Impact of adult atopic dermatitis on topical drug penetration: assessment by cutaneous microdialysis and tape stripping.

Abstract

Appropriate methodologies for the determination of drug penetration in diseased skin have not yet been established. The aim of this study was to determine the cutaneous penetration of a metronidazole cream formulation in atopic dermatitis, employing dermal microdialysis and tape strip sampling techniques. Non-invasive measuring methods were used for the quantification of the severity of the dermatitis. Skin thickness and the depth of the microdialysis probes in the skin were measured by 20 MHz ultrasound scanning. Metronidazole concentration, sampled by microdialysis, was 2.4-fold higher in the atopic dermatitis compared with uninvolved skin (p<0.001). Tape stripping methodology did not disclose this difference in penetration. Thus, the skin layer of interest and the integrity of the skin barrier should be considered when selecting sampling methodology. Microdialysis sampling is the method of choice whenever the dermis is the target tissue for topical treatment and a skin disease affecting the barrier function is present.

Full text

Page 1

© 2009 The Authors. doi: 10.2340/00015555-0562
Journal Compilation © 2009 Acta Dermato-Venereologica. ISSN 0001-5555
Acta Derm Venereol 89
INVESTIGATIVE REPORT
Acta Derm Venereol 2009; 89: 33–38
Appropriate methodologies for the determination of
drug penetration in diseased skin have not yet been esta-
blished. The aim of this study was to determine the cuta-
neous penetration of a metronidazole cream formulation
in atopic dermatitis, employing dermal microdialysis
and tape strip sampling techniques. Non-invasive mea-
suring methods were used for the quantification of the
severity of the dermatitis. Skin thickness and the depth
of the microdialysis probes in the skin were measured
by 20 MHz ultrasound scanning. Metronidazole concen-
tration, sampled by microdialysis, was 2.4-fold higher
in the atopic dermatitis compared with uninvolved skin
(p < 0.001). Tape stripping methodology did not disclose
this difference in penetration. Thus, the skin layer of
interest and the integrity of the skin barrier should be
considered when selecting sampling methodology. Micro-
dialysis sampling is the method of choice whenever the
dermis is the target tissue for topical treatment and a
skin disease affecting the barrier function is present. Key
words: atopic dermatitis; dermatopharmacokinetics; met-
ronidazole; microdialysis; skin penetration; tape stripping;
topical formulations.
(Accepted June 30, 2008.)
Acta Derm Venereol 2009; 89: 33–38.
Patricia García Ortiz, MD, Department of Dermatology,
Gentofte Hospital, University of Copenhagen, Niels
Andersens Vej 65; DK-2900 Hellerup, Denmark. E-mail:
patriciagarcia@dadlnet.dk
Atopic dermatitis (AD) is a frequent skin disease with
a biological skin barrier defect determined by genetic
factors. The clinical picture of AD varies over the years
and it is often limited to childhood. In a proportion of
patients the disease lasts into adulthood (1).
For treatment of this skin disease the first choice is
a topical corticosteroid, accompanied by antibiotics or
antimycotics whenever an infection is present.
Few techniques are available for the assessment of
in vivo topical drug penetration in human skin. The
bioavailability and potency of topical corticosteroids
can be investigated by the skin blanching technique (2).
Other methods, such as punch biopsies, suction blisters
and shave biopsies, can be useful for the determination
of cutaneous drug penetration but have no place in hu-
man studies due to their invasive nature and the limited
information provided.
Recently two methods have been evaluated for the
investigation of drug penetration from topical formula-
tions: microdialysis (MD) sampling and tape stripping
(TS) methodology (3, 4). MD is a technique for in vivo
sampling of endogenous and exogenous substances in the
extracellular fluid, which represents up to 20% of the tis-
sue volume. The technique has developed into a valuable
tool for pharmacokinetic, pharmacodynamic and bioequi-
valence studies in human skin (5–11). For a more detailed
description of this technique see Groth et al. (7)
TS of the skin relies on sampling of the stratum
corneum (SC) cells by successive tape application and
removal. This is conducted a short time (usually 30 min)
after topical drug application and following removal of
excess formulation. The drug content in the SC material,
collected by ten consecutive tape strips, is then analysed
(yielding a single end-point measurement). The percu-
taneous absorption of the compound over the following
days has been predicted by linear extrapolation (12).
TS, also called the dermatopharmacokinetic method,
has previously been considered the method of choice
for studies of topical drug penetration by the US Food
and Drug Administration (2).
However, the usefulness of these two methodologies
for the determination of topical drug penetration in the
presence of skin disease is not known. Both methods
have been evaluated in experimentally induced irritant
dermatitis as a model for endogenous skin disease (4).
The aims of this study were: (i) to investigate whether
MD and TS can be used for bioavailability studies in skin
with AD; (ii) to determine if the methods would be corre-
lated when sampling in diseased skin; (iii) to study the
correlation between the severity of the skin disease and
the drug concentration obtained by either technique; and
(iv) to evaluate the variability obtained by both methodo-
logies in the presence of endogenous skin disease.
The formulation chosen for the investigation was
metroni dazole in a cream formulation (Flagyl® 1%
cream), since this drug is accessible to sampling by
both meth ods and the drug concentration in the samples
can be analysed by high-performance liquid chromato-
graphy and mass spectometry (HPLC-MS) with a very
low limit of quantification (4).
Impact of Adult Atopic Dermatitis on Topical Drug Penetration:
Assessment by Cutaneous Microdialysis and Tape Stripping
Patricia GARCíA ORTIz1, Steen H. HANSEN2, Vinod P. SHAH3, Torkil MENNé1 and Eva BENFELDT1
1Department of Dermatology, Gentofte Hospital, University of Copenhagen, Denmark, 2Department of Pharmaceutics and Analytical Chemistry, Pharma-
ceutical Faculty, Copenhagen University, Copenhagen, Denmark and 3Office of Pharmaceutical Science, Food and Drug Administration, Rockville, MD,
USA. *Current address: Pharmaceutical consultant, North Potomac, MD, USA.

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P. Garcia Ortiz et al.
MATERIALS AND METHODS
The study was approved by the Copenhagen County ethics com-
mittee (ref. KA 05032) and was undertaken in accordance with
the Declaration of Helsinki. Subjects were given a detailed de-
scription of the study and their written consent was obtained.
Study population
Six subjects with adult AD, 5 men and one woman, with a mean
age (± standard deviation (SD)) of 30 ± 9 (range: 19–45 years)
participated in the study. All had active AD on the skin of the
volar aspect of both forearms. The volunteers did not have any
disease (skin or systemic) other than the AD, and they refrained
from taking medication for 2 weeks prior to the study. Topical
moisturizers were allowed up to 12 h prior to the experimental
day. The severity and duration of flare-up of their eczema varied
from erythema with scattered papules, to more chronic changes
represented by lichenification, dryness and scaling of the skin
(see Fig. 1 as an example).
Study formulation
Flagyl® cream 1% (Aventis Pharma A/S, Hørsholm, Denmark)
was used as is. For the local anaesthesia prior to the insertion
of the probes, subcutaneous injections of lidocaine (Xylocaine®
10 mg/ml, Astrazeneca, Albertslund, Denmark) were used. The
perfusate for the MD experiment consisted of sterile isotonic
saline solution (sodium chloride 9 g/l, 308 mmol/l).
Experimental protocol
At the beginning of the experimental day non-invasive measu-
rements (transepidermal water loss (TEWL) and erythema) of
barrier integrity were performed and followed by a standardized
gentle wash of both forearms. The penetration areas were de-
marcated on the skin by drawing with a permanent marker pen:
two areas measuring 2 × 2.6 cm on the left forearm (one with
changes from the AD, and one with uninvolved skin) for Dermal
Microdialysis (DMD) sampling; on the right forearm four round
areas with a diameter of 2.2, two in uninvolved skin and two in
skin with AD for TS harvesting (Figs 1 and 2). A control area
was placed above the wrist and stripped at the beginning of the
experiment to establish a baseline of drug content in the skin.
The content was below the limit of detection (0.2 ng/ml of the
final sample solution) in all tape strip control samples.
In vivo microdialysis
Local anaesthesia was injected in the dermis/subcutaneous
tissue around the demarcated areas (volume < 20 ml), and two
linear MD probes were inserted in each area parallel to each
other and to the skin surface by means of a 22G guide cannula.
The outlet was placed in a 300-µl glass vial, and perfusion was
started at a low flow (0.4 µl/min). MD sampling at a 1.173 µl/
min flow was started following an equilibration period of 1 h
after probe insertion, in order to allow the insertion trauma to
subside, as shown necessary by Groth & Serup (13). Following a
20-min baseline sample the formulation was applied to the skin
above the two investigational areas and samples were collected
every 20 min for 3 h. The length of the membrane accessible
to microdialysis was 3 cm in all experiments. From a previous
study (García Ortiz et al., submitted to Skin Pharmacology),
the in vivo relative recovery (RR), determined by in vivo loss,
of metronidazole with the present experimental set-up was
known to be 34%.
In vivo tape stripping
The metronidazole (MTz) formulation was applied to the de-
marcated areas on the right forearm for TS harvesting at the
same time as application to the MD locations. Thirty minutes
after application two of the penetration areas were stripped (one
in uninvolved skin, and one in skin with AD), and 120 min later
the other two areas were harvested. D-squame® tapes with a dia-
meter of 22 mm (CuDerm®, Texas, USA) were used. The skin
surface was wiped twice with cotton gauze to remove residual
formulation. Tape discs were applied and removed by pincers,
using gentle pressure with the blunt end after application to as-
sure good skin contact, and alternating strip removal directions
(N, S, E and W). The first two discs were discarded, discs 3–12
stacked and placed in an airtight glass container and stored at
–35ºC until analysis.
Drug application
Flagyl® cream was applied at t = 0 to the 6 investigational areas
of both forearms, using pre-weighed glass spatulas with a pre-
weighed (Mettler Toledo PB303) amount of formulation 3 mg
in excess of the intended dose; a dose of 4 mg/cm2 was applied
(as per AAPS/FDA Workshop Report, 1998 (2)) directly and
with no occlusion to the skin surface.
Fig. 1. Application area for microdialysis sampling. Area 2 × 2.6 cm with
atopic dermatitis on the left forearm.
Fig. 2. Application area for tape stripping. Round areas on the right forearm
with atopic dermatitis for harvesting after 30 and 120 min (S30 and S120,
respectively).
Acta Derm Venereol 89

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Topical drug penetration in atopic dermatitis
Non-invasive measurements of skin barrier function, erythema,
skin thickness and probe depth
To establish the severity of barrier impairment in the presence of
AD compared with the uninvolved skin of the patients, TEWL
and erythema were measured at the beginning of the experimental
day. TEWL measurements were performed by an evaporimeter
(Dermalab Cortex technology, Hadsund, Denmark), according
to guidelines (14) and recorded in triplicate. A colorimeter
(Minolta Chromameter® CR300, Osaka, Japan) was used to mea-
sure erythema (the a* value) (15) in accordance with the standar-
dized Commission Internationale de l’Ecleirege Guidelines.
At the end of the experiment the depth of the microdialysis
probes in the skin and the skin thickness of the volunteers
were measured by 20 MHz ultrasound scanning (Dermascan-
C, Cortex, Hadsund, Denmark) in three separate scans along
the length of the probe in situ (near probe entry, middle and
near probe exit).
Sample analysis
The concentration of MTz in dialysates was measured, without
pre-treatment, by HPLC-MS.The content of MTz in the tape
strips was analysed following extraction procedures by LC-MS.
For a detailed description of the analysis method see García
Ortiz et al. (4).
Data analysis
All data are presented as means ± SD or standard error of mean
(SEM). Logarithmic transformation of the results was required
for the statistical analysis to achieve normalization and stabilize
the variance of the data. This was checked graphically. The area
under the concentration-vs.-time curve (AUC) was used for
the pharmacokinetic analysis of MTz dermal concentration in
both normal and diseased skin. The statistical significance of
differences in MTz penetration between normal skin and AD
was analysed using one-way analyses of variance (ANOVA)
followed by pair-wise comparisons. The relationship between
both methods in both situations was studied by linear regression
analysis. A p-value < 0.05 was considered significant.
RESULTS
Metronidazole penetration by dermal microdialysis
The dermal concentration of MTz sampled by MD
was 2.4-fold higher in AD (671.7 ± 127.9 ng/µl×min)
(mean ± SEM) than in uninvolved skin (285.5 ± 66.3
ng/µl×min), p = 0.004 (see Fig. 3 and Table I).
The intra-subject coefficient of variation in normal
skin was 26%, and 83% in eczematous skin, whereas
the inter-subject coefficient of variation (CV) was 264%
and 120%, respectively.
Metronidazole penetration by tape stripping
methodology
Results obtained by tape strip harvesting are shown
in Fig. 4 and Table II. The content of MTz in the SC
sampled by this technique was approximately 15%
lower in diseased skin (30 min: 34.3 ± 13.0; 120 min:
24.3 ± 6.4) (mean ± SD) than in uninvolved skin (30 min:
39.3 ± 11.0; 120 min: 29.7 ± 11.9), p = 0.334.
The inter-individual CV of TS was 18.6% in unin-
volved skin and 25.8% in skin with AD.
Measurements of probe depth and skin thickness
The MD probes were all placed inside the dermis and
approximately at the same depth in both uninvolved
skin (0.79 mm ± 0.001) (mean ± SD) and skin with
AD (0.86 mm ± 0.06), as measured by high-frequency
ultrasound scanning, p = 0.64 (Table I).
The thickness of the skin of the volunteers was measu-
red at the locations of MD sampling. Involved skin with
AD was found to be significantly 0.12 mm thicker (equal
to an increase of 8%) than uninvolved skin (p = 0.015).
Non-invasive measuring methods of skin barrier
function and erythema
Both TEWL and erythema measurements significantly
(p < 0.05) confirmed the barrier impairment and increased
redness due to skin inflammation in skin with active
dermatitis (Tables I and II). One exception was the me-
asurement of erythema over the area for TS after 120
min, where the difference was not significant (p = 0.11).
Table I. Quantification of metronidazole penetration by
microdialysis
Barrier perturbation UninvolvedAtopic dermatitisp
Number of subjects
Number of probes
TEWL (g/m2h)a
Erythemaa
Skin thickness (mm)a
Probe depth (mm)a
AUC (ng/µl×min)b
95% CI (ng/µl×min)
6 6
12
8.9 ± 5.5
3.8 ± 1.3
1.59 ± 0.1
0.79 ± 0.001
285.5 ± 66.3
(142.3; 428.7)
12
30.6 ± 15.6
8.3 ± 2.5
1.71 ± 0.1
0.86 ± 0.06
671.7 ± 127.9
(395.7; 947.7)
0.013
0.01
0.015
0.6
0.004

aResults are expressed as mean ± standard deviation.
bResults expressed as mean ± standard error of mean.
AUC: area under the time vs. concentration curve of the topical
penetration of metronidazole; CI: confidence interval; TEWL:
transepidermal water loss.
Fig. 3. Microdialysis. Concentration vs. time curve for the cutaneous
penetration of metronidazole (MTz). Mean concentrations are presented for
n = 6. The area under the time vs. concentration curve was significantly larger
in atopic dermatitis (eczema) compared with uninvolved skin (p = 0.004)
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P. Garcia Ortiz et al.
Here the redness was discrete, probably because in most
volunteers the eczema was in a chronic phase.
Correlation between metronidazole concentrations and
non-invasive bioengineering methods
For the left arm, where MD sampling was underta-
ken, we found the concentrations of MTz sampled in
uninvolved skin were positively correlated with their
corresponding TEWL (r = 0.7) and erythema (r = 0.69)
values, but these correlations were not significant. In
areas with active eczema, no correlation between MTz
penetration and measurements of TEWL and erythema
(r = 0.005 and r = –0.16, respectively) was found.
For the right arm, where TS sampling was performed,
no significant correlation between the content of MTZ
and TEWL and erythema was found (neither for ecze-
matous nor uninvolved skin).
DISCUSSION
This is the first study of topical drug penetration in-
vestigated in atopic dermatitis by dermal MD sampling
and TS methodology.
Dermal MD demonstrated a 2.4-fold increased pe-
netration of the MTz topical formulation in active AD
compared with uninvolved skin. The most authoritative
studies concerning topical drug penetration in patients
with AD are those performed by Turpeinen and co-
workers (16–18), in which the percutaneous absorption
of hydrocortisone (HC) was investigated in 38 children
by 4-h HC absorption tests. Here the cutaneous and percu-
taneous penetration of HC was significantly 2.6 times
increased in the acute phase of AD, and this increase
correlated significantly (rs = 0.991) with the severity of
the skin disease as measured by TEWL. Similarly, the
percutaneous absorption of HC during exacerbation
and remission of AD in 18 adults and 16 children was
investigated using the direct HC absorption test and 4-h
HC absorption test, respectively (17, 18). During the con-
valescence period the percutaneous penetration measured
by exogenous cortisol in plasma samples was 1/8 of that
in the acute phase of the disease. This reduction can be
attributed to the restoration of the skin barrier function
and, as a consequence, reduced penetration of HC.
Working with irritant dermatitis as a model for en-
dogenous skin disease and intradermal MD sampling
of topical penetration, the cutaneous penetration of
salicylic acid has been shown to be positively and
significantly correlated with the degree of barrier im-
pairment as measured by TEWL and erythema (3). In a
recent study (4) our group have reported a significantly
(p < 0.001) three-fold increased penetration of a MTz
formulation sampled by dermal MD in skin with mild
irritant dermatitis (sodium lauryl sulphate 1%) compa-
red with normal skin.
In the present study we could not find a correlation
between MTz penetration and skin barrier function eva-
luated by TEWL and erythema measurements. The expla-
nation probably lies with the modesty of the skin changes
in the AD as well as the small study population.
In contrast to these findings, TS methodology yielded
samples with reduced MTz content from application of
the formulation onto eczematous skin compared with
uninvolved skin. However, the reduction of approxi-
mately 16% was not significant. In a recent in vivo TS
study of percutaneous penetration in AD compared with
normal skin, Jakasa et al. (19) showed an increased dif-
0
10
20
30
40
50
60
MTZ content (µg/cm2)
p = 0.75
p = 0.22
U30
E30 U120E120
Fig. 4. Tape stripping. Metronidazole (MTz) concentrations (mean ± SD) in
tape strip samples of the stratum corneum from uninvolved skin (U30 and U120)
and eczema (E30 and E120), after 30 and 120 min, respectively, n = 6.
Table II. Quantification of metronidazole (MTZ) penetration by the tape stripping (TS) methodology
Barrier perturbation
Uninvolved skinAtopic dermatitis
TS30minTS120minTS30minTS120min
Number of subjects
TEWL (g/m2h) a
Erythemaa
Content (µg/cm2) a
95% CI (µg/cm2)
6
9.5 ± 5.6
4.3 ± 1.6
39.3 ± 11.0
(29.6; 49.0)
6
8.6 ± 3.6
4.3 ± 1.7
29.7 ± 11.9
(19.1; 40.3)
66
27.3 ± 14.9
7.6 ± 1.6
34.3 ± 13.0
(22.8; 45.8)
28.2 ± 17.4
6.9 ± 2.1
24.3 ± 6.4
(18.7; 29.9)
p-values concerning the comparison between uninvolved and eczematous skin:
Location: TS30 min Location: TS120min
MTz concentration 0.75 MTz concentration 0.22
TEWL 0.02 TEWL
Erythema 0.03 Erythema
aResults are expressed as mean ± standard deviation.
TEWL: transepidermal water loss; CI: confidence interval.
0.03
0.11
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Topical drug penetration in atopic dermatitis
fusion coefficient of polyethylene glycols of different
molecular weight in both involved and uninvolved
eczematous skin compared with normal skin. Thus,
considering the findings of this recent publication as
well as the consistency of the results from investigations
referred to earlier, where dermal and systemic drug
concentrations were increased in the presence of AD,
it can be speculated that a faster initial penetration into
and through the epidermis can account for the lower
concentrations found in the SC. Furthermore, this expla-
nation is corroborated by the dermal pharmacokinetics
of MTz penetration as sampled by MD (Fig. 3), where
the maximum concentration of MTz is found at 30 vs.
150 min, respectively. Despite this “direct evidence” of
a faster MTz penetration in AD, we admit that a follow-
up experiment where TS was conducted at, for example,
5, 10, 15 and 20 min following drug application would
further clarify our finding of reduced MTZ content in
TS samples from eczematous skin.
The intra-individual variation of MD results in skin
with AD (CV of 83%), was higher than in uninvolved
skin (CV of 26%), possibly due to variability in the ex-
tent and severity of eczema across the application sites.
The inter-individual variability was notably high both
in eczema (120%) and normal skin (264%); differences
in the severity of the AD of the volunteers as well as
in the degree of the impairment of barrier function in
the uninvolved skin must account for these results. Nu-
merous publications (20–23) have shown an increased
susceptibility of straightforward impaired barrier func-
tion of apparently uninvolved skin of subjects with AD
compared with healthy subjects. Little variation was
found in the results obtained by TS in both the AD (CV
of 25.8%) and uninvolved skin (CV of 18.6%). This is
concordant with our previous investigation of topical
penetration in irritant dermatitis (4).
Hence, this is the first study of topical drug penetration
where microdialysis and tape stripping methodologies
have been used in skin with atopic dermatitis. This study
has demonstrated that: (i) both sampling techniques are
able to sample cutaneous drug penetration in the presence
of skin disease. Even sparse AD resulted in a 2.4-fold
increase in dermal MTz concentration as measured by
MD sampling, whereas TS method did not detect this
increase in penetration. (ii) Consequently, the two metho-
dologies did not correlate when sampling in diseased
skin. (iii) An absence of correlations between the drug
penetration sampled by both techniques and non-inva-
sive measurements of skin barrier function was found.
This could be explained by the fact that the eczema of
the partici pants was in a chronic stage, thus giving only
a modest increase in TEWL and erythema values, in
combination with the small study population size. (iv)
The presence of atopic eczema resulted in a doubling of
inter-individual varia bility in MD results, whereas TS
variability was largely unaffected.
In conclusion, dermal MD seems to be the most sui-
table technique for the study of topical drug penetration
in atopic dermatitis. However, further investigations
are needed for a more detailed characterization of the
results obtained by this technique and their correlation
with the measurements of the integrity of the skin by
non-invasive methods.
ACKNOWLEDGEMENTS
This study was conducted in the Phase I Unit at Gentofte Hos-
pital, and we thank Chief Physician Jesper Sonne for his hospi-
tality. Aage Vølund, PhD, is thanked for statistical support, and
laboratory technicians Kirsten Andersen and Eva Tiedemann for
excellent technical assistance. Consultant Dermatologist Tove
Agner is also thanked for lending us access to the Evaporimeter,
Minolta Chromameter and Dermascan.
Financial support for this study was provided by Aage Bang’s
Foundation, Hans and Nora Buchard’s Foundation, the Danish
Hospital Foundation for Medical Research, Region of Copen-
hagen, The Faeroe Islands and Greenland Foundation, The Leo
Foundation, Mrs Liv Bryhn’s Foundation, The Danish Psoriasis
Foundation and the Foundation for Scientific Research without
Animal Experiments.
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