v

Research Report

Ketoprofen Tissue Permeation in

Swine Following Cathodic
Iontophoresis
Background and Purpose. Pharmacokinetic assessment of drug tissue
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permeation following iontophoresis is limited. The depth of ketopro-

fen tissue permeation following cathodic iontophoresis (4 mA, 40
minutes) and the stereoselectivity of drug delivery were examined in
this study. Subjects. Ketoprofen (750 mg) was iontophoresed onto one
porcine medial thigh, with passive drug permeation conducted on the
other thigh. Methods. Skin, subcutaneous fascia, and muscle biopsies
from the drug delivery sites were harvested and stored separately, and
the “R” and “S” ketoprofen enantiomers were determined. Results.
Iontophoretic and passive applications yielded equivalent total keto-
profen concentrations in the skin and fascia. In contrast, multivariate
analysis demonstrated that the ketoprofen concentration in the first
centimeter of muscle following iontophoresis was greater than the
drug concentration in the deeper underlying muscle layers and greater
than that delivered to any muscle layer following passive delivery. No
transcutaneous stereoselective delivery of ketoprofen was detected.
Conclusion and Discussion. Compared with passive delivery, ionto-
phoresis enhances nonstereoselective ketoprofen permeation into the
fascia-muscle interface. With delivery to deeper tissue sites, however,
there is no apparent enhancement over passive application. @Panus PC,
Ferslew KE, Tober-Meyer B, Kao RL. Ketoprofen tissue permeation in
swine following cathodic iontophoresis. Phys Ther. 1999;79:40– 49.#

Key Words: Cutaneous administration, In vivo, Iontophoresis, Ketoprofen, Nonsteroidal anti-

inflammatory drug.
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Peter C Panus

Kenneth E Ferslew

Brunhilde Tober-Meyer

Race L Kao

40 Physical Therapy . Volume 79 . Number 1 . January 1999


M
ultiple pharmaceutic methods exist for
delivering medications transcutaneously.
The most common and widely used
method is injection. Alternative methods
include the use of electromotive force (iontophoresis or
ionization), mechanical force (phonophoresis or sono-
phoresis), or passive permeation. Parenteral transcuta-
neous delivery of pharmacologic agents has potential
benefits. Avoidance of adverse effects on the gastrointes-
tinal system when these same agents are introduced into
the body orally is one example. Following oral adminis-
tration and alimentary absorption of drugs, they are
transported through the portal system to the liver where
they may be metabolized. Parenteral drug delivery min-
imizes the metabolism of drugs by the liver, thus avoid-
ing this first-pass effect.1 Transcutaneous pharmacologic
delivery also provides the potential for localized delivery
to the tissues underlying the site of application.
Both electromotive and mechanical forces have been
documented to transcutaneously deliver a wide variety of
pharmacologic agents under either experimental or
clinical conditions. Transcutaneous deliveries of polar
and nonpolar agents of either small or large molecular
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weights have been documented.2–10 Phonophoresis may
have an additional advantage in the transcutaneous
permeation of nonpolar agents due to the utilization of
mechanical rather than electromotive force.4,9,10 In con-
trast to iontophoresis and phonophoresis, passive per-
meation is limited in the variety of pharmacologic agents
delivered.2,5,9,11,12 Passive administration has not been
shown to efficiently deliver proteins or hydrophilic
anionic compounds transcutaneously. The potential for
transcutaneous delivery of a wide variety of agents is
clearly documented.
A recent literature review documented that both protein
and nonprotein pharmacologic agents have been admin-
istered transcutaneously with the purpose of achieving
therapeutic systemic pharmacologic levels while avoid-
ing first-pass effects of the gastrointestinal system.13
In contrast, transcutaneous administration of anti-
inflammatory drugs has focused on local tissue perme-
ation at the application site, regardless of whether the
permeation was attempted by iontophoresis,13,14 phono-
phoresis,8,15 or passive administration.16,17 The pharma-
cologic agents that have been transcutaneously adminis-
tered for local tissue anti-inflammatory effects include

PC Panus, PhD, PT, is Assistant Professor, Department of Physical Therapy, College of Public and Allied Health, East Tennessee State University,
Box 70624, Johnson City, TN 37614-0624 (USA) (panus@access.etsu.edu). Address all correspondence to Dr Panus.

KE Ferslew, PhD, is Professor, Section of Toxicology, Department of Pharmacology, James H Quillen College of Medicine, East Tennessee State
University.

B Tober-Meyer, DVM, is Director, Division of Laboratory Animal Resources, James H Quillen College of Medicine, East Tennessee State University.

RL Kao, PhD, is Professor, Department of Surgery, James H Quillen College of Medicine, East Tennessee State University.

All animal experimentation in this study received prior approval from the Institutional Animal Care and Use Committee of East Tennessee State
University.

This research was supported by East Tennessee State University Research and Development Committee Grants 96-001/GIA and 96-065/MJR) and
by endowments from EMPI Inc (Minneapolis, Minn) and Waters Corporation (Milford, Mass).

This article was submitted December 31, 1997, and was accepted September 20, 1998.

Physical Therapy . Volume 79 . Number 1 . January 1999 Panus et al . 41

steroidal and nonsteroidal drugs.13 The advantages of
transdermal delivery of anti-inflammatory drugs into the
local tissue underlying the application site are reduced
systemic drug levels and reduction of adverse gastro-
intestinal effects when these drugs are administered
orally.18
Ketoprofen is an anionic nonsteroidal anti-inflammatory
drug (NSAID) with approximately 160 times the anti-
inflammatory potency of aspirin on a per weight
basis.18,19 Commercially, ketoprofen is formulated as a
racemic mixture of “R” and “S” enantiomers, which are
equivalent on a per weight basis. Enantiomers of a
chemical are mirror images of the structure about a
chiral center. Because of this difference, ketoprofen
exhibits enantiomeric selectivity, with only the “S” enan-
tiomer demonstrating pharmacodynamic activity.20 Both
oral and transcutaneously administered ketoprofen
reduced carrageenan-induced acute inflammatory
edema in the rat paw.21 Passive transcutaneous adminis-
tration of the ketoprofen gel at the inflammatory site was
equivalent to oral administration of the drug in the
degree to which the edema was reduced. The ketopro-
fen concentrations required for decreasing the inflam-
mation by 50% for the topical gel and the oral formula-
tion were 2.2 mg and 6.0 mg, respectively, per kilogram
of animal weight. Thus, the topical formulation was
nearly 3 times more effective on the basis of the drug
weight than that of the oral formulation.
In humans, repeated daily passive topical administration
of a ketoprofen gel for 3 days achieved detectable
ketoprofen in the synovial fluid of the knee.22 Ketopro-
fen has also been measured in venous blood flow from
the application sites following 160-mA 3 min cathodic
iontophoresis in humans.23,24 Following these passive or
iontophoretic applications, the detectable ketoprofen
concentrations in the synovium and return venous blood
were within the 0.4- to 6-mg/mL potential therapeutic
range of ketoprofen.19,20
Several clinical investigations25–27 have also demon-
strated positive clinical outcomes when ketoprofen was
delivered transcutaneously. In these investigations, a
twice-daily application of a ketoprofen gel for a mini-
mum of 3 days resulted in reduced subjective assessment
of pain, stiffness, movement limitations, or swelling
associated with either acute soft tissue injuries or rheu-
matic dysfunction. Whether the positive clinical benefits
observed with passive transcutaneous permeation of
ketoprofen, and potentially with iontophoretic perme-
ation of ketoprofen, were truly a local tissue effect or an
effect following systemic distribution, however, remains
open to discussion.11,12,20,22,28,29
42 . Panus et al
Investigations have documented the potential for trans-
cutaneous ketoprofen permeation following both pas-
sive administration22 and iontophoretic administra-
tion.23,24 The in vivo depth of local transcutaneous
permeation for anionic anti-inflammatory drugs
(eg, dexamethasone, ketoprofen) following a single ion-
tophoretic or passive administration under clinically
relevant conditions, however, has not been reported.
Our investigation, therefore, was undertaken (1) to
examine the depth of ketoprofen tissue permeation
following a single iontophoretic or passive application
and (2) to determine whether transcutaneous ketopro-
fen permeation following these applications was stereo-
selective to either the pharmacodynamically active “S” or
the inactive “R” enantiomers. Ketoprofen was used as a
model anionic anti-inflammatory drug due to previous
evidence of transcutaneous permeation following pas-
sive administration22 or cathodic iontophoresis,23,24
edema reduction when applied passively,21 reduction in
the complaints of pain by patients when applied passive-
ly,25–27 and detection of the drug in muscle tissue
samples.30
Materials and Methods
Our experimental protocol using a swine model has
been described previously,23,24,30 and only additional
relevant methods will be elaborated on in this report.
Materials
Ketoprofen, fenoprofen, S-(-)-a-phenylethylamine, and
anhydrous isopropanol were obtained from Sigma
Chemical Company.* Acetonitrile (Optima), iso-octane,
isopropanol, methylene chloride, methanol, glacial ace-
tic acid, NaHCO3 , and HCl were obtained from Fisher
Scientific.† All borosilicate test tubes, Teflon‡-sealed 50-
and 25-mL tubes and uncapped 20-mL tubes, and 30-mL
polypropylene homogenization tubes were also obtained
from Fisher Scientific.† Polypropylene tubes (250 mL)
were from Cole Palmer International.§
Solution Preparation
Standards of ketoprofen and fenoprofen (internal stan-
dard) were dissolved in methanol at 1 mg/mL and
stored in Teflon-sealed brown glass containers (25°C).
The internal standard was a quality-control variable for
ketoprofen quantification. The internal standard was
added to all biologic standards and unknown samples to
document the efficiency of ketoprofen extraction,
dimerization, and chromatographic detection from the
tissues in question. Ketoprofen used for in vivo ionto-
phoresis was prepared at 300 mg/mL in phosphate-
buffered saline with 20% ethanol (volume per volume).
*
Sigma Chemical Co, PO Box 14508, St Louis, MO 63178-9916.
†
Fisher Scientific, PO Box 4829, Norcross, GA 30091.
‡
EI du Pont de Nemours & Co Inc, 1007 Market St, Wilmington, DE 19898.
§
Cole Palmer International, 7425 N Oak Park Ave, Niles, IL 60714.
Physical Therapy . Volume 79 . Number 1 . January 1999

These reagents and all tissue extraction, derivatization,
and high-performance liquid chromatography (HPLC)
reagents were prepared as described previously.23,24,30
Water used for all solutions was 18-MV eluant from a
Barnstead Nanopure system.i
Animal Surgery and Tissue Sampling
Surgical anesthesia in the pigs was induced by atropine
(0.05 mg/kg, administered intramuscularly), ketamine
(25 mg/kg, administered intramuscularly), aceproma-
zine (0.5 mg/kg, administered intramuscularly), and
sodium pentobarbital (15 mg/kg, administered intrave-
nously). Anesthesia in one animal was induced by succi-
nylcholine (0.4 mg/kg, administered intravenously).
Anesthesia was maintained by inhalation of 0.5% to 1.0%
halothane in oxygen. The animals were ventilated at 12
to 16 respirations per minute at 300 to 400 mL per
breath. At the termination of the experiment, the mean
heart rate was 94 beats per minute, and the systolic and
diastolic blood pressures were 70 and 38 mm Hg,
respectively. These results confirm that the cardiovascu-
lar system was intact during the application period. The
medial surface of each thigh was prepared by removal of
hair with clippers, so as not to damage the stratum
corneum. The application areas were precleaned with
isopropyl alcohol swabs.
Iontophoresis was conducted using a medium EBIE
applicator electrode and Dupel iontophoretic device.# A
total of 2.5 mL of the ketoprofen (750 mg) solution was
applied to the applicator. The first drug delivery appli-
cator was placed on the medial surface of the animal’s
thigh and attached to the cathodic electrode of the
Dupel device. The return anodic electrode was placed
on the animal’s abdomen 25 cm from the delivery
applicator. The iontophoretic dosage was 160 mA 3 min
(4 mA for 40 minutes) at a current density of
0.28 mA/cm2. The second drug delivery applicator was
placed on the contralateral medial thigh of the animal,
but without connection to the Dupel device, and served
as the passive delivery system. Following the iontophore-
sis, the electrode was removed. A 2.54-cm-diameter key-
hole bit with the center mandril removed was placed at
the center of the iontophoretic application, and the cir-
cumference of the bit was scribed with a scalpel. The skin
underlying the electrode was then excised from the under-
lying fascia and surrounding skin and stored for analysis.
The fascia was then clipped from the underlying muscle
and stored separately for later analysis. Finally, a core
sample of muscle was taken. The keyhole drill was cleaned
and placed on the surface of the muscle and then run in
reverse to the lateral surface. The muscle biopsy specimen
was stored separately for later analysis.
i
Barnstead Thermolyne, 2555 Kerper Rd, PO Box 797, Dubuque, IA 52007-0797.
# **
Empi Inc, 599 Cardigan Rd, St Paul, MN 55126-3965.
Physical Therapy . Volume 79 . Number 1 . January 1999
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The procedure was repeated on the contralateral thigh
receiving the passive ketoprofen delivery. Serum samples
were obtained from the systemic circulation, either by
cardiac puncture or from one of the great vessels in the
central cavity, and then coagulated on ice, centrifuged
(850 times per gram, 5 minutes, 25°C) (Beckman
TJ-6),** and stored as described below. All instruments
were cleaned so as to be free of visual tissue residue
between obtaining tissue samples following ionto-
phoretic and passive ketoprofen deliveries. Finally, all
samples were transported vertically on ice prior to
storage (280°C).
Sample Preparation
Spiked tissue from skin, fascia, and muscle for standard
curved determinations were processed as described pre-
viously.30 Tissue standards were homogenized in 1 nor-
mal NaHCO3 , methylene chloride was added, and the
homogenates were vortexed and centrifuged. The upper
aqueous layer was acidified with 10 normal HCl, then
combined with iso-octane:propanol. The samples were
vortexed and centrifuged, and the organic layer was
evaporated. Except for the following differences, tissue
samples from the passive and iontophoretic application
sites were processed in the same manner as in the spiked
tissue standards. Upon thawing, the skin and fascia
samples were weighed and sufficient nonexposed tissue
was added to result in a final wet tissue weight of 0.5 g.
Fenoprofen was added (40 mL, 1 mg/mL), and the
samples were subsequently processed as the standards.
The muscle core sample was laid on a metal rule.
Samples were obtained by slicing the core perpendicu-
larly at 1-cm depths. Each 1-cm aliquot was weighed, and
sufficient 1 normal NaHCO3 was added to make the final
sample weight:volume ratio 0.1 g/mL. Each muscle
sample was homogenized as described earlier, and
2.5 mL of the homogenate was transferred to a 50-mL
round-bottom flask. An additional 2.5 mL of 1 normal
NaHCO3 was added to make the final concentration 0.05
gm/mL, fenoprofen was added (40 mL, 1 mg/mL), and
the samples were subsequently processed as the stan-
dards. The total ketoprofen concentration in serum
samples was determined23,24 and compared with a stan-
dard curve generated by spiking 1 mL of serum with 10
mL of ketoprofen (1 mg/mL) and 4 subsequent 1:1
(volume per volume) serial dilutions. The internal stan-
dard was 40 mL of fenoprofen (1 mg/mL).
Sample Derivatization and High-Performance Liquid
Chromatographic Determination
The extracted ketoprofen and fenoprofen were derivat-
ized with ethylchloroformate/phenylethylamine in tri-
ethylamine. Sample derivatization and chromatographic
separation and determination were conducted at 25°C.
Beckman Inc, PO Box 10200, Palo Alto, CA 94304.
Panus et al . 43
 skin (y50.122x 10.010, r 25.992 and
N57), fascia (y50.160x 2 0.010,
r 25.999 and N57), muscle
(y50.086x 2 0.061, r 25.982 and
N512), and serum (y50.176x 2
0.058, r 25.978 and N57). Unless
stated otherwise, all data are pre-
sented as means (6standard devia-
tion). Analyses between matched
data from the same animals were
determined by a 2-tailed paired t test
(P#.05). A multivariate analysis of
variance was used to detect differences
among depths of drug delivery within
a treatment protocol and differences
between delivery protocols at a given
depth. To determine specific differ-
ences among drug delivery depths
within a protocol and differences
between delivery protocols at a given
Figure 1. depth, a Tukey post hoc groupwise
Total ketoprofen concentrations recovered in the skin and underlying fascia following either 40 comparison (P#.05) was conducted.
minutes of cathodic iontophoresis at 4 mA or 40 minutes of passive delivery. No differences were All statistical analyses were conducted
observed between iontophoretic and passive ketoprofen permeation at either skin or fascia depths,
as previously described31 using the
as determined by a 2-tailed paired t test (N55).
SAS System for the PC‡‡ on a Pentium
Millennia Transport.§§
Samples were transferred to 250-mL polypropylene vials, Results
injected via a 717 autosampler,†† and analyzed on Both cathodic iontophoresis (160 mA 3 min; 4 mA for
an HPLC device, which consisted of an M45 solution 40 minutes) and passive permeation for 40 minutes
delivery system,†† a Lambda Max Model 480 variable- demonstrated measurable and equivalent total ketopro-
wavelength ultraviolet monitor,†† a 746 data module,†† fen concentrations in the skin and fascia underlying the
and a Z-module.†† The 717 autosampler injected a 50-mL application sites (Fig. 1). Total ketoprofen concentra-
sample for analysis, and the injector was rinsed with tions in the fascia underlying the iontophoretic and
methanol:water (1:5, volume per volume) between sam- passive delivery sites were 70% to 79% lower than the
ples. Analytes were eluted from a Waters Nova-Pak C18 drug concentrations in the skin.
radial compression column†† (8 mm 3 100 mm 3 4 m)
with 10-m Bondapak C18 Guard-Paks†† and detected at Unlike the skin and fascia, the muscle tissues underlying
255 nm. The mobile phase was acetonitrile:water:glacial the drug application sites were divided into sections 1 cm
acetic acid:triethylamine (43:57:0.1:0.03, volume per vol- in depth and analyzed separately. The total ketoprofen
ume) with a flow rate of 2.5 mL/min. concentrations in these different muscle samples were
influenced by both the depth of the sample from the
Calculations and Data Analysis surface of the muscle and whether ketoprofen was
The peak areas under the curve for “R” and “S” diaste- applied passively or by iontophoresis (Tab. 1). An inter-
reomers of ketoprofen or fenoprofen were added action between the depth of the muscle sample and
together for calculation of total ketoprofen or fenopro- ketoprofen delivery protocol was also detected in the
fen concentrations. Sample-to-sample variation was nor- statistical modeling. The ketoprofen concentrations at
malized by dividing the total ketoprofen peak area by the the various tissue depths following iontophoretic and
total fenoprofen peak area. Regression analyses were passive administration are represented graphically in
calculated by plotting ketoprofen concentration on Figure 2.
the abscissa and ketoprofen-to-fenoprofen peak area
ratios on the ordinate. Calculations for unknown At the iontophoretic site, the total ketoprofen concen-
ketoprofen concentrations were determined by utiliz- tration in the initial centimeter of muscle tissue was
ing a linear regression function for each tissue. The greater than the drug concentrations at depths of 2 to 5
regression functions for the tissues were as follows:
‡‡
SAS Institute Inc, Box 8000, Cary, NC 27511-8000.
†† §§
Waters Inc, 34 Maple St, Milford, MA 01757. Micron Electronics Inc, 900 E Karcher Rd, Nampa, ID 83687.

44 . Panus et al Physical Therapy . Volume 79 . Number 1 . January 1999


cm. No differences in total ketoprofen concentrations
were detected at depths of 1 to 5 cm following passive
ketoprofen administration. Comparisons of the ketopro-
fen concentrations at each 1-cm muscle depth following
iontophoretic or passive administration were also con-
ducted (Fig. 2). Only the first centimeter of muscle
depth demonstrated an increase in total ketoprofen
concentrations following iontophoresis when compared
with passive delivery. At depths of 2 to 5 cm, the total
tissue ketoprofen concentrations following ionto-
phoretic and passive administration were equivalent.
Subsequent power analyses were conducted to deter-
mine the number of additional experiments required to
detect statistical differences in total ketoprofen concen-
trations between the first centimeter and deeper layers
under passive administration and between iontophoresis
and passive administration at the second centimeter and
deeper layers. The common standard deviation of these
analyses was derived from the mean square error shown
in Table 1, which represented the pooled variation for
all of the means under consideration. In brief, the
analyses predicted that an additional 30 animals would
be required to detect statistically significant differences
in ketoprofen between the first and second layers under
passive application. Additionally, approximately 6 times
as many animals would be required to detect differences
in ketoprofen between iontophoretic and passive admin-
istration at the second-centimeter muscle layer. These
results suggest that further experiments to determine
statistically significant differences in total ketoprofen
concentrations for these variables were prohibitive both
ethically and economically.
The ketoprofen levels measured in the skin, fascia, and
muscle may represent ketoprofen that was absorbed into
the systemic circulation and redistributed back to the
tissue underlying the iontophoretic or passive applica-
tion sites. To differentiate whether ketoprofen present
within the tissue underlying the application sites repre-
sented local tissue drug delivery or was the result of
systemic drug distribution, ketoprofen levels in the sys-
temic blood from these animals were measured. Total
ketoprofen concentrations from systemic serum were
detectable in only 2 of the 5 animals. The detected
ketoprofen concentrations in these animals were 0.44
and 0.5 mg/mL. These results suggest that the ketopro-
fen tissue concentrations observed at the application
sites represent delivery of local drug concentrations and
are not the result of systemic distribution.
The ratio of “R” and “S” enantiomers of ketoprofen was
determined in skin, fascia, and muscle (Tab. 2). The
percentage of “S” enantiomer in the skin was statistically
greater than the percentage of “R” enantiometer follow-
ing both iontophoretic and passive administration. In
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Table 1.
Statistical Modelinga of Total Ketoprofen Permeation Into
Muscle Tissue
Variable df SS MS F P>F
Model 9 1914 213 7.45 .0001
Depth 4 1304 326 11.42 .0001
Treatment 1 208 208 7.27 .01
Treatment 3 depth 4 402 100 3.52 .015
Error 40 1142 28.6
Corrected total 49 3056
a
Statistical modeling examined the effects of ketoprofen permeation depth
into muscle tissue (depth) by either iontophoretic or passive administration
(treatment) and the interaction between drug administration protocol and
depth of drug permeation (treatment 3 depth). Both main effect variables
and their interaction were all found to demonstrate an influence on total
ketoprofen concentration in the muscle tissue.
the fascia and muscle tissues, however, the “R” and “S”
enantiomer percentages were equivalent. Thus, trans-
cutaneous delivery of ketoprofen by iontophoresis or
passive application was not stereoselective.
Discussion
We examined the in vivo tissue depth for passive and
iontophoretic transcutaneous permeation of ketoprofen
and whether the transcutaneous permeation was stereo-
selective. The pig was the animal model selected due to
similar iontophoretic permeation properties between
porcine and human skin. Riviere et al32 demonstrated
parallel pharmacokinetic variables between human and
porcine skin during in vitro cell diffusion experiments.
In our investigation, nonstereoselective transcutaneous
permeation of ketoprofen was observed for both ionto-
phoretic and passive applications.
Positive clinical outcomes observed following transcuta-
neous application of anti-inflammatory agents might
result from a local effect of a drug or from systemic
absorption and redistribution back to a tissue. Investiga-
tors have examined the tissue permeation of anionic
anti-inflammatory agents following both passive perme-
ation11,12,16 and iontophoretic permeation.14,28,33 Local
passive transcutaneous permeation has been shown for
diclofenac, hydrocortisone, indomethacin, naproxen,
piroxicam, and salicylic acid, but ketoprofen tissue per-
meation following passive or iontophoretic application
has not been documented. When these anionic anti-
inflammatory drugs were applied passively, the maximal
tissue depth for local transcutaneous permeation was
down to the superficial muscles.11,12 Drug concentra-
tions in deeper tissues were equal to or less than systemic
blood levels, and the drug probably was delivered to
these tissues by the systemic blood flow.
The restrictive properties of the epidermis toward pas-
sive transcutaneous permeation of these agents are
significant. Singh and Roberts,11,12,28 using a rat model,
Panus et al . 45
 tion, limited investigations have
examined the depth of tissue perme-
ation following iontophoretic appli-
cation of these agents. Dexametha-
sone phosphate iontophoresed from
the anode (ie, positive electrode)
has been shown to penetrate to deep
periarticular structures in several dif-
ferent joints in a single-monkey
experiment.14 In that experiment,
the investigators concluded that the
presence of dexamethasone in these
deep tissues was the result of local
tissue permeation during the ionto-
phoresis, not an effect of drug deliv-
ery by the systemic circulation. In
contrast, cathodic iontophoresis of
salicylic acid resulted in local trans-
cutaneous tissue permeation down
to superficial muscles.28 Salicylate
Figure 2. reached the deeper tissue structures
Total ketoprofen concentrations recovered at the various muscle depths following 40 minutes of
below the application site only by the
cathodic iontophoresis at 4 mA or 40 minutes of passive administration. Asterisk (*) indicates
differences (P#.05) were demonstrated for total ketoprofen concentrations between the initial 1 cm systemic bloodstream. In our investi-
of muscle tissue and subsequent depths within a given delivery protocol. Plus sign (1) indicates gation, iontophoresis delivered
differences (P#.05) were demonstrated for total ketoprofen concentrations between iontophoretic more ketoprofen to the first centi-
and passive delivery protocols at a given tissue depth. Significance was determined by Tukey post meter of muscle than did passive
hoc multiple comparison (N55).
application. Ketoprofen detected in
deeper muscle layers may have
resulted from passive or ionto-
obtained 80-mm sections from the epidermal surface phoretic transport from the superficial muscle layer,
with a microtome in order to achieve the passive perme- focal circulatory distribution, or other as of yet unexam-
ation of anti-inflammatory agents. Piroxicam was also ined experimental variables. The tissue permeation that
delivered to similar tissue depths in the rat, without we found following ketoprofen iontophoresis agrees
epidermal debridement, but with a permeabilizing agent with the results of a previous investigation of tissue
incorporated into the gel.16 In our investigation with permeation following salicylate iontophoresis.28 Our
ketoprofen, the epidermis was not sectioned for passive results and those of previous salicylate studies contrast
application, but isopropyl alcohol was applied to the with the results of Glass and colleagues’ study of dexa-
surface vigorously as a preparatory step, and the keto- methasone iontophoresis.14
profen was prepared in a 20% ethanol solution and
applied to both the passive and iontophoretic sites. Methodological variances may account for the differ-
Short-chain alcohols have been shown to reduce the ences in the results of Glass and colleagues’ study of
restrictive properties of the epidermis, allowing greater dexamethasone,14 and the results of our investigation
transcutaneous permeation of hydrophilic agents under and Singh and Roberts’ research on salicylate ionto-
both passive and iontophoretic conditions.33–35 Our phoresis.28 In the study of dexamethasone,14 the
results are in agreement with previous findings as to the iontophoretic current density was calculated to be
depth of tissue permeation of anionic anti-inflammatory 0.94 mA/cm2 , whereas in our study and in the study of
drugs following passive application. In our investigation, salicylate iontophoresis,28 the current densities were 0.28
passive permeation of ketoprofen to tissue depths previ- and 0.38 mA/cm2 , respectively. With commercial elec-
ously reported for animals with epidermis removed may trodes and iontophoretic devices, the current densities
be accounted for by animal model differences (ie, the obtained during clinical iontophoresis in physical ther-
use of rats in previous investigations compared with the apy parallel those used in our study and in the study of
use of pigs in our investigation) or the epidermal salicylate iontophoresis.28 The current density used dur-
permeabilizing potential of short-chain alcohols. ing clinical iontophoresis may be calculated by dividing
the current setting on the iontophoretic device by the
In contrast to the multiple anionic anti-inflammatory active surface area of the electrode. According to Banga
drugs that have been examined during passive applica- and colleagues,13,36 patients generally can tolerate cur-

46 . Panus et al Physical Therapy . Volume 79 . Number 1 . January 1999

 IIIIIIIIIIIIIIIIIIIIIIIII
rent densities of less than 0.5 mA/cm2. The aggregate Table 2.
Comparison (X# 6SD) of Ratios of “R” and “S” Enantiomers of
results of our investigation and previous studies of
Ketoprofen at Different Tissue Depths Following Either Cathodic
salicylate and dexamethasone suggest that the depth of Iontophoresis or Passive Administrationa
drug tissue permeation may be positively correlated to
the current density. This conclusion is supported by Iontophoresis Passive Delivery
observations of the effect of current density on other
Tissue %S %R %S %R
in vivo iontophoretic variables. The concentration of
total ketoprofen delivered following cathodic ionto- Skin 5360.5 4760.5* 5360.6 4760.6*
phoresis in humans appeared to be directly proportional Fascia 5060.4 5060.4 5160.7 4960.7
to the current density.23,24 Iontophoresis of ketoprofen Muscle
1 cm 5468 4768 5160.6 4960.6
at 4 mA for 40 minutes (0.28 mA/cm2) resulted in 2 cm 5569 4569 5162 4962
approximately twice the ketoprofen delivery when com- 3 cm 4967 5167 5261 4861
pared with 2 mA for 80 minutes (0.14 mA/cm2). The 4 cm 5061 5061 43618 57618
enhanced iontophoretic delivery of ketoprofen 5 cm 5162 4962 4961 5161
appeared to be dependent on the higher current density a
Statistical comparison of the ratios of “R” and “S” enantiomers was done at
(ie, 0.28 versus 0.14 mA/cm2), as the dosage for these 2 each tissue depth and for each delivery modality. Significant differences (*)
iontophoretic regimens remained constant at 160 mA 3 between the percentage of “S” and “R” isomers at each tissue depth and
within a given delivery modality were determined by a 2-tailed paired t test
min. Thus, the high current density used in Glass and with P#.05 (N55).
colleagues’ study of dexamethasone14 may have resulted
in deeper iontophoretic drug permeation, but this den-
sity exceeds that normally used during current clinical
iontophoresis. We therefore question the value of the arthropathy. The therapeutic range for ketoprofen in
study of dexamethasone to current clinical use of the blood has been characterized as 0.4 to 6 mg/mL.19
iontophoresis. The specific density of heart muscle has been docu-
mented as 1.05 g/mL,38 and we believe that skeletal
Regardless of the exact tissue permeation depth of muscle should demonstrate a similar specific density.
anionic anti-inflammatory drugs following either passive Based on our data, therapeutic ketoprofen concentra-
or iontophoretic application, investigators13,17,25–27 have tions may occur throughout the first 5 cm of muscle
documented local anti-inflammatory effects when under both passive and iontophoretic applications. Keto-
applied transcutaneously. Controversy exists, however, as profen is also highly concentrated within the vascula-
to whether the anti-inflammatory effects from these ture.19 Nonvascular compartments, such as the syno-
transcutaneously administered agents are the result of vium, may achieve ketoprofen concentrations of only
local tissue permeation or delivery via systemic circula- 20% of systemic blood levels.29 Thus, therapeutic keto-
tion. Local tissue anti-inflammatory effects have been profen concentrations at nonvascular inflammatory sites
documented when anionic anti-inflammatory agents may actually be less than the 0.4 to 6 mg/mL observed in
were applied passively21 or by iontophoresis.37 Locally the systemic vascular compartment.19 Radermacher
applied and passively delivered ketoprofen was 3-fold et al,39 however, documented that transcutaneously
more effective at inhibiting in vivo carrageenan-induced applied NSAIDs reached nonvascular compartments,
rat paw edema compared with oral administration of the such as the synovium, after systemic absorption from the
drug.21 Additionally, in vitro iontophoretic flux of diclofe- application site and vascular distribution to the joints.
nac across the skin parallels the inhibitory effect of diclofe-
nac, when applied by iontophoresis, on carrageenan- The clinical implications of our current research and the
induced rat paw edema.37 In a study by Byl et al,8 a single previous research of other investigators suggest that
phonophoretic application of dexamethasone demon- anionic anti-inflammatory agents, in general, and keto-
strated subcutaneous anti-inflammatory effects at the appli- profen, in particular, have the potential to penetrate the
cation site. When submuscular or subtendinous tissue superficial skeletal muscle tissue at pharmacologically
below the application site was examined, no such anti- relevant concentrations so as to alleviate inflammation
inflammatory effects were observed. Yet, Byl et al also in soft tissue. Under clinical conditions, however, these
reported reduced anti-inflammatory effects at sites distal to drugs must be used within the methodologies estab-
the phonophoretic application, suggesting systemic distri- lished in the clinical and experimental research. With
bution of the dexamethasone. our current research, the ketoprofen concentration
used was 300 mg/mL in a 20% ethanol solution, and the
Grahame17 contended that local application of anionic iontophoretic dosage was 160 mA 3 min. The extrapo-
anti-inflammatory agents may have potential usefulness lation of these results to the current clinical use of
in the treatment of patients with soft tissue injuries, but dexamethasone phosphate would be inappropriate. The
not in the treatment of patients with inflammatory dexamethasone phosphate concentration available for
iontophoresis is either 4 or 24 mg/mL, and device

Physical Therapy . Volume 79 . Number 1 . January 1999 Panus et al . 47

manufacturers recommend a dosage equal to or less
than 100 mA 3 min. The relative mobilities of these 2
drugs in an electrical field are also unknown. Thus,
which drug is optimal for iontophoretic applications
remains a question. To determine the value of ketopro-
fen for clinical iontophoresis, further experimental or
clinical investigations would be required to document a
cause-and-effect relationship between drug concentra-
tions within the tissue and their anti-inflammatory effect
on soft tissue injury. Additionally, tissue compartments
surrounding joints with a different vascular network may
also demonstrate distinctly different drug tissue perme-
ation profiles following iontophoretic or passive anionic
anti-inflammatory drug administration. Further pharma-
cokinetic and pharmacodynamic investigations would be
required to document the clinical value of anionic
anti-inflammatory drugs at these application sites.
Conclusion
Panus and colleagues23,24 have previously examined ion-
tophoretic factors that would optimize ketoprofen ion-
tophoresis in vivo, and they documented iontophoretic
transcutaneous delivery of clinically relevant ketoprofen
concentrations in humans. Our current investigation
examined the local tissue permeation depth during in
vivo cathodic ketoprofen iontophoresis using previously
established optimal variables. The results indicate that
transcutaneous permeation of ketoprofen following ion-
tophoretic or passive permeation is not stereoselective at
subcutaneous sites. Under the described experimental
conditions, iontophoretic and passive application
resulted in equivalent permeation of the drug down to
the fascia. Iontophoresis delivered greater amounts of
ketoprofen to the superficial muscle layer compared
with passive delivery. Our results suggest the clinical
value of local transcutaneous application of anionic
anti-inflammatory agents to muscular sites, with ionto-
phoresis demonstrating superior permeation capabilities
over passive application. Following a single application,
however, submuscular sites would appear to be beyond
the local permeation potential of transcutaneously
applied anionic anti-inflammatory drugs.
References
1 Benet LZ, Kroetz DL, Sheiner LB. Pharmacokinetics: the dynamics of
drug absorption, distribution, and elimination. In: Hardman J, Lim-
bird L, eds. Goodman and Gilman’s: The Pharmacological Basis of Thera-
peutics. 9th ed. New York, NY: McGraw-Hill; 1996:3–28.
2 Kari B. Control of blood glucose levels in alloxan-diabetic rabbits by
iontophoresis of insulin. Diabetes. 1986;35:217–221.
3 Meyer BR, Kreis W, Eschbach J, et al. Successful transdermal admin-
istration of therapeutic doses of a polypeptide to normal human
volunteers. Clin Pharmacol Ther. 1988;44:607– 612.
4 Kim A, Green PG, Rao G, Guy RH. Convective solvent flow across the
skin during iontophoresis. Pharm Res. 1993;10:1315–1320.
48 . Panus et al
5 Thysman S, Preat V. In vivo iontophoresis of fentanyl and sufentanil
in rats: pharmacokinetics and acute antinociceptive effects. Anesth
Analg. 1993;77:61– 66.
6 Li LC, Scudds RA. Iontophoresis: an overview of the mechanisms and
clinical application. Arthritis Care Res. 1995;8:51– 61.
7 Miyazaki S, Mizuoka H, Kohata Y, Takada M. External control of
drug release and penetration, VI: enhancing effect of ultrasound on
the transdermal absorption of indomethacin from an ointment in rats.
Chem Pharm Bull (Tokyo). 1992;40:2826 –2830.
8 Byl NN, McKenzie A, Halliday B, et al. The effects of phonophoresis
with corticosteroids: a controlled pilot study. J Orthop Sports Phys Ther.
1993;18:590 – 600.
9 Mitragotri S, Edwards DA, Blankschtein D, Langer R. A mechanistic
study of ultrasonically enhanced transdermal drug delivery. J Pharm Sci.
1995;84:697–706.
10 Mitragotri S, Blankschtein D, Langer R. Ultrasound-mediated trans-
dermal protein delivery. Science. 1995;269:850 – 853.
11 Singh P, Roberts MS. Skin permeability and local tissue concentra-
tions of nonsteroidal anti-inflammatory drugs after topical application.
J Pharmacol Exp Ther. 1994;268:144 –151.
12 Singh P, Roberts MS. Deep tissue penetration of bases and steroids
after dermal application in rat. J Pharm Pharmacol. 1994;46:956 –964.
13 Banga AK, Panus PC. Clinical applications of iontophoretic devices
in rehabilitation medicine. Critical Reviews in Rehabilitation Medicine.
1998;10:147–179.
14 Glass JM, Stephen RL, Jacobson SC. The quantity and distribution
of radiolabeled dexamethasone delivered to tissue by iontophoresis.
Int J Dermatol. 1980;19:519 –525.
15 Byl NN. The use of ultrasound as an enhancer for transcutaneous
drug delivery: phonophoresis. Phys Ther. 1995;75:539 –553.
16 McNeill SC, Potts RO, Francoeur ML. Local enhanced topical
delivery (LETD) of drugs: Does it truly exist? Pharm Res. 1992;9:
1422–1427.
17 Grahame R. Transdermal non-steroidal anti-inflammatory agents.
Br J Clin Pract. 1995;49:33–35.
18 Insel P. Analgesic-antipyretic and anti-inflammatory agents and
drugs employed in the treatment of gout. In: Hardman J, Limbird L,
Molinoff PB, et al, eds. Goodman and Gilman’s: The Pharmacological Basis
of Therapeutics. 9th ed. New York, NY: McGraw-Hill Inc; 1996:617– 658.
19 Ketoprofen—American Hospital Formulary Services: Drug Information.
Bethesda, Md: American Society of Health-System Pharmacists Inc;
1996:1416 –1422.
20 Jamali F, Brocks DR. Clinical pharmacokinetics of ketoprofen and
its enantiomers. Clin Pharmacokinet. 1990;19:197–217.
21 Chi SC, Jun HW. Anti-inflammatory activity of ketoprofen gel on
carrageenan-induced paw edema in rats. J Pharm Sci. 1990;79:974 –977.
22 Ballerini R, Casini A, Chinol M, et al. Study on the absorption of
ketoprofen topically administered in man: comparison between tissue
and plasma levels. Int J Clin Pharmacol Res. 1986;6:69 –72.
23 Panus PC, Campbell J, Kulkarni B, et al. Effect of iontophoretic
current and application time on transdermal delivery of ketoprofen in
man. Pharmaceutical Sciences. 1996;2:467– 469.
24 Panus PC, Campbell J, Kulkarni SB, et al. Transdermal ionto-
phoretic delivery of ketoprofen through human cadaver skin and in
humans. Journal of Controlled Release. 1997;44:113–121.
25 Matucci-Cerinic M, Casini A. Ketoprofen vs etofenamate in con-
trolled double-blind study: evidence of topical effectiveness in soft
tissue rheumatic pain. Int J Clin Pharmacol Res. 1988;8:157–160.
Physical Therapy . Volume 79 . Number 1 . January 1999

26 Baixauli F, Ingles F, Alcantara P, et al. Percutaneous treatment of
acute soft tissue lesions with naproxen gel and ketoprofen gel. J Int Med
Res. 1990;18:372–378.
27 Airaksinen O, Venalainen J, Pietilainen T. Ketoprofen 2.5% gel
versus placebo gel in the treatment of acute soft tissue injuries. Int
J Clin Pharmacol Ther Toxicol. 1993;31:561–563.
28 Singh P, Roberts MS. Iontophoretic transdermal delivery of salicylic
acid and lidocaine to local subcutaneous structures. J Pharm Sci.
1993;82:127–131.
29 Netter P, Bannwarth B, Lapicque F, et al. Total and free ketoprofen
in serum and synovial fluid after intramuscular injection. Clin Pharma-
col Ther. 1987;42:555–561.
30 Panus PC, Tober-Meyer B, Ferslew KE. Tissue extraction and
high-performance liquid chromatographic determination of ketopro-
fen entantiomers. J Chromatogr B Biomed Appl. 1998;705:295–302.
31 Kleinbaum DG, Kupper LL. Applied Regression Analysis and Other
Multivariable Methods. North Scituate, Mass: Duxbury Press; 1978.
32 Riviere JE, Sage B, Williams PL. Effects of vasoactive drugs on
transdermal lidocaine iontophoresis. J Pharm Sci. 1991;80:615– 620.
33 Sanderson JE, de Riel S, Dixon R. Iontophoretic delivery of non-
peptide drugs: formulation optimization for maximum skin permeabil-
ity. J Pharm Sci. 1989;78:361–364.
Physical Therapy . Volume 79 . Number 1 . January 1999
IIIIIIIIIIIIIIIIIIIIIIIII
34 Srinivasan V, Higuchi WI, Sims SM, et al. Transdermal ionto-
phoretic drug delivery: mechanistic analysis and application to
polypeptide delivery. J Pharm Sci. 1989;78:370 –375.
35 Srinivasan V, Su MH, Higuchi WI, Behl CR. Iontophoresis of
polypeptides: effect of ethanol pretreatment of human skin. J Pharm
Sci. 1990;79:588 –591.
36 Banga AK, Chien YW. Iontophoretic delivery of drugs: fundamen-
tals, developments, and biomedical applications. Journal of Controlled
Release. 1988;17:1–24.
37 Varghese E, Khar RK. Enhanced skin permeation of diclofenac by
iontophoresis: in vitro and in vivo studies. Journal of Controlled Release.
1996;38:21–27.
38 Helak JW, Reichek N. Quantitation of human left ventricular mass
and volume by two-dimensional echocardiography: in vitro anatomic
validation. Circulation. 1981;63:1398 –1407.
39 Radermacher J, Jentsch D, Scholl MA, et al. Diclofenac concentra-
tions in synovial fluid and plasma after cutaneous application in
inflammatory and degenerative joint disease. Br J Clin Pharmacol.
1991;31:537–541.
Panus et al . 49