Content uploaded by Elisabete Pereira dos Santos
Author content
All content in this area was uploaded by Elisabete Pereira dos Santos on Aug 28, 2015
Content may be subject to copyright.
1304
KEY WORDS: antifungal, in vivo retention studies, release studies, tape stripping, topical release
* Author to whom correspondence should be addressed. E-mail: ricci@pharma.ufrj.br
Latin American Journal of Pharmacy
(formerly Acta Farmacéutica Bonaerense)
Lat. Am. J. Pharm. 34 (7): 1304-10 (2015)
Regular article
Received: February 24, 2015
Revised version: April 29, 2015
Accepted: May 1, 2015
Development and Characterization of Ketoconazole Cream
To Treat Superficial Mycoses:
In Vitro Permeation and In Vivo Retention Studies
Zaida M.F. DE FREITAS
1
, Eduardo RICCI-JUNIOR
1
*, Carolina S. BEMVINDO
1
,
Elisabete P. DOS SANTOS
1
, Silvia STORPIRTIS
2
, Chang CHIANN
3
& Nadia M. VOLPATO
4
1
Faculty of Pharmacy, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil
2
Faculty of Pharmaceutical Sciences, University of São Paulo (USP), São Paulo, Brazil
3
Institute of Mathematics and Statistics, University of São Paulo (USP), São Paulo, Brazil
4
Faculty of Pharmacy, Federal University of Rio Grande do Sul (UFRGS), Porto Alegre, Brazil
SUMMARY. The ketoconazole cream developed in the laboratory was compared with the reference prod-
uct and a generic product in relation to physical-chemical characterization (pH, viscosity and content), in
vitro release, permeation through a biological membrane, and in vivo retention by tape stripping. The re-
sults of in vitro release study indicated the ketoconazole flux from reference product (RP), generic product
(GP) and ketoconazole cream (KC) was 2.41, 2.16 and 2.49 μg/cm
2
/h, respectively. In the permeation stud-
ies, the three formulations were similar in relation to drug retention in the skin layers and did not promote
drug release in the receptor solution. Statistical analysis showed no statistical difference between RP and
GP (p > 0.05) and RP and KC (p > 0.05) in relation to ketoconazole retention in the stratum corneum.
Thus, we conclude that the ketoconazole cream that was developed in this work promoted drug retention
in the stratum corneum in similar amounts to the reference product.
RESUMEN. La crema de ketoconazol desarrollada en el laboratorio se comparó con el producto de referencia y
un producto genérico en relación a la caracterización físico-química (pH, viscosidad y contenido), la liberación
in vitro, la permeación a través de una membrana biológica y la retención in vivo por arrastre de cinta. Los resul-
tados del estudio de liberación in vitro indican que el flujo de ketoconazol del producto de referencia (RP), pro-
ducto genérico (GP) y crema de ketoconazol (KC) fue 2,41, 2,16 y 2,49 μg/cm
2
/h, respectivamente. En los estu-
dios de penetración, las tres formulaciones fueron similares en relación con la retención de fármaco en las capas
de la piel y no promueven la liberación del fármaco en la solución receptora. El análisis estadístico no mostró
ninguna diferencia estadística entre RP y GP (p > 0,05) y RP y KC (p > 0,05) en relación con la retención de ke-
toconazol en el estrato córneo. Por lo tanto, llegamos a la conclusión de que la crema de ketoconazol que se desa-
rrolló en este trabajo promovió la retención del fármaco en el estrato córneo en cantidades similares a las del pro-
ducto de referencia.
INTRODUCTION
Skin mycoses affect millions of people
worldwide every year. There are several antifun-
gal agents used in the treatment of this group of
diseases and ketoconazole is the most widely
used drug. The clinical efficacy of the antifungal
agent depends on factors like concentration of
the drug in the formulation, formulation type,
time of contact with the skin, and penetration
ability of the drug in the cutaneous layers
1
. Ke-
toconazole (KTZ, MW = 531.44) is a broad-spec-
trum antifungal agent. It is administered orally
in a dose of 200 or 400 mg per day or topically
in the form of 2% cream twice daily
2,3
. General-
ly, topical administration is chosen for the treat-
ment of superficial cutaneous fungal infections
to avoid the systemic effects of oral administra-
tion
2
. It is effective against several of these su-
perficial infections, such as candidiasis, pitiriasis
versicolor, tinea cruris and dermatophytosis
2-3
.
In the case of superficial cutaneous infections
by dermatophytes, where the microorganism re-
sides on the outermost layers of the skin, the
antifungal must be delivered in adequate con-
centrations to inhibit the fungal growth.
The 2% ketoconazole cream (KC) was devel-
oped in the Galenic Development Laboratory
(LADEG) of the Faculty of Pharmacy of Federal
ISSN 0326 2383 (printed ed.)
ISSN 2362-3853 (on line ed.)
1305
Latin American Journal of Pharmacy - 34 (7) - 2015
University of Rio de Janeiro. KC has been pre-
scribed by dermatologists for the patients of the
dermatology sector of Clementino Fraga Filho
University Hospital. In vitro permeation and in
vivo retention studies are important to verify
whether the ketoconazole cream has character-
istics similar to the reference product.
In vitro release studies can be used to evalu-
ate the release rate of the drug from the formula-
tion. These studies are based on diffusion cells
and synthetic membranes
4
. These membranes do
not offer resistance to drug diffusion from donor
chamber to receptor chamber, allowing determi-
nation of the release rate. This study is simple
and allows verifying similarities between the re-
lease rate and flow of the studied samples
5
.
In vitro permeation studies are used to as-
sess the degree of penetration and drug reten-
tion in the skin layers. These studies are based
on diffusion cells and natural membranes. Pig
ear skin has been used as a natural membrane
due to its anatomical and physiological similari-
ty with human skin
5
.
In the case of superficial fungal infections,
microorganisms live and grow in the stratum
corneum (SC). Thus, a semisolid formulation
must release the ketoconazole in amounts suit-
able for retention in the SC and inhibition of
fungal growth. This approach can be investigat-
ed by in vivo retention studies using the tape-
stripping technique. In this technique, the SC is
removed from the total skin by repeated appli-
cations of adhesive tapes. Thus, the drug is ex-
tracted from the SC and quantified
6,7
.
Our group developed and characterized 2%
ketoconazole cream (KC). The samples were
characterized for pH, viscosity, content, in vitro
release, in vitro permeation using a natural
membrane, and in vivo retention by tape strip-
ping. The results obtained with KC were com-
pared with the results of the reference product
(RP), a generic product (GP) and compounded
product (CP) to verify the similarity of the for-
mulations.
MATERIALS AND METHODS
Materials
The reference product (RP) and generic
product (GP) were 2% ketoconazole creams and
were purchased from a drugstore. The com-
pounded product (CP) (2% ketoconazole cream)
was purchased from a compounding pharmacy.
Octyl stearate, petroleum jelly, lauryl glucoside
polyglyceryl, 2-dipolihidroxiestearato, propylene
glycol, imidazolinidyl urea, cetostearyl alcohol,
sodium alkyl sulfate and ketoconazole, butylhy-
droxytoluene, all pharmaceutical grade, were
purchase from Fragon (Brazil). Methanol,
ethanol, acetonitrile and K
2
PO
4
was purchased
from Tédia (Brazil).
Formulation
The formulation of the Reference Product
(RP) (official information) contains stearyl alco-
hol, cetyl alcohol, isopropyl myristate, sorbitan
monostearate, polysorbate, sodium sulfite,
propylene glycol, purified water and ketocona-
zole. The generic product (GP) contains the
same composition as RP (official information).
The 2% ketoconazole cream (Compounding
Product) was purchased from a compounding
pharmacy. The compounding product (CP) con-
tains octyl stearate, liquid petrolatum, methyl-
paraben, propylparaben, petrolatum, sodium
metabisulfite, cetostearyl alcohol, sodium alkyl
sulfate, purified water, and ketoconazole. The
manufacturer did not provide the amount of
each component of the formulations.
The formulation of the 2% ketoconazole
cream (KC) is described in Table 1. Reactors
containing a heating jacket and overhead stirrer
were used to prepare this cream. The compo-
nents of the oil phase (1-8) and aqueous phase
(9) were separately heated to 75 and 80 °C, re-
Component
Ketoconazole
cream (g)
1. Octyl Stearate 15
2. Petroleum Jelly 5
3. Lauryl glucoside polyglyceryl/2-dipolihidroxiestearato (1:1) 2
4. Propylene glycol 20
5. Imidazolinidyl urea 0.5
6. Cetostearyl alcohol/sodium alkyl sulfate (1:1) 10
7. Ketoconazole 2
8. Butylhydroxytoluene 0.2
9. Distilled water 47.5
Table 1. Components of the formulation: 2% ketoconazole cream.
1306
DE FREITAS Z.M.F., RICCI-JUNIOR E., BEMVINDO C.S., DOS SANTOS E.P., STORPIRTIS S., CHIANN C. & VOLPATO N.M.
spectively. The aqueous phase was added to the
oil phase with constant stirring to form the KC.
Characterization
RP, GP, CP, and KC were characterized by
determination of pH, viscosity and content. The
pH was determined using a potentiometer with
glass electrode for semi-solid systems without
dilution of the product. Viscosities were mea-
sured with Brookfield digital viscometer (DV-II)
using a Helipath model D apparatus, using 100
g of sample at room temperature (25 oC), F
spindles, and speeds of 0.3 and 0.6 rpm.
The extraction method of the drug from the
formulation is described below. One gram of
sample and 15 mL of tetrahydrofuran were
added to a 100 mL volumetric flask. The mixture
was homogenized for 6 min and then the vol-
ume was completed with methanol, after which
2 mL of the methanol solution were diluted with
the mobile phase in another volumetric flask (25
mL). The solution was filtered using a Millex
Millipore filter (syringe filter, hydrophilic PVDF
membrane, pore size of 0.45 m).
The concentration of ketoconazole in the
samples was measured by HPLC (Waters Corpo-
ration, Milford, MA, USA) using a C-18 column,
mobile phase of acetonitrile and K2HPO4 (0.01
M, pH 6.8, 50:50), flow of 0.7 mL/min and de-
tection at 242 nm
8
.
In vitro release study
In vitro release studies were based in diffu-
sion cells and a synthetic membrane. The area
for diffusion was 1.13 cm
2
and the receptor
chamber volume was 6.5 mL. The receptor solu-
tion was a hydro-alcoholic mixture (70:30 v/v).
This medium was selected in order to raise the
solubility of the KTZ from 5 µg/mL in pure wa-
ter to 100 µg/mL in the mixture, thus allowing
the establishment of a drug flux in the system
without drug saturation in the receptor cham-
ber. This receptor medium has been used in
other release studies
9,10
. Cellulose acetate mem-
branes (Sigma, Germany) were used as support
for the samples. Each receptor compartment
was submitted to magnetic stirring. One gram of
sample was added in the donor compartment of
the diffusion cell. At pre-determined time inter-
vals, 600 µl of the receptor solution was re-
moved and replaced with the same volume of
hydro-alcoholic solution. The samples were fil-
tered using a Millex Milipore filter and analyzed
by HPLC.
Release profiles were constructed by the re-
lationship between the amount released as a
function of time and area. The mass transport
was expressed in terms of flux. The results are
the mean ± SD of six experiments (n = 6 deter-
minations) and the data were submitted to sta-
tistical analysis (one-way ANOVA, α = 0.05) em-
ploying the SS for Windows 1.0 software.
In vitro permeation studies
The permeation studies were performed us-
ing a diffusion cell. Six diffusion cells were used
in this study (n = 6 determinations). The experi-
mental conditions were established according to
the literature
11
. The excised porcine skin was
placed on the diffusion cell with the stratum
corneum oriented toward the donor compart-
ment. The receiver compartment was filled with
PBS pH 7.4. Diffusion cells were maintained at
37 ± 0.5 °C using a thermostatted circulating wa-
ter bath and continuously stirred with a magnet-
ic bar. The skins remained in contact with the
receptor medium for 1 h to reach equilibrium of
the system. One gram of sample was added to
the donor compartment and thus the studies oc-
curred under the condition of infinite amount of
drug. After application of the formulation, an
aliquot of 600 µL of the receptor medium was
removed for analysis every 2 h. Then 600 µL of
PBS pH 7.4 was replaced in the receptor cham-
ber of the diffusion cell. The drug’s presence in
the receptor medium was monitored for 8 h.
Aliquots of receptor medium were filtered and
the amount of ketoconazole permeated was de-
termined by HPLC.
After 8 h, the system was dismounted and
the skin was removed from the cell. The formu-
lation was removed from the skin surface with
cotton. The skin was cleaned with moistened
cotton and dried with paper towels (three
times). The epidermis was separated from the
dermis with a scalpel by a method developed
by our group
5,12
. Ketoconazole was extracted
from the epidermis or dermis following an ex-
traction protocol: 1.5 mL of extractor solvent
(water:acetonitrile:methanol - 40:30:30) was
added to a test tube containing epidermis or
dermis. The tubes were shaken for 30 s (three
times) and centrifuged at 10,000 × g for 10 min.
The solutions were filtered and the amount of
ketoconazole retained in the epidermis or der-
mis was determined by HPLC.
The method for extraction of ketoconazole
from epidermis and dermis described above was
validated by our group in triplicate (n = 3 deter-
minations). Twenty µL of ethanol solution of ke-
1307
Latin American Journal of Pharmacy - 34 (7) - 2015
toconazole at 80, 160, or 320 µg/mL was added
in the tubes containing epidermis or dermis. Af-
ter drying of the ethanol, 1.5 mL of the extractor
solvent mixture (water:acetonitrile: methanol -
40:30:30) was added to the tissues. The tubes
were agitated for 30 s (three times), centrifuged
(10,000 × g for 10 min), and filtered using a
Millex Milipore filter The amount of drug ex-
tracted from tissues was measured by HPLC.
The permeation data were analyzed by two-
way ANOVA. The drug retentions in the epider-
mis and/or dermis were compared for evalua-
tion of similarities or differences between the
three formulations: reference, generic and keto-
conazole cream developed in the study.
In vivo retention studies by tape stripping
Eight healthy women volunteers, aged 22-53,
were included in the in vivo tape stripping
study. This experiment was approved by the
ethics committee of Federal University of Rio de
Janeiro (Protocol numbers CEP:035/02 and
CIC:067/02). Written consent was obtained from
each volunteer. The sites of application of the
samples were the surfaces of the ventral fore-
arms. The volunteers’ forearms were washed
with water and neutral soap one h before the
experiment. Six areas of 2.5 cm2 were marked
in both ventral forearm surfaces to study the
same formulation. Fifteen mg of sample was ap-
plied in each marked site and left for times of
15, 60, 120 and 240 min (Fig. 1)
13
. After each
period, the excess formulation was removed
with three applications of cotton. The SC was
removed by application of eleven adhesive
Figure 1. Diagram of the marked areas on the volun-
teers’ ventral forearms: the formulation was applied
in the central area of the hexagon; the square of 1.56
cm
2
corresponds to removal field of SC. C(+): positive
control and C(-): negative control.
Right ventral forearm Left ventral forearm
Products’ application
tapes (Transpore, 3M, 1.56 cm
2
). The tapes were
pressed on the skin six times using a glass rod
and then they were quickly removed. The first
one was discarded to avoid overestimation of
the amount of drug. Ten tapes were analyzed
with the objective of quantifying the total drug
mass retained in the SC. They were fixed on a
metal staff, which was introduced in a glass
tube with 10 mL of the extractor solvent mixture
(water:acetonitrile:methanol - 40:30:30). Keto-
conazole was quantified by HPLC.
As controls, two untreated skin areas were
also marked: negative control and positive con-
trol. In the positive control, 0.5 µg of ketocona-
zole (10 µL of a solution of 50 µg/mL) was
added to each adhesive tape, yielding 5 µg of
drug (n = 10 tapes). The extraction technique
described above was applied in the tapes con-
taminated with ketoconazole to calculate the re-
covery. The recovery results were expressed in
µg of ketoconazole/mg of stratum corneum.
Thus, the mass of the stratum corneum removed
by tape stripping was determined using the pro-
tocol described below.
The tissue weight was measured to detect
the amount of stratum corneum removed by
each tape. The weight of the adhesive tape was
measured with an analytical microbalance (AD-
4, Perkin Elmer) before and after the tape strip-
ping. Eleven tapes were removed from both
ventral forearms of eight volunteers. Each tape
was pressed against the area marked by a glass
rod (to roll the rod under the area six times).
Then, the tapes were removed and individually
weighed. The first tape was discarded. The aver-
age weight of the stratum corneum was ob-
tained for each volunteer and normalized by the
area (mg/cm
2
). These data were used to calcu-
late the retention of the ketoconazole in µg per
mg of stratum corneum
14,15
.
The in vivo retention data were analyzed by
two-way analysis of variance (ANOVA). The
drug retention in the SC after application of RP
(reference product) was compared with the
drug retention afforded by GP (generic product)
or KC (ketoconazole cream) in the hypothesis
test (α = 0.05).
Analytical methods
The drug was analyzed by a method adapted
from the literature
8
. Ketoconazole content was
quantified by HPLC (Waters Corporation, Mil-
ford, MA, USA) using a C18 column, injector
with loop of 50 µL, mobile phase of 50:50 (v/v)
acetonitrile: 0.01 M K
2
HPO
4
(pH 6.8), flow rate
1308
DE FREITAS Z.M.F., RICCI-JUNIOR E., BEMVINDO C.S., DOS SANTOS E.P., STORPIRTIS S., CHIANN C. & VOLPATO N.M.
of 0.7 mL/min, and detection wavelength of 242
nm. The analytical curve was linear of 0.2 at 10
µg/mL with R
2
> 0.9998. Detection Limit (DL)
and Quantification Limit (QL) were 25 and 80
ng/mL, respectively.
RESULTS AND DISCUSSION
The results of characterization of the formu-
lations are shown in Table 2. The formulations
were characterized for pH, viscosity and drug
concentration. According to the literature, skin
pH can vary from 4.0 to 7.0
16
. The pH of RP,
CP and KC were within the pH range of the
skin, however the GP formulation exhibited pH
above 7.0. The pH of GP was within the range
specified by the manufacturer and near the pH
range of the skin. GP exhibited higher viscosity.
The formulations contained concentrations with-
in the range of acceptable limits of 90 to 110%
recommended by the USP 34
th
edition
17
.
Fig. 2 shows the curves of the in vitro re-
lease studies of the formulations using synthetic
membrane of cellulose acetate and vertical dif-
fusion cell. The release profiles were linear, in-
dicating steady-state flux (J
ss
) (Table 2). The ke-
Figure 2. Amount of KTZ released per area during
the in vitro release studies. RP-Reference Product,
GP-Generic Product, CP-Compounding Product and
KC-Ketoconazole Cream. Mean ± SD of n = 6 deter-
minations.
Viscosity (x10
3
cP)
Formulation pH Content (%) (p/p) Flux (µg/cm
2
/h)
a
0.3 rpm 0.6 rpm
RP 6.85 100.9 741 534 2.41 ± 0.16
GP 7.64 107.6 1470 1135 2.16 ± 0.09
CP 5.68 97.9 593 463 5.85 ± 1.00
KC 5.68 105.0 567 386 2.49 ± 0.11
Table 2. Characterization of the formulations.
a
Mean ± SD of n = 6 determinations.
Drug recovery
Epidermis (%) Dermis (%)
1.6 96.4 ± 3.1 96.0 ± 10.8
3.2 103.64 ± 7.6 95.49 ± 3.9
6.4 106.9 ± 8.5 98.2 ± 6.4
Table 3. Drug recovery (%) from the skin layers and
coefficient of variation. Mean ± SD of n = 3 determi-
nations.
Ketoconazole
mass (µg)
toconazole flux was defined from the slope of
the release curve between the first and third
hour. These data indicated that the drug release
from CP was greater than from RP and GP. The
composition of the formulation CP is different
RP. CP does not have propylene glycol in their
composition. The difference in the composition
creates an unfavorable environment to the drug
increasing its flow to the receiver solution dur-
ing the release studies. The preparation proce-
dure and formulation components resulted in
higher thermodynamic activity for the drug from
the CP. In the experimental conditions em-
ployed, we obtained a flux twice that achieved
by commercial RP and GP products. The RP and
GP contained the same components (label dec-
laration), with high content of propylene glycol.
According to Florence & Attwood
18
, propylene
glycol behaves as co-solvent and can modify the
thermodynamic activity of the drug by increas-
ing its solubility in the vehicle. Moreover, keto-
conazole presented high affinity for the vehicle,
resulting in small partition to receptor medium.
The formulations RP and GP had similar fluxes
and the differences were not significant (p >
0.05). Ketoconazole cream (KC) was developed
from the reference product and had high con-
tent of propylene glycol. The drug flux from the
KC formulation was similar to that of the RP and
differences were not significant (p > 0.05). Thus,
only KC was used in the studies using natural
membranes for evaluation of drug retention in
the skin layers.
Time (h)
Ketoconazole release (μg/cm
2
)
RP
GP
KC
CP
1309
Latin American Journal of Pharmacy - 34 (7) - 2015
The results of the drug recovery of the skin
layers are exhibited in Table 3. The drug recov-
ery was excellent (recovery > 95%). Thus, the
extraction technique was suitable for drug re-
covery from the epidermis and dermis. The pos-
sibility of drug diffusion through the skin into
the receptor compartment of the diffusion cell
was monitored during the permeation studies.
The detection limit (DL) of the chromatographic
method was 25 ng/mL. Thus, it was not possible
to detect the ketoconazole concentration below
the LD. Ketoconazole concentrations above the
quantification limit (80 ng/mL) were efficiently
detected and quantified.
Fig. 3 shows the amount of ketoconazole re-
tained in the epidermis or dermis 8 h after ap-
plication of the formulations. The amount of
drug in the dermis was smaller than in the epi-
dermis for the three formulations, but the differ-
ence was not significant (p > 0.05). The drug re-
tention in the epidermis was not statistically dif-
ferent between the three formulations (p >
0.05). Moreover, a similar result was observed
for the dermis and ketoconazole retention was
not statistically different for the three formula-
tions (p > 0.05). The three formulations were
similar in relation to drug retention in the skin
layers. Another important fact that should be
noted is that the formulations did not promote
drug release in the receptor solution during the
permeation studies. Thus, the three formulations
were selected for the in vivo tape stripping.
Figure 3. Amount of ketoconazole retained in the
epidermis or dermis 8 h after application of the for-
mulations. RP-Reference Product, GP-Generic Product
and KC-Ketoconazole Cream. Mean ± SD of n = 5 de-
terminations.
Figure 4. Amount of stratum corneum removed by
adhesive tape. Mean ± SD of n = 16 determinations.
Formulations
RP GP KC
Epidermis
Dermis
Ketoconazole retained (μg/cm
2
)
Order of removal of the stratum corneum by tapes
Amount of stratum corneum (μg)
The tape stripping method is useful to deter-
mine the in vivo drug retention in the stratum
corneum (SC). Fungal growth on or within the
SC can be inhibited when there are adequate
amounts of ketoconazole at the target site. The
amount of drug from formulations retained in
the stratum corneum can be compared by the
tape stripping technique. For that, it is essential
to optimize the extraction condition of the drug
from adhesive tapes. Here this extraction tech-
nique provided recovery of 91 ± 1.1% of keto-
conazole. Moreover, in the negative control, no
compound was detected after the retention time
of the ketoconazole (around 7.6 min).
Fig. 4 shows the amount of stratum corneum
removed by adhesive tape. This study was car-
ried out to determine the amount of stratum
corneum removed by each adhesive. The stra-
tum corneum (SC) of both ventral forearms of
volunteers was removed by the tapes and
weighed to normalize the drug retention per
mass of SC removed (Fig. 4). The average
weight was 0.474 ± 0.0034 mg of SC removed
by application of 10 tapes. The mean weight
was 0.3038 ± 0.0033 mg of SC per 1 cm
2
after
normalization for area (1.56 cm
2
).
Fig. 5 shows the ketoconazole retention per
mass of stratum corneum. This retention in-
creased as a function of time for the three for-
mulations up to 240 min. The KC was compared
statistically with RP in relation to drug retention
in the stratum corneum. Based on ANOVA,
there was no statistical difference between RP
and GP (p > 0.05) and RP and KC (p > 0.05),
except for the time of 15 min (p < 0.05, Fig. 5)
in relation to ketoconazole retention in the SC.
1310
DE FREITAS Z.M.F., RICCI-JUNIOR E., BEMVINDO C.S., DOS SANTOS E.P., STORPIRTIS S., CHIANN C. & VOLPATO N.M.
Acknowledgements. We would like to thank Dr. Lú-
cia Maria Azevedo (Clementino Fraga Filho University
Hospital - Federal University of Rio de Janeiro) for
clinical investigation of the volunteers and Simone
Pereira da Silva (Chemical Institute/Federal University
of Rio de Janeiro) for measurements with the mi-
crobalance. We also acknowledge the research grant
from Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior (CAPES/PICDT).
REFERENCES
1. Kaur, I.P. & S. Kakkar (2010) Expert Opin.
Drug Deliv. 7: 1303-27.
2. Lesher, J.L. (1996) Dermatol. Clin. 14: 163-9.
3. Heel, R.C., R.N. Brodgen, A. Carmine, P.A.
Morley & T.M. Speight (2012) Drugs
3: 1-36.
4. Silva, F.F.F., E. Ricci-Júnior & C.R.E. Mansur
(2013) Drug Dev. Ind. Pharm. 39: 1378–88.
5. Santis, A.K., Z.M.F. Freitas, E. Ricci-Júnior, L.
Brito-Gitirana L, L.B. Fonseca & E.P. Santos
(2013) Drug Dev. Ind. Pharm.
39: 1098-106.
6. Pershing, L.K. (2000) Cosm. Toiletries 115: 43-
51.
7. Reddy, M.B., A.L. Stinchcomb, R.H. Guy &
A.L. Bunge (2010) Pharm. Res.
19: 292-8.
8. Gonçalves, J.R.O., C.S. Bemvindo & N.M. Vol-
pato (2010) Pharmatech 1: 358-62.
9. Shah, V.P., C.R. Behl, G.L. Flynn, W.I.
Higuchi & H. Schaefe (1992) J. Pharm. Sci.
81:
1051-4.
10. Shah, V.P., J.S. Elkins & R.L. Williams (1999)
Pharm. Dev. Tech.
4: 377-85.
11. Silva, E.R., Z.M.F. Freitas, L. Brito-Gitirana &
E. Ricci-Júnior (2011) Drug Dev. Ind. Pharm.
37: 569-75.
12. Mota, A.V., Z.M.F. Freitas, E. Ricci-Júnior,
G.M.D. Ortiz, R.S. Oliveira, R.A. Ozzetti, et al.
(2013) Int. J. Nanomed. 8: 4689-701.
13. Shah, V.P., G.L. Flynn, A. Yacobi, H.I.
Maibach, C. Bon, N.M. Fleischer, et al. (2008)
Pharm. Res.
15: 167-71.
14. Weigmann, H.J., J. Lademann, R.V. Pelchrzim,
W. Sterry, T. Hagemeister, R. Molzahn, et al.
(1999) Skin Pharmacol. Appl. Skin Physiol.
12:
46-53.
15. Weigmann, H.J., J. Lademann, S. Schanzer, U.
Lindemann, R.V. Pelchrzim, H. Schaefer, et al.
(2001) Skin Pharmacol. Appl. Skin Physiol.
14:
98-102.
16. Wilhelm, K.P., A.B. Cua & H.I. Maibach
(1991) Arch. Dermatol. 127: 1806-9.
17. United States Pharmacopeia. 34. ed. (2013)
Rockville: U.S.P. Convention.
18. Florence, A.T. & D. Attwood (2012) Physical
Pharmacy, Pharmaceutical Press, London, pp.
1-232.
Moreover, there was no statistical difference be-
tween right and left forearms in relation to drug
retention.
CONCLUSION
The ketoconazole cream developed in this
study showed similar characteristics to the RP.
The release profile of the drug from the KC for-
mulation was similar to the profile obtained
from the RP. Moreover, similarity in the drug re-
lease profile was also achieved by the GP for-
mulation in relation at RP. However, the keto-
conazole release profile from the CP was differ-
ent and was excluded in the other experiments.
RP, GP and KC provided similar retention of
the drug in the epidermis and dermis without
significant differences. Furthermore, it was not
possible to detect the ketoconazole concentra-
tions below the LD. We were not interested in
passage of the drug into the bloodstream, so the
three formulations were selected for studies by
tape stripping.
In vivo tape stripping showed similarities be-
tween the formulations. The ketoconazole reten-
tion in the stratum corneum increased with
time. There were no significant differences be-
tween drug retention in the stratum corneum
from KC and RP. Thus, we can conclude that
the 2% ketoconazole cream (KC) developed in
this study has similar characteristics to the refer-
ence product (RP) available in the market.
Figure 5. Amount of ketoconazole retained per mass
of stratum corneum (SC) in function of time during
the in vivo retention studies by tape stripping. RP-Ref-
erence Product with mean ± SD of n = 14 determina-
tions; GP-Generic Product with mean ± SD of n =15
determinations; KC-Ketoconazole Cream with mean ±
SD of n = 14 determinations; * significant difference
(p < 0.05).
Time
15 min 60 min 120 min 240 min
RP GP KC
μg of ketoconazole/mg of SC
