such expectations also must be accompanied by rational
and realistic evaluation of its potential, since the repositioning process still
requires proof of efficacy and safety of the compound. This implies different
3
challenges to be dealt with, such as the physicochemical properties of the drug,
its oral absorption process and clinical outcomes. Therefore, this review aims to
discuss challenges involving niclosamide repositioning for cancer diseases, in
addition the prospect of therapeutic benefits that nano-based formulations may
provide.
4
Fig. 1. Molecular structure of niclosamide (M.W. 327.12 g/mol).
5
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.
6
nonpolar surface area showed a correlation to aqueous solubility, indicating that
these drugs would be “grease balls”, with solvation limited solubility [25].
For niclosamide, the existence of N–H···O intramolecular interaction
favours a near planar conformation for the structure, whereas O–H···O (from
hydroxyl to carbonyl group) align the molecules along an axis, being connected
and stabilized by interactions between Cl···NO2 groups and between aromatic
rings, promoting the formation of layered molecular chains [26,27]. The
presence of hydrogen-bonding sites (NO2, OH, carbonyl groups), and two Cl
atoms contribute to solvation by water, and the anhydrous crystal lattice allows
the entry of water or other molecules in the cavities of the structure, forming
hydrates or solvates [26,27].
Solvation and disruption of the crystal packing are among the processes
related to solubilization of crystalline compounds, which requires energy to
occur [28]. Besides, the presence of water molecules may provide stability to a
crystal lattice, comparing to the anhydrous form [28]. Thus, a niclosamide
crystalline structure might restrict interactions with water and breaking of the
crystal, which is reflected in the low solubility of the anhydrous form (13.32
µg/mL). In addition, formation of stabilized hydrates might limit even more
niclosamide interactions with water, reflecting the lower aqueous solubility for
HA and HB monohydrates (0.95 and 0.61 µg/mL, respectively). Therefore,
niclosamide water solubility could also be associated with a solvation limited
phenomenon. However, since it presents a stable crystalline structure (M.P.
228-230ºC), its profile could be more similar to tolfenamic acid. As referred to
by Bergström and colleagues, specifically for this also small molecule (261.1
g/mol), the combination of lipophilicity and stability of the crystal (M.P. 212 ºC)
was likely related to its low solubility [25].
7
binding to proteins [29,30]. The LogD parameter also refers to lipophilicity, but
the pH variable is included in the calculation. Hence, the value of 3.6 at pH 6.9
(Fig. 2) indicates a decreasing trend in its partition in the lipid phase due to a
higher proportion of ionized species at this pH. The high lipophilicity also might
favour the development of lipid-based formulations [29]. However, not all poor
water-soluble compounds may present good solubility in lipids, since the high
stability may also be an obstacle to solubilization in these components [30].
Therefore, care should be taken with the development of the formulation, such
as a rational approach for the selection of the excipients, and use of design of
experiments (DOE) [30].
A possible mechanism by which lipid formulations might be associated
with niclosamide oral absorption was studied by Alskär and colleagues, when
lipolysis-triggered supersaturation and precipitation of eight compounds were
evaluated, and correlated with their physicochemical properties [31]. Three
types of formulations were prepared: one containing long-chain lipids (soybean
oil and Maisine® 35-1), a second containing medium-chain lipids (Capmul®
MCM EP and Captex® 355), and a third containing only surfactant and
cosolvent (Cremophor® EL and Carbitol™). After in vitro lipolysis tests, using
intestinal simulated fluid as medium, solubility and solid characterization were
performed. In brief, niclosamide significantly precipitated as a crystalline hydrate
after addition of pancreatic extract and, despite the decrease in solubility due to
the precipitation, the compound remained in a supersaturated state during the
tests [31].
As a comparison, according to the authors, the compound showed poor
saturation solubility in the medium with enzymes (30 µg/ml). However, during
lipolysis, the values related to the aqueous phase dropped from 250 to around
80 µg/ml after 60 minutes of test, using the medium-chain rich lipid-based
formulation as drug carrier [31]. Obviously that solubility values may vary
according to conditions of the test, but this range is clearly higher than a
reported value for the most water-soluble anhydrate form (13.32 µg/ml) [17].
Therefore, although the possibility of solid formation and the subsequent
dissolution-rate dependence for absorption, lipid-based formulations might
improve niclosamide bioavailability due to the ability to maintain some degree of
supersaturation in the gastrointestinal fluids [32]. This could be optimized if
maintained as long as possible during the passage of the drug throughout the
8
intestine [32]. Using multivariate analysis, Alskär and colleagues observed that
compounds with low molecular weight (< 350 g/mol) and high melting point (>
200 ºC), among them niclosamide and tolfenamic acid, tended to precipitate in
crystalline forms, also indicating some similarity between these two molecules
related to the solid state [31].
During the digestion of a formulation, the lipids can be absorbed or
partitioned into micelles or mixed micelles, composed of bile salts and/or lipidic
excipients [33,34]. Then, the drug released is also subjected to partition into the
micellar species formed [33,34]. Thus, supersaturation might not be the only
phenomenon related to a possible enhancement of niclosamide oral absorption.
For instance, lipid particles can adhere to the membrane of the enterocytes,
releasing the drug within the cells, which can be optimized by reducing the
particle size, providing a higher surface area for adhesion [34]. Additionally,
inhibition of drug efflux transporters by surfactants and other excipients, such as
Tween® 80 and Cremophor® EL, were already proposed as a possible
mechanism, increasing the permeability and oral absorption of drugs [35-37].
Therefore, these examples reflect the need for a better understanding of the
phenomena that may be associated with oral absorption of niclosamide and
other drugs.
Regarding the lipophilicity of drugs, increasing logP leads to an increase in
the volume of distribution of the compound, and also in central nervous system
(CNS) penetration [29]. Nonetheless, a high logP value (> 5) is not desirable for
drug candidates because it may be reflected in poor aqueous solubility and
absorption, and likelihood of in vivo toxicity, which could compromise approval
in clinical trials [29]. Thus, because niclosamide is a drug candidate, the
possibility of systemic action raises the question about its drug-like features,
that is, if its physicochemical properties are related to likelihood of successful
approval [38].
Based on molecular descriptors and physicochemical properties, different
rules of thumb or guidelines have been proposed to guide the design and
development of drug candidates [38,39]. One of the first, and most known, was
the work of Lipinski, which proposed a cLogP (calculated logP) ≤ 5 for good oral
candidates, based on a statistic evaluation of compounds that reached, at least,
Phase II clinical trials [38,39]. Another example was the work of Gleeson, who
used a set of compounds from GlaxoSmithKline (GSK) to evaluate the influence
9
of molecular descriptors in ADMET properties (absorption, distribution,
metabolism, excretion, toxicity). The author suggested that molecules with both
M.W. < 400 and clogP < 4 have a better chance of presenting desirable ADMET
properties [40]. Lastly, Waring proposed an optimum range between 1 and 3
and for lipophilicity, since high hydrophilic compounds also present undesirable
properties, such as high renal clearance and low permeability and tissue
distribution [41].
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.
10
4. Repositioning clinical studies for colon and prostate cancer
11
Table 2. Niclosamide repositioning clinical studies for cancer diseases. For this search, the keyword “niclosamide” was used at the ClinicalTrials.gov website.
Duke University Niclosamide (PO QD), during 7 days, prior Dose limiting toxicity,
2017 I Niclosamide Colon 18 2022
USA (NCT02687009) to surgical resection of primary tumour. niclosamide blood levels
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.
12
Table 3. Treatments and first results from clinical trials for niclosamide repositioning. The study from the University
of Washington was completed in 2017 and published in 2018 [81]. University of California, Davis [82] and Charité
University [83] published preliminary results in 2018.
Niclosamide
Study Drug Patients
Blood levels (ng/mL) Adverse Results
(niclosamide treatment) substances (ages)
Target Achieved effects
Nausea,
University of Washington Niclosamide, 5
> 163.5 35.7-182 * diarrhea, colitis No PSA reduction
(500 – 1000 mg/PO TD) enzalutamide (60-84)
Niclosamide,
University of California, Davis 6 PSA reduction for
abiraterone, > 32.71 100-162 ** Nausea, diarrhea
(1600 mg/PO TD) (74-83) at least four patients
prednisone
Possibility of longer
Charité University No drug related
Niclosamide 5 – 429-1777 * time until disease
(2000 mg/PO QD) toxicity reported
progression
* Corresponds to C max values. ** Corresponds to trough levels. PO: by mouth, QD: once a day, BID: twice a day, TID: three-times-daily; PSA:
prostate-specific antigen.
13
considered the maximum tolerated dose [81]. Complementary studies showed
that even after administrating high doses of niclosamide no clinical evidence of
activity is observable [81]. This fact can be assigned to the inability to overcome
the poor bioavailability and consistently achieve the plasma concentrations
necessary to inhibit tumour growth, based on CRPC models [55-57]. However,
despite the clinical failure of the compound, the authors recognized some
limitations of the study, such as the small number of patients and non-evaluated
drug-drug interaction and the need for the development of alternatives with
improved oral bioavailability [81]. In this context, as long as niclosamide shows
striking anticancer activity, a better understanding of the potential for
repositioning can be obtained from the ongoing clinical trials.
What reinforces this argument is that different findings were described in
preliminary results from the University of California, Davis (NCT02807805)
(Table 3), when niclosamide was evaluated in combination with abiraterone and
prednisone [82]. In a preliminary phase, CRPC patients were evaluated, with
dose escalation of niclosamide from 400 mg PO BID to 1,600 mg PO TID
(Table 3). According to results, the 1,600 mg PO TID regimen was well
tolerated in five patients, considering the recommended dose for Phase II trial,
which is, intriguingly, even higher than those assessed in the previous study
from the University of Washington, with nausea and diarrhea being reported.
Although the niclosamide blood levels are in a range comparable to the
failed study from the University of Washington (Table 3), the trial from the
University of California used “trough level” as a pharmacokinetic parameter,
which refers to the lowest blood concentrations during therapeutic drug
monitoring, obtained at the end of a dose interval [84]. Thus, these Universities
adopted different strategies to address the relationship between the anticancer
activity and niclosamide blood levels. The University of California trial
emphasized that therapeutic blood levels are achievable. Hence, this might
indicate that higher concentrations can be obtained, that would be necessary for
some anticancer effect. An indication for this is that, in this study, PSA reduction
was reported for two patients (<0.01 ng/mL). In addition, two other patients
presented reduction of ≥ 50% in PSA response [82]. In summary, the authors
considered that the combination of niclosamide with abiraterone and prednisone
presented promising preliminary safety and efficacy results [82].
14
For colon cancer, initial results from the Cherite University trial
(NCT02519582) (Table 3) were described in the study of Burock and colleagues
(2018) [83]. Patients with metastasized colorectal cancer received 2 g of
niclosamide in tablets (Yomesan®), once a day, until observation of disease
progression or toxicity. In brief, no toxicity was reported. In addition, Cmax values
were clearly higher than values from the University of Washington trial (Table
3), which further indicates that higher niclosamide blood levels might be
achieved. A patient with the highest median plasma level (598 ng/mL) showed
stable disease at 4 months and, according to the authors, preliminary results
indicated that those with higher plasma concentrations might present longer
time until disease progression, justifying more investigation [83].
In summary, niclosamide showed consistent anticancer activity in
preclinical studies, which justified initiating clinical trials for its repositioning.
Initial clinical results might show the importance of the blood levels to present
some effect against colon and prostate cancer. However, high doses were
administered to achieve target concentrations, as predicted by its
physicochemical properties. This raises the question, in the case of approval, as
to which doses will be therapeutically applied. Although it is well tolerated when
used for tapeworm infection, generally a single dose is administered in these
cases. Thus, the impact of its administration during longer periods for patients
with cancer diseases has still not been clarified. In this case, nausea, diarrhea
and gastrointestinal irritation will likely be commonly observed adverse effects.
Therefore, taken together, these considerations further justify the need for
nano-based drug delivery systems.
15
commonly described in the literature is that they have a higher surface to
volume ratio [86]. This means that the reduction of size provides a higher
surface area for the system, compared to the bulk material, which also means
that a higher proportion of atoms or molecules are present on the surface of the
particle [86]. Hence, at the nanoscale, altered properties, such as optical,
electrical, magnetic and thermal, can emerge, which can be explored for
pharmaceutical or biomedical applications, for instance as therapeutic or
diagnostic purposes [86-88].
One example is related to drug substances in their pure solid state, when
the reduction of particle size provides physicochemical improvements supported
by the Noyes-Whitney equation (1) [89]:
(1)
According to this model, dC/dt is the dissolution rate, S is the surface area,
D is the diffusion coefficient, h is the diffusion layer thickness, V is the volume of
dissolution, Cs is the saturation solubility, and C is the concentration at time t
[89]. Then, an increase of the surface area enhances dissolution rate. Another
commonly reported effect is that particle size also affects the diffusion layer
thickness and saturation solubility [89,90]. Thus, increased saturation solubility
and dissolution rate are two important features of solid particles at the
nanoscale, which is a valuable strategy to improve absorption and bioavailability
of poor water-soluble drugs.
Other advantages are usually attributed to nanomaterials. One example is
that a higher surface area contributes to adhesiveness of the particles to
biological membranes, which can improve their uptake by cells [91]. In addition,
incorporation of drugs into nanocarriers can reduce degradation and toxicity
[92]. Drug targeting, in turn, consists of coating the surface of nanocarriers with
ligands that bind to specific receptors, generally overexpressed by target cells,
which is useful for cancer diseases [93]. This strategy, therefore, can provide
specificity for treatment, with the prospect of reducing damage to normal tissues
[93].
16
However, if nanoparticles provide interesting advantages for
pharmaceutical products, on the other hand one of the biggest challenges
related to their development is the stability of the system. The higher surface to
volume ratio also means that the higher proportion of chemical species on the
surface are not surrounded by their counterparts (comparing to the inner
species of the particle), being in contact with the external medium [86].
Therefore, these systems present high total surface energy, being
thermodynamically unstable, and phenomena like agglomeration and Ostwald
ripening reflect the tendency of the particles to aggregate, increasing the
particle size [94]. Thus, the use of polymers (to promote steric hindrance)
and/or surfactants (to provide electrostatic repulsion or reduction of interfacial
tension) are strategies usually adopted to preserve the stability of the system
[94].
Different strategies have been adopted to develop nanostructured
systems. Using nanocrystals is the simplest approach. As previously
mentioned, this consists of reducing the particle size of the micronized
compound to the nanoscale, being stabilized in water by the presence of
polymers or surfactants [95]. Among the advantages related to them is the
simplicity of the formulation, and the fact that established techniques for large-
scale production are used for their preparation, which supports their approval
for clinical use [90,96]. In fact, different products are already on the market [89].
One marked feature is related to nanocrystals: the formulation does not require
incorporation of the drug into a matrix system, providing then 100% drug
loading to the nanoparticle [95].
The other types may be defined as matrix systems or nanostructured
carriers. These structures are classified differently, such as lipid-based,
polymeric-based, carbon or magnetic based [96]. Briefly, lipid-based
formulations include nanoemulsions, composed of mixture of two immiscible
liquids, with submicron droplets of a liquid dispersed in another liquid, stabilized
by the presence of surfactants [97]. The two types of nanoparticles that are
solid at ambient temperature are: solid-lipid nanoparticles, composed of a solid
lipid matrix containing the drug; and the nanostructured lipid carriers, composed
of a mixture of solid and liquid lipids [98,99]. Polymeric nanoparticles include the
use of natural or synthetic polymers to encapsulate drugs in vesicular reservoirs
17
or in solid polymeric matrix [100], whereas polymeric micelles are composed of
amphiphilic polymers (two or more in the case of mixed micelles) which self-
assemble into nanocarriers [101]. Still considering polymers, the application of a
strong electric field is used to produce nanofibers, which can also act as drug
carriers [102]. Examples of carbon derived nanomaterials include nanotubes,
graphene and fullerenes, which can also be functionalized with ligands [103].
Magnetic nanostructures are composed of metal or metal oxides, which are
directed to the targeted tissue by an external magnetic field [104].
Two types of methods of production are used to prepare nanostructured
systems: bottom-up and top-down. A third method is a combination of these two
[105,106]. In bottom-up techniques, the drug are dissolved in a solvent, and the
production of nanoparticles are carried out by its precipitation, by adding an
antisolvent. The advantages include the low cost and use of low energy for
preparation. On the other hand, drawbacks include the use of toxic solvents,
and the challenge of large-scale production [105,106]. In contrast, top-down
techniques start from the micronized drug, and application of mechanical forces
reduces the particle size to the nanoscale. Therefore, these are high energy
techniques [105,106]. The disadvantages include the use of high energy
equipment, and possibility of alteration of the crystal form of the starting
compound. Among the advantages, established techniques (media milling and
high pressure homogenization) have good reproducibility, with the prospect of
industrial production. The combination of these techniques is generally done by
preparing nanoparticles through a bottom-up technique, and then reducing the
size by a top-down method [105,106].
18
in formulations. However, promising improvements in pharmacokinetic
parameters were described in some of these studies. In addition, considering
niclosamide’s diverse anticancer activity, the possibility of synergy with other
compounds and drug targeting were also explored.
19
Table 4: Nanostructured systems containing niclosamide.
Regression in
Polymeric PEGCE, PS-b-PAA, ~ 2 (MCF-7, tumour growth from
2018 100 ± 25 – – – [108]
(solvent evaporation) anti-CD44-peptide MDA-MB231) MCF-7 cells in mice
Self-assembly
0.94 Reduced tumour volume
2017 polypeptidic micelles Elastin-like polypeptide 30-81 – – – [109]
(HCT116) from HCT116 cells in mice
(conjugation synthesis)
11.08-fold increase in
Solid lipid Stearic acid,
2017 204.2 ± 3.2 0.328 ± 0.02 –33.16 ± 2 89.1 ± 0.03 – bioavailability in rabbits, [111]
(micro-emulsion) polysorbate 80, PEG400
compared to marketed drug
Reduction of 50%
Pristine carbon nanoparticles
2016 Agave nectar, cucurbit[6]uril 88 ± 5 – –18 ± 5 – 21 ± 2 (MCF-7) in tumour size in mice, [113]
(Facile hydrothermal)
comparing to control
20
Mixed micelles ®
2016 Pluronic , biotin 31.8 ± 1.7 0.131 –3.37 ± 1.08 91.9 ± 1.9 < 0.9 (A549) – [115]
(thin-film hydration)
Polymeric 5 (A549),
2015 Albumin, glutaraldehyde 199.9 – –34.2 92.36 – [117]
(desolvation) 2.6 (MCF-7)
1 ± 0.5 (MDA-MB231),
Polymeric Hyperstar polymers, 30 ± 5 (MCF-7),
2015 90 ± 10 – – – – [119]
(solvent evaporation) amonafide 20 ± 5 (SKBR-3),
5 ± 1 (BT549)
12 ± 2 (MDA-MB231),
Polymeric
2015 PEGCE, PS-b-PAA ~ 69 0.2 –12 86.9 5 ± 1 (MCF-7), – [120]
(solvent evaporation)
1 ± 0.5 (C32)
a: 2.44 (CP70),
a: nanocrystals a: 105 ± 21 5.52 (SKOV-3);
a: PVA
2013 b: polymeric b1: 662 ± 121 – – – b1: 1.37 (CP70), – [123]
b: PLGA ****
(electrospray) b2: 584 ± 110 7.81 (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.
21
Examples of pharmacokinetic improvements were reported by Zhang and
colleagues when nanoemulsions were designed for evaluating niclosamide
pharmacokinetic properties in rats [121]. In this study, two types of
nanoemulsions were prepared: with and without poly(ethylene glycol)
monooleate (PEGM), in order to assess the ability of this polymer to provide
“stealth” properties, that is, to avoid lipolysis by digestive enzymes. Among the
results, no significant differences were observed in the lyposis test, considering
the presence of PEG, which the authors associated with the insufficient
molecular mass of the polymer (~1200) in to promote “stealth” properties.
Regarding pharmacokinetic evaluation, niclosamide was administered by
oral gavage in Sprague-Dawley rats (250 ± 20 g), at the dose of 20 mg/kg.
Then, Cmax values for nanoemulsions were 0.726 and 0.432 µg/mL, for with and
without PEGM, respectively, whereas 0.195 µg/mL for the suspension [121].
Despite differences in particle size between the nanoformulations, similar
bioavailability values were observed: ~2.5 (AUC0-t µg.h/mL) for nanoemulsions,
whereas ~0.5 for the suspension, resulting in a 5-fold increase. Hence,
considering the possible mechanisms related to niclosamide oral absorption,
these results support the prospect of increasing bioavailability and reducing the
dose by lipid-based matrix systems.
The authors still note that while blood levels in rats are not directly related
to humans, a low dose (20 mg/kg) was administered to the animals, which for
them could be an indication that target blood levels in humans could be easily
achieved. In this context, the pharmacokinetic of niclosamide had also been
evaluated in a preclinical study from Duke University [44]. In this study,
niclosamide was mixed into a polyethylene glycol system (90% polyethylene
glycol-300, 10% 1-methyl-2-pyrrolidone) for oral administration of 200 mg/kg,
using NOD/SCID mice (23-25 g) as in in vivo model. Thus, Cmax was 0.893
µg/mL (tmax = 15 minutes), whereas blood levels ranged from ~0.04 to ~0.08
µg/mL (0.5 to 12h). According to the authors, high doses were administered to
the animals, which for them could be associated with complex absorption
behavior of the compound in different regions of the gastrointestinal tract [44].
Lipid-based nanoformulations were also designed and prepared in
Rehman et al. [111]. In this study, solid lipid nanoparticles were prepared by
micro-emulsion technique, using stearic acid (SA), polysorbate 80 and
22
polyethylene glycol (PEG). Four variables were evaluated: concentrations of
SA, polysorbate-80 and PEG, and stirring time. Then, optimized formulations
were prepared varying concentrations of the drug. The objective was to
evaluate the phamacokinetic profile of the compound by oral route in rabbits (2
±0.3 kg), comparing with marketed drug (Mesan ®). In brief, Cmax values for the
nanoparticles and marketed drug were 3.97 and 1.84 µg/mL, respectively,
whereas reported bioavailability (AUC0-t µg.h/mL) were 16.74 for
nanoformulation, and 1.51 for Mesan ® [111].
One of the justifications for this study was that the solid lipid nanoparticles
could improve absorption of niclosamide to the lymphatic system, reducing the
first-pass effect by the liver. This possibility is attributed to lipid formulations in
general, and it is based on the way that lipids from the diet are digested, and
then absorbed in the intestine [124]. This phenomenon is mediated by the
synthesis and secretion of chylomicrons by enterocytes, which secrete the
lipoproteins to the lymphatic capillaries. The hypothesis for the absorption and
transport of drugs by lymphatics is that, after the digestion of the lipid
components of the formulations (a combination of lipids, surfactants and or co-
solvents), drug and lipids (fatty acids and monoglycerides) are uptaken by the
enterocytes [124-126]. Then, long-chain triglycerides are synthetized in the cell
and usually incorporated into chylomicrons, along with the drug and proteins.
Thus, after secretion by the enterocytes, the size of these large lipoproteins
limits their entry into blood vessels, which favours the entry into lymphatic
capillaries. Hence, it is believed that drug and chylomicrons follow the
unidirectional flow of the lymphatic vessels, reaching the systemic blood
circulation by subclavian vein [124-126]. Therefore, despite a possible
crystalline solid precipitation, lipid-based formulations might enhance
niclosamide oral absorption not only due to phenomena such as
supersaturation in intestinal fluids, but also due its partition in the lipid phase. In
this case, the possibility of solubilization in triglycerides and incorporation into
chylomicrons could favour its absorption toward the lymphatic system. This
could be a strategy to achieve therapeutic benefits, as increasing bioavailability,
reduction of the first pass-effect by the liver and drug-drug interaction.
Still considering lipid-based formulations, a pharmaceutical advantage that
might be achieved could be related to the crystal transformations of
23
niclosamide. This phenomenon might be critical to nanocrystals, since the lack
of a matrix allowed the nanosized crystalline structure to be in contact with
water, being subject to hydration. As previously discussed, this implies changes
in physicochemical properties, such as saturation solubility and intrinsic
dissolution. Considering the in silico and experimental logP values (3.89 and
4.45, respectively), niclosamide has a much higher tendency to dissolve in lipid
phase than in aqueous phase. Hence, incorporation and solubilization of this
drug substance in a lipidic matrix system could diminish the influence of
niclosamide crystal transformations (anhydrous to monohydrate HA, and then to
HB) in stabilizing the nanoparticles. On the other hand, possible disadvantages
include lower drug loading, compared with nanocrystals, and the required
compatibility among the excipients. In the case of solid lipid nanoparticles,
polymorphic changes of the solid lipid may be a critical factor in stabilizing the
system [99].
Considering nanocrystals, this strategy was presented in the work of Lin
and co-workers, when niclosamide nanoparticles were prepared by electrospray
technique to evaluate its anticancer activity against ovarian cancer cells [116].
The nanosuspension contained 1% polyvinyl alcohol (PVA) in a phosphate-
buffered saline solution, and formulation test presented an average particle
diameter of 105 nm. Among the results, the nanosuspension was able to
suppress the metabolism and the in vitro growth of CP70 and SKOV3 cells, with
IC50 (µM) of 3.59 and 3.38, respectively. Besides, using NOD/SCID mice, and
24
nanocrystals might work as if they “brought” the compound closer to a desirable
drug-likeness region.
Still considering their study, a second peak was observed in the plasma
concentration profile by oral and IV route, which for them could be associated
with an enterohepatic circulation phenomenon [116]. In the study of Duke
University, the authors associated the oral absorption of the compound in rats
with complex behavior in different regions of the gastrointestinal tract, but which
was less likely to be related to enterohepatic circulation [44]. Lastly, in the study
of Zhang and colleagues, a similar pattern, with a second peak, is also
observed in the pharmacokinetic profiles of the nanoemulsions, although a
hypothesis for this phenomenon was not discussed [121].
Drugs that reach the bloodstream are eliminated mainly by two pathways:
by the kidneys or the liver. In the latter case, compounds may be removed in an
unchanged form or as metabolites, and some of them may be excreted via bile
[127]. Hence, enterohepatic circulation is the elimination of drugs or other
substances from the bile to the small intestine, which are available for
reabsorption, being subject to new elimination by the liver or reaching systemic
circulation [127,128]. Examples of cases already observed in humans include
the drugs diltiazem, irinotecan, nevirapine, meloxicam and piroxican [127,128].
Molecules that undergo this phenomenon usually show multiple peaks in
plasma concentration versus time profiles. However, obviously drug metabolism
in animals is not directly related to humans, and it is difficult to attribute a single
cause for multiple peaking in pharmacokinetic profiles [128].
Therefore, it is still not clarified if niclosamide is subject to enterohepatic
circulation. If it is another challenge for its oral absorption, bioavailability
improvements provided by nanostructured systems might be achieved by two
different strategies. Nanocrystals may promote a higher amount of dissolved
drug that reaches the portal circulation, that might provide therapeutic
concentrations more easily. The other strategy is the drug delivery based on
lipid formulations. This could contribute to niclosamide absorption not only due
to supersaturation, but also due to the transport by the lymphatic system,
reducing the first-pass effect by the liver. These considerations are summarized
in Fig. 3.
25
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.
26
process, further considering that a combination therapy is a common approach
for serious diseases like cancer [130,131].
This idea was evaluated in the study of Misra and colleagues, when
polymeric nanoparticles were prepared to encapsulate niclosamide, a STAT3
blocker, and amonafide, a topoisomerase-II inhibitor, using them against triple
negative breast cancer (TNBC) cells [119]. The strategy was to prepare
“hyperstar polymers” (HSP) as nanocarriers, when hyperbranched macro-
initiators were polymerized to produce a core-shell structure to contain the two
compounds. The spherical nanostructure was composed of protonable tertiary
amine groups in the shell (for water dispersion), and acid-degradable acetal
groups in the core (for drug release under acid conditions). Then, the
nanoparticles were compared with the compounds alone, and with a
conventional formulation containing the two drugs substances, in MTT assays.
Among the results, the combination were able to produce synergistic effect
against TNBC cells (IC50) (~5 µM for BT549, and ~1 µM, for MDA-MB231)
compared to MCF-7 and SKBR-3 cells (~30 and ~20 µM, respectively) [119].
Later, Misra and colleagues also adopted the strategy of drug targeting for
breast cancer stem cells, which could be also associated with synergy [108].
The concept of cancer stem cells (CSC), briefly, is based on evidence that, from
a population of cancer cells, a small percentage present stemness features,
which can self-renew or differentiate into rapidly proliferating ones [132,133].
Thus, conventional chemotherapy would have an effect against the majority of
the cells, but the remaining unaffected CSCs would be responsible for
repopulation, therapy resistance and metastasis formation [133,134]. In the
study, polyethylene glycol cetyl ether (PEGCE) and poly(styrene)-block-
poly(acrylic) acid (PS-b-PAA) were used to prepare polymeric nanoparticles
containing niclosamide, being marked with a CD44-targeting peptide. Among
the results, from a population around 10% of CSC MCF-7 cells (CD44+),
maximum in vitro reduction was observed with targeted nanoparticles: around
60%, comparing with 20-30% using niclosamide alone or encapsulated in non-
targeted structures. In addition, the reduction of tumour growth in xenograft
mice, the targeted nanostructures reduced the CD44+/CD24- CSC population in
vivo and downregulated stemness marker genes. Mechanistically, the authors
27
attributed the anticancer effects to inhibition of STAT3 phosphorylation by
niclosamide [108].
Different strategies against CSCs include inhibition of cellular mechanisms
(Wnt, Hedgehog, Notch, NFκB) [134]. Niclosamide may block three of these
pathways (Wnt, Notch, NFκB) [12,13]. Besides, Misra and colleagues attributed
its action by inhibition of STAT3 signaling. Hence, since this compound may
present activity against CSCs, which can be optimized with nanostructured
systems, treatments including the combination of this molecule with
conventional drugs could provide better results considering recurrence, therapy
resistance and metastasis.
7. Conclusion
28
combination with other compounds, in order to achieve synergistic effects.
Approaches that are more sophisticated may include the use of ligands for drug
targeting. In this case, treatments including a combination with conventional
chemotherapy against CSCs might provide therapeutic benefits related to drug
resistance and recurrence.
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Graphical abstract
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