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Original Paper
Skin Pharmacol Physiol 2013;26:155–159
DOI: 10.1159/000348876
Chronological Age Affects the Permeation
of Fentanyl through Human Skin in vitro
R.Holmgaard a,d E.Benfeldt b J.A.Sorensen c J.B.Nielsen d
Departments of
a Plastic Surgery and
b Dermatology, University of Copenhagen, Roskilde Sygehus, Roskilde ,
c Department of Plastic and Reconstructive Surgery, Odense University Hospital, and
d Environmental Medicine,
Institute of Public Health, University of Southern Denmark, Odense , Denmark
Introduction
The human skin changes during a lifespan. In addition
to the effect on the visual appearance of the skin, also
physiological and structural changes are seen. These
changes are not only a consequence of intrinsic aging but
also related to the cumulative exposure to extrinsic harm,
such as sunlight or smoking, during a lifespan. Chrono-
logically aged skin appears thinner
[1] , paler, rougher [2]
and drier
[3] compared to young skin. The dry skin is
caused by alterations of the stratum corneum lipid bar-
rier
[4] as the stratum corneum lipid content decreases
with age
[5] and the lipid composition changes [3] . Skin
permeability and barrier integrity have been demonstrat-
ed to change with age in relation to some specific sub-
stances
[6–10] . Thus, transepidermal water loss decreases
in aging skin
[7, 11, 12] , the echogenicity changes [13]
and the permeability of hydrophilic penetrants increases
[7, 8, 10] . Only a few researchers have studied how these
skin changes affect the penetration of highly lipophilic
molecules into and through the stratum corneum of hu-
man skin
[14] .
In the treatment of chronic pain, transdermal admin-
istration [i.e. patch, transdermal therapeutic delivery sys-
tems of fentanyl (TTS-F)] has become widely used. The
majority of individuals treated with TTS-F are elderly
cancer patients, and this group of patients is increasing
in number due to an increasing life expectancy
[15] . El-
derly patients are considered more sensitive to opioids
Key Words
Human skin · Penetration · Age · Fentanyl · Static diffusion
cells
Abstract
Aim: To study the influence of chronological age on fenta-
nylpermeation through human skin in vitro using static dif-
fusion cells. Elderly individuals are known to be more sensi-
tive to opioids and obtain higher plasma concentrations fol-
lowing dermal application of fentanyl compared to younger
individuals. The influence of age – as an isolated pharmaco-
kinetic term – on the absorption of fentanyl has not been
previously studied. Method: Human skin from 30 female do-
nors was mounted in static diffusion cells, and samples were
collected during 48 h. Donors were divided into three age
groups: <30 years of age (n = 6), ≥ 30 and <60 years of age
(n= 18) and ≥ 60 years of age (n = 6). Results: The young-
estgroup had a significantly higher mean absorption (3,100
ng/cm
2 ) than the two other groups (2,000 and 1,475 ng/cm
2 ,
respectively) and a significant larger AUC (young age group:
9,393 ng; middle and old age groups: 5,922 and 4,050 ng,
respectively). Furthermore, the lag time and absorption rate
were different between the three groups, with a significant-
ly higher rate in the young participants versus the oldest par-
ticipants. Conclusion: We demonstrate that fentanyl perme-
ates the skin of young individuals in greater amounts and at
a higher absorption rate than in middle-aged and old indi-
viduals in vitro. Copyright © 2013 S. Karger AG, Basel
Received: November 2, 2012
Accepted after revision: February 11, 2013
Published online: May 28, 2013
Rikke Holmgaard
Department of Plastic Surgery, University of Copenhagen
Koegevej 7–9
DK–4000 Roskilde (Denmark)
E-Mail rikkeholmgaard @ gmail.com
© 2013 S. Karger AG, Basel
1660–5527/13/0263–0155$38.00/0
www.karger.com/spp
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Holmgaard/Benfeldt/Sorensen/Nielsen
Skin Pharmacol Physiol 2013;26:155–159
DOI: 10.1159/000348876
156
[16] and have increased vulnerability to adverse events
and interactions
[15] . A reason for these adverse events
has been suggested to be associated with the higher se-
rum concentration found after the use of TTS-F, indicat-
ing an age-related increase in absorption [17] or a de-
crease in excretion.
Acquiring knowledge about the influence of age on ab-
sorption of fentanyl as an isolated pharmacokinetic term
is therefore relevant.
Materials and Methods
Penetrant
The physicochemical properties of fentanyl makes it highly
qualified as a transdermal drug. It has a relatively low molecular
weight of 336 g/mol, which is required for transdermal use
(<1,000 g/mol), a low melting point of 87.5
° C [18] favoring
transdermal delivery, a water solubility of 200 mg/l
[18] and a
high lipophilicity (logPow ∼ 4) [19] . Following transdermal de-
livery, fentanyl has an in vivo bioavailability close to 100%
(92%), which means that the drug is neither significantly de-
graded by the skin’s microflora nor by the cutaneous enzyme
metabolism
[20] .
C h e m i c a l s
We used a phosphate buffer (0.05
M Na 2 HPO 4 , 2H 2 O, pH 7.4;
Merck, Darmstadt, Germany) as receptor fluid (17.22 ml). A com-
mercially available fentanyl citrate in water for injection (50 μg/ml,
pH 4.5) was added to the donor chamber (1 ml).
S k i n
The skin samples used were obtained from the Department of
Plastic and Reconstruction Surgery, Odense University Hospital,
Odense, Denmark. They were collected from women undergo-
ingbreast reduction or breast reconstruction. Skin from a total
of 30 female donors were used and divided into three age gro-
ups:<30 years of age (n = 6), ≥ 30 and <60 years of age (n = 18)
and ≥ 60 years of age (n = 6). The skin was frozen immediately
following surgery and kept at –20
° C for periods not exceeding
12 months. This has proven to maintain the barrier properties
with no significant change in water permeability
[21] , whereas
lower temperatures have been shown to have a damaging effect
[22] . Skin samples were allowed to thaw for 1 h at room tem-
perature before it were gently cleaned with tap water and paper
tissue, cut into suitable pieces and mounted in Franz diffusion
cells. The donors were given complete anonymity with registra-
tion of only age and skin site of the donors, and the local ethics
committee approved the use of the skin for this study.
Study Design
Franz diffusion cells were used in this in vitro study. The meth-
od is well established in the laboratory in Odense, Denmark, where
it has shown good reproducibility
[23] . Full-thickness skin (thick-
ness between 0.7 and 1.0 mm) was mounted on the metal grid be-
tween the donor and the receptor chamber. The receptor chamber
was filled with buffer, and 2 ml of buffer was added to the donor
chamber for capacitance measurement. The receptor chambers
were placed in a water bath with a temperature of 37
° C, keeping
the temperature at the skin surface at 32
° C. Capacitance measure-
ments were performed 1 h before as well as right after the experi-
ment to evaluate the integrity of the barriers. Cells with a capaci-
tance above 55 nF prior to the experiment were replaced. The cells
were left to equilibrate for 1 h before a ‘clean’ buffer replaced the
buffer in the receptor chamber; additionally, the buffer in the do-
nor chamber was replaced by the fentanyl solution. At this time
point, the experiment began (t = 0), and at specified time intervals
throughout the experiment, 1 ml was sampled from the receptor
chamber using a 2-ml syringe and replaced by an equal volume of
new phosphate buffer. The samples from the receptor chamber
were analyzed for fentanyl. The donor chambers and sampling
tube from the receptor chambers were covered with Parafilm
throughout the experiment to avoid evaporation ( fig.1 ). The skin
diffusion area was 2.12 cm
2 /cell, and the average receptor chamber
volume was 17.2 ml.
A n a l y s i s
Fentanyl was assayed by high-performance liquid chromatog-
raphy (HPLC). The chromatographic system was produced by
Kontron (BioTek Instruments, Milan, Italy) and consisted of a
HPLC 420 pump, a HPLC 360 auto sampler and a HPLC 430 UV
detector. Separation was performed on a LiChrospher 60 RP-
Select B (5 mm) 125 × 4 mm column equipped with a RP-Select
B (5 mm) 4 × 4 guard column (Merck). The mobile phase con-
sisted of 0.23% perchloric acid in Milli-Q water: acetonitrile (60:
40 vol/vol) [24] and was used with a flow of 1.5 ml/min. System
control and data handling were carried out on a personal com-
puter equipped with Kontron MT 450 software. Detection was
performed at 205 nm, an absorbance range of 0.005 and a re-
sponse time of 2 s. All samples were analyzed in duplicates, and
the average concentration was reported. The limit of detection
was 10 ng/ml. Standard solutions were prepared in water, and
standard curves were produced on each day of analysis and cov-
Fig. 1. Drawing of the static diffusion cell. Circles indicate pene-
trating molecules.
Color version available online
Parafilm
Magnetic stirrer
Metal grid
Skin
Parafilm
Receptor chamber
Donor chamber
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Chronological Age Affects Fentanyl
Premeation through Human Skin
Skin Pharmacol Physiol 2013;26:155–159
DOI: 10.1159/000348876
157
ered a range from 0 to 5,000 ng/ml. Correlation coefficients of
the standard curves were >0.999. All organic solvents were pur-
chased from Riedel-de Haën and were of CHROMASOLVA pu-
rity (Sigma-Aldrich Laborchemikalien, Seelze, Germany); fur-
ther, perchloric acid was of pro-analysis quality and was provid-
ed from Merck.
D a t a A n a l y s i s
Data was plotted as the cumulative amount of drug collected in
the receptor compartment as a function of time. The specific sam-
ple points were at 0, 3, 6, 12, 24 and 48 h. The lag time was deter-
mined by back extrapolation from the steepest linear part of the
permeation curve. The absorption rate was established by linear
regression on the same part of the permeation curves showing the
cumulative amount of fentanyl absorbed per square centimeter of
skin sample per time.
Data from 30 individuals were used. For each donor, the me-
dian amount absorbed, median absorption rate and median per-
meability coefficients were calculated. Then the mean amount ab-
sorbed, absorption rate and permeability of each age group were
calculated, and statistical calculations were made. The reason for
using the median within the individual is the small sample sizes of
3–5 samples per donor. The age variation was studied by dividing
the donors into three groups: a young group (<30 years of age; n =
6), a middle group ( ≥ 30 and <60 years of age; n = 18) and an older
group ( ≥ 60 years of age; n = 6).
One-way ANOVA was performed before testing the groups
two by two using Student’s t test. The level for statistical signifi-
cance between the groups was set at p < 0.05. Data were handled
using the Excel software program from Microsoft.
R e s u l t s
The dermal permeation of fentanyl in individuals old-
er than 60 years (n = 6), between 30 and 60 years (n = 18)
and younger than 30 years (n = 6) were compared. The
graphic presentation of the mean amount of fentanyl
sampled over time showed differences between the age
groups ( fig.2 ). The differences in the AUC, shown for the
three age groups individually ( fig.3 ), were significantly
higher in the youngest group than in the middle-aged
(p= 0.04) and in the old group (p = 0.02).
The mean AUC ± SD was 9,393 ± 3,990 ng fentanyl for
the young age group, 5,922 ± 3,040 ng for the middle age
group and 4,050 ± 2,493 ng for the old age group. The lag
times tended to increase with increasing age ( table 1 ),
though differences between groups did not reach statisti-
cal significance. The mean absorption rate ± SD of fen-
tanyl was 78 ± 30 ng/cm
2 /h for the youngest age group,
54 ± 26 ng/cm
2 /h for the middle age group and 43 ±
16ng/cm
2 /h for the old age group. The absorption rate
was significantly higher in the young age group compared
to the old age group (p < 0.05) ( table1 ).
D i s c u s s i o n
Elderly individuals are systematically excluded from
most clinical trials due to the potential presence of co-
morbidities and polypharmacy
[25, 26] . Consequently,
the pharmacokinetics of numerous medications in el-
derly patients is not known
[27] . We need to draw atten-
tion to this issue as the demographic development pre-
dicts an increase in the group of elderly patients in future
societies.
Even though patients treated with TTS-F benefit
from an increased compliance and a better efficacy in
pain treatment
[28, 29] , elderly patients may experi-
encemore severe adverse events than younger patients
Color version available online
Fig. 2. Absorption of fentanyl (50 μg/ml) through human breast
skin from three different age groups. * Significantly different from
other groups (p < 0.05).
Color version available online
Fig. 3. Histogram demonstrating the mean AUC of the three age
groups as well as their SD. The young group absorbs significantly
more fentanyl than the two other groups (p < 0.05).
Fentanyl (ng/cm2)
5,000
4,500
4,000
3,500
3,000
2,500
2,000
1,500
1,000
500
0
50403020100
Time (h)
*
<30 years of age (n = 6)
30 and <60 years of age (n = 18)
60 years of age (n = 6)
0
2,000
4,000
6,000
8,000
10,000
12,000
14,000
16,000
Mean AUC of fentanyl (ng)
<30 years of age 30 and <60
years of age
60 years of age
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Skin Pharmacol Physiol 2013;26:155–159
DOI: 10.1159/000348876
158
[30] and show a less predictable response to the medica-
tion
[31] .
In this study, we used static diffusion cells to investi-
gate the in vitro skin permeation of fentanyl in relation
to different age groups. How age affects the skin perme-
ability is an area under debate since different studies
point in different directions
[6, 32, 33] . We found a sig-
nificant difference between the accumulated absorption
of fentanyl over 48 h in young individuals and in middle-
aged individuals as well as between young individuals
and older individuals. Our results demonstrate that per-
cutaneous permeation of fentanyl decreases with in-
creasing chronological age of the donor individual. This
tendency was also seen when we studied the absorption
rate and lag time, though these differences were not sta-
tistically significant.
Meidan and Roper [32] recently published a retrospec-
tive study of the influence of age on the percutaneous flux
of tritiated water. No correlation with age was found, but
compared to our age groups the study by Meidan and
Roper contained only 2 young and 2 elderly skin donors,
whereas the majority of the donors (11 individuals) was
grouped in the middle-aged group. Roskos and Guy
[33]
compared the skin barrier function by measurements of
transepidermal water loss measured in one body site in
young and older individuals and found no relation be-
tween the barrier function and the age of the individuals.
In 1964, Christophers and Kligman
[6] demonstrated an
increase in permeability of the hydrophilic fluorescein in
cadaver skin from elderly individuals and a decrease in
the permeability of testosterone in elderly people in vivo.
Later, Roskos et al.
[8] also studied testosterone, estradiol,
benzoic acid and hydrocortisone and found no significant
changes in penetration of testosterone and estradiol in the
elderly group, whereas the more hydrophilic penetrants
demonstrated an increased penetration in aged skin.
In the case of fentanyl, in vivo studies in humans have
shown that the fentanyl penetration from TTS, assessed
as time to doubling of plasma concentration, appears to
be delayed in elderly patients compared to children (11
vs. 4 h). The C
max and t
max were not significantly different
between the two groups, but a prolonged elimination
phase T
1/2 was observed in the elderly patients (31 vs.
21h, respectively)
[34] . In these human in vivo studies,
an effect of altered elimination capacity such as reduced
renal clearance will have an effect on the result
[35] .
We found an inverse relation between the amount of
fentanyl absorbed and the chronological age of the do-
nors. Thus, this study corroborates with results found by
plasma concentration measurements of fentanyl perme-
ation from a TTS. Our data therefore indicate that an in-
creased risk of adverse events, which has been proposed
for fentanyl administration in the elderly
[15, 17, 36] ,
does not relate to an increased percutaneous permeation
of fentanyl in elderly people but may be caused by a slow-
er excretion or a decreased metabolic capacity towards
opioids in the elderly compared to younger populations.
A future study of fentanyl excretion and metabolism in
relation to age is therefore highly relevant.
These results need to be substantiated with more
chemicals/drugs with varying lipophilicities for the con-
clusions to be applied more generally. The relevance is
that the group of older individuals is increasing in most
societies these years, and testing of transdermal drugs
before acceptance for clinical use does not, in most cas-
es, include older individuals. Maibach’s group
[37] has
also given emphasis to this issue and suggests inclusion
of elderly in future studies to determine if this age group
should have different topical dosing regimens to ensure
drug efficaciousness with minimal adverse effects.
Table 1. Patient characteristics of the three age groups (total n = 30)
Characteristics <30 years (n = 6) >30 and ≤60 years (n = 18) >60 years (n = 6)
Mean age, years 24 44 65
AUC of fentanyl, ng 9,393±3,990a,b 5,922±3,040 4,050±2,493
Amount of fentanyl, ng/cm23,099±1,152a,b 2,021±945 1,474±782
Lag time, h 10±3 11±4 15±5
Absorption rate, ng/cm2/h 78±30b54±26 43±16
Values represent mean ± SD, unless otherwise indicated. aSignificantly different from the middle age group
(Student’s t test <0.05). bSignificantly different from the old age group (Student’s t test <0.05).
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Chronological Age Affects Fentanyl
Premeation through Human Skin
Skin Pharmacol Physiol 2013;26:155–159
DOI: 10.1159/000348876
159
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