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Skin Research and Technology 2006; 12: 211–216 Copyright & Blackwell Munksgaard 2006

Printed in Singapore. All rights reserved Skin Research and Technology

Determination of the in vivo bioavailability of

iontophoretically delivered diclofenac using a
methyl nicotinate skin inflammation assay
Renzo Lambrecht1, Peter Clarys1, Ron Clijsen2 and André O. Barel1
Faculty of Physical Education and Physiotherapy, Vrije Universiteit Brussel, BIOM, Brussel, Belgium and
Internationale Akademie für Physiotherapie, ‘Thim van der Laan’, Landquart, Switzerland

Background/aims: In this study, we investigated the bioa- measurements for the following treatment modalities: (1) elec-
vailability of iontophoretically delivered diclofenac with the trically assisted delivery: respectively, 65% and 100%, (2)
methylnicotinate (MN) test. The inhibition of an erythema application under a contact sponge: 66% and 97% and (3)
provoked by MN is proportional to the bioavailability of passive diffusion without any covering: 32% and 65%. A sig-
diclofenac in the skin. It was our aim to use this procedure nificant reduction was equally observed for the site treated with
in the determination of the contribution of, respectively, the current alone: 19% and 42%. There was no significant
passive diffusion, occlusion and electrically assisted deliv- difference between the response after iontophoretic-delivered
ery during an iontophoretic procedure as used in physiother- diclofenac (mode A) and application of diclofenac under a
apy. contact sponge (mode B).
Method: A total of six application sites were marked on the Conclusion: The procedure used enabled us to evaluate
volar forearms of each volunteer (n 5 12), for the following the bioavailability of a non-steroidal anti-inflammatory drug
treatment and/or control modes: A 5 cathodal iontophoresis in the skin. Under the conditions used, we did not find an
of 12 mg/cm 2 Voltaren Emulgel (diethylammonii diclofenac increased bioavailability after electrically assisted delivery
1%) for 20 min; B 5 passive diffusion under a contact of diclofenac compared with the passive percutaneous
sponge; C 5 passive diffusion without any covering; D 5 penetration under the contact sponge.
current alone; E 5 standard MN response; and F 5 blanco
site. Tristimulus surface colorimetry and Laser Doppler flow-
metry were used to measure, respectively, the skin color
and the perfusion of the microcirculation. Bioavailability was Key words: iontophoresis – methylnicotinate – occlusion –
assessed by quantification of an MN-induced erythema passive diffusion
under the different conditions.
Results: A significant reduction of the MN-induced er- & Blackwell Munksgaard, 2005
ythema was observed with the Chromameter and Laser Doppler Accepted for publication 11 July 2005

N PHYSIOTHERAPY, iontophoresis is used to en- pared with a placebo without an active ingredi-
I hance the penetration of drugs in the topical
treatment of muscles, tendons and joints. In
ent. As the passive penetration can be substantial
during the delivery, it is important to include this
practice, the drug is applied under a wet cellulose control to estimate the enhancement factor.
sponge, which covers the electrode. The current The methylnicotinate (MN) assay in vivo has
intensity and treatment time used are rather already been proposed to assess the bioavailabil-
limited: up to maximum 0.5 mA/cm2 for 20 min. ity of corticosteroids and non-steroidal anti-in-
When screening the literature on iontophoretic flammatory drugs (NSAIDs) (6, 7). It has been
delivery as used in physiotherapy, we observed demonstrated that the reduction of the MN-in-
that in most human in vivo studies, some basic duced erythema is related to the NSAIDs con-
principles of percutaneous penetration were not centration in the skin and the potency of the drug
always taken into account (1–5). Indeed, ionto- (6, 8). It was also possible to discriminate between
phoretic delivery is seldom evaluated vs. passive different formulations (9). This method can be
diffusion. Iontophoretic delivery is usually com- useful for predicting the clinical performance of a

Lambrecht et al.

formulation or to test bioequivalence in the de-

velopment of a generic product (7). The MN
response can easily be determined with colori-
metric evaluation of the skin and determination
of the perfusion of the microcirculation (10).
In this experiment, we tried to estimate the
contribution of the passive percutaneous pene-
tration during electrically assisted delivery of
Therefore, we used the method as proposed by
Duteil et al. (6) and Treffel and Gabard (7) to
estimate the bioavailability sof diethylammonii
diclofenac (Voltaren Emulgel ) in the skin after
iontophoretic delivery vs. several controls on the
same subject. Fig. 1. Overview of the application sites.
Hence, the contribution of different factors
such as passive diffusion, semi-occlusion and
electrical assistance can be estimated. Therefore, Belgium) using a pen electrode covered with a
the results obtained may better reflect the real wet circular cellulose sponge of a 7 cm2 surface.
contribution of the iontophoretic stimulation. The anodal sponge of 42 cm2 was placed perpen-
dicularly under the application site at the coun-
terpart of the forearm (Fig. 1). The sponges and
Method electrode were obtained from Gymna (Bilzen,
Twelve volunteers (six males and six females, Belgium).
mean age 5 22.3  3.4 years) participated in this Spot B (passive diffusion under contact sponge):
study. A total of six application sites (from A to F, application of 12 mg/cm2 diethylammonii diclo-
surface 7 cm2) were marked on both volar fore- fenac 1%, covered with a wet circular cellulose
arms. contact sponge on the same surface of 7 cm2 for
An acclimatization period of 30 min prior to the 20 min.
measurements was followed. Temperature (20  Spot C (passive diffusion without any covering):
2 1C) and relative humidity (45  5%) were kept application of 12 mg/cm2 diethylammonium
constant during the experiment. diclofenac 1% for 20 min.
Skin color was evaluated with the Minolta Spot D (current effect on barrier): direct current:
Chromameter-CR200 (Minolta Camera Benelux 0.2 mA/cm2, cathode, for 20 min.
BV, Kontich, Belgium). The a* parameter, ex- Spot E (standard MN response): no treatment
pressed in arbitrary units, which typically mea- with diclofenac.
sures the redness of the skin, is a good indicator Spot F: (blanco): no treatment.
of skin erythema. In addition, local blood flow, Sponges were saturated with distilled water
expressed in dimensionless perfusion units, was before application.
evaluated using Laser Doppler flowmetry (Laser A randomization schedule was followed in
Doppler, Järfälla, Sweden) (Periflux PF3). order to exclude regional effects.
After the 20 min treatment period, all skin sites
were cleansed with distilled water using a wet
Experiment part 1: diclofenac application soft tissue.
After baseline measurements, the following
applications were performed:
Spot A (iontophoretic delivery): application of Experiment part 2: MN application
12 mg/cms2 diethylammonii diclofenac 1% Because the current application induced a skin
(Voltaren Emulgel, Novartis, Novartis Consu- erythema, a resting period of 90 min was allowed
mer Health, Brussels, Belgium), iontophoretically after current application. At that time, a nicoti-
stimulated by a direct current: 0.2 mA/cm2 at the nate test was performed using paper filter disks
cathode, for 20 min. Current was generated with (18 mm, Epitest Ltd Oy, Tuusula, Finland) satu-
a Sonopuls 992 (Enraf Nonius NV, Aartselaar, rated with an MN solution (0.005 M) and applied

In vivo bioavailability of iontophoretically delivered diclofenac

for 30 s on all skin sites, except the blanco site rest period
(spot F). Bioengineering measurements were car- ± 90 min.
ried out every 5 min post-MN application for 20 min.
65 min. ionto (A) occlusion (B) passive (C)
MN (E) blanco (F) current (D)

Statistical analysis 9
All data were tested on normality using the

a* (a.u.)
Kolmogorov–Smirnov goodness-of-fit test. 6
Kinetics were compared using the MANOVA
procedure, while areas under the response curve MN application 30 sec.
were compared using an ANOVA test followed by 0
Sheffé test. −40 −20 0 20 40 60
Time (min.)
Inhibition, corrected for blanco response, was
expressed in percentage of the standard MN Fig. 2. Colorimetric evaluation of a methylnicotinate-induced skin
erythema after diclofenac application under different application and
inflammation assay. Significance level was set
controls modes (A, iontophoretic delivery; B, passive diffusion under a
at 5%. contact sponge; C, passive diffusion without covering; D, placebo
iontophoresis; E, standard MN response and F, blanco).

Results 350
81%,* 100%,
During iontophoresis, a mild prickling sensation 300
was experienced on the skin at the cathode side
a* (A.U.)

when current was turned on but the sensation 35%,** 34%,** standard MN
150 current
diminished during the treatment. A mild er- passive alone
ythema was visible after iontophoretic stimula- iontophoretic
50 occlusion
tion but this was no longer detectable with color 0
or microcirculation evaluation 90 min after cur-
Fig. 3. Colorimetric evaluation of the microcirculation: response to an
rent cessation.
MN application; comparison of the different application modes
After a resting period of 90 min, the MN test expressed in percentage of the standard MN response. Data presented
was performed. as total kinetic response (area under the curve) corrected for blanco
For the colorimetric measurements, we ob- values. *Po0.05, **Po0.001, compared with standard MN response.
served a significant inhibition of the MN response
on the three skin sites that were pre-treated with
diclofenac. The inhibition after iontophoretic de- the iontophoretically delivered diclofenac com-
livery was significant compared with the stan- pared with delivery under the contact sponge
dard MN protocol (A vs. E, P 5 0.001), with (P 5 0.79). After placebo iontophoresis we ob-
delivery under a contact sponge (B vs. E, P 5 served a significant inhibition of the MN-induced
0.001) and with passive diffusion alone (C vs. E, redness (19%) compared with the standard MN
P 5 0.04) (Fig. 2). The response after iontophoretic response.
delivery did not differ from the response under For the perfusion of the microcirculation (Laser
contact sponge occlusion (A vs. B, P 5 0.13). Also, Doppler), we observed similar results (Fig. 4).
after placebo iontophoresis, the erythema was Inhibition of the MN response was significant for
inhibited compared with the standard MN- all diclofenac pre-treated sites: inhibition after
induced erythema (D vs. E, P 5 0.04). iontophoresis (A vs. E, P 5 0.0001), passive pene-
With the data presented as areas under the tration under a contact sponge (B vs. E, P 5
curve, we calculated the percentage of inhibition 0.0001) and passive diffusion without any cover-
compared with the standard MN response (Fig. ing (C vs. E, P 5 0.002).
3). Total inhibition was 65% and 66% for ionto- Again, there was no significant difference be-
phoretic and passive penetration under the con- tween the response obtained after iontophoretic
tact sponge, respectively, while passive diffusion delivery and passive penetration under the con-
without any occlusion resulted in a 32% inhibi- tact sponge (A vs. B, P 5 0.440).
tion compared with the standard MN response. Presenting the data, corrected for blanco va-
Again, no significant difference was observed for lues, as area under the curve (Fig. 5), we observed

Lambrecht et al.

rest period cyclooxygenase (COX), also called prostaglandin

± 90 min. endoperoxide H synthetase. Our interest lies in
20 min. COX-2 because this enzyme is responsible for the
ionto (A) occlusion (B) current (D) formation of inflammatory prostaglandins. It has
passive (C) MN (E) blanco (F)
already been demonstrated that diclofenac inhi-
bits this COX-2, resulting in a reduction of the
perfusion units

MN application 30 sec. inflammation (11). We used this property to

evaluate the tissue bio-availability of diclofenac,
which is proportional to the inhibition of an MN-
induced erythema. It can be assumed that the
reduction is a function of the amount of NSAIDs
available at the site of inflammation. It has
−40 −20 0 20 40 60 already been demonstrated that there is a good
Time (min.) correlation between the in vitro uptake of NSAID
in the skin and the in vivo inhibition of the
Fig. 4. Evaluation of the microcirculation of a methylnicotinate-
induced skin erythema after diclofenac application under different provoked inflammation (12).
application and controls modes and (A, iontophoretic delivery; B, The iontophoretically delivered diclofenac was
passive diffusion under a contact sponge; C, passive diffusion without compared with the passive diffusion when the
covering; D, placebo iontophoresis; E, standard MN response and F, application site was left uncovered or occluded
blanco). with the contact sponge electrode. This allowed
us to compare the iontophoretic delivery with the
4500 passive diffusion and to estimate the occlusive
perfusion units (A.U)

4000 100%
3500 effects.
3000 35%,* For the Laser Doppler measurements, we ob-
2000 0%,** standard MN
served a complete inhibition of the signal when
1500 3%,**
response the test sites were pre-treated with diclofenac. It
1000 iontophoretic
delivery occlusion passive alone seems that there is a strong inhibition of the
0 microcirculation at a measuring depth of 1 mm.
Fig. 5. Evaluation of the microcirculation: response to an MN appli- The Laser Doppler data represent the result from
cation; comparison of the different application modes expressed in the blood flow in the superficial dermal plexus
percentage of the standard MN response. Data presented as total and capillary loops, and do not reflect the blood
kinetic response (area under the curve) corrected for blanco values. flow in the deeper regions.
*Po0.05, **Po0.001, compared with standard MN response.
The results therefore differ from the color
evaluation. Treffel et al. (12) hypothesized that
the redness measured by the Chromameter was
that the inhibition of the perfusion of the micro- mainly because of the blood increase in the
circulation was complete after iontophoretic de- capillary loops and in the arteriovenous shunts
livery (100%) and did not differ significantly from of the subpapillary plexus. Skin redness gives
the response after passive penetration under the information from different depths and is there-
contact sponge, which resulted in a 97% reduc- fore more appropriate for this type of measure-
tion of the erythema response (P 5 0.13). The ments.
perfusion of the microcirculation after passive Diclofenac inhibits the induced inflammation
penetration without covering was inhibited redness, resulting in lower a* values obtained
(65%) compared with the standard MN response. with the Chromameter. Hence, we may assume
Again, after placebo iontophoresis, we observed, that lower values represent higher diclofenac
using the Laser Doppler, a significant inhibition concentrations in deeper tissue layers. The inhi-
of the MN-induced response (42%) compared bition of the erythema was stronger after ionto-
with the standard MN response. phoresis and passive diffusion under contact
sponges compared with the passive diffusion
alone, pointing to an enhanced delivery under
Discussion iontophoretic and occlusive conditions. We ob-
The anti-inflammatory properties of diclofenac served no significant difference between the MN
are because of the inhibition of the enzyme responses when diclofenac was iontophorized,

In vivo bioavailability of iontophoretically delivered diclofenac

compared with passive diffusion under the con- tions. Moreover, it has the potentials to evaluate
tact sponge. As a consequence, the enhanced the bioavailability after iontophoretic delivery
delivery compared with the passive diffusion of NSAIDs. Colorimetric evaluation seemed to
seems to be explained by an occlusive effect be appropriate for evaluating the inhibition,
and not by a current-induced mechanism. We whereas evaluation of the microcirculation gives
were not able to demonstrate any advantage of additional information that can support the col-
the current for a single application of diclofenac. orimetric data. In our in vivo experiment, we
The major factor influencing the penetration demonstrated that for a single application, the
seems to be the occlusion. During iontophoresis, presumed enhancing effect of a current is negli-
the treated area is covered with a water-humidi- gible compared with passive penetration under a
fied sponge electrode and this occlusion causes contact sponge. In fact, the wet occlusion seemed
an increase in the hydration of the skin. It is well to be the most important factor explaining the
known that occlusion of the treated area im- enhanced percutaneous penetration.
proves the percutaneous absorption (13). We recommend that in future experiments
A current pre-treatment seems to have an effect where iontophoresis is investigated, the inclusion
on the passive penetration of MN. In a previous of several controls needs to be considered, be-
study, we demonstrated that a current pre-treat- cause the passive penetration and the effect of the
ment influences the passive uptake of MN, re- occlusion or other manipulation effects may be
sulting in shorter time-to-peak values and lag the dominant factors compared with the current-
times (14). An increased passive uptake after a induced delivery.
current pre-treatment was also observed in other
studies (15, 16). The increased uptake may be
explained by a significant decrease of the skin
impedance, which persists several hours after the References
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Related Interests