Journal of Pharmaceutical Sciences xxx (2016) 1-9

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Journal of Pharmaceutical Sciences

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Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Transdermal Delivery of Etoposide Phosphate I: In Vitro and

In Vivo Evaluation
Hiren Patel, Abhay Joshi, Amit Joshi, Grazia Stagni*
Division of Pharmaceutical Sciences, Arnold and Marie Schwartz College of Pharmacy, Long Island University, Brooklyn, New York 11201

a r t i c l e i n f o a b s t r a c t

Article history: Cancer chemotherapy frequently requires long periods of multiple intravenous infusions that often
Received 18 March 2016 results in patients opting out of treatment. The main purpose of this study was to investigate the
Revised 14 April 2016 feasibility of delivering one of these anticancer agents: etoposide phosphate (ETP) transdermally using
Accepted 15 April 2016
iontophoresis and a combination of iontophoresis/microporation. The iontophoresis conditions for ETP
were first optimized in vitro then tested in vivo in a rabbit model. Both ETP and its active form etoposide
(VP) were quantified in dermis (via microdialysis sampling) and in plasma, with a specially developed
Keywords:
high-performance liquid chromatography method. In vitro, the amount of total etoposide permeated and
etoposide phosphate
etoposide
the steady state flux increased (p < 0.05) with increase in iontophoretic current densities (100-400 mA/
microdialysis cm2). At 300 mA/cm2, microporation/iontophoresis further improved both parameters by 2- and 2.8-fold,
microporation respectively. In vivo, exposure increased proportionally to current density in plasma, whereas dermal
anti-cancer concentration dropped significantly at the highest current density. Microporation led to a 50% increase in
iontophoresis Cmax and AUClast values in both skin and plasma. In conclusion, a mild current density (300 mA/cm2) and a
chemotherapy small surface area (10.1 cm2) achieved and maintained the minimum effective concentration for the
rabbit entire duration of electrical current delivery; microporation further increased the plasma concentrations
transdermal
at the same current density.
© 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Introduction length of administration; however, the oral bioavailability (~50%,

Etoposide (VP; Fig. 1) is a semisynthetic glycoside derivative of
the bioactive natural product podophyllotoxindan extract from the
plants Podophyllum Peltatum and Podophyllum Emodi.1 VP is a front-
line chemotherapy agent active against various cancers including
small-cell lung cancer, testicular carcinoma, lymphoma, and Kaposi
sarcoma.2 In spite of its good pharmacological activity, its poor
water solubility hinders the therapeutic use of VP. To overcome
poor solubility, the water-soluble phosphate salt, in which the
phosphate moiety is attached covalently to VP, was approved by the
US Food and Drug Administration for intravenous use in 1996.3
Endogenous phosphatase enzymes efficiently convert etoposide
phosphate (ETP; Fig. 1) to VP in vivo. As a result, ETP has an anti-
tumoral effect and toxicity profile equivalent to those of VP.2,4,5 An
advantage of ETP over VP is the elimination of problems related to
inactive ingredients and solubilizers in the parenteral formulation.6
The improved water solubility of ETP increases the convenience of
intravenous (IV) infusion, that is, reduced volume of fluid and
* Correspondence to: Grazia Stagni (Telephone: 718 488 1231; Fax: 718 780 4586).
E-mail address: gstagni@liu.edu (G. Stagni).
range: 25%-75%) is only slightly improved and remains highly
variable presumably because of conversion into VP in the gut.7
Once VP is eliminated, cancer cells recover quickly8; therefore, to
maintain VP plasma concentration in the therapeutic range, IV
infusions must be administered frequently. Because cancer treat-
ments often require administration of more than one chemother-
apeutic agents as IV infusion, multiple IV infusions have a
significantly negative impact on the quality of life of patients that
often discourages them to continue the chemotherapy and opt out
of the treatment. Thus, there is a need to develop an alternative
route of administration for VP or ETP, which would increase pa-
tients' compliance and quality of life.
In this study, we explored the possibility to deliver therapeuti-
cally relevant doses of VP through the skin via iontophoresis and a
combination of microporation/iontophoresis in vivo, in a rabbit
model. Among all transdermal drug delivery systems, iontopho-
resis has the advantage of easily controllable and faster delivery.9
Iontophoresis uses a mild electrical current to drive ionic drug
molecules of the same polarity through the stratum corneum.10
Because ETP is easily ionizable, it is more suitable for iontopho-
retic delivery than VP and thus it was used for the study. To further
increase the extent and rate of ETP delivery, we also evaluated the

http://dx.doi.org/10.1016/j.xphs.2016.04.014
0022-3549/© 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
2 H. Patel et al. / Journal of Pharmaceutical Sciences xxx (2016) 1-9
Figure 1. Chemical structure of etoposide (left) and etoposide phosphate (right).
impact of microneedle pretreatment (microporation) on ionto-
phoretic delivery. Indeed, in the past decade, several studies
demonstrated a positive synergism between the two techniques to
further enhance delivery of either high-molecular-weight11 or low-
molecular-weight compound12,13 in vitro as well as in vivo.14
Microneedles are micron-size needles that pierce the skin's outer
barrier (stratum corneum); however, they are too short to reach the
nerves15,16 and therefore do not cause pain.17 To understand better
the mechanism of transdermal delivery of ETP, we measured the
kinetics of ETP and VP in dermis via microdialysis sampling as well
as systemic delivery by conventional plasma sampling. Simulta-
neous sampling from dermis and blood provides in-depth transport
mechanism insights in the process of drug delivery through the
skin. Among all the available sampling techniques to measure
dermal concentration, microdialysis permits the continuous, real-
time sampling of drug molecules present in the skin extracellular
fluid with minimal tissue damage.18,19 In a previous study,14 we
confirmed the suitability of the rabbit model to use microdialysis
sampling with a combination of microneedles pretreatment and
iontophoresis.
Materials and Methods
Materials
All chemicals were of analytical grade or higher in quality. VP for
standards was from U.S. Pharmacopeial Convention (Rockville,
MD). ETP for standards and iontophoretic solutions was from
Chem-Impex International Inc. (Wood Dale, IL) and SimagChem
Corporation (Xiamen, China). Sodium acetate trihydrate was from
VWR International, LLC (Chester, PA). Acetonitrile was from
Pharmaco-AAPER (Brookfield, CT) and high-performance liquid
chromatography (HPLC) water was from Fisher Scientific (Fair
Lawn, NJ). Glacial acetic acid was from EM Science (Gibbstown, NJ).
Lactated ringer's solution (LRS) was from Abbott Laboratories
(North Chicago, IL). ETOPOPHOS® Injection was from Bristol-Myers
Squibb Co. (Princeton, NJ). Sterile water for injection was from
Hospira Inc. (Lake Forest, IL).
Analytical Method
VP and ETP concentrations were determined using a reverse-
phase HPLC method. The HPLC equipment consisted of a Waters
717 Auto sampler (Waters Corp., Milford, MA), Hitachi L-4250
UV-VIS Detector (Hitachi, Ltd., Tarrytown, NY), Shimadzu LC-20AT
Pump (Shimadzu Scientific Instruments, Inc., Columbia, MD),
Perkin Elmer Nelson 900 Series Interface (PerkinElmer, Inc., Wal-
tham, MA), and the Perkin Elmer software Turbochrome Navigator
(PE Nelson, Version 6.3.2.0645) data-handling system. A Waters
SunFire C-18 column (3.0
150 mm, 3.5 mm, 100 Å; Waters Corp.,
Milford, MA) protected with a Phenomenex Analytical Guard Car-
tridge System (KJ0-4282; Phenomenex, Inc., Torrance, CA) was used
for all samples.
VP concentrations were determined with a method modified
from Chen et al. and You et al.20,21 VP samples were eluted using a
mobile phase that consisted of 0.01-M sodium acetate buffer (pH
5.2): acetonitrile (70:30% v/v) for microdialysis as well as plasma
samples. Samples were kept at 4 C in the autosampler and eluted at
a flow rate of 0.5 mL/min. Injection volume was 10 mL, and the
detection wavelength was 210 nm. Microdialysis samples were
injected directly onto HPLC, whereas plasma samples were purified
by liquideliquid extraction. Briefly, 100 mL of plasma was added
with 50 mL of the internal standard Teniposide (VM; 5 mg/mL) and
400 mL of dichloromethane (Sigma-Aldrich Corp., St. Louis, MO) and
vortexed thoroughly for 2 minutes. Then, the mixture was centri-
fuged at 12,000 rpm for 15 minutes at 4 C. The organic layer (300
mL) was transferred into a separate Eppendorf and evaporated to
dryness with nitrogen gas at 40 C. The residue was reconstituted
with 40 mL of mobile phase and again vortexed for 2 minutes. In-
jection volume was 10 mL. The typical retention time was 6.2 mi-
nutes for both microdialysis and plasma samples. This assay was
applied in experiments where VP was tested alone (e.g., in vitro
calibration, in vitro microdialysis, stability, and binding studies).
When ETP was present along with VP, the following assay was used.
ETP and VP were quantified simultaneously using a new
RP-HPLC method developed in our laboratory. Dialysate samples
were injected directly onto the column and eluted with a mobile
phase that consisted of 0.01-M sodium acetate buffer (pH 5.2):
methanol: acetonitrile (63:25:12% v/v/v) for in vitro experiments
and of (64:25:11% v/v/v) for in vivo experimentsdmobile phase
was modified to separate the ETP's peak from that of endogenous
substances. The isocratic flow rate was 0.35 mL/min, the autosam-
pler temperature was 4 C, and the detection wavelength was 210
nm. Typical retention times were 5.2 and 31 minutes for ETP and
VP, respectively, for in vitro studies and 6.1 and 36 minutes for ETP
and VP, respectively, for in vivo microdialysis studies. Standard
curves were linear over the range 45-150,000 nM for both ETP and
VP analytes (R2 > 0.99). Plasma samples were purified by adding
100 mL of plasma with 100 mL of methanol and vortexed thoroughly
for 2 minutes. Then, the mixture was centrifuged at 12,000 rpm for
15 minutes at 4 C. The upper layer (75 mL) was transferred into a
separate Eppendorf and again centrifuged at 12,000 rpm for
15 minutes at 4 C. A 50-mL aliquot of the upper layer was collected
and 10 mL was injected onto the HPLC. The mobile phase consisted
of 0.01-M sodium acetate buffer (PH 5.2): methanol (68:32% v/v).
All the other parameters were the same as for the dialysate sam-
ples. Standard curves were linear over the range 45-150,000 nM for
both ETP and VP analytes (R2 > 0.99). The lower limit of quantifi-
cation was 45 nM in all the conditions for ETP and VP. Plasma
extraction efficiency was performed with three different concen-
trations in plasma and compared with same concentrations
prepared in ringer's solution.
In Vitro Experiments
Microdialysis
Apparatus. The microdialysis apparatus consisted of a pump
(Fusion 400, Chemyx Inc., Stafford, TX) equipped with 3-mL plastic
syringes (Becton Dickinson & Co., Franklin Lakes, NJ). Disposable
microdialysis probes were made in our laboratory according to the
procedure previously described.22 They consisted of two 7-cm arms
of polyimide (0.00500 ID
0.007500 OD; RiverTech Medical LLC,
Chattanooga, TN) tubing glued with cyanoacrylate glue (Henkel
 H. Patel et al. / Journal of Pharmaceutical Sciences xxx (2016) 1-9
Corp., Rocky Hill, CT) to a semipermeable hollow membrane of
polyacrylonitrile with a molecular-weight cutoff of 50 kDa (AN69
HF Gambro Industries, France). The membrane window was 15
mm. An internal stainless steel wire (0.001500 in diameter; Molecu-
Wire, Wall Township, NJ) reinforced the probes. The probe was
inserted into a standard jacketed type-1 Franz diffusion cell via flow
ports (PermeGear, Inc., Hellertown, PA; internal volume: 8 mL) in
such a way that the semipermeable membrane of the probe
remained exactly at the center of the cell. The temperature was
maintained at 37 ± 0.5 C with a water bath circulator, (VWR In-
ternational, LLC, Radnor, PA). The probe recovery was studied
in vitro for (1) ETP, (2) VP, and (3) a combination of ETP and VP in
concentration of 45,000, 15,000, and 1500 nM in LRS. For “gain”23
studies, the probes were perfused with plain LRS and the inner
chamber of the glass cell was filled with the analyte solutions,
whereas for “loss” studies, the glass cells were filled with LRS and
the probe was perfused with known concentrations of the analytes.
Loss and gain were calculated with the following equations: % re-
covery by loss (%RL) ¼ (Cperfusate  Cdialysate)/Cperfusate
100; and %
recovery by gain (%RG) ¼ Cdialysate/Cbulk
100.
Permeability Studies
In vitro permeability studies were performed with type-1 Franz
diffusion cells (PermeGear, Inc., Hellertown, PA) with an exchange
area of 1 cm2 (diameter of 11.28 mm). The temperature of receiving
compartment was maintained at 37 ± 0.5 C throughout the ex-
periments. The receiver was filled with 8 mL of LRS. Porcine ear skin
was used as membrane. Porcine ears were obtained from a local
slaughterhouse (New York, NY) within maximum of 2 days after
sacrifice of the animal. Skin was prepared for permeation study
according to the reported methods by Yamamoto et al.24 Briefly,
porcine ears were cleaned under running cold water and hair were
removed with animal hair clipper. Ear was cut into (3 cm
3 cm)
pieces, and then, the outer region of the ear was removed from the
whole skin and separated from the underlying cartilage with a
scalpel. Thickness of the skin was measured with Traceable digital
stainless steel caliper (Control Co., Friendswood, TX) and then, each
piece was individually wrapped in Parafilm M (Bemis Company Inc.,
Neenah, WI) and stored for not more than a week at 20 C until
used. The average thickness of the excised porcine ear skin was 1.03
± 0.08 mm (n ¼ 24). On the day of experiment, the required pieces
of skin were thawed at room temperature for 15-20 minutes.
Integrity of skin was evaluated by visual inspection to ensure that
no holes or other imperfections were present. Skin was let to hy-
drate with LRS for 30-35 minutes before each experiment. The
solution in the receiving compartment was continuously stirred
with a magnetic stirrer at 500 rpm to maintain a constant hydro-
dynamic condition in the diffusion medium.
Iontophoresis. The following electrodes were tested sequentially:
(1) silver wire (Sigma-Aldrich Corp.), (2) steel electrode encased in
a plastic shell with a constant surface area of 3.14 cm2 (donated by
Transport Pharmaceutics, Inc., Framingham, MA), (3) Empi Dupel
B.L.U.E. (EMPI, St. Paul, MN), and (4) TransQE (IOMED Inc., Salt Lake
City, UT). Preliminary experiments showed that cathodal ionto-
phoresis was more suitable than anodal for ETP delivery. ETP is an
acid with a pKa probably between 1 and 2.25 Therefore, the cathode
was attached to the electrode filled with ETP solution, and the
anode was attached to a silver wire 0.5-mm diameter placed in the
receiving compartment26 for all experiments. The following current
densities were applied for an hour 0, 100, 200, 300, or 400 mA/cm2
using an IOMED Phoresor II, Model No. PM 700 (IOMED Inc., Salt
Lake city, UT). At the end of the current delivery period, the patch
was removed from the donor compartment; however, sampling
continued for an additional 5 hours. Aliquots (50 mL) from the
3
receiving compartment were taken and replaced with equal vol-
ume of fresh LRS every 20 minutes for the first 3 hours and then
every 30 minutes from 3 to 6 hours. The strength of the donor
compartment solution was kept constant at 30 mg/mL (44.873 mM)
for all types of electrode used and it contained 6 mg/cm2 of ETP
solution. The pH of ETP solution in the donor compartment was
measured at the beginning and at the end of the experiments.
During the iontophoresis experiments, the receiving compartment
of a separate Franz diffusion cell was filled with a known concen-
tration of ETP solution, and samples were taken every hour to
assess possible conversion of ETP to VP under such experimental
conditions.
Microporation and Iontophoresis. The same apparatus and condi-
tions were used in this set of experiments with the only difference
that porcine skin was pretreated with a 3M Microchannel skin
system (3M Drug Delivery Systems, St. Paul, MN) consisting of 351
microneedles/cm2 and each microneedle having a 650-micron
length. A current density of 300 mA/cm2 was applied for 1 hour.
All the experimental procedures including sampling intervals were
the same as described in previous section.
In Vivo Experiments
The Institutional Animal Care and Use Committee at Long Island
University, Brooklyn, NY, approved all animal procedures. The ex-
periments were performed on female, pathogen-free, New Zealand
albino rabbits. Rabbits were housed under standard laboratory
conditions (22 ± 1 C; relative humidity: 40%-60%) and were fed
with normal rabbit chow and tap water to drink. At the time of
experiments, the animal's age ranged from 9 to 15 months and
weighed 3.4-5.2 kg. After each experiment, animals were allowed
at least 1-week period of full recovery before being used for another
study.
The day before the experiment, the rabbit's dorsal hair was
removed as close as possible to the skin surface with the help of an
electrical animal hair clipper.27 The shaved skin was then wiped
with alcohol swab pads. On the day of the experiment, the rabbits
were tranquilized with 1-mg/kg intramuscular injection of ace-
promazine maleate (10 mg/mL; Vedco, Inc., St. Joseph, MO) and
allowed to tranquilize for 25-30 minutes. An indwelling catheter
(24G
¾; Exelint International, Co., Los Angeles, CA) was inserted
into the central ear artery to collect blood samples. The exposed
artery and catheter were irrigated with 100 U/mL of heparin
solution (Hospira., Inc.) in sterile water for injection (Hospira, Inc.).
Microdialysis probes were implanted according to the technique
described by Stagni et al.28 as superficially as possible, using a
25G
1½ BD PrecisionGlide needle (Becton Dickinson and Co.) as
a guide. Particular care was taken that the needle was inserted as
superficially as possible (dermis) and apart from blood vessels.
Before insertion, microdialysis probes were tested for leakage. The
probe inlet and outlet were taped with surgical 3M Durapore Tape
(3M Health Care, St. Paul, MN) in such a way that the 15-mm
dialysis window was positioned exactly in the center. Skin was
allowed to recover from the insertion trauma for about 45 mi-
nutes. Inlet of the probe was connected to the microdialysis pump
with Tygon tubing (0.01000 ID, 0.03000 OD; Norton Performance
Plastics, Akron, OH) using cyanoacryl glue (Henkel Corp.,
Rocky Hill, CT).
Retrodialysis
In vivo retrodialysis was performed to determine probe per-
centage loss in vivo. Probes were perfused with 3 different
concentrations (45,000, 15,000, and 4500 nM) of VP and ETP, in four
different rabbits. Flow rate was 1 mL/min. Dialysate samples were
4 H. Patel et al. / Journal of Pharmaceutical Sciences xxx (2016) 1-9
collected every 20 minutes for 3 hours. Twelve experiments (4
rabbits and 3 concentrations) were performed to estimate
percent in vivo recovery by loss. Microdialysis samples were
analyzed on the same day without any further extraction.
Iontophoresis
Two microdialysis probes were inserted in parallel, approxi-
mately 2 cm apart, on the dorsal skin of the tranquilized rabbit. Skin
transepidermal water loss (TEWL) was measured with a Vapometer
(Delfin Technologies, Inc., Stamford, CT) on the skin above the
microdialysis probes before starting the probes' perfusion. The
GelSponge pad was saturated with 2 mL of ETP solution (60 mg
equivalent to 89.747 mmol of ETP dissolved in 2 mL of HPLC water)
in such a way to eliminate any dry spots. Then, the iontophoresis
patch (pH stabilized, IOMED TransQE disposable electrode with a
contact surface area of 10.1 cm2) was applied making sure that the
microdialysis probe's inlet and outlet were outside the contact
surface of the patch. An IOMED Phoresor II provided electrical
current. Excessive pressure was avoided on the drug delivery pad to
prevent leaking of the ETP, and no tapes were used to stick pad with
the skin. A dispersive pad was applied about 4 inches away from the
active site. On separate occasions, the following electrical current
was applied for 1 hour: 0, 100, 200, 300, or 400 mA/cm2 as cathodal
iontophoresis. The patch was removed at the end of the current
application period. The skin was gently dried with a soft paper
towel without rubbing to remove possible residual traces of ETP.
The pH of the patch loaded with ETP solution was measured before
and after application. Microdialysis samples were collected every
20 minutes for 2 hours and then every 30 minutes for following
4 hours. Blood samples (0.6 mL) were collected at 0, 15, 30, 45, 60,
70, 80, 90, 105, 120, 140, 160, 180, 210, 240, 300, and 360 minutes,
and plasma was separated and stored at 20 C until further anal-
ysis. Each of the three rabbits received four different current den-
sities and one passive diffusion (current density: 0 mA/cm2)
treatment. Studies were separated by at least 1 week of washout
period.
Microporation
On separate occasions, the effect of microporation on the
iontophoretic delivery of ETP was tested. Rabbit dorsal skin was
microporated with a 3M Microchannel skin system (3M Drug De-
livery Systems, St. Paul, MN) that opens 351 micropores per cm2,
followed by the immediate application of the IOMED TransQE
loaded with ETP. A current density of 300 mA/cm2 was applied for
1 hour. All the remaining experimental procedures were the same
as described in the previous section.
Skin Irritation
The skin response to the iontophoretic administration was
assessed by visual inspection. Visual scoring of any redness, ery-
thema, edema, and papules was performed using US Food and Drug
Administration Guidance for Industry: Skin Irritation and Sensiti-
zation Testing of Generic Transdermal Drug Products.29 Dermal
response was observed either at least for a week or until completely
recovered from any side effects.
IV Infusion
Each rabbit received 2 mg/kg (2.99 mmol/kg) of ETP (ETOPO-
PHOS® Injection) as 10 minutes of IV Infusion. Two microdialysis
probes were inserted in the rabbit skin as previously described.
Dialysate samples were collected every 20 minutes for 2 hours
and then every 30 minutes for following 4 hours. Blood samples
(0.6 mL) were collected at 0, 5, 10, 15, 20, 30, 45, 60, 80, 100, 120,
150, 180, 210, 240, 300, and 360 minutes, and plasma was
separated and stored at 20 C until further analysis.
Data Treatment and Statistical Analysis
Total VP was calculated by addition of molar concentration of
ETP and VP at the same time point.
In Vitro Skin Permeation
The cumulative amount (nmol/cm2) of total VP permeated
through porcine skin was plotted versus time. Steady state flux (Jss)
was calculated as the best-fit slope of the apparent linear portions,
for each individual data set. Statistical analysis was performed with
Microsoft Excel 2013 (Microsoft, Seattle, WA). The data were
considered to be significant at p < 0.05.
In Vivo Pharmacokinetics
Skin dialysates were corrected by the recovery factor to obtain
the actual concentration of ETP/VP in extracellular fluid and plotted
versus the midpoint of the collection time interval for pharmaco-
kinetic analysis.18 Pharmacokinetic analysis was performed by
noncompartmental analysis using Phoenix 6.3 (Certara, Princeton,
NJ) for both plasma and skin samples. Statistical analysis was per-
formed with Phoenix 6.3 and Microsoft Excel 2013. The following
parameters were estimated for all experimental conditions: AUC0et
(area under the time concentration curve from time 0 to time t, the
last data point), AUC0-∞, Cmax (maximal plasma drug concentra-
tion), Tmax (time to maximal plasma drug concentration), half-life,
Tlag, and clearance. The amount delivered by iontophoresis was
calculated as Xi ¼ CL
AUCionto; where CL is the total body clear-
ance calculated from the IV infusion and AUCionto is the area under
the curve observed following the administration of each ionto-
phoresis treatment. The absolute bioavailability fraction (%F) was
calculated as: F ¼ Xi/Dosei.
100.
Results
In Vitro Experiments
Microdialysis
ETP: the percentage in vitro recovery by loss was 59.49 ± 5.83
(n ¼ 11) when alone and 62.07 ± 4.39 (n ¼ 9) when in combination
with VP; the percentage in vitro recovery by gain was 69.08 ± 5.51
(n ¼ 11) when alone and 70.03 ± 2.91 (n ¼ 9) when in combination
with VP.
VP: the percentage in vitro recovery by loss was 73.25 ± 1.41 (n ¼
9) when alone and 71.75 ± 4.53 (n ¼ 9) when in combination with
ETP; the percentage in vitro recovery by gain was 88.87 ± 3.52 (n ¼
9) when alone and 89.82 ± 3.67 (n ¼ 9) when in combination with
ETP. All relative recoveries were independent of the drug concen-
tration and of the presence of VP or ETP in the range tested. The fact
that in vitro recoveries were similar between “gain” and “loss” ex-
periments for either ETP, VP, or combination of ETP and VP supports
the hypothesis that the percentage loss estimated in vivo with
retrodialysis (loss) can be applied to convert the dialysate con-
centrations into the actual interstitial free-drug concentration.30
Permeability Studies
Selection of Iontophoretic Conditions. Preliminary experiments
demonstrated that cathodal iontophoresis was more effective for
ETP iontophoresis than anodal iontophoresis based on the amount
of ETP detected in the receiving compartment. Screening of ionto-
phoretic electrodes showed that among all electrodes tested,
IOMED TransQE was more suitable for delivery of ETP in terms of
both amount delivered and reproducibility. This result may be due
to the stabilized pH of the IOMED TransQE electrode. The initial pH
of the ETP solution in the donor compartment was 2.38 ± 0.32 (n ¼
24), and no significant change in pH was observed after current
 H. Patel et al. / Journal of Pharmaceutical Sciences xxx (2016) 1-9 5

Figure 2. Effect of current density on the in vitro iontophoresis of ETP through porcine ear skin. Both ETP and VP were detectable in the receiver compartment. Graph's bars
represent arithmetic mean (n ¼ 4); error bars represent standard deviation. MP indicated skin pretreatment with microneedles.

application (1.19 ± 0.29; n ¼ 20) compared to no current application
(0.96 ± 0.18; n ¼ 4).
Iontophoresis. Both ETP and VP were detected in the receiving
compartments of Franz diffusion cells in all experiments. However,
VP was not detectable in the control cells, where a solution of ETP
was exposed to the same experimental conditions. This supports
the hypothesis that VP was formed from ETP in the presence of the
porcine skin. Because of this conversion, the total VP delivered was
calculated by adding the molar concentrations of ETP and VP. The
cumulative amount and flux of VP, ETP, and total VP (VP þ ETP)
permeated across porcine skin increased significantly (analysis of
variance [ANOVA]; p < 0.05) with current densities (0, 100, 200,
300, and 400 mA/cm2 for 1 hour) as shown in Figure 2. In general,
the ratio of ETP and VP to total VP was consistent at all current
densities with an average of 71.94 ± 5.27% of ETP and 30.55 ± 5.29%
of VP in the receiving compartment (n ¼ 24) at steady state.
Figure 3 shows the time course of cumulative amount of total VP in
the receiver compartment.
Microporation and Iontophoresis. The microporation pretreatment
significantly increased the total amount of VP permeated compared
to in vitro iontophoresis alone. At current density of 300 mA/cm2,
the amount permeated increased from 412 ± 39 to 798 ± 39 nmol/
cm2 (n ¼ 4; ANOVA; p < 0.01), and steady state flux increased from
157 ± 23 to 436 ± 8 nmol/cm2/h (n ¼ 4; ANOVA; p < 0.01).
passive diffusion and that iontophoresis effectively enhances the
ETP permeability. The dialysate concentrations were corrected with
the in vivo retrodialysis recovery to obtain the actual concentration
of ETP and VP in interstitial fluid (IF). VP was detectable from the
first samples in both skin and plasma, which shows that there was
not much of a lag time involved in delivery. Figure 4 (upper panel)
shows the mean total VP concentration time course during and
after iontophoretic current applications in skin and plasma,
respectively. Plasma data are reported as total VP concentration,
whereas skin concentrations represent the unbound VP present in
the dermal IF.
VP plasma protein binding determined in this study in rabbits
(90%) is similar to that observed in humans (93%).31 Plasma
extraction efficiency was 89 ± 3 (n ¼ 9). The skin and plasma levels
of total VP increased gradually during iontophoresis and reached a
maximum value at 60 minutes when current was terminated. VP
was detectable in skin and plasma up to 360 minutes. Pharmaco-
kinetic parameters are listed in Table 1. TEWL data showed that the
stratum corneum barrier properties were not significantly modified
by iontophoresis. In addition, TEWL data did not show any signif-
icant correlation with the extent of delivery of ETP.
Microporation and Iontophoresis
Figure 4 (lower panel) shows the total VP concentration versus
time compared with the same current density with and without
microporation in skin and plasma, respectively. Pharmacokinetic

In Vivo Experiments

All rabbits well tolerated the microdialysis, microporation, and

iontophoresis procedures, and no indication of skin irritation, rash,
erythema, or edema were observed during or immediately after the
end of the experiments. However, redness (score level up to 2)
gradually increased for the following 24-36 hours and eventually
disappeared within 10 days. Microdialysis in vivo percentage re-
covery (%RL) was 64.79 ± 7.74 (n ¼ 12) and 70.01 ± 6.12 (n ¼ 12) for
ETP and VP respectively.

Iontophoresis
Both ETP and VP were detected in skin dialysates, whereas only
VP was detected in plasma probably because of the rapid and
complete conversion of ETP to VP in blood, fact that is well docu-
mented in literature.5 Conversely, neither ETP nor VP was detect-
able in skin dialysate and plasma when no electrical current was Figure 3. In vitro permeability profiles of total etoposide (ETP þ VP) at different current
applied (0 mA/cm2), which confirms that ETP does not permeate by density (mA/cm2) through porcine ear skin. Each point represents the mean ± SD (n ¼ 4).
6 H. Patel et al. / Journal of Pharmaceutical Sciences xxx (2016) 1-9

Figure 4. Upper panels: Average total etoposide concentrations detected in skin (n ¼ 6 for 100, 300 mA/cm2 and n ¼ 5 for 200 and 400 mA/cm2) and plasma (n ¼ 3) after transdermal
iontophoresis of etoposide phosphate. Error bars represent standard error. Lower panels: Average total etoposide concentrations detected in skin (n ¼ 6) and plasma (n ¼ 3) after
transdermal iontophoresis of etoposide phosphate with and without microporation (MP). Error bars represent the standard error.

parameters are reported in Table 1. VP exposure increased signifi- IV Infusion

cantly in plasma and not in skin. Microporation in combination of After IV administration of ETP, skin dialysate samples contained
iontophoresis enhances Cmax and AUClast values by approximately both ETP and VP, whereas only VP was detectable in blood samples
50% in plasma (paired t test; p < 0.01). (Fig. 5). Table 1 reports pharmacokinetic parameters in skin and

Table 1
Pharmacokinetic Parameters of Total Etoposide in Skin and Plasma Calculated After Iontophoretic Delivery of ETP at Different Current Densities and IV Infusion in New Zealand
Female Albino Rabbits

Parameter Current Densities (mA/cm2) IV Infusion

100 200 300 MP þ 300 400

Skin
AUClast (mmol
min/L) 256.1 ± 59.5 562.1 ± 169.2 1110.1 ± 263.3 1553.7 ± 1241.8 944.9 ± 1375.9 63.0 ± 7.5
AUC∞ (mmol
min/L) 271.3 ± 56.3 626.6 ± 167.2 1126.1 ± 262.3 1566.1 ± 1243.8 983.0 ± 1406.1 67.8 ± 7.0
Cmax (mmol/L) 3.2 ± 0.5 7.2 ± 1.3 14.9 ± 2.4 23.1 ± 18.1 9.2 ± 8.3 1.0 ± 0.3
Half-life (min) 56.5 ± 30.1 49.8 ± 12.1 60.2 ± 28.1 48.8 ± 8.7 55.9 ± 18.1 39.9 ± 14.4
Tmax (min) 30-70 30-70 30-70 30-50 30-70 10-30
Plasma
AUClast (mmol
min/L) 13.6 ± 6.1 26.8 ± 16.4 44.6 ± 6.7 68.3 ± 6.7 53.8 ± 10.5 177.5 ± 25.0
AUC∞ (mmol
min/L) 14.4 ± 6.6 29.3 ± 15.6 44.9 ± 6.6 70.6 ± 6.0 55.1 ± 11.4 178.6 ± 24.6
Cl (L/min) 7.2 ± 3.2 3.7 ± 1.7 2.1 ± 0.3 1.3 ± 0.1 1.7 ± 0.3 0.073 ± 0.013
Cmax (nmol/L)a 171.6 ± 57.8 342.6 ± 148.3 572.3 ± 77.3 809.5 ± 28.1 675.2 ± 106.6 9529.3 ± 1046.7
Half-life (min) 41.3 ± 23.0 51.6 ± 27.9 33.2 ± 7.3 92.4 ± 40.6 38.2 ± 19.6 45.3 ± 6.8
Tmax (min) 60 60 30-60 45-60 60 10
% F (Bioavailability) 1.0 ± 0.1 2.0 ± 0.4 3.5 ± 0.3 5.4 ± 0.4 4.2 ± 0.5

Data are reported as mean ± SE (n ¼ 3 for plasma; n ¼ 6 for skin except at current densities 200 and 400 mA/cm2 where n ¼ 5) except for Tmax and F which are reported as ranges
and percentage, respectively.
MP, microporation; AUC, area under the curve; Cl, clearance; Cmax, maximum concentration; Tmax, time to reach maximum concentration; F, absolute bioavailability.
a
Note that plasma concentrations are reported in nmol/L.
 H. Patel et al. / Journal of Pharmaceutical Sciences xxx (2016) 1-9
Figure 5. Average total etoposide concentration detected in skin (n ¼ 6) and plasma
(n ¼ 3). Data represent mean ± SE.
plasma. IV infusion clearance was used to calculate the total
amount delivered by iontophoresis.
Discussion
This study represents the first attempt to deliver VP via trans-
dermal route. The specific goals were (1) to assess the feasibility to
deliver therapeutically relevant doses of VP via iontophoresis or a
combination of iontophoresis/microporation and (2) to elucidate
the role of skin in the mechanism of ETP transport to systemic
circulation. Therapeutically important reasons support the
hypothesis that transdermal delivery of ETP/VP would improve its
efficacy and patient compliance.32,33 The minimum effective con-
centration for VP is estimated at approximately 300 ng/mL, corre-
sponding to 500 nmol/L, although the specific threshold may vary
depending upon the type of tumor.34 This study demonstrates that,
among the current densities tested, the 300 mA/cm2 was able to
achieve plasma concentrations above the minimum effective con-
centration (500 nmol/L), shortly after application of electrical cur-
rent and to maintain it above minimum effective concentration for
the entire duration of the iontophoresis without causing any visible
sign of local toxicity. Although the highest current density (400 mA/
cm2) also achieved the goal without any immediately observable
side effects, it triggered a delayed toxicity. Indeed an increasing
redness appeared at the site of application within 24 hours from the
experiment and lasted for at least a week before it eventually dis-
appeared. The reason for this toxicity is probably related to the high
iontophoretic current and possibly due to the cytotoxic nature of
ETP. Indeed, microdialysis data (Fig. 4) show a sustained high
dermal concentration at this particular current density.
To further improve the transdermal delivery of ETP and to make
the transdermal route viable for a larger number of cancer types,
the effect of microporation was investigated. Before the application
of the iontophoretic patch, hundreds of microchannels <100 mi-
crons deep35 were opened in the delivery area by gently pressing a
commercially available, disposable, microneedle patch into the
skin. The mechanical opening of micropores in the skin16 is an
emerging technique to enhance transdermal drug delivery and
offers the possibility to deliver a broader spectrum of molecules.
The microchannel system was preferred over microroller applica-
tion (Dermaroller) because it requires a single application thereby
improving reproducibility.14 The microporation/iontophoresis
combination significantly increased VP plasma concentrations by
50%. However, the length of time spent above the minimum
7
effective plasma concentration did not change significantly. Indeed,
the plasma concentration decreased dramatically after the
discontinuation of electrical current. This is probably because of the
very short half-life of VP (Table 1) in rabbits; in humans, the half-
life of VP is approximately 6.4 hours34; therefore, a slower elimi-
nation may prolong the length of time the plasma concentration is
above the minimum effective concentration. Because the efficacy of
VP therapy correlates with the length of time the plasma concen-
tration stays above the minimum effective level, it is probably
necessary to prolong the iontophoresis application to further
optimize delivery. This is a proof-of-principle study, and the length
of the iontophoretic current application was 1 hour because rabbits
must be restrained during the experiments and, as per our Insti-
tutional Animal Care and Use Committee guidelines, they cannot be
restrained continuously for >8 hours. Within this parameter, the
experimental time profile included 2 hours for experiment prepa-
ration, 1 hour of iontophoresis, and 5 hours to characterize the
elimination phase.36
An interesting discovery of this study is the finding that ETP is
converted to VP in the skin, even in vitro. Permeability studies
through cellulose membranes (data not reported) as well as the
stability studies with electrical current demonstrated that under
these experimental conditions, ETP does not convert to VP. In
general, the most relevant in vitro membrane to evaluate trans-
dermal permeability is human cadaver skin; however, human skin
specimens of sufficient size and quality are only available in limited
amounts. Therefore, porcine ear skin is often used as a membrane
for in vitro permeability experiments. Porcine skin is readily
obtainable from abattoirs and has histologic and biochemical
properties similar to human skin.37-39 Porcine ear skin is often used
for permeation studies for passive as well as active type of trans-
dermal formulations.40,41 When porcine ear skin was used as a
membrane in the Franz diffusion cell apparatus, each sample
collected from the receiving compartment contained approxi-
mately 30% of VP. These results support the hypothesis that excised
porcine ear skin still contains active phosphatase enzymes which
might be responsible for conversion of ETP to VP. A simple, sensi-
tive, and accurate, new HPLC method for the simultaneous deter-
mination of ETP and VP with UV detection was developed in our
laboratory. Previously, simultaneous determination of ETP and VP
was carried out with micellar electrokinetic chromatography and
near-field thermal lens detection42 or capillary electrophoresis
using laser-induced native fluorescence detection.43 It is interesting
to note that in all in vivo experiments, microdialysis samples con-
tained both ETP and VP, whereas plasma samples contained only
VP. Indeed, skin dialysate contained both ETP and VP also following
the ETP IV infusion when only VP was detectable in plasma. This
confirms that the conversion occurs in vivo44 as well as during
plasma storage. Dialysate samples are protein/enzyme free, and
thus, molecules collected do not undergo further degradation
during storage.
In vitro permeability profiles show a substantial lag time varying
from approximately 40 minutes for the highest current density to
60-80 minutes for passive delivery (Fig. 3). Conversely, in vivo, no
lag time was detected in both skin and plasma. This may be because
drug molecules have greater affinity for the lipophilic components
of the skin membrane rather than for the hydrophilic receiver so-
lution (LRS) of the Franz cell apparatus. Iontophoretic delivery oc-
curs mostly via skin appendages such as hair follicles and sweat
glands. These features may differ among animal species (e.g.,
porcine skin versus rabbit) or may be affected differently by the
electrical current between ex vivo and in vivo skin. Once ETP and VP
appear in the cell receivers, delivery continues for an additional 1-
3 hours after the 1-hour period of electrical current application,
whereas in vivo experiments show that the skin concentrations
8 H. Patel et al. / Journal of Pharmaceutical Sciences xxx (2016) 1-9
start to decline shortly upon discontinuation of electrical current
(Fig. 4).
The microdialysis technique samples drug molecules that are
freely moving in the dermal IF. The probes are inserted in dermis at
approximately 1-2 mm from the surface of the skin, and therefore,
the path length that a molecule must travel to reach the probe is
similar to that travelled in vitro across the porcine skin (~1 mm).
However, there are some substantial differences between the Franz
cells' receiver solution and the dermal IF. In dermis, the drug
molecules freely soluble in the IF are in dynamic equilibrium with
those bound to IF components, (1) mostly proteins, or (2) cell sur-
faces, and the molecules that penetrated inside the cells. Whereas
some molecules will permanently leave the IF by crossing into
blood capillaries or the subcutaneous tissues. Therefore, the time
course of drug concentration in dermis is the sum of input and
elimination profiles resulting into a time course like that observed
in Figure 4, upper panel. The in vitro permeability profiles such as
those presented in Figure 3 show the cumulative input of drug
reaching the receiver solution, from where there is no elimination.
Plasma concentrations increased proportionally with current
densities as expected.45,46 However, this was not the case for skin
where exposure increased proportionally at the 100, 200, and 300
mA/cm2 but not at current density of 400 mA/cm2. As previously
described, there was no visible signs of skin damage at the end of
the experiments. In addition, skin burning could cause coagulation
of proteins and collapse of appendage's pathways that would
hamper delivery to dermis as well as plasma. A reasonable expla-
nation is that at the highest current density, most of the ETP mol-
ecules are eliminated from dermis into the blood circulation before
they can be collected by the microdialysis probe. Hence, the dermal
kinetics measured by microdialysis sampling apparently mirror the
plasma concentrations up to 300 mA/cm2 current density and not at
the highest current density. The proper identification of a linear
range is a prerequisite for an accurate modulation of safe delivery,
and it is helpful for an informed development of formulations and
in vitro in vivo correlation.47 We developed an in vitro in vivo
correlation model from these data48 that will be presented in a
forthcoming manuscript.
In transdermal delivery, dermis represents the interface be-
tween the barrier of the stratum corneum and epidermis and the
systemic circulation that will bring the drug to the site of action.
There is an increasing evidence that also the viable epidermis offers
a substantial barrier to the transdermal delivery of drugs.49 How-
ever, even once a molecule reaches the lower layers of the skin, the
dermis, it may bind or partition into the skin components. This can
lead to the formation of deposits and affect the input in the sys-
temic circulation resulting in unpredictable plasma profiles and
effect. Microdialysis data from this study show that ETP and VP are
not retained in the skin because the skin concentration declines
immediately after discontinuation of the electrical current appli-
cation for the 100, 200, and 300 mA/cm2.
Conclusions
The present study is the first to assess the suitability of the
iontophoresis technique for ETP. We identified the current density
range in which delivery is linear in skin as well as in plasma. In the
current density range of 100-300 mA/cm2, no visible signs of toxicity
were observed regardless of VP cytotoxicity. This research dem-
onstrates that iontophoresis of ETP results in therapeutically rele-
vant plasma levels of VP with moderate current densities and that
microporation in combination with iontophoresis further enhance
the extent of ETP delivery in vitro as well as in vivo. The results
obtained in this study can be further expanded to develop more
sophisticated iontophoresis/microneedles devices to maintain
sustained concentrations of ETP. An additional discovery of this
study is that ETP is converted to VP also in vitro by porcine ear skin.
Acknowledgments
The Division of Pharmaceutical Sciences, Arnold and Marie
Schwartz College of Pharmacy, Long Island University, Brooklyn,
New York, sponsored this research.
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