Permeation of topically

applied caffeine through

human skin a comparison
of in vivo and in vitro data
Sindy Trauer,
1,2
Alexa Patzelt,
1
Nina Otberg,
1
Fanny Knorr,
1
Christel Rozycki,
3
Gabor Balizs,
3
Rolf Bttemeyer,
4
Michael Linscheid,
5
Manfred Liebsch
2
& Jrgen Lademann
1
1
Department of Dermatology, Charit-Universittsmedizin Berlin,
2
Zebet at The Federal Institute of Risk
Assessment (BfR),
3
Federal Institute of Risk Assessment (BfR),
4
Department of Surgery,
Charit-Universittsmedizin and
5
Institute of Chemistry of the Humboldt University, Berlin, Germany
Correspondence
Alexa Patzelt, MD, Department
of Dermatology, Charit
Universittsmedizin Berlin, Charitplatz 1,
10117 Berlin
Tel.: + 49 30 450 518 106
Fax: + 49 30 450 518 918
E-mail: alexa.patzelt@charite.de
----------------------------------------------------------------------
Keywords
caffeine, follicular penetration, hair follicle
----------------------------------------------------------------------
Received
18 December 2008
Accepted
5 May 2009
WHAT IS ALREADY KNOWN ABOUT
THIS SUBJECT
The hair follicles represent important shunt
routes into the skin for a multiplicity of drugs and
chemicals. Recently, it has been shown that the
hair follicles are responsible for a fast delivery of
topically applied substances. After topical
application, caffeine was already detected in the
blood of the volunteers after 5 min, whereas,
when the hair follicles were selectively blocked
utilizing the newly developed Follicular Closing
Technique (FCT), caffeine was detectable only
after 20 min. Because of ethical reasons, in vivo
investigations are not always applicable.
Therefore, appropriate in vitro methods have to
be developed and compared with the available in
vivo data, in order to identify their transferability.
WHAT THIS STUDY ADDS
In the present study, the FCT was adapted for in
vitro use in the Franz diffusion cell and the
penetration of caffeine was investigated and
compared with the previously obtained in vivo
data. It was shown that the combination of FCT
and Franz diffusion cell represents a valuable
method to estimate the follicular penetration
process in vitro, which revealed comparable
results in vivo, whereas the kinetics of caffeine
penetration were signicantly different. These
ndings are of importance and need to be kept
in mind when evaluating the results obtained in
in vitro studies.
AIMS
Due to ethical reasons, in vivo penetration studies are not applicable
at all stages of development of new substances. Therefore, the
development of appropriate in vitro methods is essential, as well as the
comparison of the obtained in vivo and in vitro data, in order to identify
their transferability. The aim of the present study was to investigate the
follicular penetration of caffeine in vitro and to compare the data with
the in vivo results determined previously under similar conditions.
METHODS
The Follicular Closing Technique (FCT) represents a method to
investigate the follicular penetration selectively. In the present study,
FCT was combined with the Franz diffusion cell in order to differentiate
between follicular and intercellular penetration of caffeine into the
receptor medium in vitro. Subsequently, the results were compared
with the data obtained in an earlier study investigating follicular and
intercellular penetration of caffeine in vivo.
RESULTS
The comparison of the data revealed that the in vitro experiments were
valuable for the investigation of the follicular penetration pathway,
which contributed in vivo as well as in vitro to approximately 50% of
the total penetration, whereas the kinetics of caffeine penetration were
shown to be signicantly different.
CONCLUSIONS
The combination of FCT with the Franz diffusion cell represents a
valuable method to investigate follicular penetration in vitro.
Nevertheless, in vivo experiments should not be abandoned as in vitro,
structural changes of skin occur and blood ow and metabolism are
absent, probably accounting for reduced penetration rates in vitro.
British Journal of Clinical
Pharmacology
DOI:10.1111/j.1365-2125.2009.03463.x
Br J Clin Pharmacol / 68:2 / 181186 / 181 2009 The Authors
Journal compilation 2009 The British Pharmacological Society
Introduction
For most topically applied pharmaceuticals and cosmetics,
penetration through the skin barrier is essential for devel-
oping their effects. However, regarding optimization and
the development of new substances, it is consequently of
the highest relevance to be familiar with the correspond-
ing penetration pathways. In principle, four different
penetration pathways are available for topically applied
substances. On the one hand, penetration can occur inter-
cellularly along the lipid layers or intracellularly. Addition-
ally, penetration via the sweat glands as well as via the hair
follicles is feasible. In the past, the intercellular penetration
pathway was supposed to represent the most important
pathway and took scientic centre stage [1-6]. On the con-
trary, the shunt routes (i.e. hair follicles and sweat glands)
have been somewhat neglected. The hair follicles were
assumed to cover only 0.1% of the skin surface and there-
fore were considered to be irrelevant for skin penetration
processes [7]. During the past years, this opinion has dras-
tically changed. Scientists recognized that the hair follicles
represent weak spots in the skin barrier. Moreover, hair
follicles represent invaginations of the epidermis extend-
ing deep into the dermis, thus providing a greater actual
area for potential absorption [8]. In the meantime, a multi-
plicity of studies has shown that the hair follicles represent
important penetration pathways, as well as a long-term
reservoir for topically applied substances [915]. However,
the development of a method to investigate the follicular
penetration selectively represents a particular challenge.
Available skin absorption tests, such as the tape stripping
procedure or the Franz diffusion cell do not allow a clear
differentiation between the different penetration path-
ways. Recently, the Follicle Closing Technique (FCT), an
in vivo method to investigate the follicular penetration
pathway, was introduced by Teichmann et al. [16]. After
closing the hair follicles with a varnish wax mixture, the
penetration of topically applied caffeine was investigated
and compared with caffeine penetration through a skin
area with open hair follicles. In the case of the open hair
follicles, the caffeine was detectable in the blood signi-
cantly earlier (detection after 5 min), whereas in the case of
closed hair follicles, caffeine was not detectable in the
blood until 20 min after administration [14]. Recently,
the FCT was also successfully established for in vitro use
in the Franz diffusion cell (FD-C) [17].
The aim of the present study was to investigate follicu-
lar penetration of caffeine in vitro utilizing the FCT in com-
bination with the FD-C and to compare these data with the
available data on in vivo follicular penetration of caffeine,
derived from the study of Otberg et al. [14].
Due to ethical reasons, in vivo studies are not applicable
at all stages of development of new substances; therefore,
the development of equivalent in vitro models seems
highly reasonable. However, as in vitro data cannot com-
pletely reect the in vivo situation, a comparison of in vivo
and in vitro results is essential in order to identify the trans-
ferability.
Methods
Preparation of test formulation
Caffeine 2.5 g (Sigma Aldrich, Steinhagen, Germany) was
added to 30 g of ethanol 70% (ethanol p.a. analytical
grade, Merck, Darmstadt, Germany). Subsequently, 67.5 g
of propylene glycol (Henry Lamotte GmbH, Bremen,
Germany) was added and the composition was homog-
enized in an ultrasonic bath for 15 min corresponding to
the formulation utilized by Otberg et al. [14].
Skin absorption test
The in vitro experiments were performed according to the
OECD Test Guideline 428 [18].
Pre-calibrated static Franz diffusion cells with an area of
1.76 cm
2
available for diffusion and receptor compartment
volume of approximately 12 ml were used for the skin
absorption tests. The receptor compartment was carefully
lled with Dulbeccos phosphate buffered saline (DPBS)
with Ca
++
and Mg
++
from PANBiotech GmbH (Aidenbach,
Germany) and stirred with a small magnetic stir bar to
ensure adequate mixing.
Skin samples
Human full thickness skin was obtained during plastic
surgery from the breast region and from four different
subjects (female, aged 3562 years). The study had been
approved by the Ethics Committee of the Charit. The hair
follicle density was 22 follicles cm
-2
on average. The hair
follicle density in the in vivo study by Otberg et al. [14] was
20 to 32 follicles cm
-2
.
Follicular closing technique
The follicular closing technique (FCT) was performed on
the skin test samples, which had been placed beforehand
between the donor and receptor chamber of the static
Franz diffusion cell.
The follicular orices of the test samples were closed by
small drops of a varnish wax mixture in accordance with
Otberg et al. [14]. The method has been described in detail
elsewhere [14, 16, 17]. The varnish wax mixture was also
applied to the control samples, but only in the vicinity of
the follicles, so that the shunts were not blocked. In both
cases, the penetration area was reduced on the same
surface.
Application protocol and sampling
For the in vitro study, 17.6 ml of the caffeine formulation
was applied to a skin area of 1.76 cm
2
. The test formulation
contained 25 mg caffeine ml
-1
. Thus, 250 mg cm
-2
of caffeine
was applied, which corresponded to a ve-fold increase in
the amount of caffeine having been applied in vivo by
S. Trauer et al.
182 / 68:2 / Br J Clin Pharmacol
Otberg et al. [14] (50 mg cm
-2
). This was inevitable as the
detection limit of caffeine for the analysis of the in vitro
samples with HPLC was signicantly lower (25 ng ml
-1
) in
comparison with the detection limit of the in vivo blood
samples (1 ng ml
-1
) determined by SI/MS.
After application of the caffeine formulation, samples
from the sampling port (400 ml receptor uid) of the static
Franz diffusion cell were taken at the time points 0, 1, 2, 5,
8 and 24 h, and immediately replaced by fresh receptor
medium of equal volume and temperature.
The recovery rate was determined after 24 h in all Franz
diffusion cell experiments for four different components
(donor, epidermis, dermis and receptor uid). Samples
were extracted using an ultrasonic bath for 1 h in isopro-
panol (Isopropanol SupraSolv analytical grade, Merck
Darmstadt, Germany) or DPBS, respectively.
High performance liquid chromatography
A WATERS liquid chromatography equipped with a
WATERS 510 high-pressure pump, as well as a 712WISP and
a WATERS photo diode array detector were employed in
combination with a Reversed Phase column TYPE WATERS
RESOLVE C18. 5 mm, 3.9 mm 150 mm.
To prepare the samples for calibration, the donor solu-
tion was used. For every run, these calibration samples
were analyzed and a calibration curve was calculated.
The HPLC detection limit for caffeine was 25 ng ml
-1
at a
wavelength of 262 nm.
For analysis, 50 ml of each test sample was used. The
elution mixture for caffeine was 40 : 60 methanol : ammo-
nium acetate buffer (pH 5.35) (both analytical grade,
Merck, Darmstadt, Germany).
The in vitro investigations were performed according to
the experimental conditions of the in vivo study conducted
by Otberg et al. [14]. In both studies, the investigations
were performed on breast skin providing a follicular
density of 2032 follicles cm
-2
in vivo and 22 follicles cm
-2
on average in vitro. The articial blocking of the hair fol-
licles was carried out in accordance with the FCT devel-
oped by Teichmann et al. [16]. In both cases, the same
varnish wax mixture was utilized. Due to the closing of the
hair follicles and the corresponding application of the
varnish wax mixture to the control area, the penetration
surface was reduced by 10%0.76%. In vivo, the penetra-
tion surface was reduced to 8%[14]. In all experiments, the
same caffeine formulation was applied, although different
amounts had to be employed due to different detection
limits of the analytical methods.
Results and discussion
Although the rst studies on follicular penetration had
already been performed 40 years ago [20, 21], in 2006,
Akomeah [19] criticized the consistent lack of an adequate
in vitro technique to investigate shunt route penetration
and to differentiate between different penetration path-
ways. Since then, follicular penetration has become more
and more important. Moreover, it has been recognized that
the hair follicles offer interesting therapeutic target sites, as
they represent complex and dynamic three-dimensional
structures [22]. In particular particulate substances, such as
nanoparticles or liposomes have been shown to penetrate
preferentially into the hair follicles. These ndings allow a
selective targeting of specic structures within the hair
follicles and offer new possibilities, for example, for selec-
tive gene therapy or topical vaccination [22, 23]. Never-
theless, the development of an adequate method to
investigate the follicular penetration selectively still repre-
sents a particular challenge.
The Follicle Closing Technique, established by Teich-
mann et al. [16], permitted the in vivo investigation of the
follicular penetration pathway selectively. Recently, Trauer
et al. [17] implemented a combination of FCT with FD-C,
enabling the quantication of the follicular penetration
pathway in vitro, for the rst time.
The aim of the study was the comparison of the in vivo
data, obtained by Otberg et al. [14] in a previous study, with
the in vitro data generated in the present experiments, in
order to assess the transferability of the in vitro data to the
in vivo situation. Therefore, the experimental conditions of
the in vitro experiments were adapted as far as possible to
the in vivo conditions.
The comparison of the in vivo and in vitro data both
revealed a number of similarities, as well as signicant dif-
ferences.
The in vivo results obtainedby Otberget al. [14] showed
a penetration of caffeine into the blood already after a few
minutes, following topical application (see Figure 1). The
maximum of caffeine penetration was reached after 1 h
(control samples) or 2 h (test samples). In the case of
0
2
4
6
8
10
12
14
16
5 10 20 30 60 120 300 480 1440
%

o
f

t
o
p
i
c
a
l
l
y

a
p
p
l
i
e
d

c
a
f
f
e
i
n
e
Time after caffeine application (min)
Figure 1
Kinetics of caffeine penetration for control and test skin sites in relation to
the topically applied amount of caffeine, determined as 100%. The in vivo
values were determined in the blood, the in vitro values were determined
in the receptor medium at different time points. in vivo test (); in vivo
control ( ); in vitro test ( ); in vitro control ( )
Follicular pathway in vivo vs in vitro
Br J Clin Pharmacol / 68:2 / 183
the open hair follicles (control skin), more caffeine was
detected in the blood of the volunteers than in the case of
the closed hair follicles (test skin). During the test period of
24 h, the caffeine concentration found in the blood in the
case of the closed hair follicles and the open hair follicles
decreased continuously.
In comparison, the in vitro investigations revealed
detectable concentrations of caffeine in the receptor
medium, initially, 2 h after topical application. In the recep-
tor medium of the test skin (closed hair follicles), only
0.09% caffeine was found. However, in the receptor
medium of the control skin (open hair follicles), 0.39%
of the topical caffeine was detected, which implies a
signicantly increased penetration rate of caffeine in the
case of the open hair follicles (U-test after Wilcoxon/
MannWhitney, P < 0.05).
A possible explanation for the faster occurrence of
caffeine in the blood in comparison with the receptor
medium might represent the still existent blood ow in
vivo. Around the infundibulum region of the hair follicles,
the blood vessels form a relatively dense capillary network
[22] being responsible for a fast evacuation of the perme-
ated substances. In vitro, this mechanism is absent and this
might explain the longer period of time needed for the
caffeine to be detectable in the receptor medium. After
permeating the hair follicle, the caffeine reaches the living
tissue.The evacuation via the bloodsystemin vivo is absent
in vitro; therefore, the caffeine has to penetrate through all
skin layers to reach the receptor medium. In 1979, Zesch
et al. [24] found that due to the absence of blood and
lymph ow, a 450-fold higher caffeine concentration could
be detected in the coriumafter 1000 min. They found com-
parable concentrations of caffeine after a 5 h penetration
time, as in the present study.
Up until the end of the in vitro experiments (after 24 h),
the concentration of the caffeine in the receptor medium
increased continuously in the control as well as in the
test skin. After 24 h, 11.82% of the applied caffeine were
detected in the receptor medium of the control skin,
whereas a signicantly lower concentration of caffeine
(5.45%) was found in the receptor medium of the test skin
(P < 0.05, F-test, t-test). In comparison, in vivo, the concen-
tration decreased continuously until the 24 h end point.
A possible explanation might again be the absence of
blood ow and metabolism in vitro. Due to a continuous
evacuation and degradation of the caffeine in vivo, the
concentration gradient is kept up, whereas, in vitro, a static
Franz diffusion cell is applied. Here, the receptor medium
does not change, as only after sampling, a small amount of
fresh medium is replaced. Thus, the in vitro data represent
cumulative values of caffeine permeation.
Additionally, the in vitro experiments allowed the bal-
ancing of the control and test skins. The total recovery rate
of caffeine in the test skin was 89.8% 3.36; and in the
control skin 88.8% 6.15%. In Table 1, the mean values
and SD of the cumulative caffeine recovery rates over 24 h
are given for the different skin compartments.
In Table 2, the follicular penetration rates were calcu-
lated for the in vivo and in vitro situation. The caffeine pen-
etration values of the test skin areas were subtracted from
Table 1
Cumulative caffeine recovery rates of different skin compartments (donor, epidermis, receptor and total) after a 24 h penetration time. Values are given as
percentage of applied concentration. Data are given as mean values and SD for control and test skins
Donor (%) Epidermis (%) Dermis (%) Receptor (%) Total (%)
Test skin 75.7 3.38 4.7 0.57 2.6 1.02 7.0 1.79 89.8 3.36
Control skin 55.3 3.68 9.6 1.09 7.2 1.34 16.9 3.90 88.8 6.15
Table 2
Mean cumulative penetration of caffeine as %of applied dose in different compartments (in vitro: epidermis, dermis, receptor; in vivo: blood) for control and
test skin sites. The follicular penetration rate was calculated by subtracting the values of the test samples fromthe control samples. The relative value is given
in relation to the total penetration rate
Compartment
In vitro experiments
Compartment
In vivo experiments
Control skin (%) Test skin (%)
Control skin
test skin = follicular
penetration (%)
Control skin (%) Test skin (%)
Control skin test
skin = follicular
penetration (%)
abs. rel. abs. rel.
Epidermis 9.6 1.09 4.7 0.57 4.9 51.0 N.d.
Dermis 7.2 1.34 2.6 1.02 4.6 63.9 N.d.
Receptor 16.9 3.90 7.0 1.79 9.9 58.6 Blood 24.9 1.05 12.4 0.90 12.5 50.2
N.d., not determined; abs., absolute value in relation to applied amount; rel., relative value in relation to permeated amount.
S. Trauer et al.
184 / 68:2 / Br J Clin Pharmacol
the caffeine penetration values of the control skin areas.
This calculation revealed comparable follicular penetration
rates in vivo and in vitro. In vitro, 58.6% of the permeated
caffeine penetrated via the follicular pathway, whereas in
vivo, 50.2%of the penetrated caffeine utilized the follicular
pathway. In vitro, the follicular penetration rate was addi-
tionally determined for the different skin compartments.
It was calculated that 51.0% of the caffeine, which pen-
etrated into the epidermis and 63.9% of the caffeine that
penetrated into the dermis utilized the follicular penetra-
tion pathway.
In the case of closed hair follicles, the penetration of
caffeine can be considered as signicantly lowered in vitro,
in comparison with in vivo, although the 24 h values were
higher. On the one hand, this might be due to the accumu-
lation of the caffeine in the receptor medium, on the other
hand it is known that the permeability of the skin in the
FD-C increases as time goes by. Signicant differences
between in vivo and in vitro penetration rates were also
found for the control areas with open hair follicles (P < 0.05,
U-test after Wilcoxon/MannWhitney). Moreover, some
further key data were investigated and signicant differ-
ences were found when comparing the in vivo and in vitro
test skin data and also for the control skin data (U-Test after
Wilcoxon/MannWhitney, P < 0.05). a) For the rst detec-
tion of caffeine in blood (in vivo) and the receptor medium
(in vitro), the detected concentration of caffeine was sig-
nicantly higher in vivo than in vitro for both test and
control samples. b) The overall highest caffeine concentra-
tion was found in vitro after 24 h. c) At the endpoint, after
24 h, the caffeine concentration was signicantly higher
in vitro in the receptor medium than in vivo in the blood.
In vivo, no signicant differences between test and control
samples (1.19% vs 1.44%) were detectable (P > 0.05, F-test,
t-test) at this time point, whereas in vitro, a signicant
difference (5.45% vs 11.82%) for test and control samples
was established (P < 0.05, F-test, t-test).
Additional explanations for the differences in the in
vivo and in vitro results might be due to differences in the
investigated skin. For the in vivo study, male volunteers
were observed, whereas the breast skin, utilized for the
in vitro experiments, was derived from female patients.
Masculine breast hair is mainly of terminal origin, which
means that the hair follicles are large and even reach into
the subcutaneous fat tissue. According to OECD TG 428
[18], full thickness skin utilized for in vitro experiments has
to be disburdened from the subcutaneous tissue, which
leads inevitably to a destruction of the pilosebaceous unit
and could possibly inuence penetration experiments.
Therefore, in the present study, skin derived from female
patients providing exclusively vellus hair follicles was uti-
lized, as these hair follicles are signicantly smaller and
do not reach the subcutaneous fat tissue. Moreover, it is
known that skin contracts after excision. By mounting the
skin sample onto the Franz diffusion cell, the skin is again
extended, but the multiple elastic bres around the hair
follicle remain contracted, which reduces the follicular res-
ervoir by up to 90% [25]. Also, this aspect might represent
a feasible explanation for the reduced and slower perme-
ation of caffeine in vitro. Additionally, the lower detection
limit of caffeine in vitro should also be taken into consid-
eration, which was probably only partially compensated by
the increased amount applied.
Inconclusion,thecombinationof theFranzdiffusioncell
and the Follicle Closing Technique represents a suitable
methodfor the investigationof the follicular penetrationof
topicallyappliedsubstances invitro.This combinedmethod
contributes helpful information in terms of risk assessment
of active agents and formulations and presents a new
opportunity to evaluate the efcacy of newly developed
substances. The comparison of the in vivo and in vitro data
revealed that the in vitro experiments were valuable for
the investigation of follicular penetration processes. In
summary, the FCT represents a suitable method to investi-
gate follicular penetration in vivo and in vitro. It was shown
that in the case of caffeine, approximately half of the per-
meated concentration utilized the follicular pathway.
Nevertheless, in vitro experiments can be applied in
order to estimate approximately the penetration pathway
preferably utilized by a test substance. However, in vivo
experiments should not be abandoned, as in vitro, struc-
tural changes of the skin occur and blood ow and
metabolism are absent. In static Franz diffusion cells, no
continuous evacuation of the test substance is available.
Therefore, the diffusion gradient between the different
compartments cannot be investigated. Thus, in vitro expe-
riments are less feasible to investigate the penetration
kinetics of a test substance.
Competing interests
None declared.
We wouldlike tothank the FoundationSkinPhysiology of
the Donor Association for German Science and Humanities
for nancial support.
We would also like to thank the Centre for Alternative
Methods to Animal Experiments ZEBET at the Federal Insti-
tute of Risk Assessment for laboratory equipment andthe unit
Residues of Medicinal Products at the Federal Institute of Risk
Assessment, for analytical support.
Finally, we would like to thank Ms Elisabeth Schmidt for
valuable advice whilst creating the study design, as well as
for the many hours of helpful scientic discussions.
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