New oligoethylene ester derivatives of 5-iodo-2′-deoxyuridine as dermal prodrugs: Synthesis, physicochemical properties, and skin permeation studies

Article abstract of DOI:10.1002/jps.1174

Five new oligoethylene ester derivatives (9-13) of 5-iodo-2′-deoxyuridine (IDU) were synthesized and assayed to determine their lipophilicity by both experimental lipophilicity indices (log K′) and calculated partition coefficients (CLOGP). In vitro exper

Full text of DOI:10.1002/jps.1174

New Oligoethylene Ester Derivatives of
5-Iodo-20-deoxyuridine as Dermal Prodrugs: Synthesis,
Physicochemical Properties, and Skin Permeation Studies
FRANCESCO PAOLO BONINA,1 MARIA GRAZIA RIMOLI,2 LUCIA AVALLONE,2 FRANCESCO BARBATO,2
MARZIA AMATO,2 CARMELO PUGLIA,1 MAURIZIO RICCI,3 PAOLO DE CAPRARIIS4
1
Á
Á
Dipartimento di Scienze Farmaceutiche, Facolta di Farmacia, Universita degli Studi di Catania, Viale A. Doria 6, 95125
Catania, Italy
2
Á
Á
Dipartimento di Chimica Farmaceutica e Tossicologica, Facolta di Farmacia, Universita degli Studi di Napoli Federico II,
Via Domenico Montesano 49, 80131 Napoli., Italy
3
Á
Á
Dipartimento di Chimica e Tecnologia del Farmaco, Facolta di Farmacia, Universita degli Studi di Perugia, Via del Liceo 1,
06100 Perugia, Italy
4
Á
Á
Dipartimento Farmaco Chimico Tossicologico, Facolta di Farmacia, Universita di Sassari, Via Moroni 23, 07100 Sassari, Italy
Received 29 February 2000; revised 7 May 2001; accepted 25 May 2001
ABSTRACT: Five new oligoethylene ester derivatives (9±13) of 5-iodo-20-deoxyuridine
(IDU) were synthesized and assayed to determine their lipophilicity by both
experimental lipophilicity indices (log K0) and calculated partition coef®cients (CLOGP).
In vitro experiments were carried out to evaluate the chemical and enzymatic stability
and ¯uxes through excised human skin of these new IDU derivatives. Esters 9±13
showed increased lipophilicity compared with the parent drug (IDU), had good stability
in phosphate buffer (pH 7.4), and were readily hydrolyzed by porcine esterase. No
correlation between lipophilicity and skin permeation ¯uxes of synthesized esters 9±13
was observed. Results from in vitro percutaneous absorption studies showed that,
among all esters synthesized, only esters 9 and 10 signi®cantly increased the cumula-
tive amount of IDU that penetrated through excised human skin compared with the
parent drug (IDU). ß 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J
Pharm Sci 91:171±179, 2002
Keywords: IDU; dermal prodrugs; SCE membranes; in vitro
INTRODUCTION
skin permeation. Approaches have included for-
mulation additives, such as chemical penetration
enhancers, and vehicles that maximize partition-
ing of IDU into the skin.2±4 The penetration
enhancers are chemical compounds that are
themselves pharmacologically inactive but can
partition into the skin and interact with the
constituents of the super®cial skin layer and
reduce the resistance of the skin to drug diffu-
sion.5 The use of penetration enhancers shows
some potential disadvantages:6 the inclusion of
these compounds within a formulation may
increase the absorption of components other than
the drug, which can provoke skin damage and
Although 5-iodo-20-deoxyuridine (IDU) is useful
in the treatment of skin herpes simplex keratitis,1
its therapeutic usefulness is limited by its poor
skin permeability, which is due to the polar
nature of this drug. To overcome this problem,
various strategies have been used to enhance IDU
Correspondence to: Francesco Paolo Bonina (Telephone:
0039-95-222258, Fax: 0039-95-222258;
E-mail: bonina®@mbox.unict.it)
Journal of Pharmaceutical Sciences, Vol. 91, 171±179 (2002)
ß 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 1, JANUARY 2002
171

172
BONINA ET AL.
toxicity. The prodrug approach represents an alter-
native and very promising method of enhancing
the skin permeability of a topically applied drug.
The prodrug concept involves the chemical
modi®cation of a drug into a bioreversible form
to change its pharmacological and pharmacoki-
netic properties and thus enhance its skin deli-
very. At the same time, the prodrug should be able
to readily regenerate enzymatically to the parent
drug in the target tissues (viable epidermis and/or
dermis). The prodrug strategy has been used to
increase IDU permeability through various biolo-
gical membranes such as blood±brain barrier7,8
and cornea.9
In the recent past, we have applied a new
promising strategy in the dermal prodrug design
that consists of conjugating the parent drug with
promoieties that have an inherent ability to
enhance skin permeability. We have obtained
several dermal prodrugs of nonsteroidal antiin-
¯ammatory drugs, such as indomethacin and
naproxen, conjugating them by ester linkage to
different penetration enhancers such as N-alkyl-
lactames,10,11 N-acyllactames12 polyoxyethylene
glycols,13 and terpenoids.14
Some of these prodrugs with good water stabi-
lity, rapid enzymatic hydrolysis, and increased
¯uxes through excised human skin can be regar-
ded as promising indomethacin and naproxen
dermal prodrugs. To increase IDU dermal deliv-
ery, we describe the synthesis of new ester
derivatives of IDU (9±13) by coupling this drug
with polyoxyethylene glycols via a succinic acid
spacer (see Scheme 1). IDU derivatives 9±13 were
tested to determine their hydrolytic stability in
water and susceptibility to undergoing in vitro
enzymatic cleavage and ¯uxes through excised
human skin.
spots was made by ultraviolet (UV) radiation,
exposure to iodine vapor, and spraying with
Ce(SO4)2 aqueous solution. Preparative separa-
tions were performed in columns containing
Merck 60 silica gel (70±230 mesh). Melting points
were determined with a Ko¯er hot stage micro-
scope and are uncorrected.
Elemental analyses were carried out with a
Carlo Erba model 1106 analyzer. The product
characterization by proton (1H) and carbon-13
(13C) nuclear magnetic resonance (NMR) spectro-
scopy was carried out with a Bruker AMX-500
spectrometer (CDCl3 and CD3OD as solvents).
Chemical shift values are reported in d units
(ppm).
General Methods of Prodrug Synthesis
For the preparation of the IDU esters 9±13
(Scheme 1), initially the 50-OH of IDU (1) was
blocked by reaction with 4,40-dimethoxytrityl
chloride (DMT-Cl) to give 50-O-dimethoxytrityl-
5-iodo-20-deoxyuridine (2); then, the 30-OH of
IDU was esteri®ed by succinic anhydride yielding
50-O-dimethoxytrityl-5-iodo-20-deoxyuridine-30-O-
succinate (3). The syntheses of 2 and 3 were
reported in the previous paper.15
The product 3 was coupled respectively with
the different glycols to give 30-O-(polyoxyethyle-
neglycol-succinyl)-50-dimethoxytrityl-5-iodo-20-
deoxyuridine derivatives (4±8). Brie¯y, dicyclo-
hexylcarbodiimide (DCC; 270 mg; 1.30 mmol) was
added to a solution consisting of 3 (1 g; 1.30
mmol), 4-N,N-dimethylaminopyridine (DMAP),
and the appropriate oligoethylene glycol (5.28
mmol) in ethyl acetate. The reaction mixture was
stirred at room temperature for ꢀ6 h under
anhydrous conditions. The progress of the reac-
tion was periodically monitored by TLC using a
chloroform±methanol mixture (99:1) as a devel-
oping solvent. At the completion of reaction, the
separated dicyclohexylurea (DCU) was ®ltered
off. The ®ltrate was washed twice with water to
remove any unreacted reagents and dried over
anhydrous sodium sulfate. The organic phase was
evaporated in vacuo, and the residue was puri®ed
by silica gel chromatography, using as eluent
chloroform containing 0.5% pyridine and then
with a step gradient up to ꢀ5% methanol in
chloroform.
EXPERIMENTAL SECTION
Chemicals and Apparatus
IDU and the diethylene glycol (PEG2), triethylene
glycol (PEG3), tetraethylene glycol (PEG4), pen-
taethylene glycol (PEG5), and hexaethylene gly-
col (PEG6) were obtained from Fluka. All the
other chemicals were of analytical or reagent
grade and were obtained from commercial sources.
The course of reactions and the purity of
products were monitored by thin layer chromato-
graphy (TLC) using precoated silica gel alu-
minium plates (Merck 60 F254s). Detection of
Successively, the dimethoxytriphenyl group
was removed by HCOOH, yielding 30-O-(polyo-
xyethyleneglycol-succinyl)-5-iodo-20-deoxyuridine
conjugates 9±13 by
a method reported in
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SYNTHESIS, PROPERTIES, AND SKIN PERMEATION OF 5-IODO-20-DEOXYURIDINE DERIVATIVES
173
Scheme 1. Synthesis of esters 9±13.
literature.16 In particular, a solution of deriva-
tives 4±8 in CH2Cl2±HCOOH (6 mL, 5:1, v/v) was
stirred for 45 min at room temperature. The
reaction mixture was chromatographed, eluting
®rst with chloroform until the dimethoxytriphe-
nyl±methanol was removed and then with chloro-
form±methanol (95:5 v/v) to obtain the required
products (9±13).
The esters 9±13 were obtained in oil forms and
they failed to crystallize. Elemental analysis (C,
H, N) of all synthesized products were within
Æ0.3% of the theoretical values and the infrared
(IR) and proton nuclear magnetic resonance
(1H NMR) spectral data were consistent with
their structures.
69.73 (b); 68.55 (5-C); 64.74 (50-CH2); 62.54 (a);
61.88 (f); 38.67 (20-CH2); 29.70, 29.52 (two succinic
spacer CH2).
PEG3-30-O-succinyl-5-iodo-20-deoxyuridine
Ester (10)
1H NMR (CDCl3): d 2.35±2.45 (m, 2H, 20-H); 2.60
(m, 4H, COCH2CH2CO); 3.30±3.40 (AB system,
2H, 50-H); 3.50±3.70 (10H, b, c, d, e, f); 4.10
(m, 1H, 40-H); 4.20 (t, 2H, a); 5.40 (m, 1H, 30-H);
6.25 (m, 1H, 10-H); 8.10 (s, 1H, C-6-H). 13C NMR
(CDCl3): d 172.06±171.61 (two succinic spacer
CO); 159.72 (4-CO, 2-CO); 143.96 (6-CH); 85.09
(40-CH); 84.34 (10-CH); 75.47 (30-CH); 72.45 (e);
70.53 (c); 70.29 (d); 68.97 (b, 5-C); 63.81 (a); 63.53
(50-CH2); 61.69 (f); 38.49 (20-CH2); 29.08, 28.86
(two succinic spacer CH2).
PEG2-30-O-succinyl-5-iodo-20-deoxyuridine Ester (9)
1H NMR (CDCl3): d 2.30±2.40 (m, 2H, 20-H); 2.65
(m, 4H, COCH2CH2CO); 3.55 (t, 2H, e); 3.65 (AB
system, 2H, 50-H); 3.75 (m, (4H, b, f); 4.05 (m, 1H,
40-H); 4.25 (t, 2H, a); 5.20 (m, 1H, 30-H); 6.15 (m,
1H, 10-H); 8.20 (s, 1H, C-6-H). 13C NMR (CDCl3): d
173.78±173.23 (two succinic spacer CO); 162.47
(4-CO); 151.69 (2-CO); 146.75 (6-CH); 86.65
(40-CH); 86.47 (10-CH); 76.56 (30-CH); 73.31 (e);
PEG4-30-O-succinyl-5-iodo-20-deoxyuridine
Ester (11)
1H NMR (CDCl3): d 2.35±2.45 (m, 2H, 20-H); 2.65
(m, 4H, COCH2CH2CO); 3.30±3.40 (AB system,
2H, 50-H); 3.60±3.70 (m, 14H, b, c, d, e, f); 4.10
(m, 1H, 40-H); 4.20 (t, 2H, a); 5.40 (m, 1H, 30-H);
6.22 (m, 1H, 10-H); 8.09 (s, 1H, C-6-H). 13C NMR
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 1, JANUARY 2002

174
BONINA ET AL.
(CDCl3): d 171.61±172.06 (two succinic spacer
CO); 160.00 (4-CO); 150.00 (2-CO); 145.45 (6-CH);
85.96 (40-CH); 85.45 (10-CH); 75.15 (30-CH); 72.49
(e); 70.47, 70.38, 70.34, 70.15 (4C, c, d); 68.98 (b);
68.95 (5-C); 63.90 (a); 62.14 (50-CH2); 61.51 (f);
38.06 (20-CH2); 29.14, 28.97 (two succinic spacer
CH2).
phosphate buffer (0.05 M, pH 7.4), which was
thermostatically controlled at 378C, and then 200
mL of the esterase solution was added. Both the
disappearance of the derivatives (9±13) and the
formation of IDU in the solution were monitored
by high-performance liquid chromatography
(HPLC) at different times.
Pseudo-®rst-order half-times (t1/2) for the che-
mical and enzymatic hydrolysis of esters (9±13)
were calculated from the linear slopes of plots of
the logarithm of remaining ester against time. In
enzymatic hydrolysis studies, the pseudo-®rst-
order times at which 50% of the total parent
compound had been formed (f50%) were deter-
mined from the linear slopes of the logarithm of
unformed parent drug (1) [log(parent drugmax
parent drugt)] over time as reported in litera-
ture.17,18
PEG5-30-O-succinyl-5-iodo-20-deoxyuridine
Ester (12)
1H NMR (CDCl3): d 2.35±2.45 (m, 2H, 20-H); 2.50
(m, 4H, COCH2CH2CO); 3.30±3.40 (AB system,
2H, 50-H); 3.40±3.60 (m, 18H, b, c, d, e, f); 4.00 (m,
1H, 40-H); 4.10 (t, 2H, a); 5.30 (m, 1H, 30-H); 6.20
(m, 1H, 10-H); 8.09 (s, 1H, C-6-H). 13C NMR
(CDCl3): d 174.87±174.11 (two succinic spacer
CO); 163.09 (4-CO); 151.39 (2-CO); 146.40 (6-CH);
85.85 (40-CH); 84.98 (10-CH); 75.04 (30-CH); 71.91
(e); 69.84, 69.78, 69.72, 69.65 (6C, c, d); 68.60 (b);
68.28 (5-C); 64.36 (a); 61.30 (50-CH2); 60.57 (f);
36.92 (20-CH2); 29.17, 29.00 (two succinic spacer
CH2).
Lipophilicity Indices (Log k0) and Calculated
Partition Coef®cients (CLOGP) of 1
and Esters 9±13
Lipophilicity indices of PEG derivatives esters 9±
13 were obtained by the isocratic HPLC method
described later, measuring compounds retention
times expressed as log k0.19 It is well known that
the ranking of elution in reversed-phase
HPLC represents a relative scale of lipophilicity
of analytes. Estimates of lipophilicity of deriva-
tives 9±13 were also obtained considering the
theoretically calculated values of log P (CLOGP;
CLOGP for Windows 2.0 version).
PEG6-30-O-succinyl-5-iodo-20-deoxyuridine
Ester (13)
1H NMR (CDCl3): d 2.35±2.45 (m, 2H, 20-H); 2.55
(m, 4H, COCH2CH2CO); 3.25±3.35 (AB system,
2H, 50-H); 3.45-3.65 (m, 22H, b, c, d, e, f); 4.00
(m, 1H, 40-H); 4.12 (t, 2H, a); 5.35 (m, 1H, 30-H);
6.19 (m, 1H, 10-H); 8.00 (s, 1H, C-6-H). 13C NMR
(CDCl3): d 171.61±172.06 (two succinic spacer
CO); 160.00 (4-CO); 150.00 (2-CO); 145.45 (6-CH);
85.00 (40-CH); 84.05 (10-CH); 76.71 (30-CH); 72.39
(e); 70.28, 70.23, 69.96 (8C, c, d); 68.80 (5-C); 68.73
(b); 63.75 (a); 63.29 (50-CH2); 61.32 (f); 38.00
(20-CH2); 28.80, 28.60 (two succinic spacer CH2).
HPLC Analysis
Compound 1 and esters 3 and 9±13 were deter-
mined by HPLC. In particular, we used a Model
600E (Waters-Millipore, Milford, MA) liquid chro-
matograph equipped with a Model 7125 Rheodyne
injection valve (®tted with a 20-mL loop) and a
Water Associates Model 486 ultraviolet (UV)
detector set at 254 nm was used. The chromato-
grams were recorded by a Data Module Model 746
(Millipore). The stainless steel column was a
Spherisorb S-5-ODS2 (4 Â 250 mm; Phase Separa-
tions, Clwid, U.K.). The eluent was a mixture of
acetonitrile±PBS (0.01 M, pH 7.0, 20/80, v/v) at a
¯ow rate of 1.5 mL/min. The aqueous portion of
eluent was ®ltered by membrane ®lters (type HA,
Millipore).
Chemical and Enzymatic Hydrolysis
Initially, solutions were prepared by dissolving an
aliquot of each ester 9±13 in methanol (20 mg/
mL). The hydrolysis rate for all esters 9±13 was
determined by dissolving 200 mL of methanolic
solution in 10 mL of phosphate buffer saline (PBS;
0.05 M, pH 7.4), which was thermostati-
cally controlled at 378C, and following the dis-
appearance of the chromatographic peak of the
esters (9±13) by the HPLC method reported later.
Enzymatic hydrolysis of esters was determined
as previously reported.10 Porcine esterase was
diluted 1000-fold with phosphate buffer, pH 7.4,
prior to use. A total of 200 mL of each ester (9±13)
methanolic solution was added to 10 mL of
The eluent mixture was obtained directly from
the chromatographic apparatus by mixing, at low
pressure, the organic modi®er and the aqueous
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SYNTHESIS, PROPERTIES, AND SKIN PERMEATION OF 5-IODO-20-DEOXYURIDINE DERIVATIVES
175
phase that had been deaerated previously by
bubbling helium. The chromatography was car-
ried out at room temperature. Chromatographic
retention data are expressed by the logarithm of
capacity factor (lipophylic index, log k0), de®ned as
log k0 ˆ log [(tr t0)/t0], where tr and t0 are the
retention time of the analyte and a non-retained
compound (methanol), respectively.
at different time intervals and replaced with fresh
solutions. The samples were analyzed for IDU and
esters 9±13 content by HPLC as already
described. In the case of esters 9±13, intact ester
was found in the receptor phase together with a
variable amount of IDU. Therefore, the ¯uxes
through the skin of these esters were obtained by
plotting the total cumulative amount of IDU
equivalents permeated (IDU free permeated ‡
IDU equivalent permeated as ester) versus time
and dividing the slopes of the steady-state
portions of the graphs by the area of the diffusion
cells.
In Vitro Skin Permeability
Samples of adult human skin (mean age 43Æ8
years) were obtained from breast reduction
operations. Subcutaneous fat was removed, and
the skin was immersed in distilled water at
60Æ18C for 2 min,20 then the stratum corneum
and epidermis (SCE) were peeled off. SCE
membranes were dried in a desiccator at ꢀ25%
relative humidity (RH) and then wrapped in
aluminium foil and stored at 4Æ18C until use.21
Dried SCE samples were rehydrated by immer-
sion in distilled water at room temperature for 1 h
before being mounted in Franz-type diffusion cells
(LGA, Berkeley, CA). The skin surface available
for absorption was 0.75 cm2 and the receptor
volume was 4.5 mL. The receiving compartment
contained ethanol±water (50:50) to ensure sink
conditions.22,23 The receiving solution was stirred
and maintained at 35Æ18C throughout the experi-
ments. IDU and its esters (9±13) were dissolved
in ethanol (5 mg/mL) and 200 mL was placed on
the skin surface. The solvent was allowed to
evaporate, and the experiment was run for 24 h.
Samples of the receiving solution were withdrawn
RESULTS AND DISCUSSION
Chemical and Enzymatic Hydrolysis
The degradations of IDU prodrugs (9±13) were
studied in buffer solution (pH 7.4) at 378C. The
degradation of each ester followed ®rst-order
kinetics, and the corresponding t1/2 values ranged
from 103 to 143 h (see Table 1). As shown in
Table 1, ester 12 presents higher chemical
stability compared with the other esters whose
values of t1/2 appear to be similar.
In the chemical hydrolysis studies we observed
the formation of only a small amount of inter-
mediate 3 (log k0 ˆ 0.60), scarcely detectable by
HPLC, and time-increased IDU concentrations.
These ®ndings suggest that hydrolytic cleavage of
esters 9±13 occurs preferentially at the 30-O-ester
linkage.
Table 1. Chemical and Enzymatic Hydrolysis, Lipophilic Index, Calculated Partition Coef®cient, and Flux
Through Excised Human Skin for IDU and Esters 9±13
t1/2 (h)
Porcine
Esterasec
FluxÆSDb
2
1
Compound
Buffer pH 7.4c
f50% (h)a
Log K0
CLOGP
(mg Á cm Á h
)
IDU
9
10
11
12
13
Ð
Ð
Ð
0.52
0.35
0.46
0.54
0.64
0.75
0.98
0.42
0.36
0.29
0.23
0.17
0.76Æ0.24
2.88Æ0.65e
2.17Æ0.53e
1.24Æ0.22
0.68Æ0.24
0.57Æ0.18
113.4Æ7.2
103.3Æ8.2
104.8Æ7.5
143.7Æ8.5d
107.3Æ8.2
1.92Æ0.34
2.32Æ0.20
1.97Æ0.31
4.53Æ0.49d
2.04Æ0.30
2.41
2.68
2.22
4.93
2.88
af50% is the time by which 50% of total IDU has been formed.
bThe in vitro skin permeation experiments were carried out in duplicate using SCE membrane samples obtained from three
different subjects.
cThe hydrolytic (chemical and enzymatic) experiments were carried out in duplicate.
dp < 0.01 ester 12 versus other esters (one-way ANOVA, Newman±Keuls Multiple Comparison Test).
ep < 0.01 esters 9 and 10 versus esters 12 and 13 and IDU; p < 0.01 ester 9 versus ester 11; p < 0.05 ester 10 versus ester 11;
p < 0.05 ester 9 versus ester 10 (one-way ANOVA, Newman±Keuls Multiple Comparison Test).
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176
BONINA ET AL.
Because an essential prerequisite for the
successful use of dermal prodrugs is their recon-
version into the parent drug within the skin, we
assessed the enzymatic cleavage of esters 9±13
with porcine liver esterases, which are regarded
as good models for skin esterase enzymatic
activity.10,24,25
ester prodrugs 9±13. These ®ndings, together
with the small amount of ester 3 detected in
enzymatic hydrolysis medium, suggest to us that
the enzymatic hydrolysis occurs too preferentially
in 30-O-ester linkage without formation of com-
pound 3.
As may be noted in Table 1, all the esters were
susceptible to undergoing enzymatic hydrolysis
by porcine liver esterase with t1/2 ranging from ꢀ2
to 4.5 h. The ester 12 appears more stable against
enzymatic hydrolysis with respect to the other
Lipophilicity
Stratum corneum is generally believed to be the
main barrier to drug skin permeation process.
Because the horny layer is basically a lipophilic
barrier, drug lipophilicity is regarded as one of the
key parameters that controls drug skin permea-
tion. One of the main objectives in dermal prodrug
design is, therefore, to obtain prodrugs with
increased lipophilicity compared with the parent
drug. Many authors27,28 have outlined that more
lipophilic drug derivatives could show better
partitioning and solubility into the stratum
corneum, which could result in enhanced skin
permeation.
To assess the lipophilicity of esters 9±13, two
different parameters were considered : CLOG
data from theoretical calculation and log K0
chromatographic indices. The esteri®cation of 30-
OH group of IDU gave esters 9±13 with increased
lipophilicity compared to the parent drug (see
Table 1).
esters that present similar values of t1/2
.
Table 1 includes f50% values for IDU, which
represent the time at which 50% of total parent
drug (1) was formed in a porcine esterase solution.
The f50% values have been reported in litera-
ture17,18,26 as useful parameters in enzymatic
hydrolysis evaluation of prodrugs that could
require more than one step to generate the parent
drug. Therefore, f50% can be considered as a more
relevant indicator of the formation of the parent
drug than the prodrug degradation rate. In
Figure 1, a typical time course of ester 9 degrad-
ation and IDU formation in enzymatic hydrolysis
experiments is reported: both these processes
exhibit a pseudo-®rst-order kinetics for a limited
time interval.
As reported in Table 1, the values of the ester
9±13 degradation (t1/2) are similar to determined
f50% values for IDU appearance: this evidence
suggests that the formation of the parent drug (1)
takes place at the same rate as the loss of these
As showed in Figure 2, both lipophilicity
parameters (CLOGP and log K0) of esters 9±13
increased as the polyoxyethylene chain length-
ened. In addition, as noted in Figure 2, the good
correlation observed between log K0 and CLOGP
suggests that both these parameters can be
suitably used to evaluate the lipophilicity of
synthesized esters 9±13.
Figure 1. Time courses for ester 9 (*) and IDU (*)
during hydrolysis of the prodrug in porcine esterase
aqueous solution (pH 7.4) at 378C.
Figure 2. CLOGP (&) and log K0 (^) values versus
number of polyoxyethylene groups of esters (9±13).
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SYNTHESIS, PROPERTIES, AND SKIN PERMEATION OF 5-IODO-20-DEOXYURIDINE DERIVATIVES
177
In Vitro Skin Permeation Study
Lipophilicity and water solubility are regarded
as the key parameters for a successful dermal
prodrug design.27,28,30 Accordingly, an effective
dermal prodrug must possess an increased lipo-
philicity, compared with the parent drug, toge-
ther with adequate aqueous solubility.
Skin permeation of IDU and esters (9±13) was
evaluated through excised human skin (SCE
membranes) because other authors reported that
the dermis in vitro can act as a signi®cant
additional barrier to the permeation of some
compounds.29In the case of esters (9±13) skin
permeation experiments, intact ester was found
in the receptor phase together with a variable
amount of IDU ranging between 4 and 8%,
compared with the amount of ester permeated.
The observed hydrolysis should be ascribed
mainly to the chemical hydrolysis given that in
previous in vitro skin permeation studies of
dermal prodrugs10,14 we observed a very poor
enzymatic activity of SCE membranes. Further-
more, the large amounts of ethanol used in the
receptor compartment for ensuring the sink
conditions could notably decrease the chemical
hydrolysis in this medium with respect to that
obtained in pH 7.4 buffer aqueous solution used
for in vitro chemical stability studies.
Plotting the total cumulative amount of IDU
(IDU free ‡ IDU equivalent permeated as ester
prodrug) against time we obtained typical plots
similar to that reported in Figure 3. steady-state
¯uxes of cumulative IDU, obtained after IDU or
esters 9±13 skin application, are reported in
Table 1. As may be noted, esters 11±13 did not
signi®cantly increase IDU cumulative amount
penetrated through the skin, whereas applying
esters 9 and 10 onto the skin resulted in the
cumulative amount of IDU penetrating through
the SCE being 3.8- and 2.8-fold higher, respec-
tively, compared with that obtained after topical
application of 1.
Because the skin is regarded as a lipophilic
rate-limiting barrier, enhancement of prodrug
lipophilicity seems to be important to improve
both diffusional characteristics and solubility of
the parent drug in the stratum corneum. The
enhancement of water solubility is equally impor-
tant for at least two reasons:27 (a) at the molecular
level, because the lipid domain of the stratum
corneum consist of multilamellar bilayers, the
transporting species must be able to repetitively
cross lipid±aqueous phase interfaces; (b) lipophi-
lic derivatization to obtain prodrugs increases
partitioning into the stratum corneum, forming a
reservoir, but the subsequent transport into the
aqueous milieu beneath may be limited by both
prodrug aqueous solubility and the ability of
epidermal enzymes to convert the prodrug into a
more polar metabolite. In this way, viable epider-
mis, rather than the stratum corneum, would be
the rate-limiting membrane31 and, therefore, pro-
drugs should possess suitable water solubility.
With regard to our synthesised esters (9±13), we
evaluated their lipophilicity by both measured
lipophilic indices (log k0) and calculated CLOGP
but we haven0t been able to determine the
experimental water solubility of these compounds
because their oil form creates dif®culties indeter-
mining this parameter. Furthermore, in attempt-
ing to calculate the theoretical water solubility,
we unsuccessfully applied the theoretical meth-
ods reported by Yalkowsky et al.32,33 and pre-
viously used to estimate the water solubility of
lipophilic dermal prodrugs.12,13
The application of these theoretical methods to
estimate the water solubility of our esters 9±13
produced unrealistic data: probably these theore-
tical models can be useful to evaluate only the
water solubility of very lipophilic compounds. So,
we are unable to evaluate the effects of water
solubility of esters 9±13 on their skin permeation
¯uxes. On the basis of the in vitro percutaneous
data, we obtained no correlation between lipophi-
licity and skin permeation ¯uxes of synthesized
esters 9±13. In our previous papers10±13 on
dermal prodrugs, we obtained an increase in the
skin permeation of drugs only for derivatives with
both increased lipophilicity and water solubility
(compared with the parent drug).
Figure 3. Plot of the cumulative amount (mg) of IDU
(*) and ester 11 (~) penetrated through excised
human skin versus time.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 1, JANUARY 2002

178
BONINA ET AL.
With regard to our results, the IDU skin per-
meation enhancement, showed by esters 9 and 10,
could be explained on the basis of their increased
lipophilicity and, probably, their moderate water
solubility with respect to parent drug (1).
7. Ghosh MK, Mitra AK. 1992. Enhanced delivery of
5-iodo-20-deoxyuridine to the brain parenchyma.
Pharm Res 9(9):1173±1176.
8. Ghosh MK, Mitra AK. 1992. Brain parenchymal
metabolism of 5-iodo-20-deoxyuridine and 50-ester
prodrugs. Pharm Res 9(8):1048±1052.
Despite the more lipophilic character of esters
11±13, these derivatives showed lower ¯uxes and
the lack of IDU skin enhancement. These ®ndings
could be attributed to both higher molecular size
and higher ability to form H-bonds (due to more
polyoxyethylene groups), which, as reported in
literature,34,35 could decrease the diffusional
properties of these esters through the skin. Fur-
thermore, the increased lipophilicity of esters
11±13 could result in a reduction of water
solubility, resulting, therefore, in a reduction of
the skin permeation of the esters.
In conclusion, synthesised IDU esters 9±13
proved moderately stable in aqueous media and
were readily hydrolyzed by porcine esterase.
Among all esters 9±13 synthesized, only 9 and
10 seem to penetrate through excised human skin
better than the parent drug (1). Further in vivo
pharmacological studies are planned to investi-
gate the potential use of esters 9 and 10 as dermal
prodrugs of IDU.
9. Narurkar MM, Mitra AK. 1989. Prodrugs of 5-iodo-
20-deoxyuridine for enhanced ocular transport.
Pharm Res 6(10):887±891.
10. Bonina FP, Montenegro L, de Caprariis P, Bous-
quet E, Tirendi S. 1991. 1-Alkylazacycloalkan-2-
one esters as prodrugs of indomethacin for
improved delivery through human skin. Int J
Pharm 77:21±29.
11. Bonina FP, Montenegro L, Guerrera F. 1993.
Naproxen 1-alkylazacycloalkan-2-one esters as
dermal prodrugs: In vitro evaluation. Int J Pharm
100:99±105.
12. Bonina FP, Montenegro L, Trapani G, Franco M,
Liso G. 1995. In vitro evaluation of N-acyllactam
esters of indomethacin as dermal prodrugs. Int J
Pharm 124:45±51.
13. Bonina FP, Montenegro L, de Caprariis P, Pala-
giano F, Trapani G, Liso G. 1995. In vitro and in
vivo evaluation of polyoxyethilene indomethacin
esters as dermal prodrugs. J Controlled Release
34:223±232.
14. Palagiano F, Arenare L, Barbato F, La Rotonda MI,
Quaglia F, Bonina FP, Montenegro L, de Caprariis
P. 1997. In vitro and in vivo evaluation of terpenoid
esters of indomethacin as dermal prodrugs. Int J
Pharm 149:171±182.
REFERENCES
1. Spadari S, Ciarrocchi G, Focher F, Verri A, Maga G,
Arcamone F, Iafrate E, Manzini S, Garbesi A,
Tondelli L. 1995. 5-Iodo-20-deoxy-L-uridine and (E)-
5-(2-bromovinyl)-20-deoxy-L-uridine:Selectivephos-
phorylation by herpes simplex virus type 1 thymi-
dine kinase, antiherpetic activity, and cytotoxicity
studies. Mol Pharmacol 47(6):1231±1238.
2. Touitou E, Segal R, Pisanty S, Milogoldzweig I.
1988. Glycirrhyzin gel as vehicle for idoxuridine
topical preparation: Skin permeation behaviour.
Drug Des Deliv 3(3):267±272.
15. Rimoli MG, Avallone L, de Caprariis P, Galeone A,
Forni F, Vandelli MA. 1999. Synthesis and char-
acterisation of poly(D,L-lactic acid)-idoxuridine con-
jugate. J Controlled Release 58:61±68.
16. Bessodes M, Komiotis D, Antonakis K. 1986.
Rapid and selective detritylation of primary
alcohols using formic acid. Tetrahedron Lett 27:
579.
17. Rautio J, Nevalainen T, Taipale H, Vepsalainen J,
È È
Gynther J, Pedersen T, Jarvinen T. 1999. Synthesis
and in vitro evaluation of aminoacyloxyalkyl esters
of 2-(6-methoxy-2-naphthyl) propionic acid as novel
naproxen prodrugs for dermal drug delivery.
Pharm Res 16(8):1172±1178.
È
3. Sheth NV, McKeough MB, Spruance SL. 1987.
Measurement of the stratum corneum drug reser-
voir to predict the therapeutic ef®cacy of topical
iododeoxyuridine for herpes virus infection.
Invest Dermatol 89(6):598±602.
4. Bravo ML, Auladell C, Navarro C. Evaluation
absorption and safety of idoxuridine after topical
administration. Methods Find Exp Clin Pharmacol
10(11):705±709.
5. BarryBW.1987.Modeofactionofpenetrationenhan-
cers in human skin. J Controlled Release 6: 85±97.
6. Hadgraft J. 1989. Formulation of anti-in¯amma-
tory agents. In: Lowe NJ, Hensby CN, editors. Non-
steroidal Anti-in¯ammatory Drugs. Basel (Switzer-
land): Karger, pp. 21±43.
J
È
È È
18. Jarvinen T, Poikolainen M, Suhonen P, Vepsalai-
nen J, Alaranta S, Urtti A. 1995. Comparison of
enzymatic hydrolysis of pilocarpine prodrugs in
human plasma, rabbit cornea and butyrylcholines-
terase solutions. J Pharm Sci 84:656±660.
19. Lambert WJ. 1993. Modelling oil±water partion-
ing and membrane permeation using reversed-
phase chromatography. J Chromatogr A 656:469±
484.
20. Kligman AM, Christophers E. 1963. Preparation of
isolated sheets of human skin. Arch Dermatol
88:702±705.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 1, JANUARY 2002

SYNTHESIS, PROPERTIES, AND SKIN PERMEATION OF 5-IODO-20-DEOXYURIDINE DERIVATIVES
179
21. Swarbrick J, Lee G, Brom J, Gensmantel NP. 1982.
Drug permeation through human skin I : Effect of
storage conditions of skin. J Invest Dermatol 78:
63±66.
22. Mueller LG. Novel anti-in¯ammatory esters, phar-
maceutical composition and methods for reducing
in¯ammation. U.K. Patent GB 2 204 869 A, 1988.
23. Toitou E, Fabin B. 1988. Altered skin permeation of
a highly lipophilic molecule: Tetrahydrocannabi-
nol. Int J Pharm 43:17±22.
28. Sloan KB. 1989. Prodrugs for dermal delivery. Adv
Drug Del Rev 3:912±922.
29. Bronaugh RL, Stewart RF. 1984. Methods for in
vitro percutaneous absorption studies. III. Hydro-
phobic compounds. J Pharm Sci 73:1255±1258.
30. Waranis RP, Sloan KB. 1987. Effect of vehicles and
prodrug properties and their interactions on the
delivery of 6-mercaptopurine through skin: Bisacy-
loxymethyl-6-mercaptopurine prodrugs. J Pharm
Sci 76:587±595.
24. Cheung YW, Li Wan Po A, Irwin WJ. 1985.
Cutaneous biotransformation as a parameter in
the modulation of the activity of topical corticoster-
oids. Int J Pharm 26:175±189.
31. Friend D, Catz P, Heller J, Reid J, Baker R. 1988.
Transdermal delivery of levonorgestrel: II. Effect of
prodrug structure on skin permeability in vitro. J
Controlled Release 7:251±261.
25. Wong O, Tsuzuki N, Nghiem B, Kuehnhoff J, Itoh
T, Masaki K, Huntington J, Konishi R, Rytting JH,
Higuchi T. 1989. Unsaturated cyclic ureas as a new
non-toxic biodegradable transdermal penetration
enhancers: II. Evaluation study. Int J Pharm 52:
191±202.
26. Rautio J, Taipale H, Gynther J, VersaÈlaÈinen J,
Nevalainen T, Jarvinen T. In vitro evaluation of
acyloxyalkyl esters as dermal prodrugs of ketopro-
fen and naproxen. J Pharm Sci 87:1622±1628.
27. Guy RH, Hadgraft J. 1992. Percutaneous penetra-
tion enhancement: Physicochemical consideration
and implications for prodrug design. In: Sloan KB,
editor. Prodrugs: Topical and ocular drug delivery.
New York: Marcel Dekker. pp. 1±16.
32. Yalkowsky SH, Valvani SC. 1980. Solubility and
partitioning I: Solubility of nonelectrolytes in
water. J Pharm Sci 69:912±922.
33. Yalkowsky SH, Valvani SC, Roseman TJ. 1983.
Solubility and partitioning VI: Octanol solubility
and octanol±water partition coef®cients. J Pharm
Sci 72:866±870.
34. Lien EJ, Gao H. 1995. QSAR analysis of skin
permeability of various drugs in man as compared
to in vivo and in vitro studies in rodents. Pharm Res
12(4):583±587.
35. El Tayar N, Tsai RS, Testa B, Carrupt PA, Hansch
C, Leo A. 1991. Percutaneous penetration of drugs:
A quantitative structure±permeability relation-
ship study. J Pharm Sci 80(8):744±749.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 1, JANUARY 2002