International Journal of Pharmaceutics 473 (2014) 148–157

Contents lists available at ScienceDirect

International Journal of Pharmaceutics

journal homepage: www.elsevier.com/locate/ijpharm

New amphotericin B-gamma cyclodextrin formulation for topical use

with synergistic activity against diverse fungal species and Leishmania
spp.
Helga K. Ruiz a , Dolores R. Serrano a , María Auxiliadora Dea-Ayuela b ,
Pablo E. Bilbao-Ramos c , Francisco Bolás-Fernández c , Juan J. Torrado a , Gloria Molero d, *
a
Departamento de Farmacia y Tecnología Farmacéutica, Facultad de Farmacia, Universidad Complutense de Madrid, Plaza Ramón y Cajal S/N, Madrid 28040,
Spain
b
Departamento de Farmacia, Facultad de Ciencias de la Salud, Universidad Cardenal Herrera-CEU, Moncada, Valencia 46113, Spain
c
Departamento de Parasitología, Facultad de Farmacia, Universidad Complutense de Madrid, Plaza Ramón y Cajal S/N, Madrid 28040, Spain
d
Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, Plaza Ramón y Cajal S/N, Madrid 28040, Spain

A R T I C L E I N F O A B S T R A C T

Article history: Amphotericin B (AmB) has a broad antifungal and leishmanicidal activity with low incidence of clinical
Received 30 March 2014 resistance. Its parenteral administration has high risk of nephrotoxiciy that limits its use. In order to treat
Received in revised form 29 June 2014 cutaneous infections, AmB topical administration is a safer therapy because of the low systemic
Accepted 1 July 2014
absorption of the drug across mucous membranes. Moreover, in some developing countries both fungal
Available online 3 July 2014
topical infections and cutaneous leishmaniosis are an important health problem. The aim of this work is
to formulate a topical amphotericin preparation and test its in vitro antifungal (against 11 different fungal
Keywords:
species) and antileishmanial activity. g-Cyclodextrin (g-CD) was chosen to solubilise AmB. Furthermore,
Amphotericin B
Cyclodextrin
g-CD has shown a synergistic effect on membrane destabilization with AmB. Topical novel formulations
Topical formulations based on AmB–CD complex have exhibited greater antifungal activity (48%, 28% and 60% higher) when
Antifungal activity compared to AmB Neo-Sensitabs1 disks, AmB dissolved in dimethyl sulfoxide (DMSO) and
Antileishmanial efficacy Clotrimazole1 cream, respectively. Furthermore, AmB–CD methyl cellulose gel has shown significantly
Biofilms higher inhibition activity on biofilm formation, larger penetration through yeast biofilms and higher
fungicidal activity on biofilm cells compared to AmB dissolved in DMSO. In addition, AmB–CD gel
exhibited both high in vitro leishmanicidal efficacy with wider therapeutic index (between 2 and 8-fold
higher than AmB deoxycholate depending on Leishmania spp.) and also in vivo activity in an experimental
model of cutaneous leishmaniasis. These results illustrate the feasibility of a topical AmB formulation
easy to prepare, physicochemically stable over 6 months, safe and effective against diverse fungal and
parasitic cutaneous infections.
ã 2014 Elsevier B.V. All rights reserved.

1. Introduction is also an important health problem, being its initial symptoms

Parenteral AmB administration is one of the drugs of choice
against life-threatening systemic fungal infections and some
parasitic diseases (Torrado et al., 2008). AmB has a wide broad
antifungal spectrum, being active against the most frequent fungus
producing systemic and cutaneous mycoses including Candida,
Cryptococcus, Aspergillus, Coccidiodis inmitis, Histoplasma capsu-
latum and Blastomyces dermatitidis (Fernández et al., 2005).
Moreover, in some countries of America cutaneous leishmaniasis
* Corresponding author. Tel.: +34 913941620.
E-mail address: gloros@ucm.es (G. Molero).
very similar to some cutaneous mycosis. Thus, a topical AmB
formulation able to treat both fungal or Leishmania cutaneous
infections might be an interesting solution. In order to prepare
topical AmB formulations, the low solubility of AmB (<1 mg L
1)
can limit the activity of the formulation. It is known that drug–CD
inclusion complexes can substantially increase the bioavailability
of poorly soluble drugs (Angelova et al., 1999a,b). According to
several authors, AmB solubility can be enhanced by g-CD due to its
high aqueous solubility and biggest cavity compared to other
parental CDs (Espada et al., 2008b; Jansook et al., 2010; Jansook,
2009; Kajtár et al., 1989; Kim et al., 2010; Rajagopalan et al., 1986;
Serrano et al., 2012; Szejtli, 1998). CDs are solubilizers and
stabilizers agents approved for topical applications that can act as

http://dx.doi.org/10.1016/j.ijpharm.2014.07.004
0378-5173/ ã 2014 Elsevier B.V. All rights reserved.
 H.K. Ruiz et al. / International Journal of Pharmaceutics 473 (2014) 148–157
drug permeation enhancers, improving topical penetration
(Cal and Centkowska, 2008). Moreover, CDs possess mucoadhesive
properties increasing their contact with the target tissue (Francois
et al., 2003).
The aim of this work was to obtain and characterize AmB–CD
inclusion complexes and to assess their antifungal and antileish-
manial efficacy as well as their cytotoxicity. AmB–CD complexes
were formulated with several excipients to obtain topical
formulations as gels and creams, adapted to the clinical need of
the patients.
2. Materials and methods
2.1. Materials
AmB was supplied by Azelis (Barcelona, Spain). Clotrimazole1 1%
w/w cream was purchased from Bayhealth (Madrid, Spain). g-CD
was obtained from Cavamax1 W8 produced by Wacker Chemie AG
(Germany) and distributed by ISP (USA). Neo-Sensitabs1 containing
AmB (10 mg) and clotrimazole (10 mg) were supplied by Rosco
Diagnostica A/S (Taastrup, Denmark). The culture media were
purchased from Panreac, Spain (Müeller Hinton Agar) and Difco,
Spain (Yeast Nitrogen Base (YNB) medium, RPMI 1640 medium,
Saboraud Dextrose Agar and Yeast-Extract Dextrose (YED) medium).
All other reagents and chemicals used without further purification
were obtained either from Panreac S.A. (Barcelona, Spain) or Sigma–
Aldrich Chemical Co. (Madrid, Spain).
2.2. Preparation and characterization of the AmB–CD inclusion
complex
2.2.1. AmB–CD inclusion complex preparation
The AmB–CD complex (0.125–12.5%, w/v respectively) was
prepared by dissolving the CD (12.5 g) in 50 mL of deionized water.
Once dissolved, sodium hydroxide 2 N was added to increase the pH
to 12.0 and AmB (0.125 g) was incorporated. As soon as AmB was
dissolved, orthophosphoric acid 2 N was added to adjust pH to 5.5.
Finally, deionized water was added up to 100 mL. AmB–CD
formulation was freeze-dried using a Lyomega L-7694 (Telstar,
Tarrasa, Spain). Freezing of the samples was achieved by decreasing
the temperature for 2 h to
60  C. In the first step of the freeze-drying
process, the shelf temperature was maintained at
30  C for 48 h and
the chamber pressure was kept at high vacuum which was followed
by an additional drying step of 6 h at 5  C.
2.2.2. Solubility studies
Solubility studies were performed according to the method
described by Higuchi and Connors (Higuchi and Connors, 1965)
using different CD concentrations with AmB at saturation. Samples
were prepared as described above and then were shaked, filtered
through 0.45 mm, diluted to 12 mg mL
1 and finally assayed by
spectrophotometry at 364 nm (Shimadzu 1700-UV spectropho-
tometer). Stability constant (Kc) of AmB–CD complex was
calculated according to the following equation (Higuchi and
Connors, 1965):
slope
Kc ¼ ; (1)
Soð1
slopeÞ
where Kc is the apparent stability constant (M
1), So is the AmB
solubility in the absence of CD and slope is the slope of the straight
line of the phase solubility diagram calculated using linear
regression.
Apart from Kc, the solubilizing efficiency of the cyclodextrins
known as complexation efficiency (CE) was also determined by
either the slope of the phase solubility profile or the complex to
149
free cyclodextrin concentration ratio using the following equation
(Loftsson and Brewster, 2012; Loftsson et al., 2005):
½D=CD slope
CE ¼ ¼ (2)
½CD ð1
slopeÞ
where [D/CD] is the concentration of dissolved complex, [CD] is the
concentration of dissolved free cyclodextrin and slope is the slope
of the phase solubility profile.
2.2.3. Aggregation state and particle size
AmB–CD complex was diluted as necessary with deionized
water. The resulting dilutions were scanned between 300 and
450 nm (Sánchez-Brunete et al., 2004) and particle size was
assayed by dynamic light scattering (Zetatrac Ultra, Microtrac Inc.).
2.2.4. FT-IR
FT-IR of the lyophilized AmB–CD complex was performed by
using a PerkinElmer Spectrum 100 FT-IR spectrometer (4000–
450 cm
1).
2.3. Preparation and characterization of the topical AmB formulations
2.3.1. AmB–CD gel
Gels were prepared with four different thickening agents
derived either from cellulose (methyl cellulose 1500 and
hydroxypropyl cellulose type H) or alkyl acrylate crosspolymers
(Carbopol1 ETD 2020 and Carbopol1 Ultrez 20). Once the AmB–CD
complex was obtained, the thickening agent was added (at 2% w/w
methyl cellulose, 3% w/w hydroxypropyl cellulose or at 1% w/w
alkyl acrylate crosspolymers). Cellulose-derived gels were left
unstirred over 24 h and then were stirred until gel was completely
homogeneous. Alkyl acrylate crosspolymers were dispersed on the
AmB–CD solution and then the viscosity was increased by adding
triethanolamine until pH 5.5.
2.3.2. AmB–CD cream
The AmB–CD complex was prepared by dissolving the CD
(12.5 g) and sodium chloride (2 g) in 50 mL of deionized water.
Once dissolved, sodium hydroxide 2 N was added to increase the
pH to 12.0 and AmB (0.125 g) was incorporated. As soon as AmB
was dissolved, orthophosphoric acid 2 N was added to adjust the
pH to 5.5. Deionized water was added up to 70 mL. Finally, 30 mL of
a mixture of silicones (Dow Corning1 3225C:Cyclomethicone
pentamer, 1:1) were slowly added while stirring with a homoge-
nizer Ultra-Turrax, IKA1 to obtain the cream.
2.3.3. Aggregation state and pH
Aggregation state was characterized as above described. The pH
of the formulations was determined by pH-meter paper strips
(range pH 4.5–10).
2.3.4. Disk diffusion halo assay
Antifungal activity was tested by agar diffusion assay according
to the European Pharmacopoeia (2009) and Fittler et al. (2010)
against Candida albicans CECT 1394, which was kindly provided by
Dr. Pérez (CAQYM, University of Alcala de Henares, Spain) in 2009.
Prior to the beginning of the study, yeast isolates were cultured on
Sabouraud dextrose agar at 30  C for 72 h to ensure viability and
absence of contamination. Antifungal sensitivity was assayed in
Müeller Hinton agar (MHA) medium supplemented with glucose
(2% w/v) and methylene blue (0.5 mg mL
1). The MHA was melted
at 50  C and then, 3 mL of yeast suspension, prepared in sterile
saline (NaCl 0.9%) and adjusted to 0.1 Abs at 600 nm, were
inoculated. AmB reference was dissolved in DMSO at the
concentrations of 15.4, 38.4, 96, 240 and 600 mg mL
1. AmB
150 H.K. Ruiz et al. / International Journal of Pharmaceutics 473 (2014) 148–157
deoxycholate prepared as previously described (Espada et al.,
2008b), was used as reference formulation too. AmB deoxycholate
and AmB–CD formulations (gels and cream) were diluted firstly in
water and then in phosphate buffer 0.2 M (pH 10.5) to a final AmB
concentration of 96 mg mL
1. Paper disks were embedded with
20 mL of each AmB formulation. Once the disks were dried, they
were placed onto inoculated agar plates. The plates were kept at
5  C for 2 h followed by 48 h at 30  C and then the diameter of the
growth inhibition halos was measured (Pujol et al., 2008).
2.3.5. Viscosity
Six methyl cellulose gels with increasing thickening agent
concentrations (1.5, 2.0, 2.5, 3.0, 3.5, 4% w/w) were prepared and
antifungal activity was assessed as described above. Viscosity
measurements were performed in a Brookfield Model DV III at
25  C using cone 41 and at speed rate from 5 to 25 rpm.
2.3.6. Long-term stability studies
Stability studies (AmB content, aggregation state, pH and
physical appearance) were conducted on methyl cellulose gel
formulations at room temperature (25  2  C) in dark conditions
over a period of 6 months. AmB content was analysed by HPLC as
previously described (Espada et al., 2008a).
The gel composed of AmB–CD (0.125:12.5% w/w) and methyl
cellulose (3% w/w) was selected being further tested in the
following experiments.
2.4. Disk-diffusion halo assay
2.4.1. AmB Neo-Sensitabs1 diffusion assay
The antifungal activity of the gel was compared to the AmB Neo-
Sensitabs1 tablets against nine different strains of Candida
(C. albicans CECT 1394, C. dubliniensis 63341, C. glabrata 60661,
C. glabrata 60750, C. guilliermondii 26863, C. krusei 52009, C. krusei
52011, C. krusei 55574 and C. parapsilosis 57744), and Saccharomyces
61978 and Trichosporon sp. 61978. C. albicans was kindly provided
by Dr. Pérez and the rest of the strains were provided by A. Sanchez
Sousa (Servicio de Microbiología Hospital Ramón y Cajal, Madrid,
Spain) in 2009. The experiment was performed as described in
Section 2.3.4.
2.4.2. Antifungal activity diffusion assay compared to AmB dissolved in
DMSO
AmB–CD gel antifungal activity was tested against all the
strains from Section 2.4.1 and compared to the AmB reference
dissolved in DMSO and to the placebo gel formulation (prepared in
the same way but without AmB). The experiment was carried out
as in Section 2.3.4.
2.4.3. Clotrimazole Neo-Sensitabs1 diffusion assay
AmB–CD gel antifungal activity was compared to Clotrimazole1
cream (1% w/w) and clotrimazole Neo-Sensitabs1 tablets against
C. albicans CECT 1394 as in Section 2.3.4, as clotrimazole is one of
the drugs of choice for the treatment of cutaneous mycoses
(Martinez et al., 2012). The disks contained the equivalent to 10 mg
of drug for all the formulations (AmB–CD gel, AmB in DMSO,
Clotrimazole1 cream dissolved in DMSO, AmB Neo-Sensitab1 and
clotrimazole Neo-Sensitab1).
2.5. Inhibition of biofilm formation assay
2.5.1. Inoculum preparation
All Candida spp., Saccharomyces spp. and Trichosporon spp. were
grown in Sabouraud dextrose agar at 30  C for 48 h. The strains
were grown in 50 mL of YNB medium supplemented with 50 mM
galactose at 37  C overnight in a rotatory shaker. After the
incubation, cells were harvested by centrifugation (4500 rpm for
5 min) and washed twice with phosphate buffer saline (PBS). Prior
to their use, washed cells of each strain were suspended in PBS and
the turbidity adjusted to an optical density of 0.2 at 600 nm
(Al-Fattani and Douglas, 2004).
2.5.2. Membrane impregnation
AmB–CD gel was diluted at the AmB concentrations of 25, 50,
75, 150, 300, 450 and 600 mg mL
1, which were used to impregnate
sterile cellulose membrane filters (Millipore1; pore size 0.22 mm)
during 10 s. Membranes were washed three consecutive times
with PBS. Prior to inoculation, membranes were dried at 37  C for
20 min.
2.5.3. Inoculation
Every membrane was placed on a plate containing Sabouraud
dextrose agar supplemented with 500 mM galactose. The surface
of every membrane was embedded with 50 mL of the suspended
cells in PBS. Plates were dried at 37  C for 1 h. Then, plates were
inverted and incubated at 37  C for 48 h. Membranes were
manually repositioned every 10–12 h. After incubation, the growth
of the microorganism on the membranes was evaluated. All the
experiments were performed by triplicate. Membranes impreg-
nated with placebo formulation were used as control.
2.6. Antifungal penetration studies and assessment of the antifungal
activity and viability
2.6.1. Inoculum preparation
See Section 2.5.1.
2.6.2. Preparation of YNB agar with AmB
AmB dissolved in DMSO and AmB–CD gel were prepared.
Formulations were added in fractions of YNB agar supplemented
with 500 mM galactose to obtain the following AmB concen-
trations: 150, 300 and 600 mg mL
1.
2.6.3. Development of yeast biofilms for penetration studies and
antifungal activity
Biofilm formation was performed according to the method
previously described by Samaranayake et al. (Samaranayake et al.,
2005).
2.6.4. Effect of AmB formulations on the viability of biofilm cells
The effect of the AmB formulations on the viability of the cells in
biofilm was studied after being exposed to the drug for 4 h at 37  C
during the penetration assay. After the incubation, the viable cells
in the biofilm were counted by a standard CFU (colony forming
Fig. 1. Solubility diagram of the AmB–CD inclusion complex. Data were obtained
using different CD concentrations with AmB at saturation. Samples were shaken,
filtered through 0.45 mm, diluted to 12 mg mL
1 and finally assayed by spectropho-
tometry at 364 nm.
 H.K. Ruiz et al. / International Journal of Pharmaceutics 473 (2014) 148–157
Fig. 2. FT-IR characterization of the AmB–CD complex prepared at a ratio 1:100
without filtration; (a) AmB; (b) g-CD; (c) AmB–g-CD (1:100) lyophilized complex;
(d) physical mixture AmB:g-CD (1:100); (e) physical mixture AmB:g-CD (1:1).
units) procedure by serial dilutions (Al-Fattani and Douglas, 2004)
and compared to the biofilm controls without treatment.
2.7. Leishmanicidal activity
2.7.1. In vitro leishmanicidal activity assay in promastigotes and
intracellular amastigotes
Promastigotes of Leishmania infantum (MCAN/ES/96/BCN150),
Leishmania amazonensis (MHOM/Br/79/Maria), Leishmania guya-
nensis (141/93) and Leishmania braziliensis (2903) strains were
used. Three AmB formulations were tested: AmB–CD (AmB 0.125%,
CD 12.5% w/w), AmB–CD gel (AmB 0.125%, CD 12.5%, methyl
cellulose 3%, w/w) and AmB deoxycholate (AmB 0.5%, sodium
deoxycholate 0.41%, w/w). The range of AmB concentrations tested
was between 0.0024 and 5 mg mL
1. The assays were carried out as
previously described (Dea-Ayuela et al., 2009; Sobarzo-Sanchez
et al., 2013).
2.7.2. In vivo leishmanicidal activity assay in an experimental
cutaneous model in golden hamster
The study was approved by the Complutense University
Institutional Animal Care and Ethics Committee. Hamsters were
randomly split into two groups of six animals. A suspension
(0.5 mL) containing 1 107 metacyclic promastigotes of L. ama-
zonensis in sterile PBS was injected subcutaneously in the footpad
of the left hind paw at day 0. Right hind paw was used as a negative
control (no infection). Disease progression was monitored weekly
using a vernier calliper to measure footpad size. After 28 days post-
infection, chronic cutaneous leishmaniasis was developed and
then AmB–CD gel (AmB 0.125%, CD 12.5%, methyl cellulose 3%,
w/w) was administered twice daily for 21 consecutive days in one
of the groups. The other group received no treatment. Progression
of the inflammation was assessed 5 week post-treatment.
2.8. Cytotoxicity assay in macrophages
J774 macrophages (EACC 80011428) in the pre-confluence
phase were seeded at the concentration of 2.5  105 cells mL
1 in
96-well flat-bottom microplates (Sarsted1) and allowed to adhere
for 24 h at 37  C in 5% CO2. Three AmB formulations were tested:
AmB–CD (AmB 0.125%, CD 12.5% w/w), AmB–CD gel (AmB 0.125%,
CD 12.5%, methyl cellulose 3%, w/w) and AmB deoxycholate (AmB
0.5%, sodium deoxycholate 0.41%, w/w). The range of AmB
concentrations tested was between 0.78 and 100 mg mL
1. The
151
assay was performed as previously described (Dea-Ayuela et al.,
2009; Sobarzo-Sanchez et al., 2013).
3. Results
3.1. AmB–CD inclusion complex
From the phase solubility profile shown in Fig. 1, it was observed
that the solubility of AmB was increased linearly as function of
g-CD concentration (y = 0.0161X
0.1876; R2 = 0.9964). Linearity is
characteristic of the AL type system suggesting that water-soluble
complexes were formed in the solution. The slope value was lower
than 1, which indicates that the inclusion complexes between the
AmB and the g-CD were formed in a 1:1 molar ratio (Challa et al.,
2005; Ghodke et al., 2010). Assuming 1:1 complexes were formed,
the Kc was calculated using the equation previously described and
was found to be 1129 M
1. Since no covalent bonds are formed or
broken during the drug–cyclodextrin complex formation, the
complexes are in dynamic equilibrium with free drug and
cyclodextrin molecules. The CE was 0.016 meaning that only
about one out of every 64 cyclodextrin molecules in solution are
forming a water-soluble complex with the AmB (Loftsson et al.,
2005). From Fig. 1, it is possible to estimate the amount of g-CD
required to prepare other complexes with different AmB concen-
trations. AmB was solubilised by a molar ratio AmB:CD 1:64.
Considering the differences in molecular weights between AmB
(924 Da) and CD (1279 Da), the weight ratio AmB:CD for
solubilisation was 1:88. This weight ratio was rounded in the
formulations to 1:100 as the addition of other excipients in the
formulation could decrease the CE of the AmB within the g-CD.
AmB was in monomer form in the AmB–CD inclusion complex
resulting in a transparent yellow solution with three maximum
absorption peaks at 363, 383 and 407 nm and particle size below
1 nm (Torrado et al., 2008).
The changes observed in the FTIR spectra were an indication of
the formation of AmB–CD complex (Fig. 2). FTIR spectra showed
that many functional groups could be involved in the physico-
chemical interaction between AmB and CD. Crystalline AmB
(Fig. 2a) showed a sharp CQO stretch band at 1692 cm
1 and a
CQC stretch band at 1556 cm
1 (Angra et al., 2009; Kim et al.,
2010). The disappearance of these two specific bands may be due to
the AmB inclusion complexation into the g-CD cavity (Fig. 2c)
(Kim et al., 2010) which has the biggest diameter (7.5–8.3 Å) among
all three parent CDs. Other representative findings were the
lengthening of the band at 996 cm
1 (b-glycosidic bond) and the
lowering of the alkene bands (C

H in plane) at 1368 and
1337 cm
1, which might be also related to the interaction of the
AmB within the apolar cavity of the CD (Prakash et al., 2006).
Formation of AmB–CD complex was also confirmed using XRD, DSC
and TEM (see Supporting information for further details).
3.2. Topical AmB–CD formulations
The main characteristics of the formulations (composition, pH,
aggregation state and antifungal activity) are shown in Table 1. All
the formulations exhibited a pH of 5.5, which is suitable for skin
administration. Besides, AmB aggregation state was kept as
monomer form. AmB methyl cellulose gel exhibited higher
antifungal activity when compared to hydroxypropyl cellulose
and Carbopol1 gels. Additionally, methyl cellulose gel and cream
formulations exhibited greater antifungal activity than both AmB
reference formulations (AmB dissolved in DMSO and AmB
deoxycholate).
The AmB–CD complex formulation based on methyl cellulose
gel exhibited the highest antifungal activity and for this reason this
formulation was chosen for further studies. The formulation
152 H.K. Ruiz et al. / International Journal of Pharmaceutics 473 (2014) 148–157

Table 1
Characteristics of the topical formulations prepared using AmB–CD complex. Antifungal activity was measured against C. albicans reference strain CECT 1394. Experiments
were performed in triplicate.

Formulation Composition (w/w) Inhibition halo diameter (mm) pH AmB aggregation state

AmB–CD methyl cellulose (MC) gel AmB (0.125%) 29.6  0.1 5.5 Monomer
g-CD (12.5%)
MC (2%)
AmB–CD hydroxypropyl cellulose (HPC) gel AmB (0.125%) 14.4  0.1 5.5 Monomer
g-CD (12.5%)
HPC (3%)
AmB–CD Carbopol1 ETD 2020 gel AmB (0.125%) 18.1  0.1 5.5 Monomer
g-CD (12.5%)
Carbopol1 ETD 2020 (1%)
AmB–CD Carbopol1 Ultrez1 20 gel AmB (0.125%) 18.7  0.3 5.5 Monomer
g-CD (12.5%)
Carbopol1 Ultrez 20 (1%)
AmB–CD cream AmB (0.125%) 26.4  0.3 5.5 Monomer
g-CD (12.5%)
NaCl (1%)
Dow Corning1 3225 C (15%)
Cyclomethicone pentamer (15%)
AmB deoxycholate AmB (0.5%) 22.5  0.1 5.5 Monomer
Sodium deoxycholate (0.41%)
Dibasic sodium phosphate (0.1%)
Monobasic sodium phosphate (0.09%)
AmB in DMSO AmB in DMSO 24.9  0.1 5.5 Monomer

containing 3% of methyl cellulose was selected because it had
the most suitable consistency for topical administration. The
chemical stability of this gel formulation was studied during 6
months at 25  2  C and chemical degradation of AmB was
lower than 1%. Consistency, colour, pH and AmB aggregation
state were kept unchanged over this period of time (data not
shown).
3.3. Antifungal activity
AmB–CD gel formulation exhibited a greater antifungal activity
against all the strains tested (Candida spp., Trichosporon spp. and
Saccharomyces spp.) when compared to the standard AmB Neo-
Sensitabs1 tablets (Fig. 3). All the strains tested were susceptible to
the AmB released from the AmB–CD gel formulation (inhibition

Fig. 3. Antifungal activity of AmB–CD gel (10 mg AmB disk
1) and AmB Neo-Sensitabs1 (10 mg AmB disk
1) against different Candida strains, Saccharomyces spp. and
Trichosporon spp. Disks were embedded with 20 mL of either AmB reference in DMSO (at the concentration of 96 and 600 mg mL
1) or AmB–CD gel (at the concentration of 48,
96, 250 and 500 mg mL
1 which corresponds to 0.96, 1.92, 5 and 10 mg of AmB, respectively). The disks were placed onto the inoculated agar plates as well as the AmB Neo-
Sensitab1 tablets (containing 10 mg of AmB). The inhibition zone diameters were measured at points where there was a complete inhibition of yeast growth. The isolates were
classified as susceptible (S) to AmB when the inhibition zone was 15 mm, resistant (R) when it was 9 mm and intermediate (I) or susceptible-dose dependent when the
inhibition zone was between 10 and 14 mm (Pujol et al., 2008). Inhibition zone diameters are expressed as mean  SD are in mm. Experiments were performed in triplicate.
 H.K. Ruiz et al. / International Journal of Pharmaceutics 473 (2014) 148–157 153

Fig. 4. Antifungal activity of AmB–CD gel (1.92 mg AmB disk
1) and AmB dissolved in DMSO (1.92 mg AmB disk
1) against different Candida strains, Saccharomyces spp. and
Trichosporon spp. Inhibition zone diameters are expressed as mean  SD. All experiments were performed in triplicate.

zone diameter >15 mm). In addition, AmB–CD gel formulation not unexpected, as C. krusei resistance to AmB has been previously
exhibited higher antifungal activity against all the strains tested reported by other authors (Ellis, 2002).
when compared to AmB dissolved in DMSO (Fig. 4). When the
antifungal activities of the placebo formulation, DMSO or 3.5. Antifungal penetration studies and assessment of the antifungal
phosphate buffer were tested, no inhibition halo was observed activity of the formulations through the biofilm
for any of the strains (data not shown).
Clotrimazole formulations (cream and Neo-sensitabs1 tablets) AmB–CD gel penetration through yeast biofilms was signifi-
were also tested in the same way. AmB–CD gel formulation showed cantly higher than AmB dissolved in DMSO (Fig. 5). The halos
a bigger halo compared to clotrimazole formulations as well as obtained after the diffusion through the fungal biofilms showed
compared to AmB Neo-Sensitabs1 and AmB dissolved in DMSO, that AmB remains in the active form after the diffusion, as showed
respectively (Table 2). by the inhibition halos >15 mm obtained against the C. albicans
reference strain. However, this was not the case for AmB dissolved
3.4. Inhibition of biofilm formation in DMSO that showed a high penetration through biofilm free
control membranes but not through the biofilms. AmB–CD gel
The efficacy of AmB–CD gel on the inhibition of biofilm showed no significant differences between biofilm free control
formation on membranes is shown in Table 3. AmB–CD gel at the membranes and the biofilms.
concentration of 300 mg mL
1 was able to inhibit the biofilm
formation in all the tested strains with the exception of C. krusei
which showed to be the least susceptible to AmB. This result was
Table 3
Inhibition of biofilm formation on membranes. Sterile cellulose membranes were
Table 2 impregnated with AmB–CD gel at different AmB concentrations and washed three
Antifungal activity against C. albicans. The amount of drug assayed (either AmB or times in PBS before inducing biofilm formation. Key: (Control) membranes
clotrimazole) was 10 mg in all the formulations. The antifungal activity of the AmB– impregnated with placebo formulation; (+) complete, () partial or (
) no biofilm
CD gel was compared to other AmB formulations such as AmB Neo-Sensitabs1 or formation on membrane. Experiments were performed in triplicate.
AmB dissolved in DMSO and other clotrimazole-based formulations such
Strain Control AmB concentration (mg mL
1)
Clotrimazole1 cream or clotrimazole Neo-Sensitabs1. Data are expressed as
inhibition zone diameter in mm (mean  SD). For AmB formulations, the inhibition 25 50 75 150 300 450 600
zone diameters were measured at points where there was a complete inhibition of
C. albicans 1394 + 






yeast growth. However, measurements of the clotrimazole formulations were
C. dublinensis 63341 + + + 




carried out where there was a prominent inhibition of fungal growth without
C. glabrata 60661 + + +





colonies of normal size. All experiments were performed in triplicate.
C. glabrata 60750 + + +  



Formulation Inhibition zone diameter (mm) C. guilliermondii 62863 + + + 




C. krusei 52009 + + +    

AmB dissolved in DMSO 26.9  0.1 C. krusei 52011 + + + + 



AmB–CD gel 34.4  0.1 C. krusei 55574 + + 





AmB Neo-Sensitabs1 23.2  0.4 C. parapsilosis 57744 + +  




Clotrimazole1 cream 21.5  0.5 Saccharomyces 61978 + +   



Clotrimazole Neo-Sensitabs1 21.4  0.5 Trichosporon 61978 + + 





154 H.K. Ruiz et al. / International Journal of Pharmaceutics 473 (2014) 148–157

Fig. 5. Penetration of AmB–CD gel and AmB dissolved in DMSO through different yeast biofilms compared to biofilm-free controls. Sterile polypropylene membrane filters
(diameter 47 mm; pore size 0.22 mm; Millipore1) were aseptically placed on a Sabouraud dextrose agar supplemented with 500 mM galactose plates. The polypropylene
membranes were inoculated with 50 mL of the overnight-incubated inoculum. The plates were incubated at 37  C for 1 h to dry the inoculum and subsequently, the plates
were inverted and incubated at 37  C for 48 h. Every 10–12 h, every membrane with the yeast biofilm was manually repositioned on a fresh area on the agar plate. The
membranes with the fungal biofilm (layer B) were placed facing outwards on the AmB incorporated YNB agar plates (layer A). The same system was prepared with
polypropylene membranes biofilm-free as a control. On the top of the layer B, it was placed a membrane filter (Millipore1) with 25 mm of diameter and 0.22 mm pore size
(layer C). On the top of layer C, it was deposited a blank antibiotic disk with 6 mm of diameter (layer D) which was moistened with 20 mL of PBS to ensure passive capillary
diffusion of the drug through the biofilm. The whole system was incubated at 37  C for 4 h. After the incubation time, the antibiotic disks (layer D) were placed on a fresh MHA
previously inoculated with C. albicans CECT 1394 as it is described in the disk-diffusion halo assay. Plates were incubated at 30  C for 48 h and then the diameters of the growth
inhibition zone were measured. Data are expressed as diameter of inhibition growth zone (mean  SD) in mm. All experiments were performed in triplicate. Halos 15 mm
show full AmB activity after diffusion through the biofilm.

The effect on the inhibition of biofilm cells viability after the
diffusion of the different formulations is shown in Table 4. The
antifungal activity, measured as % of CFU inhibition, was higher
than 94% for all the strains when exposed to AmB–CD gel at
600 mg mL
1. In general, all the biofilms showed higher suscepti-
bility to the AmB–CD gel antifungal activity compared to AmB
dissolved in DMSO. Especially remarkable are the results with C.
parapsilosis 57744, only susceptible to AmB–CD gel at 600 mg mL
1
or the % of CFU inhibition obtained with the different C. krusei
strains, more resistant than the other species assayed to the biofilm
growth inhibition effect, but killed by AmB–CD penetration once
the biofilm has been formed.
3.6. In vitro leishmanicidal activity and cytotoxicity assays in
macrophages
The leishmanicidal activity in promastigotes and amastigotes
and cytotoxicity in macrophages of AmB formulations are shown in
Table 5. No statistical significance (P > 0.05) was observed between
the reference formulation (AmB deoxycholate) and AmB–CD liquid
complex when it was assessed the antileishmanial activity in all
the parasite strains tested except for L. infantum and amazonensis.
However, AmB–CD gel formulation showed a diminished activity
compared to the liquid AmB–CD, which it could be related to a
slower AmB release from the gel structure. Nevertheless, AmB–CD

Table 4
Effect of the AmB formulations on the viability of biofilm cells. Cells were scraped from the biofilm (2 mm2), suspended in PBS (5 mL) and vortexed thoroughly. Serial dilutions
were performed and plated on YED agar plates. After 48 h at 30  C, CFU were counted. Biofilms without treatment were used as controls. Data were expressed as the
percentage of CFU inhibition per mm2 of biofilm compared to the control. The results are the mean of triplicate determinations. Statistical significance (P < 0.05) between AmB
dissolved in DMSO and AmB–CD gel is shown as *.

Strain CFU inhibition (%)

AmB in DMSO AmB–CD gel

DMSO 150 mg mL
1 DMSO 300 mg mL
1 DMSO 600 mg mL
1 GEL150 mg mL
1 GEL 300 mg mL
1 GEL 600 mg mL
1

C. albicans CECT 1394 97.3 97.9 98.3 100.0* 98.9* 100.0*

C. dubliniensis 63341 86.7 88.9 99.9 89.3 99.8 99.9
C. glabrata 60661 98.0 98.3 99.0 100.0* 100.0* 100.0*
C. glabrata 60750 98.0 94.0 92.8 97.6 96.8 97.4
C. guilliermondii 62863 40.0 95.0 91.7 16.7 96.0 97.9
C. krusei 52009 74.3 94.3 96.6 95.4 96.0 96.6
C. krusei 52011 0.0 25.0 82.5 25.0 96.0 96.5
C. krusei 55574 94.9 95.7 96.6 94.3 96.0 96.6
C. parapsilosis 57744 0.0 0.0 0.0 0.0 0.0 94.0*
Saccharomyces 61978 0.0 50.0 78.5 50.0* 70.0* 99.4*
Trichosporon sp. 61978 98.0 99.8 100.0 99.8* 100.0* 99.9
 H.K. Ruiz et al. / International Journal of Pharmaceutics 473 (2014) 148–157 155

Table 5
In vitro anti-leishmanial activity and cytotoxicity in macrophages. In vitro anti-Leishmania activity assays were performed on both log phase promastigotes and intracellular
amastigotes. The percentage of parasite viability and the inhibition concentration 50% (IC50) values as well as the percentage of macrophage viability and the 50% cytotoxic
concentration (CC50) values were calculated by probit analysis (SPSS v. 15.0). The selectivity index (TI) of the AmB formulations was determined by calculating the ratio
between the 50% cytotoxic concentration (CC50) over the 50% inhibitory concentration on promastigotes (IC50). All experiments were performed in triplicate.

Assay Strain AMB deoxycholate AMB–CD AmB–CD gel

IC50 (mg mL
1
) on promastigotes L. infantum 0.03  0.004 0.06  0.001 0.21  0.001
L. amazonensis 0.23  0.011 0.26  0.078 0.35  0.006
L. guyanensis 0.12  0.001 0.13  0.002 0.37  0.003
L. braziliensis 0.03  0.001 0.02  0.008 0.20  0.001

CC50 (mg mL
1) Macrophages J774 10.94  1.704 3.72  0.387 86.60  3.670

SI on promastigotes L. infantum 364.7 62.0 412.4

L. amazonensis 47.6 14.3 247.4
L. guyanensis 91.2 28.6 234.1
L. braziliensis 364.7 186.0 433.0

IC50 (mg mL
1) on amastigotes L. infantum 0.48  0.05 0.22  0.01 0.499  0.013
L. amazonensis 0.086  0.007 0.174  0.004 0.375  0.032

SI on armastigotes L. infantum 22.8 16.9 173.6

L. amazonensis 127.2 21.4 230.9

gel exhibited the highest CC50 and selectivity index values when it
was compared to both AmB–CD and AmB deoxycholate. Bearing in
mind that a long-term treatment is required to cure cutaneous
leishmaniasis, AmB–CD gel seems to be a safer AmB therapy with a
selectivity index between 2 and 8-fold higher than the reference
AmB deoxycholate depending on Leishmania spp.
the untreated group. However, parasites were not completely
eradicated since inflammation progressed when AmB topical
administration was interrupted indicating that prolonged treat-
ments are required.
4. Discussion

3.7. In vivo leishmanicidal activity
Prior to topical treatment, disease progression was similar in all
the animals and no statistical significant differences were observed
(Figs. 6 and 7). Topical administration of AmB–CD gel for 21 days
resulted in a significant reduction in the lesion size compared to
The association between AmB and CD is characterized by the
stability constant (Kc) (11) which assumes a ratio drug–CD 1:1
(Higuchi and Connors, 1965). When Kc is <100 M
1, the interaction
drug–CD is weak, whereas, if Kc is >1000 M
1, the interaction drug–
CD is strong. The experimental value of Kc of the AmB–CD complex
was 1129 M
1 which is convenient bearing in mind that Kc values

Fig. 6. In vivo leishmanicidal activity in an experiment model of cutaneous leishmaniasis. Hamsters were randomly split into two groups of six animals. After 28 days post-
infection, AmB–CD gel was administered twice daily for 21 consecutive days in one of the groups. The other group received no treatment. Lesion size (mean  SEM) is
expressed as the difference between the diameter of the infected left hind paw and the non-infected right hind paw from each animal. Statistical significant differences
(P < 0.05) are expressed as: * (U-Mann–Whitney). Key: AmB–CD gel (-^-); untreated group (-&-).
156 H.K. Ruiz et al. / International Journal of Pharmaceutics 473 (2014) 148–157
Fig. 7. Effect of AmB–CD gel on infected hamster with L. amazonensis. After 28 days
post-infection, AmB–CD gel (AmB 0.125%, CD 12.5%, methyl cellulose 3%, w/w) was
administered twice daily for 21 consecutive days in one of the groups.
Macroscopical evaluation of lesions in untreated (a) and treated hamster (b) 15
days post-treatment.
extremely high are related to lack of activity because the
interaction drug–CD is so strong that the drug does not dissociated
from the CD and then it cannot elicit its pharmacological effect
(Szejtli, 1997). Complexation between drug and CD is a dynamic
process based on hydrophilic and van der Waals interactions
instead of covalent and ionic bonds where it coexists equilibrium
between AmB and CD complex, free AmB and free CD in the
medium (Uekama, 2004). This interaction AmB–CD was confirmed
by FT-IR.
Among all the excipients used to prepare the formulations, AmB
gel containing methyl cellulose exhibited the highest antifungal
activity (2-fold and 1.6-fold higher) compared to hydroxypropyl
cellulose and Carbopol1 gels, respectively. The structural formula
of methyl cellulose is characterized by

OCH3 groups, whereas
Carbopol1 is composed of acrylic acid monomer units (

COOH)
and hydroxypropyl cellulose is composed of

OH and

OCH2CH
(OH)CH3 groups (Rowe et al., 2009). The diminished antifungal
activity can be the consequence of the impaired AmB diffusion
from the formulation due to the great formation of hydrogen bonds
between the AmB and the Carbopol1 or hydroxypropyl cellulose
functional groups.
AmB–CD methyl cellulose gel exhibited a higher antifungal
activity when compared to standard AmB deoxycholate (31%), AmB
Neo-Sensitabs1 disks (48%), reference AmB dissolved in DMSO
(28%) and was 60% higher than both clotrimazole formulations (1%
w/w cream and clotrimazole Neo-Sensitabs1 disks). We hypothe-
sized that the higher activity obtained with the AmB–CD gel could
be due to the greater drug solubility and physicochemical stability
inside the inclusion complex with CD and/or enhanced diffusion
through the MHA compared to the free AmB inside the Neo-
sensitabs1 disks which is probably less soluble in water than AmB–
CD; but also, the greater activity could be the consequence of
higher AmB concentrations held by the gel structure around the
disk. In addition, AmB reference formulation dissolved in DMSO
showed a lower antifungal activity compared to the AmB cream or
methyl cellulose gel formulations. This is a very interesting fact
bearing in mind that both CD and DMSO are absorption promoters
and AmB is in monomer state in both formulations. CD can interact
with membranes of living organisms causing destabilization and
altering their physical stability (Lopez et al., 2011). This effect can
modify the permeability of the membranes and lead to cell
disintegration (Puskas and Csempesz, 2007). We hypothesize that
the synergistic effect between AmB and CD on membrane
destabilization is the cause of the enhanced activity of the
AmB–CD formulations.
AmB–CD gel exhibited higher inhibition on fungal biofilm
formation as well as, higher penetration through these biofilms
compared to the AmB dissolved in DMSO. The capacity for
inhibition of the biofilm formation can be especially useful to
prevent CRBSIs in hospitals. The AmB–CD gel kept its inhibitory
effect on biofilm formation even when the formulation was
washed three consecutive times before the inoculation with
microorganisms. As prophylactic agent, AmB–CD gel could be used
to impregnate catheters or prosthesis before being inserted in the
patient to prevent blood stream fungal infections. However,
further toxicological and clinical studies should be performed
before its use in humans. Also crucial for the success of an
antifungal agent is the ability of killing the cells inside the mature
biofilm, which can be interesting for the eradication of certain
fungal infections, for instance onychomycosis.
From a therapeutic point of view, the fact that the same topical
formulation could be useful for the treatment of both fungal and
topical leishmaniasis infection is very interesting. Table 5 shows
that the in vitro antileishmanial activity of the liquid AmB–CD
complex was not significantly (P > 0.05) different than the
reference AmB deoxycholate formulation in most of Leishmania
spp. Once formulated in the semisolid gel formulation, the in vitro
activity was decreased probably related to the slower AmB release
from the formulation. Interestingly, the cytotoxicity of the complex
was clearly decreased by incorporation in the gel structure as
shown by the highest CC50 and selectivity index values (between 2
and 8-fold higher than AmB deoxycholate depending on Leish-
mania spp.) when it was compared to both AmB–CD liquid
formulation and the reference AmB deoxycholate. In addition,
AmB–CD gel has shown to be an effective treatment after topical
administration in an in vivo model of cutaneous leishmaniasis.
AmB–CD gel was able to penetrate across the epidermis and kill
Leishmania parasites localised in the dermis diminishing the
disease progression.
5. Conclusions
In conclusion, AmB–CD methyl cellulose gel is an inexpensive
and physicochemically stable formulation that can be used as safe
and effective topical therapy against cutaneous infections caused
by fungi and Leishmania parasites and as prophylaxis against
bloodstream fungal infection by catheters.
Acknowledgements
This work has been partially funded by a grant from UCM and
CCAA Madrid to the research group 910939 and by BIO 2012-31767
from the Ministerio de Economía y Competitividad. D.R. Serrano
acknowledges the Ministry of Education for her FPU fellowship. We
thank ISP for the gift of g-CD.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ijpharm.
2014.07.004.
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