International Journal of Pharmaceutics 478 (2015) 429–438

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International Journal of Pharmaceutics

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

Personalised medicine

Dilution of semi-solid creams: Influence of various production

parameters on rheological properties and skin penetration
C. Nagelreiter a , E. Kratochvilova a , C. Valenta a,b, *
a
University of Vienna, Department of Pharmaceutical Technology and Biopharmaceutics, Althanstraße 14, 1090 Vienna, Austria
b
University of Vienna, Research Platform ‘Characterisation of Drug Delivery Systems on Skin and Investigation of Involved Mechanisms’, Vienna, Austria

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

Article history: In order to customise treatment for patients, topical formulations are often diluted with drug-free cream
Received 15 September 2014 bases to adjust the drug dose and thereby the formulations' activity to the patients' needs. However, the
Received in revised form 28 November 2014 process of dilution influences properties of the formulations. Stability can be reduced as well as the
Accepted 29 November 2014
microbial stability and most importantly, efficacy and skin penetration behaviour can be severely and
Available online 2 December 2014
unpredictably changed.
The present study investigates the effects of production parameters on creams, namely incorporation
Keywords:
of an API (active pharmaceutical ingredients) into an OW cream with prior mixing with propylene glycol
Manual mixing
Microscopic characterisation
or without and subsequent automated or manual dilution of the resulting creams with three different
Rheological characterisation cream bases. Effects were measured by influence on microscopic appearance, measurement of chemical
Chemical stability stability, skin penetration and rheological behaviour.
Result: suggest strong influence of the cream bases used for dilution of the formulations. Mixture of equal
amounts of the employed OW and WO cream proved unfavourable due to inferior penetration behaviour
and less appealing microscopic and macroscopic appearance. Prior mixing with PG was of negligible
importance for the characteristics of the dilutions, however, the type of API and manner of dilution had an
influence on the viscosity of the formulations.
ã 2014 Elsevier B.V. All rights reserved.

1. Introduction like lipophilicity or drug content, for example. This therapeutic

Dermal drug delivery is a favourable way of treatment for
localised afflictions as it alleviates the conditions and simulta-
neously minimises the potential of systemic adverse effects
(Hengge et al., 2006). Therefore, in Europe this approach is widely
used for an eclectic variety of conditions and APIs (active
pharmaceutical ingredients) including non-steroidal anti-inflam-
matory drugs in osteoarthritis (Barthel et al., 2009), antibiotics
against acne vulgaris (Feucht et al., 1980) and foremost corticoste-
roids in the treatment of atopic dermatitis (Lucky et al., 1997; Maia
et al., 2000).
In order to customise topical treatment for the individual,
dermatologists often vary parameters of prescribed formulations
Abbreviations: API, active pharmaceutical ingredient; OW, oil in water; PG,
propylene glycol; SC, stratum corneum; WO, water in oil.
* Corresponding author at: University of Vienna, Department of Pharmaceutical
Technology and Biopharmaceutics, Althanstrasse 14, 1090 Vienna, Austria. Tel.: +43
1 4277 55 410; fax: +43 1 4277 9554.
E-mail address: claudia.valenta@univie.ac.at (C. Valenta).
concept aims to adjust the properties of formulations in order to
meet the patients' needs and therefore, well-established works
summarise and collect pharmaceutical formulations in order to
guide pharmacists in the preparation of such formulations and to
ensure certain quality standards in personalised topical medical
care (DAC/NRF, 2014). Accordingly, topical formulations are often
diluted with cream bases not containing an API (active pharma-
ceutical ingredient) with the intention of adjusting the drug dose
and thereby the formulations' activity to the patients' needs. In
theory, this has the additional advantage of minimising the risk of
adverse effects due to a dosage of drug reduced to the dose
required. However, the process of dilution strongly influences
various properties of the formulations. Stability of the resulting
formulations can be reduced as well as the microbial stability and
most importantly, the efficacy and skin penetration behaviour can
be subject to severe and mostly unpredictable changes (Busse,
1978; Demana, 2014; Magnus et al., 1981; Refai and Müller-
Goymann, 2002; Wiedersberg et al., 2008). These changes can lead
to loss of effect or, contrary, to an effect much more pronounced
than expected and wanted, which in turn may prevent the desired
outcome of the treatment and lead to negative side effects of

http://dx.doi.org/10.1016/j.ijpharm.2014.11.069
0378-5173/ ã 2014 Elsevier B.V. All rights reserved.
430 C. Nagelreiter et al. / International Journal of Pharmaceutics 478 (2015) 429–438

the drugs and thereby reduce patient compliance due to lack of
success with the treatment. Nevertheless, aforementioned practice
of dilution of topical creams is frequently employed in dermal drug
delivery, mostly without proof of concept and despite the
references indicating unpredictable changes in the diluted systems
and therefore advising against dilution of API-containing creams
(Demana, 2014; Mitriaikina and Müller-Goymann, 2009; Refai and
Müller-Goymann, 2002).
In consideration of this, the present study is aiming at gaining
further insight into the effects of various production parameters,
namely (I) prior mixing of the API for a model formulation with PG,
(II) mixing the model formulation with different cream bases to
simulate a dilution process and (III) mixing by hand or
mechanically. The effects of these parameters on the homogeneity
of the resulting cream systems was evaluated microscopically and
furthermore on the chemical stability of three incorporated APIs
being fludrocortisone acetate, diclofenac sodium, and erythromy-
cin belonging to the commonly used drug classes corticosteroids,
non-steroidal anti-inflammatory drugs, and antibiotics was
studied. Moreover, the influence of the listed parameters on the
rheological properties of the resulting formulations was studied.
In a last step the influence of the aforementioned production
parameters on the skin penetration of fludrocortisone acetate was
investigated. This drug was chosen as a model drug to simulate the
use of glucocorticoids in dermatological formulations. These have
an important role in dermatological therapy and are amongst the
most often prescribed drugs in dermatological treatment (Demana,
2014; Ring et al., 2012).
2. Materials and methods
2.1. Materials
Standard Corneofix F 20 adhesive tapes with a surface area of
approximately 4.0 cm2 were purchased from Courage+ Khazaka
GmbH (Cologne, Germany).
The APIs fludrocortisone acetate (CAS: 514,363), erythromycin
(CAS: 114-07-8) and diclofenac sodium (CAS: 15,307-79-6) were
purchased from Sigma–Aldrich (St. Louis, USA), as well as
ammonium acetate (CAS: 631-61-8). Potassium dihydrogen
phosphate (KH2PO4) was purchased from ACROS Organics (Geel,
Belgium).
All solvents as methanol (CAS: 67,561) and acetonitrile (CAS:
75-05-8) were of analytical grade and used as obtained from Sigma
Aldrich.
The industrial cream bases representing the WO, OW and
amphiphilic cream were kind gifts from Bayer Austria GesmbH
(Vienna, Austria). According to the manufacturer, the ingredients
for the cream bases are as follows:
WO cream: purified water, white petrolatum, liquid paraffin,
Dehymuls E (dicocoyl pentaerythrityl distearyl citrate, sorbitan
sesquioleate, cera alba, aluminium stearate), white wax, perfume
oil. Water content is approximately 30% (w/w). This corresponds to
Ultrabas1 of Bayer Austria GesmbH (Vienna, Austria).
OW cream: purified water, white petrolatum, liquid paraffin,
stearylalcohol, macrogolstearate 2000, polyacrylic acid, sodium
EDTA, sodium hydroxide, methyl-4-hydroxybezoate (E 218),
propyl-4-hydroxybenzoate (E 216), perfume oil. Water content is
approximately 70% (w/w). This corresponds to Ultrasicc1 of Bayer
Austria GesmbH (Vienna, Austria).
Amphiphilic cream: purified water, white petrolatum, liquid
paraffin, glycerol distearate, glycerol monostearate, polyoxyethy-
lene 100 stearate, polyoxyethylene-2 and polyoxyethylene-
21 stearyl alcohol, benzyl alcohol, perfume oil. Water content is
approximately 40% (w/w). This corresponds to Ultraphil1 of Bayer
Austria GesmbH (Vienna, Austria).
2.2. Skin tissue
For the skin penetration studies, fresh porcine ears were
purchased from a local abattoir (EU Schlachthof Gantner,
Hollabrunn, Austria). The age of the sacrificed pigs was about
6 months. The ears were removed before exposure of the carcass to
high-temperature cleaning procedures to ensure integrity of the
skin barrier (Herkenne et al., 2006). The excised ears were cooled
during transport, carefully rinsed with cold water and dabbed dry
before storage at 18  C, which is a suitable storage method and
does not affect the stratum corneum (SC) in ways that would
interfere with the envisioned experiments (Hahn et al., 2010; Klang
et al., 2011b; Stracke et al., 2006). All experiments were performed
at room temperature after allowing the ears to thaw. The skin
remained on the porcine ears for easier handling and prevention of
skin contraction (Breternitz et al., 2007; Lademann et al., 2009).
2.3. Production of the formulations
In Table 1 codes for each formulation are presented. Various
production parameters were investigated: the influence of mixing
the API with PG prior to incorporation into the cream, the influence
of manual (M) or automated (T) dilution on the homogeneity of the
formulations and the type of cream base used for dilution of the

Table 1
Codes for the investigated cream dilutions. Based on the 1% API standard formulation from the OW basic cream containing 5% PG, 1:1 dilutions with WO, amphiphilic and OW
creams were produced. OW is the first abbreviation in the code because of the standard formulation contained in every dilution. PG indicates prior mixing with PG, no means
no prior mixing with PG, T means mechanical mixing with the TopiTec1 system, M stands for mixing by hand, cream bases are indicated as follows: OW oil in water cream, A
amphiphilic cream, WO water in oil cream.

Prior mixing with PG Means of mixing Cream for dilution to 0.5% API

Code Yes No Manually Automated WO A OW

OW/PG/T/WO + + +
OW/PG/M/WO + + +
OW/no/T/WO + + +
OW/no/M/WO + + +
OW/PG/T/A + + +
OW/PG/M/A + + +
OW/no/T/A + + +
OW/no/M/A + + +
OW/PG/T/OW + + +
OW/PG/M/OW + + +
OW/no/T/OW + + +
OW/no/M/OW + + +
 C. Nagelreiter et al. / International Journal of Pharmaceutics 478 (2015) 429–438
standard formulation. The influence of the type of model drug in
terms of the quality of mixing was also investigated.
2.3.1. Preparation of 1% standard formulations
In the first step, standard formulations were prepared with the
used OW cream base. In this cream 1% (w/w) of API was
incorporated by carefully weighing and on one hand thorough
mixing with 5% (w/w) PG before adding the OW cream base. On the
other hand, 1% API was incorporated directly into the OW cream
and PG was added as the last ingredient. To achieve a distribution
of the API crystals as homogeneous as possible, both the
formulations with or without prior mixing of the API and PG
were then thoroughly mixed by means of the automated TopiTec1
system (WEPA Apothekenbedarf, Hillscheid, Germany) following
an existing programme of the software in which the formulations
were first mixed for 30 s at 2000 rpm, then for another 3 min at
1000 rpm (Mueller-Goymann and Alberg, 1999; Refai and Müller-
Goymann, 2002).
The thereby obtained standard formulations containing 1% API
served as model formulations and were processed further to
investigate the influence of dilution with various cream bases and
the manner of mixing.
2.3.2. Preparation of the 0.5% formulations
The OW-based standard preparations were then further diluted
by mixing with a WO cream, an amphiphilic cream and the OW
cream, respectively, in a ratio of 1:1, resulting in 0.5% API in each
formulation. The mixing step was performed by hand or
mechanically. Dilution by hand was done by quickly mixing the
two creams with a pestle in a bowl for about 10 s. The formulations
will be referred to by the code stated in Table 1, with the addition of
a postpositive letter indicating the incorporated API, with F for
fludrocortisone acetate, E for erythromycin, and D for diclofenac
sodium. Accordingly, the formulation labelled “OW/PG/T/WO-F”
was produced with prior mixing of the API fludrocortisone acetate
with PG in the OW cream base and subsequent dilution by means
of the TopiTec1 system with the WO cream base, for example.
2.3.3. Microscopic evaluation and concluding macroscopic inspection
After preparation, small samples of each formulation were
transferred onto a microscope slide, covered with a cover slip and
examined at 10-fold magnification with a Nikon Labphot-2 micro-
scope (Nikon Corporation, Tokio, Japan). Representative pictures of
the microscopic image of each formulation were taken with a
Canon Powershot SD630 (Canon, Tokio, Japan) for further
comparison of the quality of mixture and stability of the
formulations as well as the solubility and distribution of visible
API-crystals.
After four months, the procedure was repeated and the new
pictures were compared to the originals for changes like belated
dissolving of API-crystals or indicators of beginning instability and
phase separation.
At the end of the study, all formulations were inspected
macroscopically for distinct signs of phase separation and
instability.
2.3.4. Measurements of API content
The content of the respective API was monitored over a period
of 4 months. Samples were drawn twice a month in the first two
months, then once a month for the following three months. For
each measurement, 5 samples from every formulation (n = 5) of the
exactly weighed amount of 10 mg were taken from varying spots in
the storage pot of the formulations and then extracted with 1 ml of
methanol. Samples were shaken at a speed of 1000 rpm (rounds
per minute) at room temperature for 2.5 h with a Thermomixer
5436 (Eppendorf, Hamburg, Germany). After centrifugation at
431
12,000 rpm for 6 min the supernatant was transferred to HPLC for
analysis of the API content.
2.4. In vitro tape stripping with semi-solid formulations
In vitro tape stripping experiments were performed for the
fludrocortisone acetate containing formulations. All experiments
were conducted by the same experimenter. As the considerable
inter-individual differences between porcine ears is well known
(Schwindt et al., 1998), a minimum of 4 experiments for each
formulation were conducted on porcine ear skin to ensure
comparability (n  4).
Representative skin areas and the exact location for the tape
stripping procedure were selected and marked with a permanent
marker. From these areas, visible hairs were carefully removed
using scissors, so as not to damage the skin and minimise
interference of hairs with subsequent measurements. To ensure
integrity of the SC, the TEWL (transepidermal water loss) of the
skin was measured with a closed-chamber device (Biox Aquaflux,
London, UK). TEWL values below 20 g/m2/h were considered as
hint for adequate barrier function. On the marked areas the
carefully weighed amount of 2 mg/cm2 of the respective formula-
tion was applied onto the skin. The finger of a latex glove saturated
in the respective formulation was used for applying and gently
distributing the formulations for 1 min by the same experimenter
to foreclose variability due to different application techniques and
care was taken to ensure a reproducible working procedure. In
contrast to a previous study (Nagelreiter et al., 2013), after an
incubation time of 1 h for all formulations, the excess formulation
was gently dabbed away with soft tissues whilst avoiding pressure
or smearing of the formulation. This was done before tape
stripping experiments were started to minimize impairment of the
adhesion of tape strips to the skin (Lademann et al., 2009; Sheth
et al., 1987; Teichmann et al., 2007; Wiedersberg et al., 2009).
Standard Corneofix F 20 tapes1 were used as the adhesive tapes
for removing the superficial layers of the SC. Constant pressure of
49 N (5 kg) on each of the tape strips was ensured by performing
the experiments on a balance. The pressure was applied with a
vinyl glove covered thumb performing three successional rolling
movements to minimise the influence of wrinkles in the skin.
Thereby, corneocytes were removed as efficiently and homo-
geneously as possible (Breternitz et al., 2007). Then the adhesive
strip was removed in a single rapid movement. In this manner,
10 sequential tape strips were removed from the skin surface as
preliminary tests indicated sufficient removal of SC with 10 strips
to analyse the entire penetrated drug. All 10 strips were taken into
account for calculation of drug penetration into the skin and for
drug recovery.
2.5. Protein quantification by NIR-densitometry
The amount of corneocytes removed with each individual strip
was analysed by means of the infrared densitometer Squame-
ScanTM 850 A (Heiland Electronic Gmbh, Wetzlar, Germany). NIR-
densitometry employing the pseudo-absorption of corneocytes at
850 nm has been validated for the model of porcine ear skin and is
widely used (Hahn et al., 2010; Klang et al., 2011a). Each tape strip
was analysed for the amount of removed corneocytes at a
wavelength of 850 nm using a fresh and clean strip as reference.
The mass of the corneocytes removed with each individual strip
can be calculated in mg/cm2 with the equation m = A/0.24 (Franzen
et al., 2012).
The stratum corneum was entirely removed from one site per
ear and a representative mean value was obtained (n  8).
Emanating from this mean value, the mean mass of the stratum
corneum removed was calculated as previously described and was
 432 C. Nagelreiter et al. / International Journal of Pharmaceutics 478 (2015) 429–438
found to be 1625.67 mg/cm2  360.58.14. Assuming an average
density of the stratum corneum of 1 g/cm3 (Anderson and Cassidy,
1973), this mean mass results in an average stratum corneum
thickness of about 16.26 mm  3.61. This calculated SC thickness
was used for further calculation.
Based on this mean value of SC thickness, SC-penetration
depth of the drug from different formulations was calculated as
follows: The mass of the corneocytes removed with each
individual tape strip (as measured by NIR densitometry) was
summed up including the last tape strip on which model drug was
found in the HPLC measurements. The exact number of tape strips
until the penetration limit of the model drug was subject to the
influence of the different tested formulations as well as inter-
individual differences between ears. The sum of corneocyte mass
removed from each test site until the penetration limit was then
correlated with the previously calculated SC thickness to obtain
the SC-penetration depth of the model drug in %SC. Thereby,
penetration depth can be calculated despite the variable amounts
of corneocytes removed with individual tape strips and irre-
spective of the influence of different vehicles on the removal of
corneocytes.
After NIR analysis, each tape strip was immersed in 4 ml of
methanol. The samples were homogenised in an ultrasonic bath at
30 kHz (US Starsonic 60, Liarre, Italy) for 12 min and centrifuged for
12 min at 3200 rpm (Hermle AG, Gosheim, Germany). For
quantification of the amount of penetrated model drug, these
methanolic solutions were analysed via HPLC and the resulting
amounts summed up from first to last strip from with drug could
be found via HPLC.
2.6. HPLC analysis
Quantification of the content of employed APIs (fludrocortisone
acetate, erythromycin, and diclofenac sodium) was performed by
HPLC (PerkinElmer Inc., Waltham, USA), consisting of an auto-
sampler, a pump and an UV-diode array detector, using a Nucleosil
100-5C18 column (250 mm  4 mm, Macherey–Nagel, Düren,
Germany) and a Nucleosil 100-5C18 pre-column (CC8/4, 40 mm
 4 mm, Macherey–Nagel, Düren, Germany). For all experiments,
the injection volume was 20 ml. Data analysis was performed using
[(Fig._1)TD$IG]
Fig. 1. Micrographs of cream dilutions at a 10-fold magnification, abbreviations according to Table 1. Depicted dilutions are (A) OW/PG/M/WO-F, (B) OW/PG/T/WO-F, (C) OW/
no/M/WO-F, (D) OW/no/T/WO-F, (E) OW/PG/M/A-F, (F) OW/PG/M/OW-F, (G) OW/PG/M/WO-E, (H) OW/PG/M/WO-D, (J) OW/PG/M/WO-F (after 4 months), and (K) OW/PG/M/
WO-D (after 4 months).
the TotalChrom Navigator 6.2.0 software. The pH of buffers was
measured with an Orion pH meter model 420A (Thermo Fisher
Scientific Inc., Waltham, USA) and adjusted as necessary. For all
APIs, standard solutions were prepared and analysed to establish
calibration curves by plotting the obtained peak values against the
dissolved drug content. Analysis of all subsequent measurements
refers to these standard curves. Details of the individual methods
and calibration curves are listed below.
2.6.1. Fludrocortisone acetate
The method is based on already published quantification
methods (Cisternino et al., 2003; Höller and Valenta, 2007;
Nagelreiter et al., 2013). The mobile phase consisted of acetonitrile
and water in the ratio 40/60 (v/v) with a flow rate of 0.8 ml/min and
an oven temperature of 50  C. Retention time was about 7 min,
detection wavelength was set to 240 nm.
Concentration of the standard solutions ranged from 0.044 to
906.00 mg/ml and the resulting calibration curve showed a
coefficient of determination of R2 = 0.9859. The limit of detection
(LoD) was found to be 0.177 mg/ml and the limit of quantification
(LoQ) was set at 0.052 mg/ml.
2.6.2. Erythromycin
The method is based on an already published quantification
method (Hilton and Thomas, 2003). The mobile phase consisted of
a 0.05 M ammonium acetate buffer, pH 5.5 and methanol in the
ratio 10/90 (v/v), with a flow rate of 0.5 ml/min and oven
temperature of 40  C. Retention time was about 6.5 min, detection
wavelength was set to 205 nm.
Concentration of the standard solutions ranged from 0.474 to
970.00 mg/ml and the resulting calibration curve showed a
coefficient of determination of R2 = 0.9995. The limit of detection
(LoD) was found to be 30.31 mg/ml and the limit of quantification
(LoQ) was found to be 15.16 mg/ml.
2.6.3. Diclofenac sodium
The method is based on an already published quantification
method (Kählig et al., 2005). The mobile phase consisted of
acetonitrile and a 0.05 M potassium dihydrogenphosphate buffer,
pH 5 in the ratio 40/60 (v/v), with a flow rate of 1 ml/min and oven
 C. Nagelreiter et al. / International Journal of Pharmaceutics 478 (2015) 429–438 433
[(Fig._2)TD$IG]
temperature of 40  C. Retention time was about 10 min, detection
wavelength was set to 230 nm.
Concentration of the standard solutions ranged from 0.0493 to
1010.00 mg/ml and the resulting calibration curve showed a
coefficient of determination of R2 = 0.9943. The limit of detection
(LoD) was found to be 0.04932 mg/ml and the limit of quantifica-
tion (LoQ) was set at 0.09863 mg/ml.

2.6.4. Calculation of accumulated API and drug recovery rate

After analysis of the tape strips by means of HPLC, the
accumulated amount of penetrated API was calculated as follows:
The amount of model drug on each of the sequential tape strips
until the LoQ was summed for each individual examination area.
The thereby achieved values were used to calculate a mean value
and standard deviation of accumulated API for each individual
formulation containing the model drug fludrocortisone acetate.
Depicted values are means and standard deviations of at least
4 individual tape stripping areas (n  4).
Calculation of the amount of fludrocortisone acetate penetrated
into the SC was likewise performed for each formulation
individually. For this purpose, the summed amount of API for
each examination area was compared to the amount of API
theoretically applied for the tape stripping process. This theoreti-
cally applied amount was derived from the weighed amount of
formulation applied on the examination area on the porcine ears.
Depicted values are means and standard deviations of at least 4
individual tape stripping areas (n  4).

2.7. Rheological characterisation–flow curve

Rheological characterisation was performed with a Rheometer

MCR 302, using a cone-plate measuring device of 50 mm diameter
and an angle of 1, employing a gap size of 0.101 mm (Anton Paar,
Graz, Austria). For all formulations measurements were carried out
at 23  C.
Samples were subjected to a shear rate slope, starting at 1 s 1 to
the final stress of 100 s 1 within 2 min. For 6 s, the shear rate was
held on its maximum and then decreased back to 1 s 1 within
2 min.
Values used for evaluation of the rheological characteristics of
the formulations were obtained at a shear stress of 1 s 1, once at
the beginning, and once at the end of each measurement. Depicted
values are means and standard deviations of at least 3 measure-
ments (n  3).

2.8. Statistical data analysis

All data were analysed with Microsoft Excel 2010 and Graph-
PadPrism3, using the student's t-test for parametric tests in case of
Gaussian distribution or the Mann–Whitney test for nonparamet-
ric tests. Differences were considered significant at a level of
significance of P < 0.05. Rheological data were analysed using the
Rheoplus software included with the Rheometer MCR 203 before
further analysis with Microsoft Excel 2010 and GraphPadPrism3.
3. Results and discussion
3.1. Microscopic evaluation and concluding macroscopic inspection
Fig. 2. Measurements of chemical stability of fludrocortisone acetate over an
observation period of 14 weeks. Fludrocortisone acetate content is shown in % in the
dilutions OW/PG/M with WO, A and OW. Values are means and standard deviations
of at least 3 measurements (n  3).
image of a mixture of the OW with the WO cream containing
fludrocortisone acetate which was mixed with PG prior to
incorporation into the cream. The dilution is shown right after
production. It serves as a reference for the other pictures included
in Fig. 1 and is enlarged for easier comparison of the images. The
individual aspects examined are discussed in further detail below.

The microscopic inspection of the formulations right after
production and at the end of the observation period together with
an additional macroscopic evaluation served to investigate the
influence of various parameters on the appearance of formulations.
Fig. 1 shows a compilation of representative microscopic pictures
of selected formulations. The codes indicating each production
step for each formulation are explained in Table 1. Fig. 1A shows an
3.1.1. Influence of the different cream bases
The principal point of this aspect was to study the effect of
dilution of the OW based 1% standard formulations with the WO
cream, the amphiphilic cream base and the OW cream diluted with
itself. Although mixing of an OW cream with a WO cream in equal
parts is not favourable from a technological point of view, this
approach was chosen to determine and document possible
434 C. Nagelreiter et al. / International Journal of Pharmaceutics 478 (2015) 429–438
[(Fig._3)TD$IG]
25 homogeneous and fine distribution of small API crystals. All
dilutions obtained with the amphiphilic and OW creams were
SC-penetration depth [% SC]

20 equally homogeneous independently of the production param-

eters (other data not shown).
15
3.1.2. Influence of prior mixing with PG and the manner of dilution
WO
10
Fig. 1A–D were taken immediately after production of the
A prepared dilutions, whereby Fig. 1A and B were prepared with
OW prior mixing of the API with PG, whereas 1C and D were not.
5
By comparing Fig. 1A and C it is noticeable that in Fig. 1C the API
crystals tend towards bigger clusters and poorer distribution.
0
Thereby, it can be concluded that prior mixing of fludrocortisone
OW/PG/T OW/PG/M OW/no/T OW/no/M
acetate with PG had a visible positive influence on the
Production parameters homogeneity, rendering the appearance more homogeneous than
without prior mixing with PG. Further advantages and possible
Fig. 3. SC-penetration depth in % of stratum corneum of fludrocortisone acetate
from the different dilutions. On the x-axis the production parameters of the effects on the homogeneous distribution of drug crystals are seen
formulations are shown and grouped according to the cream base used for in the mechanically mixed samples compared to the mixtures by
1:1 dilution. WO (white), amphiphilic (light grey), and OW (dark grey). Values are hand, as indicated by the comparison of Fig. 1A with B and 1C with
means and standard deviations of at least 4 measurements (n  4). D.

negative effects. Moreover, this mixture is still en vogue in Austria 3.1.3. Influence of the different APIs
in the production of individual preparations. Pictures of formulations containing the three different drugs
Inspection of the dilution of OW with WO by hand in Fig. 1A fludrocortisone acetate (1A), diclofenac sodium (1H) and erythro-
shows large API-crystal clusters. Also, a distinct striation can be mycin (1G) are shown shortly after production.
seen and points to an inferior homogeneity of this mixture As all three formulations were diluted with the WO cream, they
compared to the dilutions prepared by mixing the OW cream with all show the aforementioned striation phenomenon most likely
OW and of the OW cream with the amphiphilic cream base (Fig. 1E derived from an incompatibility of ingredients. Apart from this,
and F). A possible explanation for this phenomenon could be the considerable aggregates of API crystals were found in all three
incompatibility between polyacrylic acid of the OW cream and Al- formulations independently of the API incorporated. Moreover,
ions of Al-stearate contained in the WO cream. In an earlier study distribution of crystals was finer and more homogeneous in
this effect between the anionic carbomer and Al-ions was dilutions containing the amphiphilic or the OW cream, respective-
investigated (Valenta et al., 1998). This study revealed that even ly, independently of the API incorporated (data not shown).
a concentration of 2 mM Al-ions in a carbomer gel leads to Throughout all the observed formulations, the APIs fludrocortisone
turbidity of the gels and a significant decrease in viscosity. Higher acetate and erythromycin showed similarly large crystals, whereas
concentrations of Al3+ between 5 and 100 mM even led to diclofenac sodium tended to form bigger crystals. A possible
precipitation. Mixing of these two cream bases therefore led to explanation for both the cruder distribution in dilutions of the WO
instability and inhomogeneity of the mixture which could be a cream and the tendency to larger crystals of diclofenac sodium can
feasible explanation for the striation visible in Fig. 1A. Comparison be found in the solubility of the employed APIs in the cream bases
with the dilutions obtained by mechanical mixing showed higher used in this study according to the logP values of the APIs obtained
homogeneity (Fig. 1B and D). Possibly, this is due to higher mixing by the software ACDLabs Reader 12.0. Due to the different logP
speed and therefore higher energy input in these formulations. values, differences in solubility and appearance were expected.
Thereby, the precipitated crystals may have been finer distributed According to the software's calculations, the smallest logP value
and therefore a more homogeneous formulation was obtained. 1.91 was calculated for erythromycin, followed by 2.72 for
As seen, the depicted dilutions of the OW with the amphiphilic fludrocortisone acetate, which corresponds well with their
and the OW with the OW cream (Fig. 1E and F) show a behaviour in the microscopic inspection of the dilutions.

Table 2
Skin penetration depth in %SC, accumulated amount of fludrocortisone acetate in mg/cm2 and in % as found in tape stripping experiments. Values are means
and standard deviations of at least 4 experiments (n  4).

SC-penetration depth Fludrocortisone acetate Fludrocortisone acetate

(%) (mg/cm2) (%)
OW/WO
PG/T 5.36  2.04 2.62  0.49 26.53  5.06
PG/M 4.41  0.83 2.36  0.27 23.77  2.64
no/T 5.67  1.08 3.06  1.19 30.40  11.73
no/M 6.25  1.00 2.62  0.54 25.62  5.39

OW/A
PG/T 18.09  1.37 7.17  1.11 72.47  10.94
PG/M 12.06  4.73 6.11  0.42 60.68  4.18
no/T 12.67  4.24 7.07  1.25. 58.04  11.39
no/M 11.03  1.46 9.69  0.42 79.28  25.31

OW/OW
PG/T 14.02  3.76 3.64  1.89 37.17  19.31
PG/M 15.01  4.25 6.20  1.66 62.56  17.09
no/T 14.83  3.64 5.62  1.28 55.83  13.20
no/M 13.35  3.60 6.18  1.88 51.28  18.73
 C. Nagelreiter et al. / International Journal of Pharmaceutics 478 (2015) 429–438 435
[(Fig._4)TD$IG]
12

10
accumulated model drug [μg/cm²]

6 OW/no/T/WO
OW/no/T/A
4 OW/no/T/OW

0
0 2 4 6 8 10 12 14 16 18 20 22
SC-penetraon depth [%]

Fig. 4. Representative presentation of the SC-penetration depth in % of stratum corneum on the x-axis plotted against the penetrated amount of fludrocortisone acetate in mg/
cm2 on the y-axis as calculated from the tape stripping experiments. Formulations shown are OW/no/T mixed with the WO cream base (dashed line, rhombi), the amphiphilic
cream base (dotted line, triangles) and the OW cream base (solid line, circles). Values are means and standard deviations of at least 4 measurements (n  4).

Diclofenac sodium, in comparison, showed a higher logP value of Fig. 2, the development of API content of the API diclofenac sodium
4.26 which indicates better solubility in lipophilic environment as, can be seen in %. Depicted are dilutions obtained after prior mixing
for example, in the dilution containing the WO cream. This also of the API with PG and dilution with an automated system; all three
corresponds well with the observation of larger diclofenac sodium cream bases examined can be found in Fig. 2 (in detail:
crystals in dilutions of the WO cream for according to the empirical formulations OW/PG/T/WO-D, OW/PG/T/A-D, and OW/PG/T/OW-
Ostwald's law of stages the most soluble substance in a mixture D). The graphs in Fig. 2 are representative for all the formulations
crystallises and re-crystallises first. Due to diclofenac sodium's examined. Comparatively high standard deviations hint at
presumably better solubility in the dilution with the WO cream microscopic clusters of API crystals and thereby a slightly
than in the formulations containing the amphiphilic or OW cream inhomogeneous dispersion on the microstructural level. Such
und according to a published work (Shekunov and York, 2000), it is clusters also have to be assumed in dilutions containing the
feasible to conclude quick recrystallisation of the diclofenac amphiphilic and the OW cream that appeared homogeneous upon
sodium crystals after dilution of the 1% API OW formulation with microscopic inspection, yet also exhibit considerably large
the WO cream as it is presumably best soluble in this dilution. standard deviations. However, the mean values are close to
100% API which suggests a negligible effect of this microstructural
3.1.4. Influence of storage time inhomogeneity.
Interestingly less and smaller fludrocortisone acetate as well as Interestingly, dilutions of fludrocortisone acetate with the WO
diclofenac sodium crystals are seen in the OW-WO dilutions after cream tended to show a lower recovery of the incorporated API
storage at room temperature for four months (compare Fig. 1A–J than the dilutions obtained with the amphiphilic and OW creams;
and Fig. 1H–K). however, the differences were not significant. Dilutions of
Moreover, the aforementioned striation of dilutions with the erythromycin and diclofenac sodium showed no differences in
WO cream was not visible anymore after 4 months of storage. An API content depending on the manner of manufacture. In addition,
explanation for this could be a state of equilibrium between the chromatography results were carefully checked for degradation
phases of the OW cream of the 1% API suspension and the WO products of the respective APIs, yet none were found over the
cream of its dilution despite the possible incompatibility of period of the present study (Ast and Abdou, 1979; Chepkwony
polyacrylic acid and Al-stearate. However, these dilutions still et al., 2000; Hajkova et al., 2002). Therefore, a satisfactory stability
exhibited an inhomogeneous appearance, but it was more finely and distribution of the incorporated drugs can be assumed,
dispersed than in the crude striation of the pictures taken right independent of the production parameters, employed cream base
after production. or API.
Dilutions containing the amphiphilic and the OW cream did not
change visibly during the observation period, but remained 3.3. In vitro skin penetration: tape stripping
homogeneous and stable as no visible phase separation was
detected (data not shown). It is therefore concluded that dilutions The outcome of the tape stripping experiments with the
containing the amphiphilic and the OW cream had reached their formulations containing fludrocortisone acetate is shown in Fig. 3.
equilibrium soon after production. The exact values for SC penetration depth of each formulation as
well as the accumulated penetrated amount of fludrocortisone
3.2. API-content and stability acetate in the analysed tape strips expressed in mg/cm2 and in
percent of the applied dose can be found in Table 2.
All formulations, regardless of production parameters and The lowest skin penetration depth independent of the
cream base, showed excellent chemical stability of the incorpo- production parameters was seen from the OW-WO cream mixtures
rated API over the investigated time period of four months. In represented by the white bars in Fig. 3. Even though the dilutions
436 C. Nagelreiter et al. / International Journal of Pharmaceutics 478 (2015) 429–438
[(Fig._5)TD$IG]

Fig. 5. Rheological characterisation: Viscosity at shear rate 1 s 1 at a temperature of 23  C. On the x-axis the cream used for dilution of the 1% API OW standard formulation is
indicated. Columns represent the exact production parameter: light grey – prior mixing with PG, automated dilution, striped – prior mixing with PG, manual dilution, white –
no prior mixing with PG, automated dilution, dark grey – no prior mixing with PG, manual dilution. Shown are (a) dilutions from 1% erythromycin formulations, (b) diclofenac
sodium, and (c) fludrocortisone acetate. Values are means and standard deviations of at least 3 measurements (n  3).
 C. Nagelreiter et al. / International Journal of Pharmaceutics 478 (2015) 429–438
with the amphiphilic cream and the OW cream showed SC-
penetration depths and amounts of penetrated drug in the same
range, a higher yet not significant trend can be observed in OW-OW
dilutions.
In Fig. 4, SC penetration depth in percent is compared to the
penetrated amount of fludrocortisone acetate in mg/cm2 from
formulations produced without prior mixing of the API with PG and
automated mixing of the 1% standard formulation with the cream
bases in order to show the kinetic of drug penetration (Herkenne
et al., 2006). This figure corroborates the trend already shown in
Fig. 3. The SC-penetration depth as read from the x-axis is higher for
the dilutions obtained with the amphiphilic and OW cream bases
and in addition these two formulations depicted in Fig. 4 were also
able to transport more of the model drug into the SC than the
formulation diluted with the WO cream base managed. Fig. 4 also
provides the interesting find that the penetration kinetic was
independent of the cream base used for dilution as the slope of the
lines indicate. These results suggest the composition of the cream
base has a minor influence on the exact kinetic of drug transporta-
tion into the skin, but a tremendous effect on the SC-penetration
depth and the amount of drug able to penetrated into the SC.
This is in excellent agreement with previous research of the
group (Nagelreiter et al., 2013) and other researchers (Cal 2006;
Jacobi et al., 2006). Moreover, it suggests a strong influence of the
components of the cream base onto penetration behaviour that
exceeds the effects of the examined production parameters.
As indicated in Table 2, the amount of fludrocortisone acetate
penetrated into the skin only reaches values between 23 and 30% of
the applied dose in case of the OW–WO dilutions and about an
average of 60% in case of the mixtures OW-amphiphilic and OW–
OW. This can be explained by our working protocol. After an
incubation period of 1 h on a defined area of the porcine ear, the
rest of the formulation was dabbed away and thereby lost for
analysis, so the low percentage of model drug in the SC indicates
that the most lipophilic formulation (OW–WO) was not able to
penetrate deep into the skin and is therefore removed. This
explanation is supported by FTIR data of recent studies in which it
was proven by a newly developed combined ATR–FTIR tape
stripping technique that especially lipophilic vehicles nearly do not
penetrate the stratum corneum to a high extent (Hoppel et al.,
2014a,b,b under review).
In contrast to this, the two more hydrophilic systems were able
to penetrate into deeper layers resulting in a higher drug recovery.
3.4. Rheological characterisation
The measurements revealed non-Newtonian behaviour for all
the tested formulations as they exhibited shear-thinning with
increased shear stress. Moreover, all samples showed pseudo-
plastic behaviour. In Fig 4 the influence of automated or manual
mixing as well as of the cream system used for dilution, prior
mixing with PG and the API on the viscosity of the resulting
formulations is presented. The similarity of the rheological pattern
can be clearly seen for the sets of formulation with each tested
model drug; however, the highest values were obtained in
formulations containing erythromycin (Fig 4a), followed by those
containing fludrocortisone acetate (4b) and diclofenac sodium
(4c), respectively. A possible explanation for these findings could
be the different interactions caused by the individual physico-
chemical behaviour of the model drugs in the various formulations.
Our microscopically gained data showed the highest amount of
crystals in the formulations containing diclofenac sodium in the
mixture of OW with WO cream, but surprisingly, the expected
friction between API crystals did not seem to have an increasing
effect on the viscosity of the formulations containing diclofenac
sodium.
437
Regardless of the API, the relatively highest viscosity values
were observed in dilutions of the OW cream with the amphiphilic
cream, which is in agreement with results from our previous
study (Nagelreiter et al., 2013), however, these higher values are
not significantly higher in case of the dilutions containing
diclofenac sodium and fludrocortisone acetate. Comparison of
the viscosity data obtained for dilutions of OW with WO cream
represented in the left group of columns of Fig 4 a, b, and c for
each API studied shows that independently of the used API and of
prior mixing with PG, manual mixing induced lower viscosity.
This effect is the exact opposite for dilutions of OW with the
amphiphilic cream as shown in the middle group of columns of
Fig 4, whereas in case of dilutions OW with OW cream as seen in
the right group of columns there were no pronounced differences
between automated and manual mixing with the exception of the
formulations containing fludrocortisone acetate, which show a
trend towards higher values after manual mixing. From these
results can be presumed that the influence of API crystals was
negligible in the examined samples and that rather the input of
energy during the process of dilution determines the major
characteristics of the vehicle matrix and thereby the viscosity of
the formulation. This also corresponds well with already
published work in which a correlation between speed and
duration of energy input and the microstructure and stability of
emulsions was found (Liu and McGrath, 2005).
In comparison to the tape stripping results, the rheological
characteristics of the examined dilutions seem to have little
influence on the skin penetration behaviour, as differences in
rheological characterisation due to the manner of production were
not correlated to differences in the skin penetration behaviour.
However, the important influence of the cream base used for
dilution was also confirmed in the rheological characterisation of
the examined formulations.
4. Conclusion
The present study deals with the influence of production
parameters, incorporated API and cream bases used for dilution of
drug-containing suspensions on appearance both microscopic and
macroscopic, the skin penetration behaviour and rheological
characteristics of the resulting formulations. Our results suggest
that prior mixing of the incorporated API with PG leads to a
smoother appearance and smaller clusters of API crystals if not all
of the API can be dissolved in the formulation. Moreover, our
results indicate that manual mixing of drug-containing creams and
dilution of these creams is comparable with mixing by automated
systems, as long as the manual production process is performed
with care. Automated systems can, however, save time and effort in
the production of pharmaceutical formulations and moreover
allow for a standardised manner of production. Even so, the
formulations should be well known and tested in order to
guarantee their correctness and equivalence. However, the
ingredients of formulations and especially the compatibility of
these ingredients among themselves play a major role in the
production and dilution of topical formulations, whereas mixing
by hand can be equal to automated mixing if certain standards are
met.
Acknowledgements
The authors would like to thank Bayer Austria GesmbH (Vienna,
Austria) for their continued support. The financial support of the
research platform “Characterisation of Drug Delivery Systems on
Skin and Investigation of Involved Mechanisms” is gratefully
acknowledged. We thank FN for assistance with the acquisition of
porcine ears.
438 C. Nagelreiter et al. / International Journal of Pharmaceutics 478 (2015) 429–438
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