Emulgel based on amphotericin B and bacuri butter Platonia insignis Mart for the treatment of cutaneous leishmaniasis characterization and in vitro

Prévia do material em texto

Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=iddi20
Drug Development and Industrial Pharmacy
ISSN: 0363-9045 (Print) 1520-5762 (Online) Journal homepage: http://www.tandfonline.com/loi/iddi20
Emulgel based on amphotericin B and bacuri
butter (Platonia insignis Mart.) for the treatment of
cutaneous leishmaniasis: characterization and in
vitro assays
Elvilene de Sousa Coêlho, Gláucia Laís Nunes Lopes, Iluska Martins Pinheiro,
Josefa Natália Policarpo de Holanda, Michel Muálem de Moraes Alves,
Naiane Carvalho Nogueira, Fernando Aécio de Amorim Carvalho & André
Luis Menezes Carvalho
To cite this article: Elvilene de Sousa Coêlho, Gláucia Laís Nunes Lopes, Iluska Martins Pinheiro,
Josefa Natália Policarpo de Holanda, Michel Muálem de Moraes Alves, Naiane Carvalho Nogueira,
Fernando Aécio de Amorim Carvalho & André Luis Menezes Carvalho (2018): Emulgel based
on amphotericin B and bacuri butter (Platonia�insignis Mart.) for the treatment of cutaneous
leishmaniasis: characterization and in�vitro assays, Drug Development and Industrial Pharmacy,
DOI: 10.1080/03639045.2018.1492610
To link to this article: https://doi.org/10.1080/03639045.2018.1492610
Accepted author version posted online: 30
Jun 2018.
Published online: 30 Jul 2018.
Submit your article to this journal 
Article views: 4
View Crossmark data
http://www.tandfonline.com/action/journalInformation?journalCode=iddi20
http://www.tandfonline.com/loi/iddi20
http://www.tandfonline.com/action/showCitFormats?doi=10.1080/03639045.2018.1492610
https://doi.org/10.1080/03639045.2018.1492610
http://www.tandfonline.com/action/authorSubmission?journalCode=iddi20&show=instructions
http://www.tandfonline.com/action/authorSubmission?journalCode=iddi20&show=instructions
http://crossmark.crossref.org/dialog/?doi=10.1080/03639045.2018.1492610&domain=pdf&date_stamp=2018-06-30
http://crossmark.crossref.org/dialog/?doi=10.1080/03639045.2018.1492610&domain=pdf&date_stamp=2018-06-30
RESEARCH ARTICLE
Emulgel based on amphotericin B and bacuri butter (Platonia insignis Mart.) for
the treatment of cutaneous leishmaniasis: characterization and in vitro assays
Elvilene de Sousa Coêlhoa, Gl�aucia La�ıs Nunes Lopesa, Iluska Martins Pinheiroa,
Josefa Nat�alia Policarpo de Holandab, Michel Mu�alem de Moraes Alvesc, Naiane Carvalho Nogueirab,
Fernando A�ecio de Amorim Carvalhoc and Andr�e Luis Menezes Carvalhoa
aPostgraduate Program of Pharmaceutical Sciences (PPGCF), Federal University of Piau�ı, Teresina, Brazil; bDepartment of Pharmacy, Federal
University of Piau�ı, Teresina, Brazil; cMedicinal Plants Research Center, Federal University of Piau�ı, Teresina, Brazil
ABSTRACT
Objective: This work aimed to develop and characterize a topical emulgel of amphotericin B (AmB) with
bacuri butter (Platonia insignis Mart.) and evaluate its antileishmanial activity using in vitro assays.
Significance: Leishmaniasis is considered an infectious disease, with high incidence and capacity to
produce deformities. The first-line treatment recommended by WHO, with pentavalent antimonials, is
aggressive and very toxic. Therefore, the development of topical treatments can emerge as a promising
and less offensive alternative.
Methods: The developed formulations were evaluated for organoleptic characteristics, centrifugation
resistance, globule size, pH, electrical conductivity, viscosity, spreadability, drug content, preliminary stabil-
ity, in vitro release profile, evaluation of antileishmanial activity using promastigotes forms of Leishmania
major as infecting agents, macrophage cytotoxicity and selectivity index (IS).
Results: Formulated emulsions presented organoleptic characteristics compatible with its constituents; pH
values were suitable for topical application, ranging from 4.73 to 5.02; introduced non-Newtonian shear
thinning system; drug content was within the established standards, and the most suitable kinetic model
of release was the first order. Regarding the in vitro assays, formulations containing both 1% and 3% of
AmB presented similar outcomes, indicating a synergism between the bacuri butter and the drug, possibly
showing a reduction on cytotoxicity to host cells.
Conclusions: It was concluded that the formulations developed showed promising antileishmanial action
and high potential for topical use.
ARTICLE HISTORY
Received 20 February 2018
Revised 21 April 2018
Accepted 30 May 2018
KEYWORDS
Biological products;
cytotoxicity; drug stability;
leishmaniasis;
quality control
Introduction
World Health Organization (WHO) highlights leishmaniasis as one
of the six major infectious diseases of highest incidence and
capacity to produce deformities, presenting an overall prevalence
of 12 million people infected [1,2]. In Brazil, the Ministry of Health
[3], reported more than 20,000 cases of the disease, being the
north and northeast region of the country the most affected.
The clinical manifestations caused by a variety of Leishmania
species comprise visceral and tegumentary forms. The tegumen-
tary form includes cutaneous, diffuse and mucocutaneous
leishmaniasis. The cutaneous leishmaniasis (CL) is the most
disseminated form, being asymptomatic or symptomatic by caus-
ing physical, social and psychological problems to patients.
Despite these adverse outcomes, there is still no satisfactory
treatment for CL that is effective, safe and comfortable for the
patient [4,5].
Since the 20s, antimonials have been used successfully in
chemotherapy for leishmaniasis. However, in the latest 10 years,
the world has experienced an increase in therapeutic failure, attrib-
uted to others factors, to the high resistance rates of infecting
agents to the used drugs. Amphotericin (AmB), a medicine used in
the treatment of leishmaniasis in patients with co-infections or
resistance to antimonials, is highly effective but relatively toxic
(cardiotoxic and nephrotoxic). This drug requires parenteral admin-
istration, have severe adverse effects, and high relapse rates.
Therefore, considering this adverse scenario, the search for new,
safer and more effective therapies is markedly relevant [6–9].
Topical treatments are a promising alternative for the treat-
ment of CL since it offers several advantages compared to the
parenteral route. These advantages are not only limited to the
practicality of administration but also can provide a better thera-
peutic efficacy and safety due to its ability to deliver the drug dir-
ectly to the target site, which leads to a reduction of the systemic
adverse effects and toxicity associated to the drug, reducing treat-
ment abandonment by patients; besides it also allows a controlled
and/or prolonged drug release [10–12].
Among topical administration pharmaceutical forms, emulgels
are the combinations of gels and emulsions that emerged as an
excellent alternative for topical drug delivery since it allows the
incorporation of lipophilic (oil-in-water systems) and hydrophilic
(water-in-oil systems) drugs. Besides, it presents an interesting vis-
ual appearance, good spreadability and high capacity to penetrate
the skin and to deliver drugs [13–15]. This pharmaceutical form
demonstrated to be equally useful in the treatment of certain dis-
eases, including formulations that use reduced concentrations of
the active principle [16].
CONTACT Elvilene de Sousa Coêlho elvilene@hotmail.com Postgraduate Program of Pharmaceutical Sciences (PPGCF), Federal University of Piau�ı, Campus
Ministro Petrônio Portella, Ininga, Teresina-PI, Brazil
� 2018 Informa UK Limited, trading as Taylor & Francis Group
DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY
https://doi.org/10.1080/03639045.2018.1492610
http://crossmark.crossref.org/dialog/?doi=10.1080/03639045.2018.1492610&domain=pdf
http://www.tandfonline.com
The association of drugs and plant derivatives with antileish-
manial activity has been shown to be an advantageous alterna-
tive, once the synergic action of these two components can
intensifythe effect, allowing the use of smaller doses of the drug,
thus reducing the chances of toxic effects, associated costs and
therapeutic resistance [5,8]. Among several plants with proven
antileishmanial activity, Platonia insignis Mart., popularly known as
bacuri stands out [17–20].
Seeing the difficulties encountered in the treatment of leish-
maniasis arouses a need to develop new therapeutic alternatives.
The application of a topical emulgel, with a modified release, pro-
motes local action with the reduction of possible adverse reac-
tions, being a formulation of easy preparation, low cost and with
possible transposition to industrial scale. This treatment may be
used in the form of monotherapy (limited to less severe forms of
cutaneous leishmaniasis without risk of dissemination) or as a
combination with systemic therapy, improving patient adherence
to treatment and promoting improved quality of life.
Thus, this work aimed to develop and characterize a topical
formulation of AmB and butter of bacuri (P. insignis Mart.) and to
evaluate its antileishmanial activity and its cytotoxicity on mam-
malian cells.
Materials and methods
Materials
Bacuri butter (P. insignis Mart.) was acquired from Amazon Oil
(Brazil), Poloxamer 407 was obtained from Embrafarma (Brazil),
Amphotericin B deoxycholate – content 844lg/mg on
Valdequ�ımica (Brazil), Oleic acid purchased on Synth (Brazil),
Propylene glycol and Methanol were supplied from Vetec (Brazil),
Potassium sorbate and Sorbic acid obtained from Nantong
(Brazil), Cellulose nitrate membrane filters purchased from Sartorius
Stedim Biotech (Germany), Ethanol – 99.5% content and methanol
– 99.8% PA – Min. were acquired from Dinamica (Brazil), AMP
ULTRA PC 2000 supplied from Agron (Brazil), distilled water,
Schneider's culture medium, RPMI (Fetal bovine serum – SFB), MTT
(3-(4,5-dimethylthiazol-2-yl) 5-diphenyltetrazolium Bromine, Alamar
Blue (ResazurinaVR ), penicillin and streptomycin antibiotics were
purchased from Sigma Chemical (Brazil), Leishmania major (MHOM/
IL/80/Friendlin) obtained from the Laboratory of Antileishmanial
Activity of the Nucleus of Research in Medicinal Plants of the
Federal University of Piau�ı (NPPM/UFPI), macrophages from male
and female BALB/c Mus musculus (four to five weeks of age),
medium of fluid thioglycollate was purchased from Biosystems
(Brazil). The animals were obtained from the NPPM/UFPI sectoral
vivarium. All protocols were approved by the Committee of Ethics
in Animal Research (CEEAPI n� 265/2016).
Technological development of formulations
Obtaining emulgel
The emulsion was applied to the phase of the aqueous phase and
the oil phase, with an aqueous phase inverted during an oily
period with stirring present. Poloxamer 407 was used as the gel-
ling agent, where the polymer and potassium sorbate (preserva-
tive) were dispersed in water and stored in the refrigerator for
24 h (8 ± 1 �C). After that, an oil phase was prepared separately,
where the bacuri butter was heated (40 �C), and after removal of
the sorbic acid (preservative) and an AmB. Oleic acid is also added
to the oil phase when necessary.
After the two-phase session, a vertical 8-degree water
cloud under an oily phase with benchtop mechanical stirrer
(brand: new ethics) model 103 with a rotation of 500 rpm for
5min [16,21,22].
Experimental design
A factorial design 23 was designed in order to evaluate the influ-
ence of alterations in the formulations of AmB and Bacuri butter
(Mb), is the altered parameters: (1) presence or absence of oleic
acid as permeation promoter; (2) change in the concentration of
bacuri butter (10% and 15%) and (3) change in the concentration
of Poloxamer gel (20% and 25%), aiming the discovery of factors
that can optimize the release properties of emulgel formulations
[23]. The concentration of each formulation was decided based on
the results obtained by a factorial design 23 (Table 1). The formu-
lations were denominated as formulation (F) with their respective
number (Examples: F1–F8). Only the services related to emulsion
development were used in factorial planning.
Physicochemical characterization and preliminary stability
Macroscopic analysis of the formulations
The macroscopic study was carried out observing the organoleptic
characteristics of the formulations, regarding the appearance,
spontaneous coalescence of the phases or alterations of color and
odor [24].
Centrifuge test
For the test, 5 g of each formulation were placed in Falcon tubes
and centrifuged at 3000 rpm for 30min at room temperature
(25 ± 1 �C). After that, each formulation was observed for presence
or absence of phase separation or any evidence of instabil-
ity [25,26].
Preliminary stability test
Emulsions were subjected to heating/cooling cycles, 45 ± 2 �C and
�5± 2 �C, every 24 h for 12 days, totaling six periods, according to
the Cosmetics Product Stability Guide and the Stability Study
Guide [27,28]. The results were expressed by the mean± standard
deviation, with n¼ 3 (mean± SD, n¼ 3). The parameters analyzed
before and after the heating/cooling cycles were: organoleptic
characteristics, pH, electrical conductivity, spreadability
and viscosity.
Measurement of globule size
Globule size analysis was determined by optical microscopy, by
observation in the immersion objective. The report was performed
at room temperature. Three slides of each formulation were ana-
lyzed, measuring the diameter of 100 droplets per slide. To pre-
pare the slides, the samples were diluted in propylene glycol and
distilled water (1:1) in a proportion of one part of the sample to
10 parts of the solution [29].
Determination of pH
The formulations pH was determined at room temperature
(25.0 ± 2.0 �C) using a Bel W3B Potentiometer by direct immersion
of the glass electrode in the sample flask (mean± SD, n¼ 3) [25].
2 E. S. COÊLHO ET AL.
Determination of electrical conductivity
The conductivity measurements were performed at room tem-
perature (25.0 ± 2.0 �C) by direct immersion of the electrode in the
sample, without prior dilution [27].
Determination of viscosity
Viscosity was determined using a Brookfield type digital rotary
viscometer at room temperature (25 ± 2 �C), and at speeds of 6,
12, 30, 60, and 100 rpm (spins per minute), with spindle two
(SP ¼ 2) [26].
Determination of spreading
To determine the rate of spreading of the developed formulations,
it was used the in vitro method of Knorst [30], and Cordeiro et al.
[31], which evaluates the relationship between the area of spread-
ing achieved by applying the crescent amount of weight over
the product.
To perform the test, a glass base plate was used, positioned on
a millimeter scale. Using a syringe, the sample was placed in the
central spot of the plate and on it a glass plate of predetermined
weight. Successive plates were placed above this initial, complet-
ing a total of twelve plates that were set at one-minute intervals
between one plate and another. After each minute, the diameters
of spreading reached by the sample were read in opposite posi-
tions (horizontal and vertical) using the millimeter scale.
Subsequently, the mean diameter was calculated, and the results
were expressed according to the equation Ei¼ d2.p/4, where: “Ei”
expresses the spreadability of the sample for a given weight in
square millimeter (mm2), and “d” corresponds to the average
diameter in millimeters (mm).
Determination of amphotericin B content by ultraviolet spectro-
photometry (UV–VIS)
The content of the drug (AmB) in the semi-solid preparation was
determined by a method previously validated by Pinheiro [29]
and reviewed by Lima [22] in the visible region using a UV–Vis
spectrophotometer at a wavelength of 405.4 nm, with the support
of the UV-Probe 2.42 software. A calibration curve was con-
structed at concentrations of 1, 2, 3, 4, 5, and 6 lg/mL and metha-
nol were used as a solvent.
In vitro release assays
In vitro release profile determination using Franz type diffu-
sion cells
The release studies were performedusing Franz type diffusion
cells, with an effective diffusion area of 1.15 cm2, using artificial
membranes of cellulose nitrate 0.45lm. The receiver compartment
was filled with a solution of propane-1,2-diol/ethanol (7:3), to fol-
low the sink conditions, in a system consisting of six individual
cells connected to a thermostatic water bath (37 �C) under con-
stant stirring at 100 rpm on a magnetic stirrer. Samples of the
receptor solution were collected at times: 0; 0.5; 1.0; 1.5; 2.0; 4.0,
and 6.0 for 6 h kinetics [32]. UV–Vis spectrophotometry deter-
mined the concentration of AmB released in 405.4 nm. The evalu-
ation of the release profile of AmB in the different formulations
was calculated from the correlation coefficients for the kinetic
models of zero order, first order and Higuchi. The values were
obtained in the last three “Steady-state” points, in which values
closer to 1 demonstrate better adaptation to the model [33,34].
Evaluation of antileishmanial activity in vitro
Obtainment of cells and parasites
Leishmania major (MHOM/IL/80/Friendlin) was used for the assess-
ment of antileishmanial activity. The promastigote forms were cul-
tured in supplemented Schneider’s medium (10% fetal bovine
serum (FBS), 10,000 IU penicillin and 1000 IU streptomycin, incu-
bated in a B.O.D. at 26 �C). Murine macrophages were collected
from the peritoneal cavities of male and female BALB/c Mus mus-
culus (four to five weeks of age), after previous elicitation (72 h) by
application of 2ml of 3% thioglycollate intraperitoneally.
The antileishmanial activity of promastigote forms of L. major
The assay was performed with promastigote forms of L. major in
log phase of growth. The parasites were seeded in 96-well cell cul-
ture plates containing Schneider's medium supplemented in the
amount of 1� 106 Leishmania/100lL of medium already contain-
ing the formulations previously added to the wells, in triplicate.
Serial dilutions were performed, reaching eight final concentration
ranges desired [800–6.25 lg/mL]. The plate was incubated in bio-
chemical oxygen demand (BOD) at 26 �C for 48 h. After 42 h of
this period, 20lL of resazurin 1� 10�3mol/l was added, and the
plate was incubated again. The plate reading was performed on
absorbance plate reader, Biotek 31(model ELx800), at a wave-
length of 550 nm, and the results were expressed concerning
growth inhibition (%) [4,35].
The positive control was performed with 2 lg/mL of
Amphotericin B (AmB). The negative control, however, was equiva-
lent to Schneider’s medium, only. In this case, it was considered
100% viability for the parasites. The blank reading for each con-
centration and the controls were essential to despise the absorb-
ance resulting from the own medium, with or without the
interference of the studied compounds [4,36].
Cytotoxicity determination
Macrophage cytotoxicity and selectivity index (SI)
Evaluation of cytotoxicity was performed in 96 well plates using
the MTT assay. 2� 105 macrophages per well were incubated in
100 ll of RPMI 1640 medium (supplemented with 10% FBS,
10,000 IU penicillin and 1000 IU streptomycin) in a 37 �C incubator
and 5% CO2, during 4 h for cell adhesion. After this time, the
supernatant was discarded for removal of nonadherent cells. The
formulations were diluted in supplemented RPMI medium, added
to the plate containing the macrophages in serial concentrations,
reaching eight ranges of final concentrations [800–6.25lg/ml],
and incubated at 37 �C and 5% CO2 for 48 h. After this time, cyto-
toxicity was assessed by the addition of 10% MTT [5mg/mL]
diluted in 100 lL of supplemented RPMI medium and the plate
was incubated again for 4 h at 37 �C and 5% CO2. Afterwards, the
supernatant was discarded, and the addition of 100 ll of DMSO
dissolved the formazan crystals. Finally, the absorbance (550 nm)
was measured using a Biotek plate reader (ELx800). The selectivity
index of each treatment was calculated by dividing the mean
cytotoxic concentration (CC50) observed for peritoneal macro-
phages of BALB/c Mus musculus by the mean inhibitory concentra-
tion (IC50) calculated for the different forms of parasite
presentation [35].
DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY 3
Statistical analysis
The results are expressed as mean± SD of at least three replicates.
The mean inhibitory concentration (IC 50), mean cytotoxic concen-
tration (CC 50) with 95% confidence limit, were calculated using
probit regression from the SPSS 13.0 program. The selectivity
index was calculated by dividing the CC50 by IC50. Analyzes of
variance ANOVA followed by the Bonferroni test were performed
using the GraphPad Prism version 5.0 program, taking the value
of p< 0.05 as the maximum level of statistical significance.
Pareto diagrams analysis
Pareto Diagrams were built to help focus improvement efforts
and identify problems. To verify which variables modified in fac-
torial design 23 (Table 1) showed a more significant influence on
the properties of pH, conductivity, viscosity, spreadability and flow
of release of the formulation, a Pareto graph was structured for
each parameter with the results obtained. Using the MinitabVR
18 software.
Results
Technological development
Newly prepared emulgel formulations were dark yellow colored,
with a smooth, homogeneous texture and glossy appearance; pre-
sented viscous sensorial, without signs of instability and character-
istic odor attributed to bacuri butter.
All formulations submitted to the centrifuge resistance test
remained without any evidence of phase separation; however,
after being introduced to heating–cooling cycles, F2, F5, and F6
presented macroscopic instability signals (phase separation), differ-
ently from the remaining formulations which did not show altera-
tions of color, odor, appearance nor homogeneity. Surprisingly,
the globules size could not be measured, due to its absence in all
formulations.
The pH values ranged from 4.73 to 5.02 (Figure 1). After the
heating–cooling cycle, pH ranged from 4.84 to 5.09 (Figure 1), with
a statistically significant difference (p> 0.05) in F2, F5, and F6.
Regarding conductivity, except for F2, all formulations showed
a decrease in conductivity after the heating–cooling cycle
(Figure 2), with F3, F5, and F7 showing a statistically significant
difference after the stability test (p> 0.05).
The viscosity test showed that the emulsions undergo deform-
ation with the application of a force, presenting decreases in
viscosity with increases of speed from 6 to 100 rpm (Figure 3).
In Figure 4, it can be seen that formulations F1, F2, F5, F6, and
F7 increased the spreadability after the heating–cooling cycle,
whereas F3, F4, and F8 decreased.
For determination of the AmB content in the prepared formula-
tions, an analytical curve was constructed, which presented an equa-
tion y¼ 0.1776x� 0.0319, with a determination coefficient (r2) equal
to 0.9983. It can be observed that the content of the drug in the
formulations ranges from 105.72% to 111.23% and that after the
heating–cooling cycle ranged from 97.71 to 103.69. A statistically
0
1
2
3
4
5
6
**
Before
After
F1 F2 F3 F4 F5 F6 F7 F8
** **
Formulations
pH
Figure 1. Average pH of emulgels formulations, before and after the heating–
cooling cycle. Legend: F: formulation; followed by an attributed number for the
correspondent composition. One-way ANOVA was performed followed by the
Bonferroni multiple-comparison post-test, assuming *p< 0.05; **p< 0.01;
***p< 0.001.
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4 Before
After
F1 F2 F3 F4 F5 F6 F7 F8
**
* *
Formulations
C
on
du
ct
iv
it
y 
(m
S/
cm
a 
25
 °
C
)
Figure 2. Average conductivity of emulgels formulations, before and after the
heating–cooling cycle. Legend: F: formulation; followed by an attributed number
for the correspondent composition. One-way ANOVA was performed followed by
the Bonferroni multiple-comparison post-test, assuming *p< 0.05; **p< 0.01;
***p< 0.001.
Table 1. Factorial design 23 for to optimizethe preparation of formulations by amphotericin B and
bacuri butter.
Variables
Factorial design
Poloxamer(%) Oleic acid (%) Butter (%) AmB (%) Sorbic acid (%) Kþ sorbate (%)
Formulations
F1 20 0 10 3 0.2 0.2
F2 20 0 15 3 0.2 0.2
F3 20 5 10 3 0.2 0.2
F4 20 5 15 3 0.2 0.2
F5 25 0 10 3 0.2 0.2
F6 25 0 15 3 0.2 0.2
F7 25 5 10 3 0.2 0.2
F8 25 5 15 3 0.2 0.2
Legend: F: formulation; followed by an attributed number for the correspondent composition.
4 E. S. COÊLHO ET AL.
significant difference regarding AmB content before and after the
heating–cooling cycle (p< 0.05) was only observed in F1 (Figure 5).
Figure 6 shows the values of the accumulated amount of AmB
released within 6h (Q6) in the in vitro release assays; where we can
6 12 30 60 100
0
300
600
900
1200
1500
F1 before
F1 after
F2 before
F2 after
F3 before
F3 after
F4 before
F4 after
Velocity (rpm)
V
isc
os
ity
 (P
a.
s)
6 12 30 60 100
0
300
600
900
1200
1500
F5 before
F5 after
F6 before
F6 after
F7 before
F7 after
F8 before
F8 after
Velocity (rpm)
V
isc
os
ity
 (P
a.
s)
Figure 3. Average viscosity of emulgels formulations, before and after the heating–cooling cycle. Legend: F: formulation; followed by an attributed number for the
correspondent composition.
77 15
7
23
7
31
6
46
6
61
6
76
5
91
5
1.0
65
1.2
15
1.5
23
1.8
33
0
5
10
15
20
25
30
F2 before
F2 after
F3 before
F3 after
F4 before
F4 after
F1 after
F1 before
Weight (g)
S
p
re
ad
ab
ili
ty
 (
m
m
2 )
77 15
7
23
7
31
6
46
6
61
6
76
5
91
5
1.
06
5
1.
21
5
1.
52
3
1.
83
3
0
5
10
15
20
25
30
F6 befor
F6 after
F7 before
F7 after
F8 before
F8 after
F5 after
F5 before
Weight (g)
sp
re
ad
ab
ili
ty
 (
m
m
2 )
Figure 4. Average spreadability of emulgels formulations, before and after before and after the heating–cooling cycle, under the application of different forces.
Legend: F: formulation; followed by an attributed number for the correspondent composition.
0
25
50
75
100
125
Before
After
Formulations
1 2 3 4 5 6 7 8
***
T
eo
r
Figure 5. Mean AmB content of the prepared emulgels (% w/w), before and
after the heating–cooling cycle. Legend: F: formulation; followed by an attributed
number for the correspondent composition. One-way ANOVA was performed
followed by the Bonferroni multiple-comparison post-test, assuming *p < 0.05;
**p < 0.01; ***p < 0.001.
0 1 2 3 4 5 6 7
0
10
20
30
40
50
C
u
m
u
la
ti
v
e
 A
m
p
h
o
te
ri
c
in
 B
 r
e
le
a
s
e
d
 (
�g
)
Time (hours)
 F1
 F2
 F3
 F4
 F5
 F6
 F7
 F8
Figure 6. Amphotericin B release profile from AmB and bacuri butter emulsions.
Legend: F: formulation; followed by an attributed number for the correspondent
composition.
DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY 5
observe that the formulations F1, F6, and F7, were the ones that
released a more significant amount of AmB at the end point (Q6).
The in vitro release profile of AmB in the emulsions is shown in
Figure 6. The three initial positions of the graph demonstrate that
the release of the drug is similar in all formulations. This fundamental
similarity in drug release is since the formulations proposed present
characteristics of a modified drug release system; a more significant
differentiation between the release profiles of the formulations is
only observed in the last three points of the curve, denominated
“steady state.” The correlation coefficient correspondent to the
applied mathematical models determines which model best
describes the release profile of AmB. However, the values were very
close (Table 2), so the residual mean square (RMS) was used to
evaluate the model that best suited. From the applied mathematical
models, the first order model is the most suitable because the RMS
was lower than the other models compared in the study (Table 3),
indicating best adequacy. Formulations F1, F3, F6, and F7 presented
the highest fluxes values (Table 2).
The analysis from the Pareto graph showed that separately the
variables of concentration of Mb, absence or presence of oleic acid
and the concentration of poloxamer have a significant effect on
the final response in relation to conductivity, pH and spreadability
(for a¼ 0.05), but when the interaction between them is evaluated
in the final formulation there is no statistically significant variance.
The other parameters did not present any significant difference in
the definitive answers for the displayed variables.
Antileishmanial activity on promastigote forms of L. major
For this test, a formulation with 1% AmB was also used to verify
the existence of synergistic action with the butter of bacuri from
the reduction of AmB presenting the same vehicle used in factor-
ial planning. The formulations of bacuri butter (Mb – Figure 7),
bacuri butter associated to amphotericin B at concentration of 1%
(MbAmB – Figure 7) and bacuri butter associated to amphotericin
B at concentration of 3% (MbAmB – Figure 7) inhibited the
growth of promastigote forms of L. major, being this inhibition
proportional to the concentration used. At the concentration of
800 lg/mL the formulations Mb, MbAmB 1%, and MbAMb 3% pre-
sented, respectively, 70%, 90%, and 100% inhibition of growth of
promastigote forms. Amphotericin B used as a positive control
(AmBc), at the concentration of 2 lg/mL, inhibited the growth of
this parasite forms in 90%, with half maximal inhibitory concentra-
tion (IC50) of 1.74; relative to the negative control. The results
found for Mb, MbAmB 1% and MbAmB 3% were respectively:
134.77, 109.89, 36.27, and 1.74lg/mL, as listed in Table 4. A pla-
cebo formulation (it contains only gel) was used, in which IC50
greater than 800 lg/mL was obtained.
Cytotoxicity on macrophages and calculation of the selectivity
index (IS)
The results obtained from the cytotoxicity assays (MTT) on murine
macrophages (Figure 8) demonstrate that the formulations
Table 2. Kinetic parameters of the release profile of the AmB and bacuri butter emulsions.
10 Zero order First order Higuchi
F1 r2¼ 0.989 J¼ 6.25 k¼ 0.00052 r2¼ 0.953 k¼ 9.6� 10-5 r2¼ 0.935 k¼ 0.00199
F2 r2¼ 0.998 J¼ 4.58 k¼ 0.00038 r2¼ 0.989 k¼ 8.8� 10-5 r2¼ 0.960 k¼ 0.00146
F3 r2¼ 0.992 J¼ 6.26 k¼ 0.00052 r2¼ 0.965 k¼ 0.00010 r2¼ 0.944 k¼ 0.00200
F4 r2¼ 0.993 J¼ 5.45 k¼ 0.00045 r2¼ 0.986 k¼ 8.8� 10-5 r2¼ 0.973 k¼ 0.00176
F5 r2¼ 0.996 J¼ 4.92 k¼ 0.00041 r2¼ 0.998 k¼ 7.9� 10-5 r2¼ 0.985 k¼ 0.00159
F6 r2¼ 0.964 J¼ 6.93 k¼ 0.00058 r2¼ 0.983 k¼ 0.00496 r2¼ 0.949 k¼ 0.00227
F7 r2¼ 0.974 J¼ 6.84 k¼ 0.00057 r2¼ 0.980 k¼ 0.00455 r2¼ 0.912 k¼ 0.00216
F8 r2¼ 0.994 J¼ 5.43 k¼ 0.00045 r2¼ 0.988 k¼ 9.6� 10-6 r2¼ 0.948 k¼ 0.00173
Legend: r2: determination coefficient; J¼ flux (lg cm�2 h�1); k¼ liberation coefficient; F: formulation; followed by an attributed number
for the correspondent composition.
Table 3. Valores de RMS (residual mean square) Para
an�alise dos modelos cin�eticos de ordem zero, higuchi e
primeira ordem.
Formulations Zero order First order Higuchi
F1 1.9512 0.0070 11.5086
F2 0.2512 0.0019 4.2500
F3 1.4398 0.0070 10.447
F4 1.3192 0.0034 4.7948
F5 0.6225 0.0003 2.1178
F6 10.2335 0.0042 14.456
F7 5.3887 0.0041 18.0106
F8 0.8646 0.0025 7.3381
Co
ntr
ol
Am
Bc 6,2
5
12
,5 25 50 10
0
20
0
40
0
80
0
0
20
40
60
80
100
**
*** ***
***
***
***
***
________________________________________
 Mb (µg/mL)
***
***
G
ro
w
th
 in
hi
bi
ti
on
 (%
)
Co
ntr
ol
Am
Bc 6,2
5
12
,5 25 50 10
0
20
0
40
0
80
0
0
20
40
60
80
100
*
**
***
***
***
*** ***
______________________________________
1% MbAmB (µg/mL)
***
G
ro
w
h 
In
hi
bi
ti
on
 (%
)
Co
ntr
ol
Am
Bc 6,2
5
12
,5 25 50 10
0
20
0
40
0
80
0
0
20
40
60
80
100
***
***
***
***
***
***
***
______________________________________
3% MbAmB (µg/mL)
***
***
G
ro
w
th
 I
nh
ib
it
io
n 
(%
)
Figure 7. Antileishmanial activity of Mb, 1% MbAmB and 3% MbAmB on L. major
promastigote forms. One-way ANOVA was performed followed by the Bonferroni
multiple-comparisonpost-test, assuming �p< 0.05 vs. control; ��p< 0.01 vs.
control; ���p< 0.001 vs. control. Subtitle: AmBc: control anfotericina B; Mb:
bacuri butter; MbAmB: bacuri butter and amphotericin B.
Table 4. Antileishmanial activity, the cytotoxic effect on macrophage and select-
ivity index.
Substances
Promastigotes Macrophages Selectivity index
CI50 (mg/mL) CC50 (mg/mL) IS
Pcb >800 >800 ND
Mb 134.77 >800 ND
MbAmB 1% 109.89 696.55 6.33
MbAmB 3% 36.27 139.57 3.84
AmBc 1.74 8.75 5.02
ND: Not determined.
6 E. S. COÊLHO ET AL.
presented low cytotoxicity on this cell type, showing high values
of mean cytotoxic concentration (CC50) when compared to the
positive control (AmBc). Increases in the concentration of AmB
increased its cytotoxicity significantly, reducing by five times its
mean cytotoxicity index.
Also, AmBc as well as in formulations containing bacuri butter,
showed to be more selective for the parasite than for murine
macrophages, as evidenced by the selectivity indexes (SI) found.
This index varied according to the concentration. MbAmB 3%
reduced in 1.64 times its selectivity index when compared to
MbAmB 1% (Table 4).
Discussion
The factorial design is a valuable statistic tool to define experi-
mental parameters because it allows evaluating the effect of a
large number of variables from a reduced number of preliminary
tests simultaneously when compared to univariate proc-
esses [37–39].
From the factorial design 23, it was possible to define the ideal
conditions for formulations production, with high efficiency, using
three independent variables. After obtaining, the emulgel pre-
sented a smooth, homogeneous texture and glossy appearance;
and the viscosity was compatible with the pharmaceutical form
emulgel. Regarding the color, it showed a dark yellow color
derived from the bacuri butter (Mb) and AmB. The formulations
containing more Mb had a darker yellow tone than the others (F2,
F4, F6, and F8). The characteristic odor of the formulation was
attributed to Mb [40].
In the centrifuge resistance test no changes were observed in
the formulations, but after the preliminary stability test (heating–
cooling cycle), F2, F5, and F6 presented visible signs of instability
(phase separation). According to Mollica et al. [41] and Silva et al.
[42], the stability of pharmaceutical products depends on environ-
mental factors such as temperature, humidity, light, and other fac-
tors related to the product itself as physical and chemical
properties of active substances and pharmaceutical excipients,
pharmaceutical form and composition, manufacturing process.
Thus, the preliminary stability studies orient the phases of the
product preparation, the choice of packaging material, allows esti-
mating the organoleptic changes that may appear, ensuring the
quality of the product.
In the optical microscopy test to observe globule size, no drop-
lets were found in any of the formulations. These results disagree
with those obtained in a study carried out by Pinheiro [29], where
3% AmB emulgels with different permeation promoters were ana-
lyzed, and it was possible to determine the droplet size by the
same method used in the present study. This divergence can be
justified by the addition of Mb, a product rich in lipids, which may
have reduced the globules at sizes not detected by the applied
method. Researchers argue that no single way is entirely sufficient
to characterize systems concerning droplet size, so a combination
of at least two techniques is recommended, one of which should
be microscopic [43].
Skin compatibility is one of the primary requirements for a
unique topical formulation (slightly acidic in the range of 4.6–5.8)
[44,45]. Also, pH compatibility between the formulation and the
incorporated drug is essential for its optimum efficacy. The max-
imum biological tolerance of skin to topical formulations ranges
from pH 4.6 to 7 [46]. Despite this range of understanding, alkalin-
isation of the formulations around pH 6.0 is required because add-
itionally to be ideal for topical application. It may improve drug
action since the pH which AmB has its maximal therapeutic activ-
ity is comprised of the range of 6–7.5 [47,48].
The formulations that presented a statistically significant differ-
ence (p> 0.05) for pH after the heating–cooling cycle (F2, F5, and
F6) also presented instability signs (phase separation) in the
macroscopic analysis.
Regarding electrical conductivity, there was a reduction of this
parameter after the heat–cooling cycle, except in F2. This fact can
be explained by a prevalence of the aqueous phase with the oil
phase, seeing that the conductivity of oily systems is about
100–1000 times inferior to the conductivity of aqueous systems.
Also, during the heating phases of the cycle water might be the
loss by the formulation due to water evaporation. However, the
fact that only F2 has increased its conductivity after the stability
test may be due to the instability presented by this formulation
after the heating–cooling cycle. The measurement of the electrical
conductivity of a formulation allows to estimate the capacity of
the dispersant phase to be formed by oil or water, and it is pos-
sible to determine the polar or apolar domains [49].
In a stable formulation, the application of a force such as the
force applied to spread a product on the skin does not induce
irreversible structural changes on the formulation that can be
interpreted as a loss of stability. The viscosity test allows analyzing
how topical pharmaceutical forms behave when submitted to dif-
ferent forces. The more viscous a formulation is, the less it will suf-
fer influence of the applied forces, and therefore the less it will
spread on the skin [50].
The formulations exhibited a non-Newtonian behavior showing
a decrease in viscosity with increasing shear velocity, which may
infer a probable pseudoplastic behavior confirmed by analyses in
a rheometer. The pseudoplastic behavior was identified when
there is a decrease in viscosity with increasing shear stress, pro-
moting fluidity on the compound. However, after cessation of the
force applied, they return to their initial state [51].
In regards to topical administration, the thixotropic characteris-
tic found in the emulsions contributes to its retention time at the
site of application. This conclusion is explained by the fact that
the formulation becomes more fluid when is under the influence
Co
ntr
ol
Am
Bc 6,2
5
12
,5 25 50 10
0
20
0
40
0
80
0
0
20
40
60
80
100 ** ***
***
***
***
***
_____________________________________
 Mb (µg/mL)
***
***
M
ac
ro
ph
ag
es
 v
ia
bi
lit
y 
(%
)
Co
ntr
ol
Am
Bc 6,2
5
12
,5 25 50 10
0
20
0
40
0
80
0
0
20
40
60
80
100
***
***
***
***
***
_____________________________________
 1% MbAmB (µg/mL)
***
M
ac
ro
ph
ag
es
 v
ia
bi
lit
y 
(%
)
Co
ntr
ol
Am
Bc 6,2
5
12
,5 25 50 10
0
20
0
40
0
80
0
0
20
40
60
80
100
______________________________________
 3% MbAmB (µg/mL)
**
***
*** *** *** *** ***
***
M
ac
ro
ph
ag
es
 v
ia
bi
lit
y 
(%
)
Figure 8. Cytotoxic effects of Mb, 1% MbAmB and 3% MbAmB against peritoneal
murine macrophages. One-way ANOVA test was performed followed by the
Bonferroni multiple-comparison post-test, assuming the �p< 0.05 vs. control;��p< 0.01 vs. control; ���p< 0.001 vs. control. Subtitle: Mb: bacuri butter;
MbAmB: bacuri butter and amphotericin B.
DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY 7
of a force, which implies in an easier spreadability in the applica-
tion region [51] and contributes to the release of the drug from
the vehicle and its bioavailability on the skin [52].
The increase in spreadability presented by F2, F5, and F6 after
the heating–cooling cycle can be justified by the loss of stability
of the emulsions (phase separation), increasing the formulations
fluidity. Changes on this parameter by F1, F3, F4, F7, and F8 did
not critically affect their dispersion properties, which shows that
polymer’sconcentration did not affect the quality of the product.
According to Nikumbh et al. [53], a good spreadability is essential
to emulsions, and Cordeiro et al. [31] emphasize its importance
for better product acceptance.
All formulations must have the drug contained within the
standards established in official compendia. According to USP
[54], the range of AmB content in topical formulations may not
comprise less than 90% or more than 125%. All formulations pre-
pared on this study, except F1, included a percentage of the drug
within the accepted range.
The release kinetics evaluation on semisolid pharmaceutical
forms of topical application is crucial to analyze its suitability as
vehicles capable of delivering the drug at the epidermal level;
besides being essential in the quality control of alternative
pharmaceutical forms. Therefore, in addition to the parameters
described above, another determining factor in the choice of a
formulation is it is in vitro release profile [55].
The release of a biologically active substance from topical for-
mulations is directly dependent on vehicle and drug properties,
generally occurring by a combination of the active diffusion mech-
anisms through the vehicle matrix and the partition coeffi-
cient [29].
Regarding the drug release profile by the in vitro release kinet-
ics, it was observed that the formulation with higher Q6 contained
a higher percentage of Mb (15%), which implies that this compo-
nent may have improved the drug release from its matrix.
From the in vitro release kinetics it was also verified the more
excellent suitability of the formulations to the first order mathem-
atical release model. This relationship can be used to describe the
drug dissolution in pharmaceutical dosage forms such water-
soluble matrices [56].
After formulation factorial planning and the parameters ana-
lysis, the formulation chosen to continue the tests was the one
that presented a proper release of the AmB and that exhibited
excellent stability. The formulation selected to keep the tests was
F7 (poloxamer 25%, oleic acid 5%, bacuri butter 10%), presented
good release flow and remained stable.
The species P. insignis, known as “bacuri” has its ethnopharma-
cological use related to the seed extract as healing and anti-
inflammatory agents. Several activities have been reported for all
parts of this fruit (seed, bark, and pulp) [17–20,57–59]. The butter
obtained from processing of bacuri seeds presents several bio-
logical properties, such as cicatrizant, antimicrobial, antitumor,
cytotoxic, antioxidant and antileishmanial [17,61,62]. The com-
pounds related to these activities are terpenes, xanthones and
phenols, reported as main constituents, as well as compounds
already isolated as Garcinialiptona FC, lupeol [17,19,60,62].
Platonia insignis, in tests performed to evaluate its antileishma-
nial activity, has been shown to be effective against free promasti-
gote forms of Leishmania in a culture medium, and against its
amastigote forms internalized in murine macrophages [17,20].
Nevertheless, this species was also able to present immunostimu-
latory properties in macrophages elicited from the peritoneal cav-
ity of mice and to solve the infection of this cell type, even
reducing its infectivity [19,20].
In this work, it was observed that Mb emulgel, as well as the
formulations of Mb associated with AmB (MbAmb 1% and MbAmB
3%), demonstrated relevant inhibitory growth activity of promasti-
gotes of L. major, after 48h of incubation, with activity dependent
on concentration. It is interesting to note that Mb presented IC50
of 134,77lg/mL showing the potential antileishmanial activity of
the bacuri butter, corroborating with the data obtained by Souza
[20] when researching this activity related to the ethanolic extract
and hexane fraction derived from the stem bark of P. insignis.
The association of Mb with AmB, resulted in a potentialized
antileishmania activity, as can be seen by the reduction in IC50 val-
ues by 1.23 times for MbAmb 1% and 3.71 times for MbAmB 3%.
This association becomes interesting, once this potential syner-
gism between the constituents of this formulation may result in
more satisfactory results on the therapy of leishmaniasis.
The MbAmb, 3% emulgel, showed to be more potent and
more effective with the others regarding its antileishmanial activ-
ity, but more toxic to the murine macrophages, as can be
observed through the CC50 values presented. The MbAmB, 1% for-
mulation, was less toxic and, therefore, more selective to the para-
site, with a selectivity index (IS) of 6.33; which is considered a
good value. This fact can be justified by the pharmacological
properties of the molecules derived from P. insignis Mart. Butter,
rich in unsaturated fatty acids (oleic and linoleic), diterpenes, and
prenylated benzophenones (Garcinialiptona – GFC), a molecule
with marked antileishmanial potential and low cytotoxicity to
mammals cells [18,57–60].
An essential criterion in the prospection of new products with
antileishmanial activity is the more significant toxicity to parasites
than to the host cells [63,64]. Thus, the developed formulations
presented an excellent outcome regarding this criterion. That is,
they showed higher toxicity for L. major than for murine
macrophages.
Commercially, there is no topical medicine containing AmB
available for the treatment of cutaneous leishmaniasis. However,
several studies have shown the efficacy of treatments with this
drug in various formulations and with varying concentrations.
Layegh et al. [65] used AmB at 5mg/mL in a topic lipophilic for-
mulation for the treatment of CL in humans obtaining good
results. Ruiz et al. [66] developed a study to verify the effect of
AmB complexed with B-gamma cyclodextrin against various fungi
and different species of Leishmania, obtaining successful out-
comes. More recently [16], developed an experimental study
involving the treatment of mice with AmB 3% emulgel for topical
application obtained excellent results, showing the benefits of
using reduced concentrations of the drug for patient safety.
Therefore, it was demonstrated that Mb is active on forms of
the parasite and the association of this natural product with AmB
may potentiate its effect due to a possible synergism between
this drug and the constituents of the butter. It has also been
shown that lower concentrations of AmB in emulgel (1%) may be
equally effective against Leishmania when compared to higher
concentrations (3%), making it more interesting from a patients’
safety point of view, since it reduces its toxicity and increases its
selectivity index. One of the limiting factors for the use of AmB in
patients with any of the forms of leishmaniasis is its adverse
effects, and thus, this association may promote the reduction of
the amount of AmB used in the treatment.
Conclusions
This study successfully developed an innovative emulgel of
amphotericin B and bacuri butter (P. insignis Mart.) for topical
8 E. S. COÊLHO ET AL.
application. The emulsions presented organoleptic characteristics
compatible with its constituents; pH values were suitable for top-
ical use and exhibited a non-Newtonian behavior. Formulations
containing 1% and 3% of AmB presented similar results on anti-
leishmanial in vitro tests, indicating a synergism between bacuri
butter and the drug, possibly showing a reduction in cytotoxicity
to host cells. Thus, the formulation developed presents a promis-
ing antileishmanial activity and high potential for topical use,
either on monotherapy, limited to less severe forms of cutaneous
leishmaniasis or as a combination therapy with a systemic drug,
facilitating patient adherence to treatment with a consequent
improvement in life quality.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
The authors are thankful to the Federal University of Piau�ı
(Teresina, Brazil) for providing necessary facilities and the Research
Program for SUS (PPSUS) forthe financial support (Edict – FAPEPI/
MS-DECIT/CNPq/SESAPI number 002/2016). They also thank the
Pharmacy School of this same University and the Antileishmanial
laboratory from the Medicinal Plant Research Nucleus for all the
support provided during the developed tests.
References
[1] Filardy AA, Silva ACC, Koeller CM. Infection with Leishmania
major induces a cellular stress response in macrophages.
PLoS One. 2014;9:e85715.
[2] WHO. Leishmaniasis fact sheet no. 375.Geneva, Switzerland:
World Health Organization; 2015. Dispon�ıvel em: http://
www.who.int/mediacentre/factsheets
[3] MS/SVS. Rede internacional de informaç~oes para a sa�ude.
Indicadores de morbidade e fatores de risco. Minist�erio da
Sa�ude: MS/SVS; 2015. Dispon�ıvel em: http://tabnet.datasus.
gov.br/cgi/tabcgi.exe?sinannet/cnv/ltabr.def
[4] Alves MMM, Brito LM, Souza AC, et al. Gallic and ellagic
acids: two natural immunomodulator compounds solve
infection of macrophages by Leishmania major. Naunyn-
Schmiedeberg's Arch Pharmacol. 2017;390:893–903.
[5] Olliaro P, Grogl M, Boni M, Carvalho EM, Chebli H, Cisse M,
Diro E, et al. Harmonized clinical trial methodologies for
localised cutaneous leishmaniasis and potential for an
extensive network with capacities for clinical evaluation.
PLoS Negl Trop Dis. 2018;12:e0006141.
[6] Pinto JG, Fontana LC, de Oliveira MA, et al. In vitro evalu-
ation of photodynamic therapy using curcumin on
Leishmania major and Leishmania braziliensis. Lasers Med
Sci. 2016;31:883–890.
[7] Didwania N, Shadab MD, Sabur A, et al. Alternative to
chemotherapy – the unmet demand against leishmaniasis.
Front Immunol. 2017;8:1779.
[8] Ponte-Sucre A, Gamarro F, Dujardin JC, et al. Drug resist-
ance and treatment failure in leishmaniasis: a 21st century
challenge. PLoS Negl Trop Dis. 2017;11:e0006052.
[9] Varikuti S, Oghumu S, Saljoughian N, et al. Topical treat-
ment with nanoliposomal Amphotericin B reduces early
lesion growth but fails to induce cure in an experimental
model of cutaneous leishmaniasis caused by Leishmania
Mexicana. Acta Trop. 2017;173:102–108.
[10] Santos AM, Noronha EF, Ferreira LAM, et al. Efeito de uma
formulaç~ao hidrof�ılica de paromomicina t�opica na leishma-
niose cutânea em pacientes com contra-indicaç~oes de tra-
tamento com antimonial pentavalentes [Effect of a
hydrophilic formulation of topical paromomycin on cutane-
ous leishmaniasis in patients with contraindications for
treatment with pentavalent antimonial]. Rev Soc Bras Med
Trop. 2008;41:444–448.
[11] Ruela ALM, Perissinato AG, Lino MES, et al. Evaluation of
skin absorption of drugs from topical and transdermal for-
mulations. Braz J Pharm Sci. 2016;52:527.
[12] Shilakari AG, Sharma PK, Asthana A. In vitro and in vivo
evaluation of niosomal formulation for controlled delivery
of clarithromycin. Scientifica (Cairo). 2016;2016:1.
[13] Khullar R, Kumar D, Seth N, et al. Formulation and evalu-
ation of mefenamic acid emulgel for topical delivery. Saudi
Pharm J. 2012;20:63–67.
[14] Supriya U, Seema C, Preeti K. Emulgel: a boon for dermato-
logical diseases. Int J Pharm Res Allied Sci. 2014;3(4):1–9.
[15] Jagdale S, Saylee P. Gellified emulsion of ofloxacin for
transdermal drug delivery system. Adv Pharm Bull.
2017;7:229–239.
[16] Pinheiro IM, Carvalho IP, Carvalho CE, et al. Evaluation of
the in vivo leishmanicidal activity of amphotericin B emul-
gel: an alternative for the treatment of skin leishmaniasis.
Exp Parasitol. 2016;164:49–55.
[17] Costa Junior JS, Almeida AAA, Ferraz BF, et al. Cytotoxic
and leishmanicidal properties of garcinielliptone FC, a pre-
nylated benzophenone from Platonia insignis. Nat Prod Res.
2013;27:470–474.
[18] Costa Junior JS, Ferraz AB, Sousa TO, et al. Investigation of
biological activities of dichloromethane and ethyl acetate
fractions of Platonia insignis Mart. seed. Basic Clin
Pharmacol Toxicol. 2013;112:34–41.
[19] Lustosa AKMF, Arcanjo DDR, Ribeiro RG, et al.
Immunomodulatory and toxicological evaluation of the fruit
seeds from Platonia insignis, a native species from Brazilian
Amazon Rainforest. Braz J Pharm. 2016;26:77–82.
[20] Souza AC, Alves MMM, Brito LM, et al. Platonia insignis
Mart., a Brazilian Amazonian plant: the stem barks extract
and its main constituent Lupeol Exert Antileishmanial
effects involving macrophages activation. Evid Based Compl
Alternat Med. 2017;2017:1.
[21] Ur-Rehman T, Tavelin S, Gr€obner G. Chitosan in situ gel-
ation for improved drug loading and retention in polox-
amer 407 gels. Int J Pharm. 2011;409:19–29.
[22] Lima MFMB. Obtenç~ao Tecnol�ogica e Perfil De Liberaç~ao in
Vitro De Emulgel Leishmanicida �a Base De Platonia Insignis
Mart.þAnfotericina B [Technological Obtaining and in Vitro
Release Profile of Emulgel Leishmanicida at the Platonia
Insignis Mart Base.þAmphotericin B.] Graduation in
Pharmaceutical Sciences.Teresina: Federal University of
Piau�ı; 2017.
[23] Lopes GLN, Nascimento MO, Cir�ıaco SL. Influência das var-
i�aveis de processo na obtenç~ao de c�apsulas gelatinosas
duras de ibuprofeno em farm�acia universit�aria [Influence of
the process variables on obtaining hard gelatin capsules of
ibuprofen in university pharmacy]. Bol Inform Geum.
2015;6:59–62.
[24] Smaoui S, Ben Hlima H, Ben ChoMba I, et al. Development
and stability studies of sunscreen cream formulations
DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY 9
http://www.who.int/mediacentre/factsheets
http://www.who.int/mediacentre/factsheets
http://tabnet.datasus.gov.br/cgi/tabcgi.exe?sinannet/cnv/ltabr.def
http://tabnet.datasus.gov.br/cgi/tabcgi.exe?sinannet/cnv/ltabr.def
containing three photo-protective filters. Arab J Chem.
2017;10:S1216–S1222.
[25] Dourado D, Barreto C, Fernandes RS. Development and
evaluation of emulsifying systems of the material grease
from Brazilian flora. J Pharm Pharm Res. 2015;3:130–140.
[26] Ochoa-Andrade A, Parente ME, Jimenez-Kairuz A, et al. A.
study of the influence of formulation variables in bioadhe-
sive emulgels using response surface methodology. AAPS
PharmSciTech. 2017;18:2269–2278.
[27] Daher CC, Fontes IS, Rodrigues RO, et al. Development of
O/W emulsions containing Euterpe oleracea extract and
evaluation of photoprotective efficacy. Braz J Pharm Sci.
2014;50:639–652.
[28] Daudt RM, Back PI, Cardozo NS, et al. Pinhao starch and
coat extract as new natural cosmetic ingredients: topical
formulation stability and sensory analysis. Carbohydr
Polym. 2015;134:573–580.
[29] Pinheiro MI. Desenvolvimento Tecnol�ogico de Emulgel de
Anfotericina B com promotor qu�ımico de liberaç~ao cutânea
[Technological Development of Emulgel of Amphotericin B
with the chemical promoter of cutaneous release.] [Master’s
Dissertation – Post-Graduation in Pharmaceutical Sciences].
Piau�ı,. Federal University of Piau�ı. Português; 2014. 123p.
[30] Knorst MT. Desenvolvimento tecnol�ogico de forma farm-
acêutica pl�astica contendo extrato concentrado de
Achyrocline satureioides (Lam.) DC. Compositae (marcela)
[Development of a pharmaceutical form containing concen-
trated extract of Achyrocline satureioides (Lam.) DC.
Compositae (marcela)] [Masters dissertation. Faculty of
Pharmacy. Federal University of Rio Grande do Sul]. Porto
Alegre, Português; 1991. 228p.
[31] Cordeiro MSF, Costa JKB, Lima CG, et al. Desenvolvimento
tecnol�ogico e avaliaç~ao de estabilidade de gel derma-
tol�ogico a partir do �oleo essencial de gengibre (Zingiber ofi-
cinalle Roscoe) [Technological development and evaluation
of dermatological gel stability from ginger essential oil
(Zingiber oficinalle Roscoe)]. Rev Bras Farm.
2013;94:148–153.
[32] Franz TJ. Percutaneous absorption. On the relevance of in
vitro data. J Invest Dermatol. 1975;64:190–195.
[33] Carvalho AL, Silva JA, Lira AA, et al. Evaluation of microe-
mulsion and lamellar liquid crystalline systems for transder-
mal zidovudine delivery. J Pharm Sci. 2016;105:2188–2193.
[34] Figueiredo KA, Neves JKO, Silva JAd, et al. Phenobarbital
loaded microemulsion: development, kinetic release and
quality control. Braz JPharm Sci. 2016;52:251–264.
[35] Oliveira LGC, Brito LM, Alves MMM, et al. In vitro effects
of the neolignan 2,3-dihydrobenzofuran against
Leishmania amazonensis. Basic Clin Pharmacol Toxicol.
2017;120:52–58.
[36] Valadares DG, Duarte MC, Oliveira JS, et al. Leishmanicidal
activity of the Agaricus blazei Murill in different Leishmania
species. Parasitol Int. 2011;60:357–363.
[37] Zeller RA, Carmines EG. Measurement in the social sciences:
the link between theory and data. Cambridge: Cambridge
University Press; 1980.
[38] Figueiredo-Filho DB, Silva-J�unior AS. Vis~ao al�em do alcance:
uma introduç~ao �a an�alise fatorial. Opin Publica.
2010;16:160–185.
[39] Soliman SM, Abdelmalak NS, El-Gazayerly ON, et al.
Novel non-ionic surfactant proniosomes for transdermal
delivery of lacidipine: optimisation using 2(3) factorial
design and in vivo evaluation in rabbits. Drug Deliv. 2016;
23:1608–1622.
[40] Canuto GAB, Xavier AAO, Neves LC, et al. Caracterizaç~ao
F�ısico-Qu�ımica de Polpas de Frutos Da Amazônia e sua
Correlaç~ao com a Atividade Anti-Radical Livre. Rev Bras
Frutic. 2010;32:1196–1205.
[41] Mollica JA, Ahuja S, Cohen J. Stability of pharmaceuticals. J
Pharm Sci. 1978;67:443–465.
[42] Silva KER, Alves LDS, Soares MFR, et al. Modelos de
avaliaç~ao da estabilidade de f�armacos e medicamentos
para a ind�ustria farmacêutica [Models of evaluation of the
stability of drugs and medicines for the pharmaceutical
industry]. Rev Ciênc Farm B�asica Apl 2009a;30:129–135.
[43] Klang V, Matsko NB, Valenta C, et al. Electron microscopy of
nanoemulsions: an essential tool for characterisation and
stability assessment. Micron 2012;43:85–103.
[44] Gonçalves GMS, Campos PMBGM. Aplicaç~ao de m�etodos de
biof�ısica no estudo da efic�acia de produtos dermo-
cosm�eticos. Braz J Pharm Sci. 2009;45:1–10.
[45] Baibhay J, Gurpreet S, Rana AC, et al. Development and
characterization of clarithromycin emulgel for topical deliv-
ery. Int J Drug Develop Res. 2012;4:310–323.
[46] Silva JA, De Santana DP, Bedor DGC, et al. Estudo de liber-
aç~ao e permeaç~ao in vitro do diclofenaco de dietilamônio
em microemuls~ao gel-like [In vitro release and permeation
study of diclofenac from diethylammonium in microemul-
sion gel-like]. Qu�ım Nova. 2009;32:1389–1393.
[47] Kaur IP, Kakkar S. Topical delivery of antifungal agents.
Expert Opin Drug Deliv. 2010;7:1303–1327.
[48] Ara�ujo GMF. Nanoemuls~oes de Anfotericina B: desenvolvi-
mento, caracterizaç~ao e atividade leishmanicida
[Amphotericin B nanoemulsions: development, characteriza-
tion and leismanicidal activity] [Master's Dissertation in
Pharmaceutical Sciences]. Campina Grande, State University
of Para�ıba. Português; 2013. 91p.
[49] Naoui W, Bolzinger MA, Fenet B, et al. Microemulsion
microstructure influences the skin delivery of an hydrophilic
drug. Pharm Res. 2011;28:1683–1695.
[50] Andrade AO, Parente ME, Ares G. Screening of mucoadhe-
sive vaginal gel formulations. Braz J Pharm Sci. 2014;50:931.
[51] Su~ner J, Calpena AC, Clares B, et al. Development of clo-
trimazole multiple W/O/W emulsions as vehicles for drug
delivery: effects of additives on emulsion stability. AAPS
PharmSciTech. 2017;18:539–550.
[52] Roggia I, Ferrony D, Alves MP, et al. Validaç~ao de metodo-
logia anal�ıtica para a determinaç~ao de benzofenona-3
nanoencapsulada incorporada em creme gel e estudo da
estabilidade f�ısico qu�ımica. R [Validation of analytical
methodology for the determination of benzophenone-3
nanoencapsulated incorporated in gel cream and study of
physical chemical stability]. Ciênc Farm B�asica Apl.
2014;35:223–232.
[53] Nikumbh KV, Sevankar SG, Patil MP. Formulation develop-
ment, in vitro and in vivo evaluation of microemulsion-
based gel loaded with ketoprofen. Drug Deliv. 2015;
22:509–515.
[54] USP – The United States Pharmacopeial Convention.
Monographs Amphotericin B. December 1, 2016.
[55] Mora-Huertas CE, Fessi H, Elaissari A. Polymer-based nano-
capsules for drug delivery. Int J Pharm. 2010;385:113–142.
[56] Ramteke KH, Dighe PA, Kharat AR, et al. Mathematical mod-
els of drug dissolution: a review. Sch Acad J Pharm.
2014;3:388–396.
10 E. S. COÊLHO ET AL.
[57] Arcanjo DD, Costa-Junior JS, Moura LH, et al.
Garcinielliptone FC, a polyisoprenylated benzophenone
from Platonia insignis Mart., promotes vasorelaxant effect
on rat mesenteric artery. Nat Prod Res. 2014;28:923–927.
[58] Silva AP, Oliveira GL, Medeiros SC, et al. Pre-clinical toxicol-
ogy of garcinielliptone FC, a tautomeric pair of polypreny-
lated benzophenone, isolated from Platonia insignis Mart
seeds. Phytomedicine. 2016;23:477–482.
[59] Uekane TM, Nicolotti L, Griglione A, et al. Studies on the vola-
tile fraction composition of three native Amazonian–Brazilian
fruits: Murici (Byrsonima crassifolia L., Malpighiaceae), bacuri
(Platonia insignis M., Clusiaceae), and sapodilla (Manilkara
sapota L., Sapotaceae). Food Chem. 2017;219:13–22.
[60] Costa-Junior JS, Ferraz ABF, Filho BAB, et al. Evaluation of
antioxidante effects in vitro of garcinielliptone FC (GFC) iso-
lated from Platonia insignis Mart. J Medicinal Plants Res.
2011;5:293–299.
[61] Nascimento JL, Coelho AG, Barros YSO, et al. Avaliaç~ao da
atividade antioxidante in vitro do extrato hexânico da
semente do bacuri (Platonia insignis Mart.) e de seu com-
plexo de inclus~ao com b-ciclodextrina. Bol Inform Geum.
2014;5:44–53.
[62] Yamaguchi KKL, Pereira CVL, Lima ES, et al. Qu�ımica e
Farmacologia do Bacuri (Platonia insignis). Scien Amazonia
2014;3:39–46.
[63] Nwaka S, Hudson A. Innovative lead discovery strategies
for tropical diseases. Nat Rev Drug Discov. 2006;5:941–955.
[64] Osorio E, Arango GJ, Jim�enez N, et al. Antiprotozoal and
cytotoxic activities in vitro of Colombian Annonaceae. J
Ethnopharmacol. 2007;111:630–635.
[65] Layegh P, Rajabi O, Jafari MR, et al. Efficacy of topical lipo-
somal amphotericin B versus intralesional meglumine anti-
moniate (Glucantime) in the treatment of cutaneous
leishmaniasis. J Parasitol Res. 2011;2011:1.
[66] Ruiz HK, Serrano DR, Dea-Ayuela MA, et al. New amphoteri-
cin B-gamma cyclodextrin formulation for topical use with
synergistic activity against diverse fungal species and
Leishmania spp. Int J Pharm. 2014;473:148–157.
DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY 11
	Abstract
	Introduction
	Materials and methods
	Materials
	Technological development of formulations
	Obtaining emulgel
	Experimental design
	Physicochemical characterization and preliminary stability
	Macroscopic analysis of the formulations
	Centrifuge test
	Preliminary stability test
	Measurement of globule size
	Determination of pH
	Determination of electrical conductivity
	Determination of viscosity
	Determination of spreading
	Determination of amphotericin B content by ultraviolet spectrophotometry (UVVIS)
	In vitro release assays
	In vitro release profile determination using Franz type diffusion cells
	Evaluation of antileishmanial activity in vitro
	Obtainment of cells and parasites
	The antileishmanial activity of promastigote forms of L. major
	Cytotoxicity determination
	Macrophage cytotoxicity and selectivity index (SI)
	Statistical analysis
	Pareto diagrams analysis
	Results
	Technological development
	Antileishmanial activity on promastigote forms of L. major
	Cytotoxicity on macrophages and calculation of the selectivity index (IS)
	Discussion
	Conclusions
	Disclosure statement
	References