European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Immunopharmacology and inflammation

Effects of novel hybrids of caffeic acid phenethyl ester and NSAIDs on experimental ocular inflammation Valeria Pittalà a, Loredana Salerno a, Giuseppe Romeo a, Maria Angela Siracusa a, Maria Nunziata Modica a, Giovanni Luca Romano b, Salvatore Salomone b, Filippo Drago b, Claudio Bucolo b,n a

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, viale A. Doria 6, 95125 Catania, Italy

b

art ic l e i nf o

a b s t r a c t

Article history: Received 25 September 2014 Received in revised form 9 February 2015 Accepted 11 February 2015

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. & 2015 Published by Elsevier B.V.

Keywords: Caffeic acid phenethyl ester Aspirin Indomethacin Heme oxygenase-1 inducer Inflammation Eye

1. Introduction Caffeic acid phenethyl ester (CAPE) is a phenolic active component 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 antiinflammatory (Michaluart et al., 1999; Natarajan et al., 1996; Toyoda et al., 2009), antioxidant (Chen et al., 2001), anti-cancer effects (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 transcription factor-erythroid 2-related factor 2 (Nrf2) in the nucleus and the n 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.: þ 39 095 7384088; fax: þ 39 095 7384236. E-mail address: [email protected] (C. Bucolo).

post-translational phosphorylation level of extracellular signalregulated 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, cerebrovascular, 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 environmental 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

http://dx.doi.org/10.1016/j.ejphar.2015.02.012 0014-2999/& 2015 Published by Elsevier B.V.

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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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 oil (861 mg, 81%): IR (KBr) cm  1 3055, 2987, 1752, 1717, 1607, 1265, 1243, 1193, 1112, 1037, and 739; 1H NMR (DMSO-d6) δ 8.07 7.90 (m, 3H, aromatic), 7.83 7.64 (m, 3Hþ1H, aromaticþCHQCHCOO), 7.53 7.45 (m, 1H, aromatic), 7.42 7.18 (m, 9H, aromatic), 6.71 (d, J¼ 16.0, 1H, CHQCHCOO), 4.38 (t, J¼6.8 Hz, 2H, OCH2CH2Ph), 2.98 (t, J¼ 6.8 Hz, 2H, OCH2CH2Ph), 2.21 (s, 3H, OCOCH3), 2.20 (s, 3H, OCOCH3). Anal. (C35H28O10) C, H.

focused our attention to CAPE that shows a better stability profile (Anand et al., 2007; Celli et al., 2007). 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 drawbacks. 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 indomethacin, 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 implemented 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.

2.1.2. Synthesis of 3-[3,4-bis[2-[1-(4-chlorobenzoyl)-5-methoxy-2methyl-1H-indol-3-yl]acetyloxy]phenyl]-(2E)-2-propenoic acid 2phenylethyl ester (CAPE–indomethacin, VP964) 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-2methyl-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-diisopropylethylamine (0.44 ml, 2.54 mmol) in 6 ml of anhydrous THF was added drop-wise to crude 1-(4-chlorobenzoyl)-5-methoxy-2methyl-1H-indole-3-acetyl chloride dissolved 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 white solid (449 mg, 74%): mp 80–81 1C; IR (KBr) cm  1 3424, 2933, 1769, 1685, 1594, 1478, 1359, 1319, 1242, 1112, 835, and 752; 1H NMR (DMSO-d6) δ 7.74  7.55 (s, 10H þ1H, aromaticþCHQCHCOO), 7.37  7.16 (m, 6H, aromatic), 7.11  7.05 (m, 2H, aromatic), 6.97  6.88 (m, 2H, aromatic), 6.77 6.71 (m, 2H, aromatic), 6.61 (d, J¼ 16.0, 1H, CHQ CHCOO), 4.36 (t, J ¼6.8 Hz, 2H, OCH2CH2Ph), 3.92 (s, 2H þ2H, CH2COOþCH2COO), 3.70 (s, 3H þ3H, OCH3 þOCH3), 2.96 (t, J ¼ 6.8 Hz, 2H, OCH2CH2Ph), 2.20 (s, 3H, CH3). 2.17 (s, 3H, CH3). Anal. (C55H44Cl2N2O10) C, H, N.

2. Materials and methods 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 within 70.4% 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 spectrometer in DMSO-d6 solution. Chemical shifts are given in δ values (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 (λ¼ 254 and 366 nm). 2.1.1. Synthesis of 3-[3,4-bis[2-(acetyloxy)benzoyloxy]phenyl]-(2E)2-propenoic acid 2-phenylethyl ester (CAPE–aspirin, VP939) Fig. 2 shows the synthesis of VP939. A solution of CAPE (500 mg, 1.76 mmol) and N,N-diisopropylethylamine (0.67 ml, 3.87 mmol) in

O

O O O O

O O

O O

O

N

O

Cl

O

O

O O

O VP939

O

O

Cl

N

O

VP964

O

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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V. Pittalà et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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O O O O

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+ O

THF, DIEA, N2

O

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Cl

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VP939 O

Fig. 2. Synthesis of caffeic acid phenetyl ester (CAPE)–aspirin (VP939) hybrid.

O N

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O

O HO OH

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SOCl2, Toluene, N2

Cl

Cl

OH

CAPE, THF, DIEA, N2

O

O

N

O

O

Cl

O

O

O O

Cl

N

O

VP964

O Fig. 3. Synthesis of caffeic acid phenetyl ester (CAPE)–indomethacin (VP964) hybrid.

2.2. Animals Male New Zealand albino rabbits (1.8–2.0 kg) were obtained from Harlan (Italy). Animals were housed under standard conditions, with food and water provided ad libitum, in a lightcontrolled (12-h light/12-h dark; lights on at 6 am) room at 217 3 1C and 5474% humidity. All animals were handled in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 2.3. Ocular inflammation model 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 inflammation 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

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 dilatation of iris and conjunctival vessels; 2¼moderate dilatation of iris and conjunctival vessels; 3¼ intense conjunctival and iridal hyperemia with flare in the anterior chamber; 4¼intense iridal hyperemia with flare in the anterior chamber and presence of fibrinous exudates. All measurements, under the same environmental conditions, were made by the same operator blind to treatment. Levels of PGE2 and TNFα in the aqueous humor were estimated with commercial ELISA kits (BD Biosciences, USA), and aqueous humor protein concentrations were measured (Protein Quantification Kit; Fluka, Milan, Italy). Polymorphonuclear leukocytes (PMNs) were quantified in aqueous humor using a hemocytometer. The PMN count was expressed as number of cells per microliter of aqueous humor.

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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4

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. Formulations (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 determination of the extent of epithelial damage because of its poor diffusion through the stromal layer (Nagelhout et al., 2005). 2.5. Statistical analysis 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 mean 7S.D. Mann–Whiteny U test was used for ordinal data. Data were considered statistically significant at P values o0.05.

Clinical Score

2.4. Ocular tolerability

3 2

*

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0. Veh 01 i % cle VP 0. 9 1% 39 V 0. 01 P9 39 % VP 0. 9 1% 64 VP 0. 1% 964 C A P 0. 1% E A 0. SA 1% IN D

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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. n Po 0.05 vs. vehicle; Mann–Whiteny U test. Data shown are expressed as the mean7 S.D. (n ¼8).

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 3.1. Chemical synthesis of hybrids CAPE–aspirin (VP939) and CAPE– indomethacin (VP964) 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 coupling 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. 3.2. Effects of hybrids on ocular inflammation Ocular inflammation elicited by paracentesis caused a remarkable 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 (p o0.05 vs. vehicle-treated group) reduced aqueous humor concentrations of TNFα (390 7101, 230 739 pg/ml at 0.01% and 0.1% respectively; Fig. 5 Panel A) and PGE2 (3.107 1.0, 2.257 0.75 pg/ml at 0.01% and 0.1% respectively; Fig. 5 Panel B) as well as PMNs number (6.57 0.7, 4.25 71.0 number/μl at 0.01% and 0.1% respectively; Fig. 5 Panel C) and protein levels (44.5 75.50, 31.27 7.25 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 PGE2 levels at the highest dose (3.75 70.5 pg/ml; Fig. 5 Panel B). The single effects of CAPE, aspirin and indomethacin on ocular

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.

4. Discussion In the present study, we described the synthesis of novel hybrids 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 inflammation, which include TNFα, PGE2, protein and infiltration of 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. CAPE–NSAIDs hybrids are conjugates of well-known and efficacious drugs, which are privileged drug-like structures. However, in the present study, only the CAPE–aspirin hybrid reduced in a significant dose-dependent manner the ocular inflammation elicited by paracentesis; in fact, CAPE–indomethacin hybrid was able to significantly attenuate the clinical grading and the PGE2 aqueous levels only at the highest dose (0.1%). Even though both hybrids are esters, easily hydrolysable by ocular esterases, different hydrolysis rate/speed, high molecular mass, high lipophilicity, and permeability can considerably affect their pharmacokinetic profile. Consequently, after topical administration, pharmacological properties of each CAPE–NSAIDs hybrid could result in different levels

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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Control 0.01% VP939 0.1% VP939 0.01% VP964 0.1% VP964 0.1% CAPE 0.1% ASA 0.1% IND

800 600 400

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8

PGE2 levels (pg/ml)

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levels (pg/ml)

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0

0

PMN levels (number/ul)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

5

80 60 40

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*

Vehicle 0.01% VP939 0.1% VP939 0.01% VP964 0.1% VP964 0.1% CAPE 0.1% ASA 0.1% IND

0

Ocular Inflammation

Ocular Inflammation

Fig. 5. Aqueous levels of TNFα (A), PGE2 (B), PMN (C), and protein (D) 2 h after the second paracentesis with or without (vehicle) CAPE hybrids treatment (0.01% and 0.1%). Data shown are expressed as the mean 7 S.D. nPo 0.05 vs. vehicle; n¼8.

Table 1 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 (n ¼4). Formulation

Conjunctiva

Iris hyperemia

Corneal opacity

Congestion Swelling Discharge Control (10 min) Control (6 h) Vehicle (10 min) Vehicle (6 h) 0.01% VP939 (10 min) 0.01% VP939 (6 h) 0.01% VP964 (10 min) 0.01% VP964 (6 h) 0.1% VP939 (10 min) 0.1% VP939 (6 h) 0.1% VP964 (10 min) 0.1% VP964 (6 h)

0 0 1 0 1

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

0

0

0

0

0

1

0

0

0

0

0

0

0

0

0

1

0

0

0

0

0 1

0 0

0 0

0 0

0 0

0

0

0

0

0

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. In summary, our data demonstrate that VP939 is an effective anti-inflammatory agent when tested in experimental paracentesis-induced inflammation models in rabbits. The data also indicate that VP964 is at least as efficacious as VP939 in terms of PGE2 at 0.1% in a model of paracentesis-induced inflammation in 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.

Acknowledgments 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. References

attained in the target tissue; such differences could explain, at least in part, the discrepancy between these two drugs pharmacodynamics profiles. Based on their pharmacological actions, it seems that VP939 possesses better drug-like properties than VP964. Furthermore, the VP964 hybrid, for some reason likely related with the physic-chemical features, is less potent than indomethacin alone. Anti-inflammatory properties of CAPE were possibly due to its specific inhibition of Nrf2, which results in decreased cytokine production and inflammatory cell activation (Toyoda et al., 2009). In our study decreased levels of TNF-α in the VP939-treated group, as well as in the CAPE-treated group, may be related, at least in part, to this mechanism. The structural analysis of CAPE suggests that the functional groups, catechol and Michael reaction acceptor in CAPE, enabled the natural product, to interfere with NFκB

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

In this study, we report the design and synthesis of novel hybrids of caffeic acid phenetyl ester (CAPE) and non-steroidal anti-inflammatory drugs (NS...
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