Aceclofenac-galactose conjugate: design, synthesis, characterization

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Aceclofenac-galactose conjugate: design, synthesis, characterization and pharmacological and toxicological evaluations. Salvatore Magliocca, Carmen De Caro, Loretta Lazzarato, Roberto Russo, Barbara Rolando, Konstantin Chegaev, Elisabetta Marini, Maria Nieddu, Lucia Burrai, Gianpiero Boatto, Claudia Cristiano, Domenica Marabello, Elena Gazzano, Chiara Riganti, Federica Sodano, and Maria Grazia Rimoli Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00195 • Publication Date (Web): 18 Jun 2018 Downloaded from http://pubs.acs.org on June 21, 2018

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Aceclofenac-galactose conjugate: design, synthesis, characterization and pharmacological and toxicological evaluations.

Salvatore Magliocca,a,

#

Carmen De Caro,a,

b, #

Loretta Lazzarato,c Roberto Russo,a

Barbara Rolando,c Konstantin Chegaev,c Elisabetta Marini,c Maria Nieddu,d, Lucia Burrai,d Gianpiero Boatto,d Claudia Cristiano,a Domenica Marabello,e,f Elena Gazzano,g Chiara Riganti,g Federica Sodano,c, * and Maria Grazia Rimoli.a

a

Department of Pharmacy, “Federico II” University of Naples, 80131 Naples, Italy;

b

Department of Science of Health, School of Medicine and Surgery, "Magna Graecia" University of

Catanzaro, 88100 Catanzaro, Italy; c

Department of Drug Science and Technology, University of Torino, 10125 Torino, Italy;

d

Department of Chemistry and Pharmacy, University of Sassari, 07100, Sassari, Italy;

e

Department of Chemistry, University of Torino, 10125 Torino, Italy;

f

Interdepartmental Center for Crystallography (CrisDi), 10125 Torino, Italy;

g

Department of Oncology, University of Torino, 10126 Torino, Italy.

# these authors contributed equally to this work

CORRESPONDING AUTHOR INFORMATION: Federica Sodano, [email protected]; Department of Drug Science and Technology, University of Torino, Via Pietro Giuria, 9; 10125 Torino, Italy Phone: +390116707140; fax number: +390116707162.


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TABLE OF CONTENTS (TOC) GRAPHIC


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ABSTRACT

Aceclofenac is a popular analgesic, antipyretic and nonsteroidal anti-inflammatory drug (NSAID) used for prolonged treatment (at least three months) in musculoskeletal disorders. It is characterized by several limitations, such as poor water solubility and low oral bioavailability. The main side-effect of aceclofenac, as well as all NSAIDs, is the gastrotoxicity; among other adverse effects, there is the risk of bleeding since aceclofenac reversibly inhibits platelet aggregation. With the aim to reduce these drawbacks, we have designed, synthesized and characterized both in vitro and in vivo, an orally administrable pro-drug of aceclofenac (ACEgal). ACEgal was obtained by conjugating carboxyl group with the 6-OH group of Dgalactose; its structure was confirmed by X-ray powder diffractometry. The pro-drug was shown to be stable at 37°C in simulated gastric fluid (SGF-without pepsin, pH=1.2), and moderately stable in phosphate buffered saline (PBS, pH=7.4). However, it hydrolyzed in human serum with a half-life (t1/2) of 36 minutes, producing aceclofenac. Furthermore, if compared to its parent drug, ACEgal was four times more soluble in SGF. In order to predict human intestinal absorption, cell permeability in a Caco-2 model of aceclofenac and ACEgal was determined. Anti-inflammatory, analgesic, and ulcerogenic activities have been investigated in vivo. In addition, oxidative stress parameters (thiobarbituric acid reactive substances, TBARS and glutathione, GSH) and platelet anti-aggregatory activity both of parent drug and pro-drug were evaluated. Results clearly showed that the conjugation of aceclofenac to a galactose molecule improves physico-chemical, toxicological (at gastric and blood level) and pharmacological profile of aceclofenac itself, without changing intestinal permeability and anti-platelet activity (in spite the new sugar moiety).

Keywords: Aceclofenac; Pro-drug approach; Pain; Inflammation; Caco-2 cell apparent permeability coefficient; Ulcerogenicity; Oxidative stress parameters; Antiplatelet activity; X-ray powder diffraction.

Abbreviations used:

NSAIDs,

nonsteroidal

anti-inflammatory drugs;

COX, cyclooxygenase; GI,

gastrointestinal; GIT, gastrointestinal tract; PPIs, Proton Pump Inhibitors; BCS, biopharmaceutical classification

system;

CYP,

cytochrome;

dichlorophenyl)amino)phenyl)acetoxy)acetic

acid;

ACEgal, TFA,

D-α,β-galactopyranose-6-yl2-(2-(2-((2,6-

trifluoracetic

acid;

EDC.HCl,

N-ethyl-N′-(3-

dimethylaminopropyl)carbodiimide hydrochloride; DMAP, N,N-dimethylpyridin-4-amine; SGF, simulated gastric fluid; PBS, phosphate buffered saline; CMC, carboxymethyl cellulose; TBARS, thiobarbituric acid reactive substances; GSH, glutathione; XRPD, X-ray powder diffraction.


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INTRODUCTION 1

Aceclofenac is a nonsteroidal anti-inflammatory drug (NSAID), developed in Spain by Grau et al. in 1991. Its structure resembles diclofenac and was once considered its pro-drug, launched with the aim of overcoming high gastric side-effects of diclofenac. Aceclofenac is mainly used in the relief of pain ailments,2 rheumatoid arthritis, osteoarthritis and ankylosing 3

spondylitis for its remarkable anti-inflammatory, analgesic and antipyretic properties. This phenyl acetic acid derivative is a cyclooxygenase (COX) inhibitor, and specifically possesses a partial selectivity towards COX4

2. Although aceclofenac has the activity and COX-2 selectivity comparable to diclofenac, it shows both an improved gastric safety (aceclofenac is slightly less ulcerogenic than diclofenac) and decreased cardiovascular risk. Recently, the use of aceclofenac has been subjected to a revision at European level; in fact, its safety profile has been carefully monitored thanks to the SOS (Safety Of nonSteroidal anti5

inflammatory drugs) project, whose aim was to compare the risk of cardiovascular and gastrointestinal events during the administration of NSAIDs. Since aceclofenac is structurally correlated to diclofenac and during its metabolism converts to it, the same precautions for diclofenac have also been introduced in package leaflet of aceclofenac. However, the gastrointestinal (GI) toxicity remains the primary concern for chronic NSAIDs-based therapies including aceclofenac.6-12 In fact, there are potentially higher deleterious effects of NSAIDs, occurring in the gastrointestinal tract (GIT) due to two reasons: the inhibition of cytoprotective COX-1, and direct irritant action of their acid groups on the GIT.13,14 Long term use (typically three months) of aceclofenac could lead to ulcer formation and bleeding

15,16

17

with observations of diarrhea, nausea, abdominal pain and flatulence.

Proton Pump Inhibitors (PPIs) are commonly co-prescribed to reduce gastroduodenal injury however recent works demonstrated the association between PPI and NSAID can exacerbate some side effects of NSAIDs, and induce other side effects such as dysbiosis.18-21 Aceclofenac belongs to the class II of Biopharmaceutical Classification System (BCS, high gastrointestinal permeability but low solubility)22 and displays a low oral bioavailability (about 15%), that could be attributable to its rapid metabolism (aceclofenac is biotransformed into 4’-hydroxy-aceclofenac and 4’-hydroxy-diclofenac via cytochrome P-450 (CYP) 2C9 mediated hydroxylation and hydrolysis, respectively; CYP 2C9 also mediates the hydroxylation of diclofenac to yield 4’-hydroxy-diclofenac and the hydrolysis of 4’-OHaceclofenac to 4’-OH-diclofenac)23. Due to its poor water solubility, no commercial intravenous preparation of aceclofenac has been developed to date.

24

Aceclofenac has an ester moiety, which makes it prone to


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hydrolysis; this hydrolysis takes place in acidic, alkaline and neutral conditions, but the extent of degradation 25

is most significant at alkaline pH.

The recommended dosage is 200 mg per day in divided doses; various novel systems for aceclofenac delivery were developed to reduce the dosing frequency, the adverse effects due to prolonged treatment and to increase patients’ compliance.26 Several pieces of evidence have shown that restricting the dosage and duration of exposure, as well as use in patients less than 65 years old, significantly reduces adverse effects, but, this therapeutic approach is not always effective or possible.

27

Consequently, adoption of the pro-drug strategy offers an interesting and

promising approach in drug design, allowing in most cases for improved and more-favourable features, pharmacological time-profile and reducing side-effects of a specific parent drug. Based on the promising results obtained with the galactosylated pro-drugs, as reported in our previous 28-30

works,

we have also proposed for aceclofenac the use of galactose molecule to provisionally hide its

acidic group.31 Consistently, in this study, we described the design, synthesis and characterization of the galactosylated prodrug of aceclofenac, D-α,β-galactopyranose-6-yl2-(2-(2-((2,6-dichlorophenyl)amino)phenyl)acetoxy)acetic acid (ACEgal). The crystalline structure was analyzed by X-ray powder diffraction. We also reported the determination of physico-chemical properties of the designed compound (e.g. lipophilicity, solubility and stability behaviour), in order to predict the ADME profile of new chemical entity. We investigated the chemical and plasmatic stability, as well as the solubility and the distribution coefficient at physiological pH; moreover, cell permeability in Caco-2 model was measured. Finally, in vivo and ex-vivo experiments demonstrated the anti-inflammatory and analgesic activity of both compounds by carrageenan-induced paw edema test and acetic acid-induced writhings test, respectively. Additionally, gastric toxicity, oxidative stress parameters (thiobarbituric acid reactive substances, TBARS and glutathione, GSH) and antiplatelet activity were measured.

EXPERIMENTAL SECTION Chemistry All chemicals used for the synthesis were purchased from commercial sources (Sigma-Aldrich). All solvents were purified and degassed before use. Chromatographic separation was carried out under pressure using


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Merck silica gel 60 using flash-column techniques. Reactions were monitored by thin-layer chromatography (TLC) carried out on 0.25 mm silica gel coated aluminum plates (60 Merck F254) using sequentially two solutions: ethanol/10% vanillin and ethanol/20% sulfuric acid as visualizing agents. Unless specified, all reagents were used as received without further purifications. Dichloromethane was dried over P2O5 and was freshly distilled under nitrogen prior to use. 1H and

13

C NMR spectra were recorded at room temperature on

a JEOL ECZ-R 600 at 600 MHz and 150 MHz, respectively and calibrated using SiMe4 as an internal reference. Chemical shifts (δ) are given in parts per million (ppm) and the coupling constants (J) in Hertz (Hz). The following abbreviations were used to designate the multiplicities: s = singlet, d = doublet, dd = doublet of doublet, t = triplet, m = multiplet, bs = broad singlet. ESI spectra were recorded on a Micromass Quattro API micro (Waters Corporation, Milford, MA, USA) mass spectrometer. Data were processed using a MassLynxSystem (Waters). Final compound purity was determined by HPLC analysis on Merck LiChrospher C18 endcapped column (250 x 4.6 mm ID, 5 µm) using CH3CN 0.1% trifluoroacetic acid (TFA)/H2O 0.1% TFA as eluent. HPLC retention time (tR) was obtained at flow rates of 1.0 mL min-1, and the column effluent was monitored using UV as the detector. The X-ray powder diffractometry pattern was collected at room temperature using an

Oxford Diffraction Gemini R

Ultra diffractometer, equipped

with mirror

monochromatized Cu-Kα radiation (λ=1.5418 Å): maximum resolution 1.4 Å, exposure time 300 s and data integration with CrysAlis Pro software.

Synthesis of 1,2,3,4-di-O-isopropylidene-D-α-galactopyranose-6-yl 2-(2-(2-((2,6dichlorophenyl)amino)phenyl)acetoxy)acetic acid (1). 1,2,3,4-di-O-isopropylidene-D-α-galactopyranose (550 mg, 2.12 mmol), N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC HCl) (500 mg, 2.6 mmol) and N,N-Dimethylpyridin-4-amine (DMAP) (13.4 mg, 0.11 mmol) were added to a solution of aceclofenac (750 mg, 2.12 mmol), in dry dichloromethane (15 mL), and stirred at room temperature. After 4 hours, the solvent was completely removed via rotary evaporation and the obtained residue was purified by flash column chromatography using Petroleum Ether/ EtOAc (80/20, v/v) as the eluents, to give 1 as a white 1

foam solid. (Yield 800 mg, 62%). H NMR (600 MHz, CDCl3) δ 7.33 (d, J = 8.1 Hz, 2H), 7.26 – 7.24 (m, 1H), 7.15 – 7.12 (m, 1H), 7.00 – 6.95 (m, 2H), 6.71 (bs, 1H), 6.55 (d, J = 8.0 Hz, 1H), 5.51 (d, J = 5.0 Hz, 1H), 4.72 (d, J =1.7 Hz, 2H), 4.59 (dd, J = 7.9, 2.5 Hz, 1H), 4.36 (dd, J =11.5, 4.7 Hz, 1H), 4.31 (dd, J = 5.0, 2.5 Hz, 1H), 4.28 (dd, J =11.5, 7.7 Hz, 1H), 4.17 (dd, J =7.9, 1.9, 1H), 4.01 – 3.98 (m, 1H), 3.94 (d, J = 5.6 Hz, 1H), 1.50 (s, 3H), 1.43 (s, 3H), 1,32 (s, 3H), 1.31 (s, 3H).

13

C NMR (150 MHz, CDCl3) δ.171.5, 167.5, 142.9,


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138.0, 131.1, 129.7, 129.0, 128.3, 124.2, 124.1, 122.3, 118.6, 109.8, 109.0, 96.3, 71.0, 70.8, 70.5, 65.9, +

64.4, 61.3, 38.1, 26.1, 26.0, 25.0, 24.6. ESI-MS [M+Na] : m/z 618.3.

Synthesis of D-α, β-galactopyranose-6-yl 2-(2-(2-((2,6-dichlorophenyl)amino)phenyl)acetoxy)acetic acid (ACEgal). The compound 1 (650 mg; 1.09 mmol) was dissolved in a solution of TFA/water 90/10 v/v and kept under stirring at room temperature for 1 minute. The solvent was subsequently evaporated under reduced pressure; the obtained residue was dissolved in toluene (15 mL) and concentrated again via rotary evaporation. This procedure was repeated for several times until, during the evaporation of the toluene, the formation of a white solid was observed. Afterwards, the crude product was dissolved in methanol (5 mL); the addition of diethyl ether (20 mL) allows the precipitation of the desired product; later, it was filtered, washed with Et2O, and desiccated producing ACEgal as a white powder. (Yield 450 mg, 80%). Mp = 166.41

167.7 °C. H NMR (600 MHz, DMSO-d6) δ 7.53 (d, J = 8.1 Hz, 2H), 7.25 (d, J =7.5 Hz, 1H), 7.21 (t, J = 8.1 Hz, 1H), 7.07 (t, J = 7.6 Hz, 1H), 6.94 (s, 1H), 6.87 (t, J = 7.4 Hz, 1H), 6.25 (d, J = 8 Hz, 1H), 4.94 (s, 1H), 4.74 (s, 2H), 4.15 (d, J = 5.4 Hz, 2H), 4.01 (t, = 5.8 Hz, 1H), 3.91 (s, 2H), 3.67 (s, 1H), 3.60 – 3.48 (m, 2H). 13

C NMR (150 MHz, DMSO-d6) δ171.1, 167.8, 142.9, 137.2, 131.0, 130.9, 129.2, 127.9, 126.1, 122.9, 120.8, +

116.0, 92.7, 69.2, 68.4, 68.5, 67.5, 65.2, 61.1, 36.6. ESI-MS [M+Na] : m/z 538.2. HPLC purity ≥ 95% (CH3CN 0.1% TFA/H2O 0.1% TFA 50/50, v/v), flow = 1.0 mL/min, tR =4.34 and 4.67 min, anomeric mixture 1/9).

Stability in SGF and PBS A solution of each compound (ACEgal and aceclofenac, 10 mM) in methanol was added to simulated gastric fluid (SGF- without pepsin, 2.0 g/L sodium chloride and 2.917 g/L HCl, pH 1.2) or PBS (pH 7.4, 50 mM) (2.3 g of disodium hydrogen orthophosphate, 0.19 g of potassium dihydrogen orthophosphate and 8.0 g of sodium chloride in sufficient water to produce 1000 mL and adjust the pH if necessary) preheated to 37°C. Resulting solutions (100 µM) were maintained at 37 ±0.5°C and at appropriate time intervals each 20 µL aliquote was withdrawn and analyzed by RP-HPLC.

Stability in human serum A solution of each compound (ACEgal and aceclofenac, 10 mM) in methanol was added to human serum (from human male AB plasma, USA origin, sterile-filtered, Sigma–Aldrich) preheated to 37°C. Resulting solutions (200 µM) were incubated at 37 ±0.5°C and at appropriate time intervals, 300 µL of each reaction


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mixture was withdrawn and added to 300 µL of CH3CN containing 0.1% TFA in order to deproteinize the serum. Samples were sonicated, vortexed and then centrifuged for 10 minutes at 2150 g. Each clear supernatant was filtered by 0.45 µm PTFE filters (Alltech) and analyzed by RP-HPLC. Pseudo-first-order half-times (t½) for chemical and enzymatic hydrolysis were calculated from the linear slopes of plots of the logarithm of remaining compounds against time.

HPLC analysis The reverse-phase HPLC procedure allowed separation and quantitation of ACEgal, aceclofenac and their degradation products (aceclofenac and diclofenac, respectively). HPLC analysis was performed with a HP 1100 chromatograph system (Agilent Technologies, Palo Alto, CA, USA) equipped with a quaternary pump (model G1311A), a membrane degasser (G1379A), a diode-array detector (DAD) (model G1315B) integrated in the HP1100 system. Data analysis were processed using a HP ChemStation system (Agilent Technologies). The analytical column was a EC Nucleosil 100-5 C18 HD (250×4.6mm, 5 µm; MachereyNagel). The mobile phase consisted of 0.1% aqueous TFA (solvent A) and 0.1% TFA CH3CN (solvent B), at flow rate = 1.0 mL/min. Elution was in gradient mode: initially 50% of solvent B until 6 min, from 50 to 70% of solvent B between 6 and 10 min, 70% of solvent B until 15 min, and from 70 to 50% of solvent B between 15 and 20 min. The injection volume was 20 µL (Rheodyne, Cotati, CA). The column effluent was monitored at 270 nm, referenced against a 700 nm wavelength. Quantitation of compounds was calculated by using 2

calibration curves, whose linearity was determined in a concentration range of 1-100 µM (r > 0.99).

Solubility assessment in SGF and PBS PBS (pH 7.4, 50 mM) and SGF-without pepsin (pH 1.2) were used to determine the solubility of the compounds at physiological pH and in an acid medium respectively. Excess amount of test compounds was added to 5 mL of PBS and SGF in glass tubes in triplicate and shaken on a magnetic stirrer for 120 minutes at 37°C. The suspensions were filtered using 0.45 µm membrane filters and were appropriately diluted; the concentration of compound in each filtrate was determined by a UV spectrophotometer Shimadzu UV2501PC, using calibration curves obtained with standard solutions. Six point calibration standards (1, 5, 10, 20, 50, 100 µg/mL) were prepared by dilution from 10 mg/mL stock solutions (in MeOH) of each compounds 2

in PBS and SGF (r > 0.99).

Distribution Coefficient


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The distribution coefficient at pH 1.2 (log D1.2) and pH 7.4 (log D7.4) of the compounds between n-octanol and SGF or PBS was experimentally obtained by shake-flask technique at room temperature. In the shake-flask experiments SGF and PBS were used as the aqueous phase; the organic (n-octanol) and aqueous phases were mutually saturated by shaking for 4 hours. The compounds were dissolved in the saturated buffered aqueous phases at a concentration equal to solubility and an appropriate amount of saturated n-octanol was added. The two phases were shaken for about 20 minutes, by which time the partitioning equilibrium of solutes was reached, and then centrifuged (10000 rpm, 10 minutes). The concentration of the solutes was measured in the aqueous phase by UV spectrophotometer Shimadzu UV-2501PC, using calibration curves obtained with standard solutions (r2 > 0.99). Each log D value is an average of a minimum of six measurements.

Caco-2 cell permeability test Chemicals Plasticware for cell culture was provided by Falcon (Becton Dickinson, Franklin Lakes, NJ). When not otherwise specified, reagents were from Sigma-Aldrich S.r.l. (Milan, Italy). Caco-2 cell culture The Caco-2 cells were a generous gift from the department of Molecular Biotechnology and Health Sciences, 4

2

University of Torino. Cells were seeded at 7.0 x 10 cells/cm for 21 days on Transwell inserts (0.4 µm diameter pore-sizes, growth area of 4.67 cm2, Corning Life Sciences, Chorges, France). Cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 20% fetal bovine serum (FBS), 1% nonessential amino acids (NEAA), 1% L-glutamine and 1% penicillin/streptomycin. Cell cultures were kept in a humidified incubator at 37°C in a 5% CO2 atmosphere. The culture medium was changed every other day until time of use. Caco-2 cell permeability Transwells with Caco-2 cells grown on them for 21 days, were rinsed twice and equilibrated with PBS at 37°C for 15 minutes before the transport experiment. Before the permeability experiments, the cell monolayers had been washed twice with HBSS (Hank’s balanced salt solution) containing 10 mM 4-(2hydroxyethyl) piperazino-1-ethanesulfonic acid (HEPES, pH 7.4). The test compounds were first prepared in DMSO solution (20 mM) and then in HBSS buffer to give a final drug concentration of 100 µM when added to the cell monolayers. Samples were obtained after 15, 30, 45, 60 and 90 minutes by moving the cell


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monolayers to a new receiver well containing fresh HBSS. Drug concentrations in the receiver compartments were determined using RP-HPLC, as previously described.

Pharmacological materials and methods Animals Ten-week-old male Swiss CD1 mice weighing 30–35 g were purchased from Charles Rivers (Calco, Italy). They were housed in cages in a room kept at 22 ± 1°C on a 12:12 h light/dark cycle. All animals were acclimated to their environment for 1 week and had ad libitum access to water and a standard rodent chow diet. All procedures involving mice were carried out in accordance with the Institutional Guidelines and complied with the Italian Ministry of Health and associated guidelines from European Communities Council Directive. The procedures reported here were approved by the Institutional Committee on the Ethics of Animal Experiments (CSV) of the University of Naples “Federico II” and by the Ministry of Health under protocol no. 2014-0084607. At the end of all experiments the animals were euthanized by CO2 overdose.

Anti-Inflammatory Activity Anti-inflammatory activity was evaluated using carrageenan-induced paw edema on mice method. Mice were divided into three groups of six animals: group I served as control group (CTRL), group II received aceclofenac 10 mg/kg, group III received ACEgal at a dose (17.5 mg/kg) molecularly equivalent to aceclofenac. A stock solution of 5 mg/mL was prepared as a homogenous suspension in aqueous solution of sodium CMC (0.5% w/v) and each animal of the group I received this vehicle orally while group II and III aceclofenac and ACEgal respectively. 60 minutes after administration, paw edema was induced by a subplantar injection of 50 µL of sterile saline containing 1% λ -carrageenan into the right hind paw.

32

Paw

volumes of all animals were measured by a plethysmometer apparatus (UgoBasile, Milan, Italy) before and after the administration of the drugs. Specifically, the paw volume was measured at 0, 2, 4, 6, 24, 48, 72 and 96 hours after carrageenan challenge. The increase in paw volume was evaluated as the difference between the paw volume measured at each time point and the basal paw volume measured immediately before carrageenan injection.

Acute pain model: acetic acid writhing Analgesic activity was carried out using the acetic acid-induced writhing method in Swiss CD1 mice. The animals were divided into three groups of six animals each. Group I served as a control group (CTRL), group


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II received aceclofenac 10 mg/kg, and group III received ACEgal 17.5 mg/kg, where the dose of the pro-drug was molecularly equivalent to aceclofenac. A stock solution of 5 mg/mL was prepared as a homogenous suspension in aqueous solution of sodium CMC (0.5% w/v) and each animal of the group I received orally this vehicle while group II and III the respective drugs. Each mouse was placed separately into a cage and allowed to acclimate for a least 10 minutes; the visceral pain was induced by intraperitoneal injection (1 33,34

mL/100g body weight of the animal; in our case 300 µL /mouse) of 0.6% acetic acid.

Tests started at 4

hours and 48 hours post oral administration of the vehicle or aceclofenac or ACEgal. In this experiment, the total number of writhes was recorded, a parameter of chemically induced pain detectable by constriction of abdomen, turning of trunk, and extension of hind legs; the above-mentioned three movements of mice were counted for 20 minutes, starting 5 minutes after the administration of the irritant agent. The analgesic effect was expressed as the number of writhes in comparison with control.

Ulcerogenicity studies NSAID-induced gastric damage in mice was evaluated following the procedure described by Russo

30

and

Chan et al.35 Acute ulcerogenic testing was performed on healthy Swiss CD1 mice, which were divided into three groups of six, with each which in turn divided into three sub-groups. Group I served as control (CTRL) and received orally the vehicle only (a stock solution of 5 mg/mL was prepared as a homogenous suspension in aqueous solution of sodium CMC, 0.5% w/v); group II received pure aceclofenac (10 mg/kg) and group III received the pro-drug in a dose equivalent to that of aceclofenac (17.5 mg/kg). The animals were fasted 16-18 hours prior to an oral single dose of the control and the test compounds; the mice belonging to three sub-groups were sacrificed after 4 hours while the others three 48 hours later from treatment. After mouse euthanasia, the stomach was excised along its greater curvature and rinsed with normal saline. The gastric mucosa of the mice was then examined by means of a magnifying glass for the presence of irritation or frank hemorrhagic lesions (ulcers). Irritation/ulcers was assigned a score from 0 to 3; 0= no irritation, 3= high irritation. The sum of total scores was used for comparison.

Evaluation of oxidative stress parameters: TBARS and GSH. Eighteen healthy Swiss CD1 mice were divided into three groups, which in turn divided in sub-groups of three each. Group I served as control (CTRL) and orally received the vehicle only (a stock solution of 5 mg/mL was prepared as a homogenous suspension in aqueous solution of sodium CMC, 0.5% w/v); group II received pure aceclofenac (10 mg/kg) and group III received the pro-drug in a dose equivalent to that of


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aceclofenac (17.5 mg/kg). 500 µL of blood was withdrawn depending on the sub-group after 4 or 48 hours from the oral administration by an intracardiac puncture; next, each blood sample was centrifugated at 3000 rpm at 4°C for 15 minutes separating so the plasma. The TBARS concentration was analyzed in these TM

plasma samples using by TBARS Parameters

Assay Kit (R&D Systems), the glutathione concentration

(both GSH, reduced specie and GSSG, oxidized specie), instead, was performed through a Glutathione Assay Kit (Cayman Chemical).

Inhibition of platelet aggregation The anti-platelet activity of aceclofenac and ACEgal was assayed ex-vivo using collagen as platelet aggregation agent. Human blood was obtained from healthy volunteers (25-45 years of age), with a restriction of no medications to be taken within two weeks prior to donation. All subjects provided informed consent and were treated according to Helsinki protocol for biomedical experimentation. Blood and blood products were handled in plastic ware, whereas siliconized glass cuvettes and stir bars were used in the aggregation assay. Inhibition of platelet aggregation was studied as elsewhere reported with some modifications.36 Platelet rich plasma (PRP) was prepared by the centrifugation of citrated blood at 200 g for 20 minutes. The transmittance of PPP was taken as 100% aggregation. PRP (500 µL) was added into the aggregometer (Chrono-log 4902D) cuvettes and preincubated at 37 °C for 10 minutes with the tested compounds (final concentrations in the PRP solutions ranging from 0.1 µM to 300 µM) or with vehicle (to eliminate the effect of the solvent on the aggregation and release reaction of platelets; the final concentration of DMSO was fixed at 0.5%, v/v). Then, collagen at submaximal concentration (0.8-1.5 µg/mL) was added to the incubated sample and aggregation was recorded as increased light transmission under continuous stirring (1000 rpm) at 37 °C for 10 minutes after addition of the aggregation inducer. The antiaggregatory activity of tested compounds is expressed as a percent inhibition of platelet aggregation compared with vehicle control samples. IC50 values (i.e., the concentration effecting 50% inhibition of aggregation), were calculated by nonlinear regression analysis (r2 > 0.80). Data are expressed as the mean of at least 3 independent experiments.

Statistical analysis All analyses were performed with GraphPad Software Inc., Version 7.0, San Diego, CA. The statistical significance was determined by Student’s t test or by one- or two-way analysis of variance (ANOVA) followed by a Bonferroni’s post hoc test for multiple comparisons.


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Page 14 of 33

RESULTS Synthesis The synthesis of ACEgal was carried out in two steps: firstly, the conjugation of aceclofenac with 1,2,3,4-di-

O-isopropylidene-D-α-galactopyranose in the presence of EDC.HCl as a coupling agent and DMAP, as catalyst in dry dichloromethane;

29

next, the deprotection about the hydroxyl groups of sugar component in

an aqueous solution of TFA;37 the targeted pro-drug (Scheme 1) was obtained as white crystalline solid and was analyzed by X-ray powder diffraction. The diffractogram shown in Figure 1, is the “finger print” of the polymorph type used for biological studies; it is characterized by main peaks at 2θ values of 7.43°, 8.59°. 9.59°, 11.06°, 11.79°, 14.67°, 16.92°, 17.90°, 18.70°, 19.86°, 20.46°, 21.82°, 22.91°, 25.45°. Scheme 1.a Synthesis of ACEgal.

a

Reaction conditions: i) EDC.HCl, DMAP, CH2Cl2, rt; ii) 90% TFA, rt.

Physico-chemical characterization of ACEgal The hydrolytic stability of ACEgal (and aceclofenac for comparison) was evaluated both at pH 1.2 in SGFwithout pepsin and at pH 7.4 in PBS at 37°C to simulate the body fluid and the gastric environment respectively; whereas, the stability against esterases was evaluated in human serum. HPLC technique was used to follow the hydrolysis of ACEgal with the consequent formation of aceclofenac and, in the meantime, the chemical degradation of aceclofenac with resulting appearance of diclofenac. The synthesized pro-drug was stable at pH 1.2, after 6 hours the percentage of hydrolysis was only 6%, and moderately stable at pH 7.4. Also, the aceclofenac resulted to be rather stable, in fact, after 24 hours of incubation both in SGF and 38-42

PBS, the formation of diclofenac was negligible.

To be defined a pro-drug, ACEgal has to regenerate the


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parent drug by enzymatic pathways, indeed its half-life (t1/2) in human serum resulted to be 36 minutes (Table 1). Also, the solubility of aceclofenac and ACEgal was evaluated both in PBS and SGF-without pepsin at 37°C.43 ACEgal resulted to be more soluble than aceclofenac in SGF, whilst it was less soluble at pH = 7.4 because, in these conditions, aceclofenac was completely deprotonated (Table 1). Additionally, we also determined the distribution coefficients (log DpH) of aceclofenac and ACEgal in n-octanol-PBS (pH 7.4) and in

n-octanol-SGF (pH 1.2). Distribution coefficient of ACEgal was almost independent of pH, reflecting the absence of ionizable groups, while aceclofenac became much more hydrophilic at pH 7.4, consistent with the deprotonation of carboxylic moiety (Table 1). However, the log D of ACEgal is included in the values range typical of compounds that show an optimal hydrophilic-lipophilic behaviour for balancing membranes permeability and dissolution in aqueous environments.

a

Table 1. Physical-chemical characterization of ACEgal. Compound

Chemical and enzymatic stability at 37 °C pH 1.2; t ½ (h)

a

pH 7.4 t ½ (h)

a

Solubility at 37 °C mg/mL

b

Lipophilicity log D (±SD)

c

Human serum a t½ (h)

pH 1.2

pH 7.4

pH 1.2

pH 7.4

ACEgal

>24

2.2

0.6

0.0125

0.023

1.70±0.07

1.68±0.07

Aceclofenac

>24

>24

>24

0.003

1.076

>3.5

0.057±0.03

a

Results are expressed as mean values (n=3; SD

Aceclofenac-galactose conjugate: design, synthesis, characterization

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