Journal of Pharmaceutical Sciences 107 (2018) 1342-1351

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Journal of Pharmaceutical Sciences

journal homepage: www.jpharmsci.org

Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Ophthalmic Econazole Hydrogels for the Treatment of Fungal Keratitis

Victoria Díaz-Tome
1, Andrea Luaces-Rodríguez 1, 2, Jesús Silva-Rodríguez 3,

Llovo-Taboada 5,
Sara Blanco-Dorado 2, 4, Laura García-Quintanilla 2, 4, Jose

Blanco-Me
Jose
ndez 1, Xurxo García-Otero 1, Rube
n Varela-Ferna
ndez 1, Michel Herranz 2,
6
María Gil-Martínez , María Jesús Lamas 2, 4
, Miguel Gonza
lez-Barcia 2, 4,
1, *
ndez-Ferreiro , 4, *
1, 2
Francisco J. Otero-Espinar , Anxo Ferna
1
Department of Pharmacology, Pharmacy and Pharmaceutical Technology and Industrial Pharmacy Institute, Faculty of Pharmacy, University of Santiago
de Compostela (USC), Santiago de Compostela, Spain
2
Clinical Pharmacology Group, University Clinical Hospital, Health Research Institute of Santiago de Compostela (IDIS), Santiago de Compostela, Spain
3
Molecular Imaging Group, University Clinical Hospital, Health Research Institute of Santiago de Compostela (IDIS), Santiago de Compostela, Spain
4
Pharmacy Department, University Clinical Hospital Santiago de Compostela (SERGAS), Santiago de Compostela, Spain
5
Microbiology Department, University Clinical Hospital Santiago de Compostela (SERGAS), Santiago de Compostela, Spain
6
Ophthalmology Department, University Clinical Hospital Santiago de Compostela (SERGAS), Santiago de Compostela, Spain

a r t i c l e i n f o a b s t r a c t

Article history: Econazole is a feasible alternative treatment in the management of fungal keratitis. Nevertheless, its low
Received 26 September 2017 water solubility is considered the main limitation to the incorporation into ophthalmic formulations. In
Revised 21 December 2017 this work, econazole nitrate is solubilized by using cyclodextrins to achieve an optimum therapeutic
Accepted 22 December 2017
concentration. Phase solubility diagrams suggest a-cyclodextrin as the most effective cyclodextrin and
Available online 3 January 2018
later the inclusion complex formed with this one was characterized in solution by 1D, 2D-NMR, and
molecular modeling. Econazole-a-cyclodextrin inclusion complex was included in 2 types of ocular
Chemical Compounds CAS/CID Identification:
k-carrageenan (CID: 11966249) hydrogels: a natural polysaccharides ion-sensitive hydrogel and a hyaluronic acid hydrogel. Both of them
gellan gum (CAS number 71010-52-1) show no ocular irritation in the hen's egg test on chorioallantoic membrane assay and a controlled
econazole nitrate (CID: 68589) econazole release over time. Permeability studies suggest that hydrogels do not modify the econazole
hyaluronic acid (CID: 3084050)
nitrate permeability through bovine cornea in comparison with an econazole-a-cyclodextrin inclusion
2-hydroxypropyl-b-cyclodextrin
(CID: 44134771) complex solution. Finally, ocular biopermanence studies performed using positron emission tomography
2-hydroxypropyl-Ƴ;-cyclodextrin show these hydrogels present a high retention time on the eye. Results suggest the developed formu-
(CID: 92043192) lations have a high potential as vehicles for the econazole topical ocular administration as fungal keratitis
a-cyclodextrin (CID: 444913) treatment.
Keywords: © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
corneal permeation
ophthalmic drug delivery
econazole
cyclodextrin
ocular permanence time
ophthalmic hydrogel
positron emission tomography

Introduction such as Aspergillus spp or Fusarium spp, are the main etiological
agents in tropical and subtropical regions, where contaminated
Fungal keratitis (FK) is a severe and difficult to treat disease vegetables (by penetrating trauma) act as the most frequent
which can be caused by different fungal species. Filamentous fungi, transmission vehicles, causing the disease later in 40%-60% of

Abbreviations used: FK, fungal keratitis; aCD, a-cyclodextrin; HPbCD, hydroxy- The authors Victoria Díaz-Tome
and Andrea Luaces-Rodríguez contributed equally.
propyl-b-cyclodextrin; HPgCD, 2-hydroxypropyl-g-cyclodextrin; PET, positron This article contains supplementary material available from the authors by request
emission tomography; PBS, phosphate buffer saline; CE, complexation efficiency; or via the Internet at https://doi.org/10.1016/j.xphs.2017.12.028.
ECN, 2 mg/mL econazole nitrate solution with 15% (w/v) aCD; ISH, ion-sensitive * Correspondence to: Anxo Fern
andez Ferreiro (Telephone: þ34-981-95-51-66)
hydrogel; HAH, hyaluronic acid hydrogel; SLF, simulated lacrimal fluid; CT, and Francisco J. Otero Espinar (Telephone: þ34-609-71-88-21).
computed tomography; MRT, mean residence time. E-mail addresses: francisco.otero@usc.es (F.J. Otero-Espinar), anxordes@gmail.
com (A. Ferna
ndez-Ferreiro).

https://doi.org/10.1016/j.xphs.2017.12.028
0022-3549/© 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
 V. Díaz-Tom
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patients.1 In colder regions, Candida albicans is the most common ratio, MW 1580) was acquired from Sigma Aldrich® (Darmstadt,
causing-FK species, and the disease incidence is related to corneal Germany); a-cyclodextrin (aCD, Cavamax® W6, MW 972.84) was
surface surgeries, abrasions caused by contaminated contact lenses, procured from Wacker Chemie AG (München, Germany); gellam
neurotrophic ulcers, or chronic use of topical corticosteroids.2 gum (Kelcogel® CG-LA molecular weight 1.5-2.5 106 Da) and
Pain, vision loss, photosensitivity, or tearing are the most k-carrageenan (Genugel® carrageenan GC-130 molecular weight
frequent symptoms in FK, leading this to a subsequent complete 3.5-8.0 105 Da) were provided by CP Kelco® (Atlanta, GA).
blindness if not treated correctly.3 The ophthalmic FK treatment
complexity requires the use of topical ophthalmic antifungal drugs
Methods
for a prolonged time period, with frequent instillations and occa-
sional corneal debridement to ensure the antifungal's penetra-
Econazole Solubilization With Cyclodextrins
tion.4,5 The current treatment mainly consists of polyene drugs
such as natamycin for the filamentous fungi and amphotericin B for Phase Solubility Diagrams. Phase solubility diagrams were used to
yeasts.6 Other treatments include azole drugs such as econazole, estimate the econazole nitrate stability constants with HPbCD,
fluconazole, or voriconazole,7,8 the latter being the most commonly HPgCD, and aCD. Solubility measurements were carried out ac-
used. Nevertheless, recent studies have shown econazole is a cording to Higuchi and Connors22 methodology following the
potential therapeutic alternative in the treatment of filamentous FK protocols previously described by Anguiano-Igea et al.23 An excess
caused by Aspergillus spp and Fusarium spp, showing therapeutic amount of econazole nitrate was added to a series of aqueous
effects similar to natamycin.9,10 solutions of increasing cyclodextrin concentrations. Econazole
However, nowadays there is a significant lack of commercial nitrate solubility in distilled water and in PBS (phosphate buffer
ophthalmic antifungal formulations due to their high cost and low saline pH 7.4) was also studied. Solutions were shaken in an orbital
stability. Therefore, a significant number of patients are in distress shaking bath (VWR) at 25 C and 100 rpm for 7 days to reach
and ophthalmologists are forced to seek alternative options such equilibrium. Afterward, an aliquot was centrifuged for 20 min at
as antifungal eye drops prepared at hospital pharmacy 16625G (SIGMA 2-16P) and 1 mL of the supernatant was diluted
departments.11 A common practice consists of using commercial 100 times. Econazole nitrate concentration was determined for
drugs for parenteral administration for the ophthalmic formula- each sample using a diode-array spectrophotometer (Hewlett
tions' preparation by simply dissolving or diluting them in phys- Packard 8452A, l ¼ 220 nm). Econazole nitrate solubility final
iological buffers. However, the poor aqueous solubility of values were shown as mean of 3 replicate measurements. Phase
econazole nitrate requires complementary preparation methods to solubility diagrams were obtained by plotting mean solubility
reach an optimum solubilization into the formulation, such as the against cyclodextrin concentration. Apparent stability constant,
complexation with the oligosaccharides derivatives cyclodex- assuming cyclodextrin inclusion complex formation with a 1:1
trins.12-14 stoichiometry (K1:1), was calculated from the slope, and drug sol-
In addition, one of the main challenges in ocular therapeutic ubility (S0) or the intercept (S0,extrap) from linear regions was
design is the formulation of ophthalmic solutions that increase obtained by least squares regression using the following equation:
drug residence time in the ocular surface as well as allowing
controlled drug release to increase bioavailability and effect dura- Slope
K1:1 ¼ (1)
tion.15 Hence, some ocular polymeric formulations, such as S0 ð1  SlopeÞ
hydrogels, are developed to improve the viscosity and mucoadhe-
Complexation efficiency (CE) parameter was produced by:
sion of eye drops. To overcome these limitations, in situ gelling
systems based on smart polymers were studied in previous works D
CD Slope
due to their capability to recognize changes in the environmental CE ¼ S0 $ K1:1 ¼ ¼ (2)
½CD 1  Slope
conditions, modifying their conformation as a result of the stimu-
lation.16-19 In addition, D:CD relation was calculated by using the CE
In this work, econazole nitrate was solubilized with cyclodex- obtained according to:
trins and incorporated into ophthalmic hydrogels, being a better  
alternative to current pharmaceutical formulations prepared at 1
D : CDrelation ¼ 1 : 1þ (3)
hospital pharmacy departments. In vitro (drug release, antifungal CE
effectiveness) and ex vivo (transcorneal permeation and hen's egg
test chorionallantoic membrane [HET-CAM]) studies were devel-
oped for validating this approach. In addition, novel in vivo studies
1
using positron emission tomography (PET) were performed to H-NMR and Molecular Modeling Studies. The inclusion complex
determine the ocular biopermanence of the econazole ophthalmic showing best econazole nitrate solubilization characteristics was
hydrogels prepared. Two different polysaccharide hydrogels were studied by carrying out 1H-NMR and molecular modeling studies.
selected to formulate econazole: an in situ ion-sensitive hydrogel Mono and bidimensional 1H-NMR spectra of econazole, aCD, and
(ISH)20 and a mucoadhesive hydrogel based on hyaluronic acid,21 the inclusion complex formed were obtained in a Bruker DRX 500
both previously developed by our group. (Karlsruhe, Germany) spectrometer at 500.13 MHz. Rotating-frame
overhauser effect spectroscopy spectra were obtained in the same
Experimental Section spectrometer. All the samples were prepared in D2O solutions.
Molecular modeling was performed by using Avogadro 20.8.0
Materials software. Econazole molecular geometry was generated by an
energy-minimization subroutine (maximum no. interactions 500;
Econazole nitrate and hyaluronic acid were obtained from minimizer cut off 0.01). aCD molecular geometry was obtained by
Acofarma®, Spain; 2-hydroxypropyl-b-cyclodextrin (HPbCD, Klep- imputing X-ray diffraction monocrystal data. Complex molecular
tose HPBCD® with a 0.65 molar substitution ratio and MW 1399 Da) geometry was based on the previous 1H-NMR results and generated
was purchased from Roquette Laisa S.A. (Valencia, Spain); using manual docking and, subsequently, by the same energy-
2-hydroxypropyl-g-cyclodextrin (HPgCD, 0.6 molar substitution minimization subroutine.
1344 V. Díaz-Tom
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In Vitro and Ex Vivo Assays

In Vitro Release of Econazole From Ophthalmic Hydrogels.
EconazoleeaCD in vitro release from the hydrogel systems (ISH and
HAH) and the econazole control solution (ECN) in contact with
simulated lacrimal fluid (SLF) was estimated by using Franz diffu-
sion cells and GVS 0.22-mm cellulose acetate membranes (mem-
brane surface area of 0.784 cm2). Five hundred microliters of each
formulation containing 1 mg of econazole were placed in the upper
compartment, adding 175 mL of SLF prepared as detailed by Ceu-
lemans et al.24 Sink conditions were maintained in the receptor
compartment, which was filled with 6 mL of SLF. During the
experiment, cell compartments were continuously homogenized
by magnetic stirring at 200 rpm in a thermostated bath at 36 C.
Serial sampling was performed at different times. The Hewlett
Packard 8452A diode-array spectrophotometer (l ¼ 220 nm) was
used for econazole content determination. Each experiment was
repeated 3 times. Release data were fitted to Higuchi diffusion
kinetics25 and Peppas-Korsmeyer26 equation using GraphPad Prism
6.0 software (GraphPad Software Inc.). Drug release was compared
among formulations by using a 1-way ANOVA with GraphPad Prism
6.0 software at different times.
Ex Vivo Corneal Permeability Studies. For this purpose,27 bovine
eyes were removed within the first hour of animal death and
transported following the BCOP test protocol. Once received, cor-
neas were isolated with 2-4 mm of surrounding sclera, washed with
an isotonic saline solution, and mounted on Franz diffusion cells. The
cornea was placed on the receptor chamber and the donor one was
attached. The receptor chamber was filled with 6 mL of SLF and the
donor one with 500 mL of each formulation containing 1 mg of
econazole (area available for permeation: 0.785 cm2) and 175 mL of
SLF. During the experiment, both compartments were continuously
homogenized as previously reported (magnetic stirring at 200 rpm
in a thermostated bath at 36 C) and serial sampling was performed
Figure 1. Phase solubility diagrams for econazole obtained with 3 cyclodextrin de-
rivatives at 25 C in water (a) and in PBS (b) (mean ± SD, n ¼ 6). The dotted lines at different times. Each experiment was repeated 3 times.
indicate the minimum CD concentration required to solubilize the required dose of the Econazole nitrate determination was performed by a HPLC
drug in hydrogels. system (Merck® Hitachi, Darmstadt, Germany), consisted of UV-VIS
photodiode array detector model L-4500 (Merck® Hitachi), a sol-
vent delivery pump system model L6200 A (Merck® Hitachi) and an
Study of the Econazol-aCD Complex Incorporated Into Ophthalmic auto injector fitted with 100-mL loop model AS4000 A (Merck®
Hydrogels Hitachi). D-7000 HMS 4.0 software (Merck® Hitachi) was used for
First, a 2 mg/mL econazole nitrate solution with 15% (w/v) aCD data processing. Analyses were performed by an isocratic method.
(ECN) was prepared in warm distilled water (40 C) under magnetic The column used was a C18 (3.9  150 mm, 5 mm of particle size
stirring for an hour. Subsequently, hydrogels were prepared by Symmetry® Waters) keeping its temperature at 25 C by using a
dispersing polysaccharides in warm distilled water (55 C), stirring thermostat model L-5025 (Merck® Hitachi). The mobile phase was
for 24 h and filtering for sterilization in a horizontal laminar flow methanol-water (85:15 v/v), using a flow rate of 1 mL min1. The
hood. The ISH was prepared by using a 0.82% (w/v) deacylated sample injected volume was 50 mL and the retention time was
gellan gum and k-carrageenan mixture in a 4:1 ratio. A volume of 4.5 min. A 320 nm wavelength was employed for the econazole
0.4% (w/v) hyaluronic acid was added to obtain a mucoadhesive quantification.
hyaluronic acid hydrogel (HAH). Polymer concentrations and ratios Apparent corneal permeability (Papp) was calculated according
were selected based on previous works.20,21 to the equation:

Table 1
Values for K1:1, CE, and the D:CD Relation Obtained From the Econazole-Cyclodextrin Complex in Water and PBS at 25 C

Econazole-CD Complex S0 (mM) S0,extrap (mM) K1:1a (M1) K1:1 extrap

b
(M1) CE D:CD (mol:mol) R2

Water
HPbCD 1.48 1.96 52.21 39.51 0.077 1:13.91 0.9187
HPgCD 1.48 2.22 54.11 36.18 0.080 1:13.46 0.8877
aCD 1.48 0.79 505.54 948.32 0.750 1:2.33 0.9936
PBS
HPbCD 0.59 1.2 86.34 42.86 0.051 1:20.5 0.7981
HPgCD 0.59 0.59 40.35 40.75 0.0245 1:42.8 0.8807
aCD 0.59 0.74 61.28 49.44 0.036 1:28.5 0.8942
a
K1:1 calculated using S0 (free drug solubility).
b
K1:1 calculated using S0,extrap (free drug solubility calculated from the phase solubility diagram).
 V. Díaz-Tom
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dQ =dt Nonparametric Kruskal-Wallis test was used to compare the

Papp ¼ econazole corneal permeability from formulations using GraphPad
A$C0
Prism 6.0 software.
where dQ/dt represents the econazole flux across corneal tissues in Irritation Ocular Assay (HET-CAM). The HET-CAM assay was
the linear portion of the representation, A the corneal surface performed to evaluate possible acute ocular irritation caused by the
(in this study 0.785 cm2) and C0 the initial drug concentration at the econazole solubilized with aCD, as described in a previous work.28 A
epithelial side. Tlag was calculated extrapolating the linear portion volume of 0.3 mL of econazole-aCD formulations (ECN, ISH, and HAH)
of the graph to the x axis. were deposited onto the egg chorioallantoic membrane (3 eggs per

Figure 2. 1H-NMR spectrum of econazole (top) and aCD (bottom) indicating the assignment of protons to different signals 1H-NMR.
1346 V. Díaz-Tom
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compound). Blood vessels were observed for 300 s with a stereo- increase in solubility with the cyclodextrins concentration, also
microscope (Olympus SZ-STN), aiming at detecting episodes of showing a constant stoichiometry of the complexes. Apparent
bleeding, coagulation, and partial lysis. The compounds' irritation stability constants (K1:1), CE, and econazole-cyclodextrin relations
score was determined as described in the INVITTOX 96 Protocol.29 with different cyclodextrins are shown in Table 1.
Antifungal Effectiveness. C albicans ATCC 90028, Aspergillus The K1:1 highest value (K1:1 ¼ 505.54) was found for aCD, indi-
fumigatus KM8001, and Paecilomyces, isolated from culture collec- cating econazole interacts more strongly with this cyclodextrin.
tion of the University Clinical Hospital of Santiago de Compostela aCD also showed the best solubilization properties for the econa-
microbiology department, were inoculated onto Mueller Hinton zole and forms the most stable complex presenting relatively high
agar and RPMI culture medium.30 Kirby-Bauer disk diffusion sus- CE (0.75) and small D:CD (1:2.33) relation.
ceptibility method was used to evaluate antifungal effectiveness. Despite eye drops were elaborated in water, the econazole-
Filter paper disks, impregnated with 20 mL of econazole, natamycin, cyclodextrins phase solubility diagram was also made in PBS to
amphotericin b, voriconazole, and fluconazole, were placed into the evaluate the effect of pH and salt concentration in the econazole
Petri dish (previously seeded) and incubated at 37 C for 24 h with C solubility once the eye drops are administered. The PBS graph
albicans and for 48 h with A fumigatus and Paecilomyces. Digital showed a considerable decrease in the econazole affinity for aCD
pictures of the discs were taken using a Leica CLS-150 stereomi- when it was dissolved in PBS (Fig. 1b). HPbCD and HPgCD were less
croscope connected to a Nikon digital camera and the econazole affected by the pH.
inhibition zone was measured and compared with other 1
H-NMR was carried out to characterize the econazole-aCD
antifungals. complex. 1D-RMN econazole and aCD spectra (with the corre-
spondent proton assignation for each signal) are shown in Figure 2.
In Vivo Assays: PET Measures of Econazole Ophthalmic Hydrogels. Chemical displacement changes were observed in the inclusion
PET studies were carried out on male Sprague-Dawley rats (500 g of complex spectra (Fig. 3), for both drug and cyclodextrin signals due
average weight), supplied by the animal facility at the University of
Santiago Compostela. Animals were kept in individual cages with
free access to food and water under controlled temperature
(22 ± 1 C) and humidity (60 ± 5%), with day-night cycles regulated
by artificial light (12/12 h) for, at least, 1 week before experiments.
Animals were treated according to the laboratory animals' guide-
lines.31 Experiments were approved by the Galician Network
Committee for Ethics Research following the Spanish and EU rules
(86/609/CEE, 2003/65/CE, 2010/63/EU, RD 1201/2005 and RD
53/2013).
Hydrogel ocular pharmacokinetic studies were performed as
described in previous works.32 Briefly, PET and computed
tomography (CT) images were acquired using the Albira PET/CT
Preclinical Imaging System (Bruker Biospin, Woodbridge, CT).
Anesthetized animals were placed in the small animal bed, and
7.5 mL of ECN or 7.5-mL econazole hydrogels labeled with
18
F-fluorodeoxyglucose were instilled into the conjunctival sac
eye by using a pipette. The administered radioactivity was 0.35
MBq per eye. Single frames of 10 min at 0.5, 1, 2, and 3 h after
instillation were acquired. Three animals (6 eyes) were tested
for each formulation. Different regions of interest were manu-
ally drawn containing the signal on each eye. Regions of interest
were replicated on different frames over time and results were
corrected for radioactive decay. Afterward, graphical represen-
tations of radioactivity versus time were obtained. Fitting of the
remaining formulation versus time to a monoexponential decay
equation using a single compartmental model was made with
pKSolver software.33 Noncompartmental analysis was also per-
formed, calculating the mean residence time (MRT) and the total
area under the curve of the remaining concentration versus
time.

Results

Econazole Nitrate Solubilization With Cyclodextrins, 1H-NMR, and

Molecular Modeling Studies

Evidence of the inclusion-complex formation was obtained from

the solubility curves for the 3 cyclodextrin hosts. In addition, the
most favorable interaction between cyclodextrin and econazole
nitrate was determined as well as the most stable inclusion com-
plex formed. Diagrams obtained in water solutions with all cyclo-
dextrins were AL type22 (Fig. 1a). These diagrams suggest the Figure 3. 2D-ROESY spectrum (details of the more significant area) of the complex
formation of high solubility complexes characterized by a linear econazole-cyclodextrin. ROESY, rotating-frame overhauser effect spectroscopy.
 V. Díaz-Tom
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Figure 4. Molecular model of aCD, econazole (upper image) and inclusion complex (lower image) obtained from NMR results.

to interactions between these molecules in solution. 2D-RMN as- cavity are observed. These results suggest the imidazole group re-
says were executed to elucidate the type of interactions. Specif- mains inside the cavity and the econazole's H2 proton is located in
ically, a 2D-rotating-frame overhauser effect spectroscopy assay the aCD cavity edge. Figure 4 contains the built molecular model,
was developed, which allows obtaining the bidimensional spectra showing how econazole is included into the aCD cavity. Chlorine,
shown in Figure 3. There, it can be seen that the interactions' for- nitrogen, oxygen, carbon, and hydrogen atoms are represented in
mation between protons of the econazole imidazolic group and green, blue, red, gray, and pale gray, respectively. Nitrogen atoms,
aCD's H5 and H3 protons (which were the ones that face the inside which are part of the econazole imidazolic ring, are located into
of the cavity). Moreover, interactions between econazole's H2 and cyclodextrin's cavity, whereas Cl atoms, which are fixed to phenyl
cyclodextrine's H2 and H4 protons located in the narrow edge of groups, remain in the external area.

Study of the Econazol-aCD Complex Incorporated Into Ophthalmic

Hydrogels

Considering the results aforementioned, aCD was selected as

the best econazole nitrate solubilizing agent and the inclusion

Table 2
Fitting of the Release Data From the Hydrogels in the In Vitro Release of Econazole to
the Diffusion Kinetics of Higuchi and Peppas and Korsmeyer

Variable Higuchi Peppas and Korsmeyer

k (mg$cm2 min0.5) R2 k (mg$cm-2 min-n) n R2

ISH 9.45 0.8624 4.58 1.05 0.9685

HAH 19.71 0.9477 29.71 0.63 0.9615
Figure 5. Release profiles of econazole in SLF from the ISH, mucoadhesive HAH, and
ECN 29.20 0.8624 77.71 0.35 0.8983
econazole control solution (ECN; mean ± SEM, n ¼ 3).
1348 V. Díaz-Tom
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Table 3
Parameters Obtained in the Ex Vivo Transcorneal Permeation Assay

Formulation Tlag (min) Flux (mg/min) (mean ± SE) Papp  106 (cm/s) % Permeation at 5 h

HAH 65 0.084 ± 0.0093 2.52 ± 0.27 0.79

ISH 100 0.083 ± 0.0044 2.50 ± 0.13 0.35
ECN 60 0.089 ± 0.0060 2.67 ± 0.18 0.63

complex was added to the ISH and HAH hydrogels. Although aCD being much bigger of those created by fluconazole, amphotericin,
minimum concentrations necessary to solubilize 2 mg/mL econa- and natamycin.
zole nitrate in accordance with solubility diagrams results were
1.2% in water and 10.5% in PBS, the cyclodextrin concentration
selected was 15% to avoid possible drug precipitation problems in Biopermanence PET Study
environments with high ion concentration such in tears. The pH The biopermanence of radiolabeled econazole hydrogels was
values for ISH, HAH, and ECN were 4.86, 5.16, and 4.52, measured in rats by using a small-animal PET system and
respectively. compared with a radiolabeled solution. A strong signal at early
times after instillation was observed for both formulations. It was
shown after 2 h of contact, 21.71 ± 10.23% of the ISH, 20.17 ±
Econazole Release Study
10.11% of HAH, and only 10.15 ± 6.34% of ECN remained in the
Figure 5 shows both hydrogels have great capacity to control
ocular surface. Data were fitted to a monoexponential model
the econazole release during the 24-h study. Peppas and Kors-
with time, as it is shown in Figure 6. The pharmacokinetic pa-
meyer kinetics fitting with n ~ 0.5 and Higuchi kinetics best
rameters obtained by fitting to the mono compartmental model
fitting of HAH suggest the drug release is produced by a diffusion
are shown in Table 5. The average half-life time (t1/2) and MRT
controlled mechanism through the polymeric network. In the ISH
were 49.3 ± 28.88 and 71.08 ± 41.66 min respectively for the
case, the n value of Peppas and Korsmeyer equation (n ¼ 1.053)
HAH, 50.16 ± 18.64 and 72.37 ± 26.90 min for the ISH and
suggests the release kinetics are close to a pseudo-zero order
20.73 ± 8.01 and 29.9 ± 11.56 min for the ECN. The hydrogels
process (Table 2). This value indicates the ISH gelation in contact
labeling was considered as optimum because the remaining
with the SLF may have an important role in the drug release. The 18
F-fluorodeoxyglucose in both hydrogels was above 95% at
2-way variance analysis, including correlations for time point
180 min after preparation.
and formulation, shows a significant influence of both factors for
Figure 7 contains axial and sagittal views of co-registered
a < 0.01. The analysis shows differences between the control
PET/CT animal head images right after the HAH hydrogel admin-
solution and the hydrogels after 10 min, and between both
istration and 1 h after instillation. CT images show the head
hydrogels after 1 h.
structure, whereas PET images show the labeled hydrogel distri-
bution. Initially, all the hydrogel is located on the ocular surface.
Ex Vivo Transcorneal Permeability
After 1 h, the amount of hydrogel on the eye is still significant,
The results from the corneal permeability study for the drug
indicating a high retention time on the ocular surface. In addition,
solution and the bioadhesive hydrogels are shown in Table 3. Eco-
the radiotracer is also detected in the nasolacrimal duct and in the
nazole nitrate shows a good permeability across the bovine cornea
nasal cavity due to the hydrogel partial clearance from the lacrimal
and the formulations do not affect the drug corneal flux or the
sac into the nasal cavity.
apparent permeability parameter (Nonparametric Kruskal Wallis a
n.s.). However, there are differences (a < 0.05) in terms of lag time
comparing ISH results with the ones obtained from ECN and the Discussion
HAH.
Topical antifungal ophthalmic formulations are not available in
HET-CAM Assay several countries. Therefore, alternatives are elaborated at hospital
Results show no damage on blood vessels after the addition of pharmacy departments, using isotonic solutions compatible with
econazole hydrogels and econazole solution after 5 min of contact the ocular surface as the main vehicle for their elaboration. These
(irritation score ¼ 0). Therefore, it seems that these compounds are manufactured solutions present many limitations, mainly related to
nonirritating for the ocular surface. their low retention time on the ocular surface.34 In addition, the
low water solubility of these drugs prevents their formulation,
Antifungal Effectiveness limiting the therapeutic arsenal in pathologies with reduced
Results obtained from the diameters (mm) of fungal growth pharmacological alternatives as FK. For this reason, achieving the
inhibition zones by each antifungal are shown in Table 4. Both solubilization of antifungal drugs is an essential task to increase the
econazole and voriconazole produce the largest inhibition zones, odds for a successful treatment.35 Many authors have performed

Table 4
Zone Diameters (mm) of Fungal Growth Inhibition by Antifungals

Antifungal C albicans A fumigatus Paecilomyces

RPMI Mueller Hinton RPMI Mueller Hinton RPMI Mueller Hinton

Econazole-aCD 36 40 >50 40 >50 >50

Fluconazole 34 39 e e e e
Amphotericine B 15 15 e e e e
Voriconazole-SBECD 55 65 >50 >50 >50 >50
Natamycin 15 13 25 15 e e
 V. Díaz-Tom
e et al. / Journal of Pharmaceutical Sciences 107 (2018) 1342-1351 1349

To determine the cause of the stronger interaction with the aCD,

1
H-NMR structural studies were performed. 1D and 2D NMR results
suggest that the econazole imidazole ring is more deeply included
into the cyclodextrin cavity and interactions between them are
stronger. The PBS graph shows a considerable decrease in the
econazole affinity for aCD. This may be due to the amino group
ionization of its imidazole ring at pH 7.4, which decreases the drug
affinity for the cyclodextrin cavity. In the case of HPbCD and HPgCD,
owing to their bigger cavity size, they would probably include the
clorophenylmethoxil or the diclorophenylethyl group in the cavity,
which are less influenced by the econazole ionization; and there-
fore, they are less affected by pH.
The importance of increasing the therapeutic arsenal in
ophthalmology is a key factor for the optimization of the FK
therapeutics. In this work, the econazole-aCD inclusion complex
effectiveness against the main fungi which caused FK was studied.
Compared with the antifungal tested, only econazole-aCD and
voriconazole solubilized with sulphobutylether-b-cyclodextrin
(Vfend®) showed activity against all the studied fungi. Therefore,
Figure 6. Clearance rate of the formulations from the ocular surface determined by the econazole-aCD complex is a very attractive therapeutic alter-
PET.
native for the treatment of these infectious diseases because of its
advantages described above. Moreover, the fact that the com-
pound is not irritating for the ocular surface is also important. The
solubility studies using cyclodextrins and co-solvents due to eco- econazole-aCD inclusion complex does not show any type of
nazole low water solubility (1.48 mM).36-39 damage in the vessels, assuming the tested formulations would
The diagrams obtained with HPgCD, HPbCD, and aCD in water not cause irritant effects in vivo, but additional assays would be
were of the AL type.22 These diagrams show the formation of high necessary.
solubility complexes characterized by a linear increase in solubility In vitro release studies using vertical Franz cells show both ISH
with the cyclodextrins concentration. In addition, complexes pre- and HAH can successfully control the econazole nitrate release
sented a constant composition. This behavior agrees with the re- over time. ECN release the drug for 250 min, where the high
sults reported by Mura et al.,40 whereas El-Gawad et al.41 obtained molecular volume of the inclusion complex could decrease the
Bs type diagrams. Bs type diagrams are typical of natural b-cyclo- drug diffusion rate through the membrane filter. The econazole
dextrins, the least soluble cyclodextrins used, showing inclusion nitrate proportion released from ISH is significantly lower than the
complexes with limited solubility. Nevertheless, HPbCD inclusion one obtained from HAH. This is due to the hydrogel's ion-sensitive
complexes usually present AL- or Ap-type diagrams. HPbCD is an characteristics, and it gels in contact with the lacrimal fluid.42,43
amorphous cyclodextrin composed by a mixture of cyclodextrin This causes the econazole-cyclodextrin complex to be trapped
molecules with different mode and substitution degree. This into the hydrogel and so the release to be slower than the HAH
structure prevents the inter- and intra-molecular hydrogen bounds network.
formation among inclusion complexes and cyclodextrins. The inter- It is important to point out that the biopermanence study of
and intra-molecular hydrogen bounds are an important factor ophthalmic formulations is an essential requirement to be able to
involved in the aggregates formation which is responsible of the determine their appropriate dosages.44 In this work, a novel in vivo
natural b-cyclodextrin's limited water-solubility. The highest values image methodology based on PET/CT for evaluating the hydrogels
of K1:1 (K1:1 ¼ 505.54 M1) and CE (0.75) as well as the smallest ocular surface residence time was used.32 PET/CT images confirm
ratio of D:CD (1:2.33) relation were found for aCD, indicating eco- the hydrogels high retention time compared with the econazole
nazole interacts more strongly with this cyclodextrin. These results solution used as control. Furthermore, PET studies allow obtaining
are in good agreement with Mura et al.40 where authors also the quantitative analysis of the ophthalmic formulations pharma-
reported the best results for aCD and econazole base. In this work, a cokinetic profile, by calculating the elimination constant, the half-
stability constant (K1:1) value of 2630 M1 was obtained. This live and the zero- and first-order pharmacokinetic parameters
higher stability constant is related to the econazole base low water AUC∞ 0 and MRT. Hydrogels show a MRT of 71-72 min in the ocular

solubility (0.013 mM) compared with econazole nitrate (1.48 mM). surface, much more than the ECN. Results from the ocular
Nevertheless, using econazole nitrate in combination with aCD biopermanence assay show both hydrogels are mucoadhesive
allows achieving higher drug concentration than using the base. compounds and have an adequate consistence to remain on the
Similar results were obtained by Mura et al.40 using different ocular surface for a long time.
organic acids in combination with cyclodextrins. The combination of the good results from the release study and
the high retention time of the hydrogels in the ocular surface
confirm the interest on these formulations for the controlled eco-
nazole release in the eye. These 2 properties of econazole hydrogels
might lead to an increase in the effect duration, decreasing the
Table 5
administration frequency. In most of the occasions, the fungal
Parameters Obtained by the Fitting of the % Formulation Remaining on the Ocular
Surface Obtained by PET Imaging to a Mono-Compartmental Model
infection reaches the aqueous humor and eye internal structures;
therefore, the drug needs to be able to cross the cornea. Hence, to
Formulation t1/2 (min) AUC∞
0 (mg/L$min) MRT (min)
verify that hydrogels were able to allow the econazole nitrate
HAH 49.3 ± 28.88 6689.02 ± 6689.52 71.08 ± 41.66 penetration into the cornea, an ex vivo transcorneal study was
ISH 50.16 ± 18.64 6812.83 ± 2426.91 72.37 ± 26.90 performed. Results show that econazole have equivalent apparent
ECN 20.73 ± 8.01 2920.13 ± 1078.09 29.9 ± 11.56
corneal permeability from both inclusion complex solution
1350 V. Díaz-Tom
e et al. / Journal of Pharmaceutical Sciences 107 (2018) 1342-1351

Figure 7. Axial (left) and sagittal (right) fusion PET-TAC images of the head of the rat. (a) Econazole HAH 10 min after administration. (b) Econazole HAH 1 h after administration.

and hydrogels. Despite the release study shows differences Acknowledgments

among formulations, transcorneal study results suggest the drug
permeation rate-limiting step in the eye is their own diffusion A.F.-F. acknowledges the support of Instituto de Salud Carlos III
across the corneal structure, and it is independent of the hydrogels (Rio Hortega research grant CM15/00188). A.L-R acknowledges the
drug release properties. financial support of the Xunta de Galicia and de European Union
The development of the proposed formulations at hospital (European Social FunddESF). This work was partially supported by
pharmacy departments is feasible when commercial alternatives grants from Fundacio
n Espan ~ ola de Farmacia Hospitalaria and
are not available or ineffective. Results obtained through this work Fundacio
n Mutua Madrilen ~ a.
demonstrate both HAH and ISH are promising as vehicles for
econazole nitrate-aCD ocular administration. Both hydrogels
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