Improvement of Topical Palmitoylethanolamide Anti-Inflammatory Activity by Pegylated Prodrugs

Article abstract of DOI:10.1021/acs.molpharmaceut.5b00397

A small library of polyethylene glycol esters of palmitoylethanolamide (PEA) was synthesized with the aim of improving the pharmacokinetic profile of the parent drug after topical administration. Synthesized prodrugs were studied for their skin accumulation, pharmacological activities, in vitro chemical stability, and in silico enzymatic hydrolysis. Prodrugs proved to be able to delay and prolong the pharmacological activity of PEA by modification of its skin accumulation profile. Pharmacokinetic improvements were particularly evident when specific structural requirements, such as flexibility and reduced molecular weight, were respected. Some of the synthesized prodrugs prolonged the pharmacological effects 5 days following topical administration, while a formulation composed by PEA and two pegylated prodrugs showed both rapid onset and long-lasting activity, suggesting the potential use of polyethylene glycol prodrugs of PEA as a suitable candidate for the treatment of skin inflammatory diseases.

Full text of DOI:10.1021/acs.molpharmaceut.5b00397

Article
pubs.acs.org/molecularpharmaceutics
Improvement of Topical Palmitoylethanolamide Anti-Inflammatory
Activity by Pegylated Prodrugs
Diana Tronino,†,∥ Roberto Russo,†,∥ Carmine Ostacolo,*,† Angelica Mazzolari,‡ Carmen De Caro,†
Carmen Avagliano,† Sonia Laneri,† Giovanna La Rana,† Antonia Sacchi,† Francesco Della Valle,§
Giulio Vistoli,‡ and Antonio Calignano†
†Department of Pharmacy, University of Naples Federico II, Via D. Montesano 49, 80131 Naples, Italy
‡Department of Pharmaceutical Sciences “Pietro Pratesi”, University of Milan, Via Mangiagalli 25, 20133 Milan, Italy
§Epitech Group s.r.l., Via Egadi 7, 20144 Milan, Italy
ABSTRACT: A small library of polyethylene glycol esters of palmitoylethanolamide (PEA) was synthesized with the aim of
improving the pharmacokinetic profile of the parent drug after topical administration. Synthesized prodrugs were studied for
their skin accumulation, pharmacological activities, in vitro chemical stability, and in silico enzymatic hydrolysis. Prodrugs proved
to be able to delay and prolong the pharmacological activity of PEA by modification of its skin accumulation profile.
Pharmacokinetic improvements were particularly evident when specific structural requirements, such as flexibility and reduced
molecular weight, were respected. Some of the synthesized prodrugs prolonged the pharmacological effects 5 days following
topical administration, while a formulation composed by PEA and two pegylated prodrugs showed both rapid onset and long-
lasting activity, suggesting the potential use of polyethylene glycol prodrugs of PEA as a suitable candidate for the treatment of
skin inflammatory diseases.
KEYWORDS: N-palmitoylethanolamide, pegylated prodrugs, topical delivery, anti-inflammatory effects, antihyperalgesic effects
an effective therapeutic tool in different skin disorders, both for
human and veterinary use, such as dermatitis, pruritus, psoriasis,
and skin barrier disruption.15−18 Finally, it has been shown that
topical application of PPAR-α activators limits their pharmaco-
logical activity to the skin, avoiding any systemic side effects.12
However, the use of topical PEA is strongly limited by short-
term activity. It is widely reported that repeated daily topical
administration of PEA ointments are necessary to achieve
remarkable pharmacological effects.12,15−18 Thus, modification
of pharmacokinetic properties of the molecule is required to
gain a sustained and prolonged biological activity, as well as a
better compliance.
INTRODUCTION
■
Fatty acid ethanolamides (FAEs) are a family of lipids that
participate in the control of multiple physiological functions,
including pain and inflammation.1−3 Palmitoylethanolamide
(PEA) is a member of this family, it has been widely reported
that PEA exerts antinociceptive and anti-inflammatory effects in
experimental animals and humans.1,2,4 Such effects are
primarily, albeit not exclusively,1 due to the ability of PEA to
bind with high affinity peroxisome proliferator-activated
receptor (PPAR)-alpha.2 The presence of PPAR-α has been
demonstrated in rodent and human keratinocytes5,6 as well in
mouse and human dermis7,8 and, in addition, in some specific
cells such as sebocytes, cutaneous macrophages, and T-
lymphocytes.9−11
Several data indicate that topically administered PEA
formulations inhibit inflammation and reduce dermatitis with
a potency comparable to the synthetic PPAR-α agonist, Wy-
14643.12−14 Moreover, PEA ointments have been proposed as
Received: May 21, 2015
Revised: August 3, 2015
Accepted: August 19, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acs.molpharmaceut.5b00397
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Figure 1. Structures of PEA (1) and its pegylated prodrugs diehtylene glycol-2-palmitamidoethyl succinate (5), triehtylene glycol-2-
palmitamidoethyl succinate (6), tetraehtylene glycol-2-palmitamidoethyl succinate (7), diethylene glycol-2-palmitamidoethyl phtalate (8),
diethylene glycol-2-palmitamidoethyl-(E)-fumarate (9), 2-(2-methoxyethoxy)ethyl-2-palmitamidoethyl succinate (10), and diethylene glycol-2-
palmitamidoethyl carbonate (11).
Figure 2. Synthesis of PEA derivatives. Reagent and conditions: (a) Succinic, phthalic, or maleic anhydride, THF, 50 °C, 3−5 h; (b) polyethylene
glycol, DCC, DMAP, dry THF, room temperature, 4 h; (c) CH3I, DMAP, dry acetone, 50 °C, 2 h; (d) CDI, diethylene glycol, dry dichloromethane,
room temperature to 60 °C, 5 h.
Prodrug technologies are commonly employed to improve
the membrane permeability or solubility of drugs, with wide
application in skin delivery.19 One of the most used strategies
for prodrug design is the direct linkage to the parent drug of
B
DOI: 10.1021/acs.molpharmaceut.5b00397
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promoieties possessing an inherent permeation enhancing
ability, in order to increase skin permeability and percutaneous
absorption.20 Among prodrugs, the use of a polyethylene glycol
(PEG) promoiety is well-described, especially for skin
delivery.21,22 PEGs are capable of conferring an adequate
aqueous stability as well as enhanced and targeted delivery to
prodrugs. Moreover, the controlled enzymatic conversion into
the parent drug and the amphiphilic properties make PEGs
privileged promoieties in prodrug design.21,23−26
Starting from these lines of evidence and from the
remarkable pharmacokinetic improvements obtained by pre-
viously investigated PEA prodrugs,27,28 the aim of the present
work was to synthesize a small library of PEA polyethylene
glycol derivatives with sustained topical delivery and prolonged
pharmacological efficacy (Figure 1). Skin accumulation was
evaluated, ex vivo, by topical application of PEA and its
prodrugs in mice. The detected PEA skin concentrations were
rationalized by molecular modeling and in vitro chemical
stability studies. Finally, PEA and its derivatives were tested in
vivo for their anti-inflammatory and antihyperalgesic effects in a
murine model of inflamed skin.
(100 MHz, CD3OD): δ 14.68, 22.81, 25.83, 29.31, 29.36, 29.42,
29.50, 29.65, 29.72, 29.75, 29.76, 32.10, 36.01, 38.15, 63.50,
172.78, 173.18, 174.12. ESI-MS (m/z): 399.56.
2-[(2-palmitamidoethoxy)carbonyl]benzoic Acid (3). Yield
1
75%. H NMR (400 MHz, CD3OD): δ 0.81 (t, 3H, J = 12.8
Hz), 1.15−1.24 (m, 24H), 1.42−1.49 (m, 2H), 2.10 (t, 2H, J =
14.9 Hz), 3.45 (t, 2H, J = 9.6 Hz), 4.26 (t, 2H, J = 9.8 Hz), 7.51
(pt, 2H, J = 6.8 Hz), 7.58 (pd, 1H, J = 4.7 Hz), 7.7 (pd, 1H, J =
4.1 Hz). 13C NMR (100 MHz, CD3OD): δ 23.76, 26.95, 30.20,
30.45, 30.49, 30.60, 30.72, 30.78, 30.80, 33.09, 37.09, 39.30,
65.20, 129.67, 130.25, 132.06, 132.44, 133.30, 134.11, 169.80,
170.61, 176.66. ESI-MS (m/z): 448.30.
(E)-4-Oxo-4-(2-palmitamidoethoxy)but-2-enoic Acid (4).
1
Yield 80%. H NMR (400 MHz, CDCl3): δ 0.90 (t, 3H, J =
13.2 Hz), 1.10−1.22 (m, 24H), 1.58−1.63 (m, 2H), 2.30 (t,
2H, J = 14.9 Hz), 3.60−3.65 (m, 2H), 4.35 (t, 2H, J = 10.0 Hz),
5.83 (bs, 1H), 6.30 (d, 1H, J = 11.7 Hz), 6.40 (d, 1H, J = 11.6
Hz). 13C NMR (100 MHz, CD3OD): δ 14.35, 22.91, 26.04,
29.45, 29.59, 29.74, 29.88, 29.93, 32.14, 32.62, 38.94, 64.47,
129.89, 131.87, 166.19, 166.81, 175.73. ESI-MS (m/z): 398.86.
Diehtylene Glycol-2-palmitamidoethyl succinates (5),
Triehtylene Glycol-2-palmitamidoethyl Succinates (6), Tet-
raehtylene Glycol-2-palmitamidoethyl Succinates (7), Dieth-
ylene Glycol-2-palmitamidoethyl Phthalate (8), and Dieth-
ylene Glycol-2-palmitamidoethyl-(E)-fumarate (9). Inter-
mediates 2−4 (2.63 mmol) were reacted with the appropriate
polyethylene glycol (diethylene glycol, triethylene glycol, and
tetraethylene glycol, 3.95 mmol) in the presence of DMAP
(0.03g, 0.26 mmol) and DCC (0.54 g, 2.63 mmol) in 30 mL of
anhydrous tetrahydrofuran. The mixture was stirred at room
temperature for 4 h. Then the precipitate formed was filtered
off, and the filtrate was evaporated and reconstituted in 15 mL
of chloroform. A saturated solution of NaHCO3 was added, and
the aqueous phase was extracted twice with chloroform.
Organic phases were collected, dried over anhydrous MgSO4,
and evaporated in vacuo. The crude products were purified by
chromatography using 9.5/0.5 chloroform/methanol as mobile
phase.
EXPERIMENTAL SECTION
■
General Procedures for the Synthesis of PEA Oligo-
ethylene Derivatives. The general synthetic procedure
adopted is described in Figure 2. Ultramicronized PEA was a
kind gift by Epitech group srl (Saccolongo, PD, Italy). Succinic,
phthalic, and maleic anhydrides, diethylene, triethylene, and
tetraethylene glycols as well N,N′-dicyclohexylcarbodiimide
(DCC), 4-dimethylaminopyridine (DMAP), 1,1′-carbonyldii-
midazole (CDI), and iodomethane were purchased by Sigma-
Aldrich. Silica gel (0.04−0.063 mm, 70−230 mesh) was
furnished by Macherey-Nagel (Duren, Germany). H and 13C
1
NMR were recorded using a Mercury plus 400 MHz
instrument (Varian Inc., Palo Alto, CA, USA). Trimethylsilane
(TMS) was used as internal standard. Chemical shifts values are
reported in δ units (ppm) relative to TMS (1%). The mass
spectra were recorded using an API 2000 instrument (Applied
Biosystem, Foster City, USA).
Diehtylene Glycol-2-palmitamidoethyl Succinate (5). Yield
75%. 1H NMR (400 MHz, CDCl3): δ 0.87 (t, 3H, J = 13.3 Hz),
1.19−1.35 (m, 24H), 1.57−1.65 (m, 2H), 2.17 (t, 2H, J = 15.7
Hz), 2.62−2.70 (m, 4H), 3.50−3.54 (m, 2H), 3.61 (t, 2H, J =
4.4 Hz), 3.69−3.74 (m, 4H), 4.20 (t, 2H, J = 9.5 Hz), 4.27 (t,
2H, J = 8.4 Hz), 5.97 (bs, 1H). 13C NMR (100 MHz, CD3OD):
δ 14.60, 23.90, 27.21, 29.97, 30.42, 30.61, 30.63, 30.80, 30.89,
30.92, 30.94, 33.23, 37.18, 39.51, 62.30, 64.35, 65.11, 70.86,
73.77, 174.08, 175.08, 176.76. ESI-MS (m/z): 487.67.
4-Oxo-4-(2-palmitamidoethoxy)butanoic Acid (2), 2-[(2-
palmitamidoethoxy)carbonyl]benzoic Acid (3), and (E)-4-
Oxo-4-(2-palmitamidoethoxy)but-2-enoic Acid (4). Inter-
mediates 2, 3, and 4 were synthesized starting from 1.00 g of
ultramicronized PEA (3.36 mmol), which was reacted with the
appropriate anhydride (succinic, phthalic, or maleic anhydride,
4.36 mmol), in 25 mL of tetrahydrofuran (THF). The mixture
was stirred at 50 °C for 3−5 h. Then, 10 mL of 2 N HCl were
added, and the mixture was cooled to 0 °C. A precipitate was
collected representing the desired intermediate. THF was then
separated from the aqueous phase, which was extracted two
more times with chloroform. The organic layers were collected,
dried over anhydrous MgSO4, and evaporated in vacuo. The
crude product thus obtained was purified by chromatography
using 9.5/0.5 chloroform/methanol as mobile phase and was
combined with precipitate previously collected. The course of
reaction and purification was monitored by glass TLC plates
(0.25 mm 5 × 10 cm, Macherey-Nagel) after development with
permanganate stain.
Triehtylene Glycol-2-palmitamidoethyl Succinate (6).
1
Yield 70%. H NMR (400 MHz, CDCl3): δ 0.88 (t, 3H, J =
13.4 Hz), 1.24−1.38 (m, 24H), 1.58−1.64 (m, 2H), 2.18 (t,
2H, J = 15.0 Hz), 2.63−2.71 (m, 4H), 3.53−3.57 (m, 2H), 3.61
(t, 2H, J= 5.0 Hz), 3.66−3.74 (m, 8H), 4.21 (t, 2H, J = 9.8 Hz),
4.28 (t, 2H, J = 8.7 Hz), 6.00 (bs, 1H). 13C NMR (100 MHz,
CD3OD): δ 15.95, 24.10, 26.13, 30.65, 30.71, 30.76, 30.84,
30.95, 31.03, 31.06, 31.08, 33.43, 37.40, 39.44, 62.35, 64.35,
64.84, 70.15, 71.54, 73.77, 174.08, 174.55, 176.76. ESI-MS (m/
z): 531.72.
Tetraehtylene Glycol-2-palmitamidoethyl Succinate (7).
1
Yield 65%. H NMR (400 MHz, CDCl3): δ 0.88 (t, 3H, J =
4-Oxo-4-(2-palmitamidoethoxy)butanoic Acid (2). Yield
83%. 1H NMR (400 MHz, CDCl3): δ 0.88 (t, 3H, J = 13.5 Hz),
1.22−1.40 (m, 24H), 1.60−1.65 (m, 2H), 2.20 (t, 2H, J = 15.1
Hz), 2.65 (t, 2H, J = 12.2 Hz), 2.71 (t, 2H, J = 12.2 Hz), 3.52−
3.55 (m, 2H), 4.22 (t, 2H, J = 9.8 Hz), 6.00 (bs, 1H). 13C NMR
7.0), 1.26−1.31 (m, 24H), 1.58−1.64 (m, 2H), 2.18 (t, 2H, J =
15.4 Hz), 2.62−2.70 (m, 4H), 3.53−3.57 (m, 2H), 3.63 (t, 2H,
J = 4.7) 3.65−3.75 (m, 12H), 4.22 (t, 2H, J = 9.7 Hz), 4.25 (t,
2H, J = 9.0 Hz), 6.0 (bs, 1H). 13C NMR (100 MHz, CD3OD):
C
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δ 15.97, 24.10, 26.15, 30.64, 30.71, 30.77, 30.85, 30.97, 31.02,
31.05, 31.09, 33.45, 37.41, 39.45, 62.40, 64.37, 64.84, 70.15,
71.54, 71.76, 73.77, 174.09, 174.57, 176.73. ESI-MS (m/z):
576.43.
Stability Studies. Prodrugs were dissolved in a solution of
Tween 80 (5 mM)/methanol 10:1 (v:v), to a final
concentration of 0.5 mg/mL. Aqueous phases were buffered
at pH 5.0, mimicking the average pH of the skin, by acetate
buffer. Solutions obtained were incubated at 37 °C. At
predetermined intervals, aliquots of 100 μL were withdrawn,
diluted 10 times with ethanol, evaporated in vacuo, and dried
overnight on P2O5. PEA and its prodrugs were isolated by
extraction and fractionation by silica gel chromatography as
elsewhere described29 and analyzed by HPLC as described
below.
HPLC Analysis. HPLC analysis was performed on a Jasco
apparatus (Jasco inc., Easton, MD, USA) equipped with a
quaternary gradient module (PU 2089 Plus), a 25 μL rheodyne
injection valve, and a multiwavelenght UV detector (MD 2010
Plus). The analytes were subjected to precolumn derivatization
by dansyl chloride as previously described by Yagen and
Burnstein.30 Analytical parameters, determined in accordance
with USP 38, are reported in Table 1. The specificity (absence
of interfering peak from skin samples) was assessed as well.
Diethylene Glycol-2-palmitamidoethyl Phtalate (8). Yield
67%. 1H NMR (400 MHz, CDCl3): δ 0.91 (t, 3H, J = 13.5 Hz),
1.22−1.36 (m, 24H), 1.60−1.65 (m, 2H), 2.22 (t, 2H, J = 15.4
Hz), 3.62−3.69 (m, 4H), 3.75−3.78 (m, 2H), 3.85 (t, 2H, J =
9.0 Hz), 4.47 (t, 2H, J = 10.2 Hz), 4.51 (t, 2H, J = 9.1 Hz), 6.3
(bs, 1H), 7,61 (pt, 2H, J = 7.2 Hz), 7.74 (pd,1H, J = 5.0 Hz),
7.82 (pd, 1H, J = 4.4 Hz). 13C NMR (100 MHz, CD3OD): δ
14.46, 23.75, 26.98, 30.24, 30.46, 30.49, 30.61, 30.73, 30.79,
30.80, 33.09, 37.09, 39.33, 62.20, 65.29, 66.10, 69.91, 73.70,
130.10, 132.47, 132.51, 133.19, 133.31, 168.97, 169.08, 176.64.
ESI-MS (m/z): 536.73.
Diethylene Glycol-2-palmitamidoethyl-(E)-fumarate (9).
1
Yield 62%. H NMR (400 MHz, CDCl3): δ 0.91 (t, 3H, J =
13.5 Hz), 1.11−1.24 (m, 24H), 1.60−1.65 (m, 2H), 2.22 (t,
2H, J = 15.1 Hz), 3.49−3.53 (m, 2H), 3.61−3.66 (m, 4H), 3.8
(t, 2H, J = 5.2 Hz), 4.32 (t, 2H, J = 9.9 Hz), 4.41 (t, 2H, J = 8.5
Hz), 5.80 (bs, 1H), 6.31 (d, 1H, J = 11.7 Hz), 6.37 (d, 1H, J =
11.9 Hz). 13C NMR (100 MHz, CD3OD): δ 13.92, 22.08,
25.15, 28.60, 28.68, 28.76, 28.81, 28,87, 28.92, 29.03, 35.28,
37.26 63.06, 64.55, 65.26, 70.98, 73.67, 128.55, 131.57, 165.10,
166.45, 172.42. ESI-MS (m/z): 486.13.
2-(2-Methoxyethoxy)ethyl-2-palmitamidoethyl Succinate
(10). To 0.260 g (0.547 mmol) of the intermediate 5 in 15
mL of dry acetone, DMAP (0.100 g, 0.820 mmol, 1.5 equiv)
and iodomethane (0.05 mL, 0.820 mmol, 1.5 equiv) were
added. The mixture was stirred at 50 °C for 2 h, then cooled to
ambient temperature and concentrated in vacuo. The resulting
solid was dissolved in chloroform, extracted with a saturated
solution of NaHCO3 and with 2 N HCl, dried over anhydrous
MgSO4, and evaporated in vacuo. Crude product was purified
by chromatography using ethyl acetate as mobile phase. Yield
71%. 1H NMR (400 MHz, CDCl3): δ 0.88 (t, 3H, J = 13.5 Hz),
1.18−1.35 (m, 24H), 1.55−1.64 (m, 2H), 2.18 (t, 2H, J = 15.5
Hz), 2.62−2.70 (m, 4H), 3.50 (s, 3H), 3.51−3.54 (m, 2H),
3.62 (t, 2H, J = 4.2 Hz), 3.71−3.75 (m, 4H), 4.22 (t, 2H, J =
9.6 Hz), 4.30 (t, 2H, J = 8.5 Hz), 6,00 (bs, 1H). 13C NMR (100
MHz, CD3OD): δ 14.62, 23.88, 27.24, 29.95, 30.50, 30.66,
30.68, 30.78, 30.91, 30.94, 30.98, 33.24, 37.19, 39.54, 58.91,
62.28, 64.32, 65.12, 70.84, 73.79, 174.01, 175.11, 176.79. ESI-
MS (m/z): 502.82.
Diethylene Glycol-2-palmitamidoethyl Carbonate (11). To
a solution of PEA (0.5 g, 1.167 mmol) in 17 mL of dry
dichloromethane, CDI (0.3 g, 1.84 mmol, 1.1 equiv) was added
portionwise. The reaction was maintained under stirring at
room temperature for 2 h. Diethylene glycol (0.35 mL, 2.5
mmol, 1.5 equiv) was then added, and the reaction was warmed
to 60 °C for 3 h. The solution was then cooled to room
temperature and extracted with a saturated solution of
NaHCO3 and 2 N HCl, dried over anhydrous MgSO4, and
evaporated in vacuo. Crude product was purified by
chromatography using ethyl acetate as mobile phase. Yield
58%. 1H NMR (400 MHz, CDCl3): δ 0.88 (t, 3H, J = 13.4 Hz),
1.10−1.25 (m, 24H), 1.54−1.62 (m, 2H), 2.17 (t, 2H, J = 15.4
Hz), 3.55−3.58 (m, 2H), 3.62 (t, 2H, J = 8.4 Hz), 3.67−3.76
(m, 4H), 4.23 (t, 2H, J = 10.4 Hz), 4.33 (t, 2H, J = 9.1 Hz),
5.85 (bs, 1H). 13C NMR (100 MHz, CD3OD): δ 14.43, 23.74,
26.01, 26.99, 30.18, 30.32, 30.39, 30.47, 30.62, 30.73, 33.76,
30.78, 33.07, 34.97, 37.12, 42.91, 61.64, 62.19, 64.58, 70.13,
73.63, 163.36, 176.65. ESI-MS (m/z): 431.97.
Table 1. Analytical Parameter for Synthesized Derivatives
Tr
linearity range
(μg/mL)
resolution
factor
% of
recovery
derivative (min)
R2
PEA
5
28.5
25.4
23.2
22.6
27.4
24.9
24.6
0.32−10.00
0.45−10.00
0.50−8.00
0.50−8.00
0.47−9.30
0.51−8.70
0.49−9.10
0.988
0.981
0.981
0.976
0.984
0.987
0.972
95−115
88−99
85−98
85−94
89−99
90−97
84−94
2.06
3.25
3.60
1.05
2.16
2.30
6
7
8
9
11
Molecular Modeling. The conformational behavior of the
PEA prodrugs was investigated by a clustered Monte Carlo
procedure, which generated 1000 conformers by randomly
rotating the rotors.31 For each molecule, the lowest energy
structure obtained was exploited in the following docking
simulations. Docking simulations involved the previously
generated hCES2 homology model32 and were carried out
using PLANTS,33 which finds plausible poses through ant
colony optimization algorithms (ACO). For all docking
simulations, PLANTS was used with default settings and
without geometric constraints. The search was focused on a
15.0 Å radius sphere around the catalytic Ser288 residue thus
encompassing the entire enzymatic cavity. Poses were scored by
the ChemPLP function using speed 1, and 10 poses were
generated for each substrate. The best poses were chosen
considering both the ChemPLP scores and the closeness
between the substrate’s labile function and Ser228. The
selected complexes were then minimized keeping fixed all
atoms outside a 15.0 Å radius sphere around the bound
substrate, and the minimized structures were finally used to
recalculate the PLANTS score functions.
Animals. Male Swiss mice weighing 25 to 30 g were
purchased from Harlan (Udine, Italy). They were housed in
stainless steel cages in a room kept at 22 1 °C on a 12/12 h
light/dark cycle. The animals were acclimated to their
environment for 1 week, and they had ad libitum access to
tap water and standard rodent chow. Animal care was in
compliance with Italian regulations on protection of animals
used for experimental and other scientific purposes (D.M.
D
DOI: 10.1021/acs.molpharmaceut.5b00397
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Figure 3. Chemical hydrolysis pattern for synthesized compounds. Pro-drug concentrations are expressed as micrograms of derivative per milliliter of
solvent SEM (n = 3). Experiments were carried on at 37 °C and at pH 5.5. §p < 0.05 concentration values significantly lower than 5, 6 and 7. #p <
0.05 concentration values significantly lower than 9. *p < 0.05, **p < 0.01 concentration values significantly lower than 8.
116192) as well as with European Economic Community
regulations (O.J. of E.C. L 358/1 12/18/1986).
4, 6, 8, 24, 48, 72, and 96 h following injection), paw edema
and mechanical hyperalgesia were evaluated (n = 6). Paw
volumes were measured by a plethysmometer (Ugo Basile,
Milan, Italy), and the increase in paw volume was assessed as
the difference between the volume measured at each time point
and the basal volume measured before carrageenan injection. In
mechanical hyperalgesia we measured paw withdrawal thresh-
old (g) by mechanical stimuli using the Randall-Selitto
analgesimeter for mice (Ugo Basile, Italy). Latencies of paw
withdrawal to a calibrated pressure were assessed on inflamed
paw 1 day before ligation, and again 2−96 h following
carrageenan injection; each paw was tested twice per session.
Cut-off force was set at 100 g.
Statistical Analyses. Results are expressed as the mean
SEM of n experiments. All analyses were conducted using
Graph-Pad Prism (GraphPad Software Inc., San Diego, CA).
The significance of differences between groups was determined
by one way (for ex vivo and stability experiments) and two-way
(for in vivo experiments) analyses of variance (ANOVA)
followed by Bonferroni posthoc tests for multiple comparisons.
Differences with P < 0.05 (*) were considered statistically
significant.
Skin Accumulation. Hydroalcoholic gels containing 0.5%
(w/v) of PEA or equimolar amounts of prodrugs were used to
assess skin permeation profiles. Gels were prepared dissolving
Carbopol 980 (1% w/v, Lubrizol Corp., Wickliffe, USA) in 85
mL of distilled water at 90 °C under stirring. PEA and prodrugs
were dissolved in 15 mL of absolute ethanol, and the resulting
solutions were added, under magnetic stirring at room
temperature, to the water phase until complete gelation. Drug
delivery was pursued using Hill Top Chambers with webril pad
(19 mm, Hill Top research, St. Petersburg, USA) loaded with
0.5 g of hydroalcoholic gel applied on the back of the shaved
animal for 2 h (n = 5). At predetermined intervals (2, 4, 6, 24,
48, 72, and 96 h following patches applications), mice were
euthanized by CO2, and skin underlying permeation area was
excised. Tissue samples were weighted and homogenized in a
solution of methanol and a serine protease inhibitor, phenyl-
methylsulfonyl fluoride (PMSF, 1 mM). Samples were
centrifuged at 4 °C, for 30 min (1000 rpm). Crude products,
obtained from the supernatant, were purified on silica gel and
analyzed by HPLC as described above. The extraction method
was validated in blank experiments by spiking freshly excised
skin with a known amount of each analyzed compound.
Recovery percentages are reported in Table 1. All the
experiments were repeated in triplicate.
In vivo experiments. Testing solutions were prepared by
dissolving PEA (2 mg/mL) or equimolar doses of prodrugs (5
= 3.25 mg/mL; 6 = 3.55 mg/mL; 7 = 3.84 mg/mL; 8 = 3.57
mg/mL; 9 = 3.24 mg/mL; 10 = 3.35 mg/mL; 11 = 2.88 mg/
mL) in absolute ethanol; moreover, a mixture (MIX)
composed of PEA (2 mg/mL) and equimolar concentrations
of prodrugs 5 and 6 in absolute ethanol was also tested. Each
solution (0.5 mL) was carefully applied by a Gilson pipet on
mice paw. Control animal received only equal volumes of
absolute ethanol. Paw edema was induced, 5 min later, by
subplantar injection of 0.1 mL of sterile saline containing 1% λ-
carrageenan into the hind paw. At predetermined intervals (2,
RESULTS
■
Chemical Hydrolysis. Results obtained from stability
studies are shown in Figure 3. Derivatives 5, 6, and 7 proved
to be the most stable to chemical hydrolysis. No statistical
differences were found in this group of derivatives. After 2 days,
almost 20% of starting material was hydrolyzed. Noticeably, this
loss did not cause the release of a comparable amount of PEA
(data not shown). It can be concluded that chemical hydrolysis
took place preferentially on the ester group distal from
acylamide moiety, releasing intermediate 2 (Figure 2) rather
than PEA. Chemical stability was strongly decreased in
derivatives 8 and 9. After 24 h in acidic media, 40% and 30%
of starting material, respectively, was hydrolyzed. Percentage of
hydrolysis increased at 48 h (65% for derivative 8 and 56% for
derivative 9). As previously evidenced, hydrolysis of prodrugs
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did not lead to comparable PEA release (data not shown). The
least stable derivative to chemical hydrolysis was derivative 11,
rapidly decreasing initial concentration over time, with an
overall hydrolysis rate of almost 80% after 48 h. Derivative 10
was not tested for chemical stability, being unsuitable for
precolumn derivatization with dansyl chloride.
Molecular Modeling. As previously mentioned, docking
simulations involved the hCES2 homology model since the
hydrolytic capacity of the mammalian skin was found to be
mostly ascribable to these isozymes.34 Although hCES2 prefers
esters with alkyl groups larger than acyl moieties, the simulated
PEA esters should suitably fit the physicochemical properties of
the enzymatic cavity since hCES2 better recognizes esters with
very hydrophobic acyl groups and more polar alkyl moieties. As
exemplified by the complex of 5 in Figure 4, all PEA prodrugs
24 and 48 h are vastly affected by chemical hydrolysis (see
above). Compound 11 was not included in the analysis since it
was found to be chemically unstable even at 6 h. In detail, Table
3 reports some relevant docking score and physicochemical
descriptors and reveals (1) the correlation between the
considered PEA concentrations and the computed docking
ChemPlp scores, thus confirming that the PEA skin release can
be mostly ascribed to the hCES2 catalyzed hydrolysis; (2) the
inverse correlation between PEA concentrations and molecular
weights, which is understandable in terms of skin permeation
according to the well-known Potts and Guy model;35 and (3)
the correlation between PEA concentrations and lipophilicity
(as computed by the MLP approach), which can be easily
explained in terms of both skin permeation and enzymatic
recognition. Notably, the same descriptors afford dramatically
worse relationships with the PEA concentrations at 24 or 48 h
thus confirming that only those at 6 h are amenable to reliable
QSAR analyses.
Skin Accumulation. Time-course skin accumulation values
for PEA and its prodrugs are depicted in Figure 5. It has to be
Figure 4. Main interactions stabilizing the putative complex between
hCES2 and 5. The residues interacting with the alkyl chain are
displayed in gold, while those contacting the acyl chain are in cyan.
assume a similar pose within the hCES2 cavity since the
palmitoyl chain is inserted into a hydrophobic subpocket where
it contacts a vast set of apolar residues (e.g., Leu151, Val152,
Phe153, Phe184, Met309, Leu349, Met415, and Phe416), while
the more polar alkyl moiety is accommodated within a
subpocket where the PEG units can stabilize H-bonds with
several polar surrounding residues (e.g., Glu253, Ser254,
Asn342, Tyr434, Tyr436, and Tyr490).
Figure 5. Compared accumulation profiles between PEA and
synthesized prodrugs. Amount accumulated is reported as average
nanomoles of PEA per milligram of excised skin (n = 5).
In all computed complexes, the catalytic Ser228 residue
conveniently approaches the carbonyl carbon atom of the ester
groups closest to the amide function thus indicating that the
hCES2 hydrolysis directly liberates PEA. While the apolar
contacts stabilized by the acyl chain are constantly observed in
all computed complexes, marked differences are seen in the
polar interactions elicited by the alkyl moieties, the number of
elicited H-bonds being roughly proportional to the observed
hydrolysis. In detail, the prodrugs, which are efficiently
hydrolyzed (see Figure 7) elicit clear H-bonds with both
Glu253 and Ser254, which may constrain the substrate in a
pose stably conducive to the catalysis. In contrast, the prodrugs
hydrolyzed with difficulty stabilize only weaker interactions
with Tyr434 or Tyr436.
Although the low number of investigated prodrugs prevents
the development of statistically robust relationships, a simple
correlative study was carried out with a view to revealing those
factors significantly influencing the PEA skin release. The study
was based on the PEA concentrations at 6 h because they
appear to be mostly due to the enzymatic hydrolysis as well as
to the prodrug skin permeation, while the concentrations after
noticed that PEA skin concentrations were constant during the
first 6 h after topical applications, were markedly reduced at 24
h, and, thereafter, only traces of PEA were detected. A different
accumulation pattern was highlighted for synthesized prodrugs.
As evidenced in Figure 5 most of the prodrugs accumulated to a
major extent (10- to 15-fold) when compared to PEA, and
some of them (derivative 6 and 7, in particular) were detected
in relatively high amount also 5 days after removal of patches.
Only derivative 11 showed a skin accumulation pattern
comparable to the parent drug. Cutaneous accumulation for
derivative 10 was undeterminable since the molecule is
unsuitable for precolumn derivatization.
The amount of PEA released from each synthesized prodrug
was assessed in the same time interval. Results obtained are
shown in Table 3. Synthesized prodrugs can be roughly divided
into three different groups.
Compounds 7, 8 and 11 released a really low amount of PEA
during the entire time-course. PEA skin accumulation
determined by prodrug 9 was significantly higher than the
parent drug only 6 h after topical administration. However, a
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Figure 6. Effect of local application of vehicle (CTR), PEA, and prodrugs 5, 6, 9, and 10 on paw edema (A,C,E) and mechanical hyperalgesia
(B,D,F). Data represents mean SEM of six mice. *p < 0.05, **p < 0.01, and ***p < 0.001 vs CTR.
remarkable skin accumulation of PEA was obtained from
derivatives 5, 6, and 10. Inside this group, prodrugs 5 and 10
showed comparable release profiles, while prodrug 6
determined a prolonged PEA release with higher accumulation
rates 48 and 96 h following application (p < 0.05 vs prodrugs 5
and 10)
Pharmacological Studies. As expected, carrageenan
injection into the mice paw produced both significant
hyperalgesia as determined by a reduction in withdrawal
latencies (s) following mechanical stimulation (Figure 6B,D,F,
black bars) and evident paw edema as determined by an
increase in paw volume (Figure 6A,C,E, black circles). PEA
treatment (1 mg/paw) markedly reduced mechanical hyper-
algesia and paw edema in a time-dependent manner, as shown
by the increase in paw withdrawal latency and by the reduction
of paw volume (Figure 6A,B). In particular, antihyperalgesic
and anti-inflammatory effects were observed at 2 and 4 h
following application, whereas no effect was later detectable. In
accordance with skin accumulation experiments, derivatives 7,
8, and 11 did not demonstrate any pharmacological effect (data
not shown). Derivative 9 (Figure 6C,D) showed moderate and
short-term effects. Hyperalgesia and paw volume were both
reduced 4 h after applications, but pharmacological activity fell
during 24 h. In contrast, derivatives 5, 6, and 10 (Figure 6E,F)
demonstrated delayed but prolonged antihyperalgesic and anti-
inflammatory effects. In particular, derivatives 5 and 10 showed
significant antihyperalgesic and anti-inflammatory effects
between 6 and 24 h following topical application. Derivative
6 further delayed the pharmacological effect to 48 h after
application, maintaining efficacy until day 5. Finally, no swelling
effect on the paw was observed when the topical application of
prodrugs was not followed by carrageenan injection (negative
control, data not shown). Aiming to obtain a formulation
characterized by early and prolonged efficacy, we tested a
mixture (MIX) composed of PEA, 5, and 6, in the same
experimental conditions. As expected, the mixture showed a
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Figure 7. Effect of local application of vehicle (CTR) and PEA + prodrug 5 + prodrug 6 (MIX) on mechanical paw edema (A) and mechanical
hyperalgesia (B). Data represents mean SEM of six mice. *p < 0.05, **p < 0.01 and vs CTR. See experimental section for methods used.
rapid onset of action along with prolonged efficacy (Figure 7),
thus dramatically improving the pharmacological profile of the
parent drug.
coefficient values, which are very similar among library
members (Table 2). It can be postulated, for derivatives 8
Table 2. Correlation between PEA Skin Accumulation at 6 h
and Some Relevant Descriptors Including Docking Scores
DISCUSSION
■
[PEA]
6 h
ChemPlp
(kcal/mol)
M.W.
(g/mol)
PEA, an endogenous PPAR-α agonist, is anticipated to become,
like the adelmidrol (azealic acid diethanolamide), a key tool of
efficacious fatty acid derivatives for topical treatment of
inflamed skin.15−17 Recently, several studies have pointed out
that PPAR-α agonist actively reduces skin lesion,12,14,36 and
some of them have already been delivered to the clinical
scenario, both for human or veterinary use.16−18,37 PEA is one
of the most attractive PPAR-α agonists, representing a naturally
occurring anti-inflammatory molecule with wide ranging
applications in prevention and treatment of skin pathologies
characterized by lack of local and systemic adverse side effects,
especially in comparison with standard steroidal anti-inflam-
matory therapies.12,16,17,36 Despite these favorable properties,
the use of topically administered PEA is strongly limited by its
disadvantageous pharmacokinetic properties, leading to short-
term effects, as also evidenced in the present work. Both anti-
inflammatory and antihyperalgesic effects of PEA rapidly
disappear after topical application in the murine model of
skin inflammation herein used (Figure 6A,B). This short-term
effect can be justified by PEA inactivation by two intracellular
lipid amidases: N-acylethanolamine acid amidase (NAAA)38
and fatty acid amide hydrolase (FAAH).39 Recently, Sasso et
al.40 showed that a potent and selective NAAA inhibitor
protects endogenous FAEs from NAAA-catalyzed degradation
in inflamed tissues following local administration. Moreover,
simultaneous topical administration of PEA and FAAH
inhibitors is capable of boosting the effects of the fatty acid
ethanolamide.41
derivative
logP_MLP
5
0.274
0.073
0.093
0.105
0.301
0.324
−92.64
−89.75
−68.75
−75.58
−101.37
−97.23
0.79
487.67
531.72
575.78
535.71
485.65
501.67
−0.84
6.18
5.57
5.99
6.16
6.13
6.94
0.73
6
7
8
9
10
correlation (r)
and 9, that molecular constraint, introduced by a malonate or
phthalate linker negatively influence skin diffusion, thus limiting
tissue accumulation. Molecular rigidity has been previously
described as a penalizing factor for skin permeation.42,43
However, while a carbonate linker has been widely described
and profitably used in prodrug design, derivative 11 showed an
unfavorable accumulation profile (Figure 5) and lacked
pharmacological effects. This prodrug proved to be very
susceptible to chemical hydrolysis (Figure 3), probably leading
to a fast and uncontrolled release of PEA, prior to reaching
suitable concentrations in the viable epidermis. As reported by
Vlieghe et al.,44 carbonates are susceptible to molecular
rearrangements in the presence of a hydroxyalkyl side chain.
As expected, the size of the polyethylene glycol promoieties
influences the pharmacokinetic profile and pharmacological
activity of synthesized prodrugs.21 An increase in the number of
ethylene glycol units leads to improved skin accumulation
(Figure 5). Tissue concentrations found for prodrug 7 are
significantly higher in comparison with derivatives 6 and 5. The
increase in molecular weight is compensated for by a more
favorable partition coefficient. Despite this suitable accumu-
lation profile, derivative 7 is totally inactive, while its lower
homologues showed a substantial pharmacological activity with
delayed and prolonged anti-inflammatory and antihyperalgesic
effects (Figure 6C−F). These results can be rationalized by
molecular modeling results, which highlighted how the catalytic
Ser228 residue of the esterase approaches the carbonyl carbon
atom of the ester groups closest to the amide function (Figure
Hence, the objective of the present work was to synthesize
pegylated PEA prodrugs for a sustained topical accumulation
and delivery as well as prolonged activity. Synthesized prodrugs
showed, for the most part, an increased accumulation in skin if
compared with the parent molecule but were discriminated by
their ability to release PEA and exert pharmacological
properties. The succinate spacer proved to be the most
suitable. In fact, derivative 5 accumulates to a major extent if
compared with its homologues (8, 9, and 11). These results
cannot be solely rationalized by molecular weight and partition
H
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a
Table 3. Comparison between PEA Skin Accumulation and Release from Synthesized Prodrugs
time course
compound
PEA
[ ] 2 h
[ ] 4 h
[ ] 6 h
[ ] 24 h
[ ] 48 h
[ ] 72 h
[ ] 96 h
0.169 0.025
0.071 0.011
0.048 0.027#
nd
0.012 0.009#
0.115 0.041#
0.062 0.029#
0.018 0.010#
0.173 0.022
0.111 0.016#
0.076 0.012#
nd
0.054 0.014#
0.235 0.029
0.120 0.012#
0.041 0.004#
0.218 0.041
0.274 0.034*
0.073 0.054
0.093 0.044#
0.105 0.025#
0.301 0.047*
0.324 0.043*
0.074 0.006#
0.093 0.011
0.212 0.050**
0.312 0.077**
0.083 0.060
0.088 0.030
0.106 0.031
0.245 0.067**
0.033 0.003#
nd
nd
nd
PEA from 5
PEA from 6
PEA from 7
PEA from 8
PEA from 9
PEA from 10
PEA from 11
0.174 0.036***
0.343 0.046***
0.034 0.006*
0.027 0.016
0.139 0.066***
0.268 0.046***
0.028 0.005*
0.051 0.023*
0.141 0.036**
0.023 0.016
nd
nd
0.009 0.003*
0.216 0.045***
0.014 0.007
nd
nd
0.147 0.042***
0.062 0.025*
nd
nd
a
Time course concentration is expressed in nanomoles of PEA per milligram of tissue SEM (n = 5) at different times. When outside the linearity
range of the analytical method used, concentrations were considered not determinable (nd). *p < 0.05, **p < 0.01, and ***p < 0.001 accumulation
#
values significantly higher than PEA. p < 0.05 accumulation values significantly lower than PEA.
4). Thus, an increase in side chain size is responsible for a
slower hydrolysis of the ester bond (Table 2). For these
reasons, despite the higher skin concentrations reached,
derivative 7 is unable to release biologically active amounts of
PEA (Table 3). Contrariwise, derivative 5 and 6 act as depot
prodrugs for sustained release, prolonging the therapeutic
efficacy of PEA after topical administration. In fact, a mixture of
compounds 5 and 6 and PEA, at equimolar dose, resulted in a
fast outbreaking and long-lasting anti-inflammatory topical
formulation, as depicted in Figure 7. Finally, the evidence
shows that derivative 10 maintains the same advantageous
pharmacological profile as prodrug 5. Taken together our data
showed (i) that prodrugs have an efficacy comparable to local
or systemic administration of standard anti-inflammatory drugs
(i.e., glucocorticoids);45,46 and (ii) that local pretreatment with
PEA and its prodrugs maintain cellular homeostasis by acting as
a mediator of resolution of inflammatory processes and by
modulating the behavior of mast cells and microglia.47
Synthesis of prodrugs: D.T. and C.O. Acquisition of data: R.R.,
C.D.C., C.A., and G.L.R. (in vivo experiments and skin
accumulation experiments); D.T. and C.O. (skin accumulation
experiments and chemical stability); A.S. (NMR and mass
spectrometry); S.L. (HPLC analysis); A.M. and G.V. (computa-
tional data). Drafting of manuscript: D.T., R.R., C.O., G.V., and
A.C. Critical revision: A.C. and F.D.V. All authors have given
approval to the final version of the manuscript.
Notes
The authors declare no competing financial interest.
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CONCLUSIONS
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In this work, we investigated a prodrug approach for topical
delivery of PEA as anti-inflammatory drug. The chemical
combination of the parent drug with specific polyethylene
glycol moieties, using the proper spacer, proved to be a viable
approach to increase skin accumulation and overcome
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AUTHOR INFORMATION
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Corresponding Author
(8) Westergaard, M.; Henningsen, J.; Svendsen, M. L.; Johansen, C.;
Jensen, U. B.; Schroder, H. D.; Kratchmarova, I.; Berge, R. K.; Iversen,
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*Address: Department of Pharmacy, University of Naples
Federico II, Via D. Montesano 49, 80131 Naples, Italy. Tel/
Fax: 0039 081 678609. E-mail: ostacolo@unina.it.
Author Contributions
∥These authors contributed equally to this work. Study
conception and design: D.T., R.R., C.O., G.V., and A.C.
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I
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