Effects of novel hybrids of caffeic acid phenethyl ester and NSAIDs on experimental ocular inflammation

Article abstract of DOI:10.1016/j.ejphar.2015.02.012

In this study, we report the design and synthesis of novel hybrids of caffeic acid phenetyl ester (CAPE) and non-steroidal anti-inflammatory drugs (NSAIDs). We assessed their effects on an experimental ocular inflammation in New Zealand rabbits. The formulations of CAPE-aspirin and CAPE-indomethacin hybrids were topical instilled in the rabbit's eye. Afterwards, the anti-inflammatory activity was evaluated by grading the clinical signs and by assessing the inflammatory cell count, protein, PGE₂ and TNFα levels in the aqueous humor. Furthermore, ocular tolerability of hybrids formulations was evaluated in a separate set of animals by using a modified Draize test. The ocular inflammation in the control group was significantly higher than in both the hybrid-treated groups, as indicated by clinical grading and biomarkers assessment. However, only the CAPE-aspirin hybrid reduced, in a significant dose-dependent manner, the ocular inflammation elicited by paracentesis. CAPE-indomethacin hybrid was able to significantly attenuate the clinical grading and the PGE₂ aqueous levels only at the highest dose (0.1%). CAPE-aspirin significantly reduced PGE₂ and TNFα levels in the aqueous humor as well as proteins and PMNs. Finally, all formulations showed no ocular irritation compared with vehicle-treated group. In conclusion, CAPE-aspirin shows full anti-inflammatory efficacy in experimental model of ocular inflammation demonstrating an optimal pharmacological and safety profile. Taken together these data indicate that CAPE-aspirin hybrid represents a valid and safe new chemical entity potentially useful for the treatment of ocular inflammation.

Full text of DOI:10.1016/j.ejphar.2015.02.012

European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Contents lists available at ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar
Immunopharmacology and inflammation
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Effects of novel hybrids of caffeic acid phenethyl ester and NSAIDs
on experimental ocular inflammation
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Valeria Pittalà , Loredana Salerno , Giuseppe Romeo , Maria Angela Siracusa ,
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Maria Nunziata Modica , Giovanni Luca Romano , Salvatore Salomone , Filippo Drago ,
Q1 Claudio Bucolo
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Department of Drug Sciences, Section of Medicinal Chemistry, School of Medicine, University of Catania, viale A. Doria 6, 95125 Catania, Italy
Department of Biomedical and Biotechnological Sciences, Section of Pharmacology and Biochemistry, School of Medicine, University of Catania,
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Article history:
Received 25 September 2014
Received in revised form
In this study, we report the design and synthesis of novel hybrids of caffeic acid phenetyl ester (CAPE)
and non-steroidal anti-inflammatory drugs (NSAIDs). We assessed their effects on an experimental
ocular inflammation in New Zealand rabbits. The formulations of CAPE–aspirin and CAPE–indomethacin
hybrids were topical instilled in the rabbit's eye. Afterwards, the anti-inflammatory activity was
9
February 2015
Accepted 11 February 2015
evaluated by grading the clinical signs and by assessing the inflammatory cell count, protein, PGE
2
and TNFα levels in the aqueous humor. Furthermore, ocular tolerability of hybrids formulations was
evaluated in a separate set of animals by using a modified Draize test. The ocular inflammation in the
control group was significantly higher than in both the hybrid-treated groups, as indicated by clinical
grading and biomarkers assessment. However, only the CAPE–aspirin hybrid reduced, in a significant
dose-dependent manner, the ocular inflammation elicited by paracentesis. CAPE–indomethacin hybrid
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Keywords:
Caffeic acid phenethyl ester
Aspirin
Indomethacin
Heme oxygenase-1 inducer
Inflammation
Eye
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was able to significantly attenuate the clinical grading and the PGE
dose (0.1%). CAPE–aspirin significantly reduced PGE and TNFα levels in the aqueous humor as well as
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aqueous levels only at the highest
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proteins and PMNs. Finally, all formulations showed no ocular irritation compared with vehicle-treated
group. In conclusion, CAPE–aspirin shows full anti-inflammatory efficacy in experimental model of
ocular inflammation demonstrating an optimal pharmacological and safety profile. Taken together these
data indicate that CAPE–aspirin hybrid represents a valid and safe new chemical entity potentially useful
for the treatment of ocular inflammation.
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2015 Published by Elsevier B.V.
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. Introduction
post-translational phosphorylation level of extracellular signal-
regulated kinase (ERK) (Kim and Jang, 2014). Heme oxygenases are
a family of ubiquitously expressed enzymes which regulate the
catabolism of heme leading to the formation of equimolar amounts
of carbon monoxide (CO), ferrous ion, and biliverdin, which in turn is
reduced to bilirubin (Abraham and Kappas, 2008). Because HO-1
induction has been shown to exert potent anti-inflammatory and
anti-apoptotic activities, this mechanisms may be exploited as a
potential therapeutic approach in inflammatory, cardiovascular, cere-
brovascular, and neurodegenerative diseases and, more recently, in
the treatment of ocular diseases (Bucolo and Drago, 2009; Pittala
et al., 2013; Salerno et al., 2013; Sorrenti et al., 2012).
HO-1 induction can be achieved following exposure to envir-
onmental factors or oxidative stress, but it may also be simulated
by a number of natural compounds, such as curcumin and CAPE
that seems to be among the most potent ones (Foresti et al., 2013;
Scapagnini et al., 2002). One major limitation of application of
curcumin is due to low bioavailability and stability; therefore, we
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Caffeic acid phenethyl ester (CAPE) is a phenolic active component
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of propolis derived from the bark of conifer trees and carried by
honeybees to their hives. Propolis is used safely in traditional
medicine as a dietary supplement for its therapeutic benefits. CAPE
possesses a variety of pharmacological activities including anti-
inflammatory (Michaluart et al., 1999; Natarajan et al., 1996; Toyoda
et al., 2009), antioxidant (Chen et al., 2001), anti-cancer effects
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(Frenkel et al., 1993; Huang et al., 1996), and immunomodulating
effect (Park et al., 2004). CAPE has been shown to stimulate heme
oxygenase-1 (HO-1) through enhancement of the nuclear transcrip-
tion factor-erythroid 2-related factor 2 (Nrf2) in the nucleus and the
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Correspondence to: Department of Clinical and Molecular Biomedicine, Section
of Pharmacology and Biochemistry, School of Medicine, University of Catania V.le A.
Doria 6 95125, Catania, Italy. Tel.: þ39 095 7384088; fax: þ39 095 7384236.
E-mail address: claudio.bucolo@unict.it (C. Bucolo).
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http://dx.doi.org/10.1016/j.ejphar.2015.02.012
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Please cite this article as: Pittalà, V., et al., Effects of novel hybrids of caffeic acid phenethyl ester and NSAIDs on experimental
ocular inflammation. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.02.012i

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focused our attention to CAPE that shows a better stability profile
(Anand et al., 2007; Celli et al., 2007).
7 ml of anhydrous tetrahydrofurane (THF) was added drop-wise to a
solution of 2-(acetyloxy)benzoyl chloride (Sigma Aldrich, Italy;
769 mg, 3.87 mmol) in anhydrous THF (2 ml) and the mixture was
stirred under nitrogen for 4 h at 22 1C. After removing the THF, the
mixture was dissolved in ethyl acetate. The obtained organic phase
was washed with water (2 ꢀ 75 ml) followed by saturated sodium
chloride solution, dried over sodium sulfate, filtered, and evaporated.
The isolated crude material was purified by column chromatography
(silica gel 60, 230–400 mesh, Merck) using ethyl acetate/cyclohexane
(3:7) as eluant. The title compound was obtained as a colorless sticky
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Nonsteroidal anti-inflammatory drugs (NSAIDs) are recognized
as the prototypical drugs in the treatment of inflammations and
ocular inflammatory diseases even if the class has several draw-
backs. In particular, indomethacin is commercially available in
Europe as topical ophthalmic preparations, and is indicated for
inflammatory ocular conditions. Also, aspirin has been explored in
experimental ocular inflammatory conditions (Joussen et al., 2002)
with very encouraging results. We therefore assumed that new
hybrids incorporating an active NSAID, such as aspirin or indo-
methacin, and a potent HO-1 inducer, such as CAPE, could
represent compounds with high anti-inflammatory potency. The
design and synthesis of hybrid drugs has been recently imple-
mented in order to improve the efficiency of using a two-drug
cocktail. The strategy, generally, involves a covalent link between
two existing drugs or pharmacophores to create a single molecule
endowed with multiple and/or simultaneous and/or synergic
pharmacological actions (Corson et al., 2008). This approach
should provide new drugs endowed with lower dosing, simpler
regimens, minimization of side effects, and patient compliance
improvement. In the present study we describe the synthesis of
CAPE–aspirin (VP939) and CAPE–indomethacin (VP964) hybrids
(Fig. 1) and assess their ocular pharmacological profile. Two drugs,
CAPE and the respective NSAIDs, are linked directly, without a
linker or a spacer through, forming an ester function that could be
easily cleaved by ocular esterases.
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ꢁ
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oil (861 mg, 81%): IR (KBr) cm 3055, 2987, 1752, 1717, 1607, 1265,
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1243, 1193, 1112, 1037, and 739; H NMR (DMSO-d
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) δ 8.07ꢁ7.90 (m,
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3H, aromatic), 7.83ꢁ7.64 (m, 3H þ1H, aromaticþCHQCHCOO),
7.53 ꢁ7.45 (m, 1H, aromatic), 7.42ꢁ7.18 (m, 9H, aromatic), 6.71 (d, J¼
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16.0, 1H, CHQCHCOO), 4.38 (t, J¼6.8 Hz, 2H, OCH
2
CH
2
Ph), 2.98 (t, J¼
).
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6.8 Hz, 2H, OCH
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CH
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Ph), 2.21 (s, 3H, OCOCH
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), 2.20 (s, 3H, OCOCH
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Anal. (C35 10) C, H.
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2.1.2. Synthesis of 3-[3,4-bis[2-[1-(4-chlorobenzoyl)-5-methoxy-2-
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methyl-1H-indol-3-yl]acetyloxy]phenyl]-(2E)-2-propenoic acid 2-
phenylethyl ester (CAPE–indomethacin, VP964)
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Fig. 3 shows the synthesis of VP964. A solution of thionyl
chloride (0.203 ml, 2.80 mmol) in 1.5 ml of anhydrous toluene was
slowly added to a solution of 1-(4-chlorobenzoyl)-5-methoxy-2-
methyl-1H-indole-3-acetic acid (indomethacin, Sigma Aldrich,
Italy, 500 mg, 1.40 mmol) in 6 ml of anhydrous toluene cooled at
0
1C and under nitrogen atmosphere. The mixture was then stirred
under nitrogen, for 4 h, at reflux. After this period, toluene was
removed and the obtained mixture was used in the subsequent
step without any further purification.
A solution of CAPE (180 mg, 0.635 mmol) and N,N-diisopropy-
lethylamine (0.44 ml, 2.54 mmol) in 6 ml of anhydrous THF was
added drop-wise to crude 1-(4-chlorobenzoyl)-5-methoxy-2-
methyl-1H-indole-3-acetyl chloride dissolved in anhydrous THF
2. Materials and methods
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2.1. Chemistry, drugs, chemicals and reagents
Melting points were determined in a Electrothermal IA9200
apparatus with a digital thermometer in glass capillary tubes.
Infrared spectra were recorded on a Perkin–Elmer FT IR 1600
spectrometer in KBr disks. Elemental analyses for C, H, and N were
within70.4% of theoretical values and were performed on a Carlo
Erba Elemental Analyzer Mod. 1108 apparatus. 1H NMR spectra
were recorded at 200 MHz on a Varian Inova Unity 200 spectro-
(2 ml) and the mixture was stirred under nitrogen for 4 h at 22 1C.
After removing the THF, the mixture was dissolved in ethyl
acetate. The obtained organic phase was washed with water
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2 ꢀ 75 ml) followed by saturated sodium chloride solution, dried
over sodium sulfate, filtered, and evaporated. The isolated crude
material was purified by column chromatography (silica gel 60,
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meter in DMSO-d solution. Chemical shifts are given in δ values
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(ppm), using tetramethylsilane as the internal standard; coupling
constants (J) are given in hertz (Hz). Signal multiplicities are
characterized as s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), br (broad signal). All the synthesized compounds were
tested for purity on TLC (aluminum sheet coated with silica gel
F254, Merck) and visualized by UV (λ¼254 and 366 nm).
230–400 mesh, Merck) using ethyl acetate/cyclohexane (3:7) as
eluant. The title compound was obtained as white solid (449 mg,
ꢁ1
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4%): mp 80–81 1C; IR (KBr) cm
3424, 2933, 1769, 1685, 1594,
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478, 1359, 1319, 1242, 1112, 835, and 752; H NMR (DMSO-d ) δ
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.74 ꢁ7.55 (s, 10Hþ1H, aromaticþCHQCHCOO), 7.37ꢁ7.16 (m,
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aromatic), 6.77ꢁ6.71 (m, 2H, aromatic), 6.61 (d, J¼16.0, 1H, CHQ
CHCOO), 4.36 (t, J¼6.8 Hz, 2H, OCH CH Ph), 3.92 (s, 2Hþ2H,
CH COO), 3.70 (s, 3Hþ3H, OCH þOCH ), 2.96 (t, J¼
COOþCH
6.8 Hz, 2H, OCH CH Ph), 2.20 (s, 3H, CH ). 2.17 (s, 3H, CH ). Anal.
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.1.1. Synthesis of 3-[3,4-bis[2-(acetyloxy)benzoyloxy]phenyl]-(2E)-
-propenoic acid 2-phenylethyl ester (CAPE–aspirin, VP939)
Fig. 2 shows the synthesis of VP939. A solution of CAPE (500 mg,
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1.76 mmol) and N,N-diisopropylethylamine (0.67 ml, 3.87 mmol) in
(C55H44Cl
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O
Cl
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VP964
VP939
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Cl
N
O
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Fig. 1. Chemical structure of the synthesized caffeic acid phenetyl ester (CAPE)–aspirin (VP939) and CAPE–indomethacin (VP964) hybrids.
Please cite this article as: Pittalà, V., et al., Effects of novel hybrids of caffeic acid phenethyl ester and NSAIDs on experimental
ocular inflammation. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.02.012i

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O
O
O
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THF, DIEA, N2
O
HO
HO
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O
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Cl
VP939
O
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Fig. 2. Synthesis of caffeic acid phenetyl ester (CAPE)–aspirin (VP939) hybrid.
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O
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O
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N
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HO
SOCl2, Toluene, N2
Cl
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CAPE, THF, DIEA, N2
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Cl
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VP964
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Fig. 3. Synthesis of caffeic acid phenetyl ester (CAPE)–indomethacin (VP964) hybrid.
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2.2. Animals
performed with a 26-gauge needle attached to a tuberculin syringe;
the needle was introduced into the anterior chamber close to the
limbus, with care taken not to damage the tissues, and 50 μl
aqueous humor was removed and discarded. After 2 h from the
first paracentesis, a second paracentesis was performed to collect
the aqueous humor for biochemical evaluation. The animals were
then euthanatized with 0.4 ml euthanatizing agent (Tanax; Intervet,
Milan, Italy). Aqueous humor samples were collected and split into
four aliquots, which were stored at ꢁ80 1C until analysis. Then both
eyes were enucleated and the iris-ciliary body was carefully excised,
placed in polypropylene tubes, and stored at ꢁ80 1C until analysis.
The clinical evaluation of both eyes was performed with a slit lamp
(4179-T; Sbisà, Firenze, Italy) at 180 min and 5 min before the
paracentesis and 115 min after the paracentesis. Clinical signs were
graded according to the following scheme: 0¼normal, 1¼discrete
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Male New Zealand albino rabbits (1.8–2.0 kg) were obtained
from Harlan (Italy). Animals were housed under standard condi-
tions, with food and water provided ad libitum, in a light-
controlled (12-h light/12-h dark; lights on at 6 am) room at
2173 1C and 5474% humidity. All animals were handled in
accordance with the ARVO Statement for the Use of Animals in
Ophthalmic and Vision Research.
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2.3. Ocular inflammation model
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Rabbits were randomly assigned to six groups (four animals per
group). Fresh aqueous formulation of VP939 and VP964 (0.01 and
0.1% w/v) containing 0.05% polysorbate 80, were prepared in PBS
(pH 7.2). We also prepared three formulations containing CAPE,
aspirin and indomethacin, respectively. We used one dose (0.1%)
for two main reasons: first of all to save animals; second of all to
quickly compare the three single molecules on ocular inflamma-
tion model, and to evaluate the pharmacological profile of each
molecule versus the hybrids.
Each formulation was instilled (25 μl) into the conjunctival sac of
each eye 180, 120, 90, and 30 min before paracentesis and 15, 30,
and 90 min after the intervention. To perform the paracentesis,
animals were anesthetized by intravenous injection of 5 mg/kg
agent (Zoletil, Virbac, Italy; 2.5 mg/kg tiletamine HCl, and 2.5 mg/kg
zolazepam HCl) and 1 drop of local anesthetic (Novesina; Novartis,
Italy) administered to the eye. Anterior chamber paracentesis was
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dilatation of iris and conjunctival vessels; 2¼moderate dilatation of 120
iris and conjunctival vessels; 3¼intense conjunctival and iridal
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hyperemia with flare in the anterior chamber; 4¼intense iridal 122
hyperemia with flare in the anterior chamber and presence of 123
fibrinous exudates. All measurements, under the same environ- 124
mental conditions, were made by the same operator blind to 125
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treatment.
Levels of PGE
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and TNFα in the aqueous humor were estimated
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with commercial ELISA kits (BD Biosciences, USA), and aqueous
humor protein concentrations were measured (Protein Quantification
Kit; Fluka, Milan, Italy). Polymorphonuclear leukocytes (PMNs) were 130
quantified in aqueous humor using a hemocytometer. The PMN count
was expressed as number of cells per microliter of aqueous humor.
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Please cite this article as: Pittalà, V., et al., Effects of novel hybrids of caffeic acid phenethyl ester and NSAIDs on experimental
ocular inflammation. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.02.012i

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2.4. Ocular tolerability
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The potential ocular irritancy and/or damaging effects of the two
formulations were evaluated according to a modified Draize test
(Giannavola et al., 2003) in a separate set of animals (4 animals/
group). A slit lamp (mod. 4179 T Sbisà, Florence, Italy) was used. The
congestion, swelling, and discharge of the conjunctiva were graded
on a scale from 0 to 3, 0 to 4, and 0 to 3, respectively. Iris hyperemia
and corneal opacity were graded on a scale from 0 to 4. Formula-
tions (25 μl) were topically administered in the right eye every
30 min for 6 h (12 treatments). At the end of the treatment, two
observations at 10 min and 6 h were carried out to evaluate the
ocular tissues. Observations were made by two independent
observers in a masked way. Methylene blue staining was used to
evaluate the corneal integrity, which allows an accurate determina-
tion of the extent of epithelial damage because of its poor diffusion
through the stromal layer (Nagelhout et al., 2005).
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Fig. 4. Clinical signs of inflammation in rabbit eyes that underwent to paracentesis.
The clinical evaluation of eye was performed 110 min after the first paracentesis.
Po0.05 vs. vehicle; Mann–Whiteny U test. Data shown are expressed as the
mean7S.D. (n¼8).
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2.5. Statistical analysis
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The software GraphPad (version 6; San Diego, CA, USA) was
used to carry out statistical analysis of results. One-way analysis of
variance (ANOVA) of data was done. Thereafter Newman–Keuls or
Dunnett post hoc test were carried out. Values are expressed as
mean7S.D. Mann–Whiteny U test was used for ordinal data. Data
were considered statistically significant at P values o0.05.
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inflammation were also evaluated. Aspirin and indomethacin were
similar to protect the eye against the inflammation elicited by
paracentesis (Fig. 5). CAPE was able to reduce significantly the
TNFα levels in the aqueous compared to vehicle group.
3
.3. Ocular tolerability of VP939 and VP964
3. Results
Both formulations based either on VP939 or VP964 with PBS
and 0.05% polysorbate 80 did not cause significant signs of ocular
inflammation or tissue alteration in the rabbit eye compared with
the vehicle-treated group. The scores of conjunctival congestion,
swelling, and discharge are showed in Table 1. Iris hyperemia and
corneal opacity scores were zero at all observations.
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3.1. Chemical synthesis of hybrids CAPE–aspirin (VP939) and CAPE–
indomethacin (VP964)
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Hybrid molecules were obtained in high yield by using an
efficient synthetic route (Figs. 2 and 3). Indomethacin was first
converted, in a quantitative yield, to the correspondent acyl
chloride by the reaction with thionyl chloride. Subsequently, the
obtained indomethacin-acyl chloride or 2-(acetyloxy)benzoyl
chloride was coupled with CAPE. This synthetic approach gave
cleaner reaction mixtures when compared with the direct cou-
pling of the free acids with CAPE in the presence of condensing
agents belonging to the carbodiimides family. The desired final
products, VP939 and VP964, were obtained by simple purification
by flash chromatography.
4. Discussion
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In the present study, we described the synthesis of novel hybrids 105
of CAPE, a potent HO-1 inducers, and NSAIDs such aspirin or
indomethacin. We evaluated the anti-inflammatory properties of
hybrids in a paracentesis-induced inflammation model of rabbit eye.
The rabbit paracentesis model is a relevant system that mimics
ocular trauma induced during surgery, such as cataract operation, in
the anterior segment eliciting an inflammation process representing
a good platform for screening anti-inflammatory agents (Shafiee
et al., 2011; Pignatello et al., 2002a, 2002b). Treatment with CAPE–
aspirin (VP939) improved clinical anti-inflammatory outcomes in
the paracentesis model. In addition, more objective measures of
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3.2. Effects of hybrids on ocular inflammation
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Ocular inflammation elicited by paracentesis caused a remark-
able damage of eye's tissues (Fig. 4).
Both doses (0.01% and 0.1%) of CAPE–aspirin hybrid (VP939)
significantly reduced clinical signs of inflammation compared with
the control group with a dose-dependent manner (Fig. 4), whereas
CAPE–indomethacin (VP964) hybrid did not show significant
effects at both doses (0.01% and 0.1%) (Fig. 4).
Furthermore, VP939, both doses, significantly (po0.05 vs.
vehicle-treated group) reduced aqueous humor concentrations of
TNFα (3907101, 230739 pg/ml at 0.01% and 0.1% respectively;
2
inflammation, which include TNFα, PGE , protein and infiltration of
5
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inflammatory cells in aqueous humor, were also favorably affected
by topical treatment with VP939. The CAPE–indomethacin hybrid
(VP964) was ineffective in reducing levels of TNFα, PMNs and
protein levels.
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CAPE–NSAIDs hybrids are conjugates of well-known and effi-
cacious drugs, which are privileged drug-like structures. However, 122
in the present study, only the CAPE–aspirin hybrid reduced in a
significant dose-dependent manner the ocular inflammation eli-
cited by paracentesis; in fact, CAPE–indomethacin hybrid was able
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Fig. 5 Panel A) and PGE (3.1071.0, 2.2570.75 pg/ml at 0.01% and
0.1% respectively; Fig. 5 Panel B) as well as PMNs number
(6.570.7, 4.2571.0 number/μl at 0.01% and 0.1% respectively;
Fig. 5 Panel C) and protein levels (44.575.50, 31.277.25 mg/ml at
0.01% and 0.1% respectively; Fig. 5 Panel D) compared with
vehicle-treated group. The VP964 hybrid did not show any
significant effect on the assessed biomarkers assessed, except for
to significantly attenuate the clinical grading and the PGE
eous levels only at the highest dose (0.1%). Even though both
hybrids are esters, easily hydrolysable by ocular esterases, different 128
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hydrolysis rate/speed, high molecular mass, high lipophilicity, and
permeability can considerably affect their pharmacokinetic profile.
Consequently, after topical administration, pharmacological prop-
erties of each CAPE–NSAIDs hybrid could result in different levels
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PGE levels at the highest dose (3.7570.5 pg/ml; Fig. 5 Panel B).
The single effects of CAPE, aspirin and indomethacin on ocular
Please cite this article as: Pittalà, V., et al., Effects of novel hybrids of caffeic acid phenethyl ester and NSAIDs on experimental
ocular inflammation. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.02.012i

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Ocular Inflammation
Ocular Inflammation
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Fig. 5. Aqueous levels of TNFα (A), PGE (B), PMN (C), and protein (D) 2 h after the second paracentesis with or without (vehicle) CAPE hybrids treatment (0.01% and 0.1%).
n
Data shown are expressed as the mean7S.D. Po0.05 vs. vehicle; n¼8.
Table 1
activation, probably via IκB (IKK) kinase inhibition, and Nrf2
activation. Nrf2 is a master regulator of the expression of a subset
of genes that code for proteins responsible for the detoxification of
electrophiles and reactive oxygen species as well as for the
removal or repair of some of their damage products.
Formulations were topically administered in the right eye every 30 min for 6 h. At
the end of the treatment, two observations at 10 min and 6 h were carried out to
evaluate the ocular tolerability (n¼4).
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Formulation
Conjunctiva
Iris
Corneal
hyperemia opacity
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In summary, our data demonstrate that VP939 is an effective
anti-inflammatory agent when tested in experimental parac-
Congestion Swelling Discharge
entesis-induced inflammation models in rabbits. The data also 100
indicate that VP964 is at least as efficacious as VP939 in terms of
PGE at 0.1% in a model of paracentesis-induced inflammation in
Control (10 min)
Control (6 h)
Vehicle (10 min)
Vehicle (6 h)
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rabbits. Altogether, these data suggest that CAPE–NSAID hybrids,
and in particular VP939, may represent the prototype of a new
class of drugs that may be useful in the treatment of ocular
inflammation in which prostaglandins and TNFα play a key role.
Clinical studies to evaluate this possibility may be warranted.
0
.01% VP939
10 min)
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(
0.01% VP939
(6 h)
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0.01% VP964
10 min)
.01% VP964
6 h)
(
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(
Acknowledgments
.1% VP939
(10 min)
0.1% VP939 (6 h)
0
1
0
0
0
0
0
0
0
0
The authors are grateful to the Italian M. I. U. R. for the financial
support. This research was supported in part by a National grant
PON01-00110.
0
.1% VP964
10 min)
.1% VP964 (6 h)
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(
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related with the physic-chemical features, is less potent than
indomethacin alone.
Anti-inflammatory properties of CAPE were possibly due to its
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ocular inflammation. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.02.012i