Synthesis, characterization and pharmacological evaluation of1-(2-chloro-6-fluorophenyl)-5-methylindolin-2-one: A new anti-inflammatory compound with reduced gastric ulceration properties

Article abstract of DOI:10.3390/molecules15118039

The new compound 1-(2-chloro-6-fluorophenyl)-5-methylindolin-2-one (1), designed using the prodrug approach, was easily obtained in 85% yield and characterized by nuclear magnetic resonance, elemental analysis, mass spectrometry and infrared spectroscopy.

Full text of DOI:10.3390/molecules15118039

Molecules 2010, 15, 8039-8047; doi:10.3390/molecules15118039
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Communication
Synthesis, Characterization and Pharmacological Evaluation of
-(2-Chloro-6-Fluorophenyl)-5-Methylindolin-2-One: A New
1
Anti-Inflammatory Compound with Reduced Gastric
Ulceration Properties
1
,
1
1
1
Jean Leandro dos Santos *, Rafael Chelucci , Richard Chiquetto , Man Chin Chung ,
2
2
Michel Leandro Campos and Rosangela Gonçalves Peccinini
1
Lapdesf - Laboratório de Pesquisa e Desenvolvimento de Fármacos, Departamento de Fármacos e
Medicamentos, Faculdade de Ciências Farmacêuticas – UNESP Rodovia Araraquara Jaú Km. 01,
1
4801-902, Araraquara, SP, Brazil
2
Departamento de Princípios Ativos, Naturais e Toxicologia, Faculdade de Ciências Farmacêuticas -
UNESP Rodovia Araraquara Jaú Km. 01, 14801-902, Araraquara, SP, Brazil
*
Author to whom correspondence should be addressed; E-Mail: santosjl@fcfar.unesp.br;
Tel.: +55-16-3301-6972; Fax: +55-16-3301-6960.
Received: 25 September 2010; in revised form: 3 November 2010 / Accepted: 5 November 2010/
Published: 8 November 2010
Abstract: The new compound 1-(2-chloro-6-fluorophenyl)-5-methylindolin-2-one (1),
designed using the prodrug approach, was easily obtained in 85% yield and characterized by
nuclear magnetic resonance, elemental analysis, mass spectrometry and infrared
spectroscopy. The lactam 1 showed anti-inflammatory and analgesic activity comparable to
that of the COX-2 inhibitor lumiracoxib, without gastro-ulceration effects. Stability studies
demonstrated that the lactam function was stable and did not hydrolyze in pH 1.2 or 7.4.
Furthermore, using a thioglycollate-induced peritonitis model, compound 1 was shown to
inhibit cell migration by 50.4%, while lumiracoxib inhibited it by 18%. This compound
represents a new non-ulcerogenic prototype for the treatment of chronic inflammatory
diseases.
Keywords: pro-drug; lactam; lumiracoxib; anti-inflammatory

Molecules 2010, 15
. Introduction
Nonsteroidal anti-inflammatory drugs (NSAIDs) are used for the treatment of inflammatory and
8040
1
rheumatic diseases. The common mechanism of NSAIDs involves the non-selective inhibition of
cyclooxygenases (COX-1 and COX-2) preventing the biosynthesis of prostaglandins (PGs). The COX-1
enzyme is the constitutive isoform, and it provides the normal production of PGs. The enzyme has an
important role in homeostasis and gastroprotection, and this enzyme inhibition, caused by the long-term
use of drugs, is associated with gastrotoxicity. This side effect produced by NSAIDs involves two
different mechanisms: inhibition of prostaglandin synthesis in the stomach and a local action exerted
by direct contact of the drugs with the gastric mucosa [1].
Scheme 1. In vivo prodrug conversion to lumiracoxib.
O
OH
H C
3
H C
3
in vivo
hydrolysis
O
N
NH
Cl
Cl
F
F
(1)
(2)
prodrug
lumiracoxib
As part of our ongoing program to identify new anti-inflammatory drug candidates with reduced gastric
ulceration properties, we describe herein the synthesis and the preliminary biological evaluation of the
compound 1-(2-chloro-6-fluorophenyl)-5-methylindolin-2-one (1), designed as a lumiracoxib prodrug.
2
. Results and Discussion
2.1. Synthesis
The title compound was prepared through an intramolecular reaction of lumiracoxib at room
temperature for 1 h using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) as
coupling reagent. After, the reaction mixture was diluted with dichloromethane and washed with 0.2 M
bicarbonate solution. The solvent was removed by evaporation, resulting in 85% yield of 1-(2-chloro-
6-fluorophenyl)-5-methylindolin-2-one (1). The purity of the synthesized compound was checked by thin
layer chromatography (TLC) and elemental analysis. The elemental analysis results were within ± 0.4%
of the theoretical values. The structure was characterized by nuclear magnetic resonance (NMR),
1
infrared spectroscopy (IR) and mass spectrometry (MS). The H-NMR spectrum showed the methyl
group and the methylene protons at δ 2.35 (s, 3H) and 3.81 (s, 2H), respectively. The aromatic protons
appeared at δ 6.83-7.4. The IR spectrum of compound 1 showed the presence of a lactam carbonyl at
−1
−1
−1
1
.736 cm . The C-F and C-Cl stretching appeared at 1.150 cm and 783 cm , respectively.

Molecules 2010, 15
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2.2. Pharmacological Evaluation
2
.2.1. Anti-inflammatory activity and ulcerogenicity studies
Figure 1 demonstrates the inhibition of swelling rat paw edema induced by carrageenan after oral
administration of the tested drugs. The in vivo result shows that 1-(2-chloro-6-fluorophenyl)-5-
methylindolin-2-one (1) presented anti-inflammatory activity comparable with lumiracoxib, the
parental drug used as control. From the first hour, the anti-inflammatory activity of lactam 1 was
statistically different when compared to the carrageenan group. All results were statistically different
when compared to an aqueous solution of sodium carboxymethylcellulose (0.5% w/v), used as control
(
data not shown).
Figure 1. Anti-inflammatory activity in carrageenan-induced paw edema in rats (data are
represented as mean ± S.E.M., n = 6, * P < 0.05 related to carrageenan group; dose of
compounds = 300 μmol/kg).
The ulcerogenicity study was carried out using rat gastric mucosa after a single oral administration
−
1
of the compounds at 300 μmol/Kg of each drug. The lesions were examined using a 4 × binocular
magnifier and the results were obtained with an average of six animals analyzed per group. Table 1
demonstrates that the lactam 1-(2-chloro-6-fluorophenyl)-5-methylindolin-2-one (1) was unable to
induce gastric ulceration. Diclofenac and lumiracoxib were used as COX-1 and COX-2 inhibitor
controls, respectively. For each stomach, the severity of mucosal damage was assessed according to
the following scoring system: 0 - no lesions or up to five puntiform lesions; 1 - more than five
puntiform lesions; 2 - one to five small ulcers; 3 - more than five small ulcers or one large ulcer;
4
- more than one large ulcer.
Table 1. Ulcerogenic effect of diclofenac, lumiracoxib and lactam (1) in rats (n = 6,
mean ± S.D.).
Compound Number of ulcers
<1 mm
1-2 mm
>2 mm
diclofenac
lumiracoxib
lactam 1
74 ± 8.1
10 ± 1.2*
0*
62 ± 6.3 (84%) 5.2 ± 1.8 (7%)
7.3 ± 2.1 (73%) 2.7 ± 1.1 (27%)
6.7 ± 2.4 (9%)
-
-
-
-
*
significant difference compared to diclofenac group; P < 0.05 (Tukey’s test)

Molecules 2010, 15
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Diclofenac induced an average of 74 ulcerogenic lesions with around 6.7 (9%) lesions larger than
mm. According to the scoring system, diclofenac was classified as class four. Lumiracoxib induced
2
an average of 10 ulcerogenic lesions with around 2.7 (27%) lesions larger than 2 mm. This result
allowed us to classify this drug as class two. The lactam 1 did not induce gastric ulceration and it was
classified as class zero. Similar results were observed when diclofenac was converted to a lactam
derivative [2]. These results demonstrated that the masking of carboxylic function of the lumiracoxib
could decrease the gastro-ulcerogenicity.
The thioglycollate-induced peritonitis in mice assay was performed in order to confirm the anti-
inflammatory profile of lactam 1 suggested by the carrageenan-induced paw edema assay results. As
depicted in Figure 2, the lactam derivative 1 was able to inhibit by 50.4% the cell migration using
thioglycollate-induced peritonitis model. Indomethacin, used as drug control, presented 72% cell
migration inhibition. In the same assay, lumiracoxib inhibited around 18% (data not shown).
Figure 2. Effect of lactam derivative 1 and indomethacin on cell migration in peritonitis
3
3
% thioglycollate-induced in mice. All compounds were administered p.o at a dose of
00 µmol/kg. Mice were killed at the time-point of 4 h after % thioglycollate-induced
peritonitis. Total cell migration was counted using a Neubauer chamber. Data represent
mean ± S.E.M. from at least six animals. * P < 0.01 (ANOVA followed by Dunnett’s test).
2
.2.2. Analgesic activity
The lactam derivative’s ability to inhibit acetic acid-induced writhing in mice was studied in order
to analyze its anti-nociceptive profile. The vehicle control exhibited 100% writhing, and the protection
of the parental drug and its derivative was calculated on a percentage basis.
According to Table 2, lactam 1 offers analgesic protection 44.2% higher than that of lumiracoxib
(
(
38.8%). Indomethacin presented 73.1% of protection in the model used. These results suggest that 1-
2-chloro-6-fluorophenyl)-5-methylindolin-2-one (1) has anti-inflammatory and analgesic activity
comparable with those of lumiracoxib.

Molecules 2010, 15
Table 2. Number of writhings and percentage of protection by indomethacin, lumiracoxib
8043
−1
and lactam 1 at 100 μmol Kg , using the acetic acid induced writhing model in mice
(
n = 6, mean ± S.D.).
Number of writhings
average)
Treatment
% protection
(
control
60 ± 1.8
-
indomethacin
lumiracoxib
lactam 1
16.1 ± 1.1
36.7 ± 1.2*
41.8 ± 0.7*
73.1
38.8
44.2
*
statistical difference compared to control group. P < 0.05 (Tukey’s test)
2
.2.3. In vitro hydrolysis of 1-(2-chloro-6-fluorophenyl)-5-methylindolin-2-one (1) at pH 1.2 and 7.4
In order to observe the stability of lactam derivative 1 at the different pHs of gastrointestinal fluid
and plasma, an HPLC (UV-Vis) method was developed. The hydrolysis study was carried out in
aqueous buffer solutions (pH 1.2 and pH 7.4). The lactam derivative 1 was stable and did not
hydrolyze at pH 1.2 and pH 7.4 during 24 h (Figure 3). These results indicated that stomach would not
be exposed to carboxylic acid function of lumiracoxib (2). The absence of hydrolysis at pH 7.4 during 24
h indicated that lactam 1 could be absorbed in intact form.
Figure 3. Hydrolysis of lactam derivatives (μg/mL) in buffer pH 1.2 and 7.4 (data are
represented as mean ± S.E.M., n = 4, P < 0.05).
Previous reports demonstrated that this stability could be related to steric and electronic factors.
Sterically, nucleophilic access to the lactam carbonyl group is blocked by fluorine and chlorine. This
factor could explain the absence of lactam hydrolysis.

Molecules 2010, 15
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3. Experimental
3.1. General
Melting points were taken with an Electrothermal melting point apparatus (SMP3 Bibby Stuart
Scientific) in open capillary tubes and they are presented as uncorrected values. Infrared spectra (KBr
−1 1
discs) were obtained on a Shimadzu FTIR-8300, and the frequencies were expressed in cm . H-NMR
spectra were scanned on a Bruker DRX-400 (400 MHz) NMR spectrometer using DMSO-d as solvent.
6
Chemical shifts are expressed in ppm (parts per million) relative to tetramethylsilane. Elemental
analysis (C, H and N) were obtained on a Perkin Elmer model 240C analyzer, and the data were within
±
0.4% of the theoretical values.
3.2. Materials
The lumiracoxib standard was donated by Novartis (São Paulo, Brazil). The diclofenac standard
was donated by EMS-Sigma Pharma (Hortolândia, SP, Brazil). 1-Ethyl-3-[3-dimethylamino-
propyl]carbodiimide hydrochloride (EDC, Sigma-Aldrich, St. Louis, MO, USA), potassium phosphate
and trichloroacetic acid (TCA) were purchased from Labsynth (São Paulo, SP, Brazil). Acetonitrile
was obtained from J.T. Baker (Phillipsburg, NJ, USA).
3
.3. Animals
Male Wistar rats (200-250 g) and Swiss albino mice (25-30 g) were housed at a constant temperature
(23 ± 1.8 ºC), humidity (55 ± 5%) and a light cycle (12/12 h) with food and water ad libitum.
Experiments were conducted during the light phase. The study protocol was approved by The Research
Ethics Committee of the School of Pharmaceutical Science, UNESP, Araraquara (Process 24/2010).
3
.4. Synthesis of 1-(2-chloro-6-fluorophenyl)-5-methylindolin-2-one (1)
Lumiracoxib (2, 315 mg, 1.07 mmol) and EDC (226 mg, 1.2 mmol) were placed in dichloromethane
(20 mL) and the reaction mixture was stirred for 1 h at room temperature. Then, it was diluted with
dichloromethane (50 mL) and washed with distilled water (3 × 20 mL). The organic phase was dried
with sodium sulfate and the solvent removed by evaporation resulting in 251 mg (85% yield) of 1-(2-
1
chloro-6-fluorophenyl)-5-methylindolin-2-one as a light yellow substance; m.p.: 130-132 °C. H-NMR:
δ 2.35 (s, 3H), 3.87 (s, 2H), 6.83 (d, 1H, J = 7.67 Hz), 6.84 (d, 1H, J = 2.15 Hz), 6.85 (dd, 1H, J = 8.26
Hz and 2.1 Hz), 6.92 (ddd, 1H, J = 7.67 Hz), 7,02 (dd, 1H, J = 7.67 Hz and 1.5 Hz), 7.4 (d; J = 7.67 Hz,
1
1
6
H) ppm; IR: 3,030 (C-H aromatic), 1,736 (C=O lactam), 1,458, 1,488 and 1,612 (C-C aromatic),
−1
,359 (C-N), 1,150 (C-F) and 783 (C-Cl) cm ; MS-EI: 275 (m/z). Calculated for C15H11ClFNO: C,
5.35; H, 4.02; N, 5.08. Found: C, 65.50; H, 4.11; N, 5.30.
3
.5. Anti-Inflammatory Activity
The anti-inflammatory activity was evaluated using the carrageenan-induced rat paw edema method [7].
Wistar rats (150-200 g) were divided into three groups of six animals each. Group I served as a control

Molecules 2010, 15
8045
−1
group without using any drug, group II received lumiracoxib (2) at 300 μmol kg , and group III
−1
received lactam 1 at 300 μmol kg in a homogeneous suspension of sodium carboxymethyl-cellulose
0.5% w/v). The dose was molecularly equivalent to that of lumiracoxib. Each animal received orally
.75-1.0 mL of the respective drugs. Thirty minutes after the administration of drugs, each rat received
(
0
a subplantar injection of 0.1 mL of 1% carrageenan solution in its left hind paw. The measurement of
the hind paw volume was carried out using a plethysmometer before each treatment (Vo) and in each
interval (Vt) after the administration of drugs. All the results are expressed as mean ± S.E.M. Statistical
analysis were performed with ANOVA followed by Tukey’s test.
3
.6. Analgesic Activity
Analgesic activity was evaluated using 1% (v/v) acetic acid solution to induce writhing [8] in Swiss
albino mice (25-30 g) of both sexes. The compounds were administered orally 1 h prior to acetic acid
injections. The numbers of writhings were counted for 30 min duration in control and test compounds.
Analgesic activity was measured as a decrease in writhings and compared with control. Mice were
divided into four groups of six animals each. Group I served as a control group without any drug, group II
−
1
−1
received indomethacin (100 μmol kg ), group III received lumiracoxib (100 μmol kg ) and group IV
−1
received lactam derivative at 100 μmol kg . Each animal received 0.3-0.4 mL orally of the respective
drugs, prepared as a homogeneous suspension of sodium carboxymethylcellulose (0.5% w/v). Acetic
acid was administered intraperitoneally at a dose of 1 mL/100 g body weight. All the results are
expressed as mean ± S.E.M. Statistical analysis were performed with ANOVA followed by Tukey’s test.
3.7. Ulcerogenicity
Gastrointestinal toxicity was determined using the method described by Cioli et al. [9]. The studies
−1
were carried out on healthy Wistar rats (150-200 g) at 300 μmol kg . The animals were divided into
four groups of six animals each. Group I served as a control and received vehicle only, group II
−1
−1
received pure diclofenac at 300 μmol kg , group III received lactam at 300 μmol kg and group IV
−1
received lumiracoxib at 300 μmol kg . The animals were fasted 8 h prior to receive the treatments,
then they received free access to food and water, and they were sacrificed 17 h later. The rats’ gastric
mucosa was examined using a 4 × binocular magnifier. The lesions were counted and divided into
large (larger than 2 mm in diameter), small (1-2 mm) and puntiform (less than 1 mm). For each
stomach, the severity of mucosal damage was assessed according to the following scoring system: 0 -
no lesions or up to five puntiform lesions; 1 - more than five puntiform lesions; 2 - one to five small
ulcers; 3 - more than five small ulcers or one large ulcer; 4 - more than one large ulcer. The mean score
of each treated group minus the mean score of the control group was considered as the ‘severity index’
of gastric damage. Statistical analysis was performed with ANOVA followed by Tukey’s test.
3
.8. Thioglycollate-Induced Peritonitis in Mice
The experiments were performed according to the method described by Savill et al. [10]. The animals
were treated with lactam derivative 1 and indomethacin (300 μmol/Kg, p.o), and 1 mL 3% thioglycollate
Difco-BD Biosciences) i.p. was administered 1 h later. After 4 h, the animals were killed by cervical
(

Molecules 2010, 15
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dislocation and the peritoneal cavity was washed with 1.5 mL cold PBS. The peritoneal exudates cells
were retrieved and their volume was measured. The levels of significance between the experimental
groups and the control were made using ANOVA in the tutorial Prisma®. The values were considered
significant when *p < 0.05. The results were expressed as average ± S.E.M of the average.
3.9. In vitro Hydrolysis of 1-(2-chloro-6-fluorophenyl)-5-methylindolin-2-one (1) in buffer (pH 1.2 and 7.4)
3
.9.1. Analytical protocol
The concentrations of 1-(2-chloro-6-fluorophenyl)-5-methylindolin-2-one (1) and lumiracoxib (2)
in buffer were measured by an HPLC method. The HPLC system used was a Shimadzu model
LC-10AD equipped with a model SPD-10A UV-Vis detector (Shimadzu). The compounds were
separated on a Shimadzu Shim-pack CLCODS (M) reverse phase C column (5 μm particle size, 250
1
8
mm × 4.6 mm I.D) running an isocratic flow of 65% acetonitrile and 35% 25 mM sodium
acetate/acetic acid aqueous buffer solution at pH 4.0, with a flow rate of 1.5 mL/min over 10 min and
2
detection at 280 nm. The calibration curve was linear (r = 0.9999; n = 6) in the range 0.5-20 μg/mL.
3
.9.2. In vitro hydrolysis of 1-(2-chloro-6-fluorophenyl)-5-methylindolin-2-one (1) in buffer (pH 1.2
and 7.4)
For buffer hydrolysis, an appropriate amount of pure 1-(2-chloro-6-fluorophenyl)-5-methylindolin-
-one (1) was weighed and diluted in sodium acetate (pH 1.2 and 7.4) to a concentration of 20 μg/mL.
The samples solutions (20 μL) were injected into the HPLC system as follows: pH 1.2: zero; 0.5; 1.0;
.0; 3.0; 4.0; 8.0; 12.0 and 24.0 h; pH 7.4: zero; 0.5; 1.0; 2.0; 3.0; 4.0; 8.0; 12.0 and 24.0 h. All
2
2
samples were done in triplicate and the results are expressed by the average of the solutions
concentrations.
3
.9.3. Statistical analysis
The data were expressed as mean ± SEM, and analyzed by one-way analysis of variance (ANOVA)
followed by Tukey’s test for multiple comparisons among groups (Sigma-Stat software). The
®
calibration curve and variation coefficients (CV %) were calculated using the Origin program.
4
. Conclusions
The lactam derivative 1 presented anti-inflammatory and analgesic activity when compared to the
lumiracoxib. The decrease of gastroulceration is the derivative’s main advantage and this effect could
be related to the masking of the carboxyl group in the lactam function. Furthermore, the lactam
derivative 1 is able to inhibit cell migration by 50.4% using a thioglycollate-induced peritonitis model,
while lumiracoxib inhibited only 18%. The stability studies demonstrate that the lactam function is
stable and does not hydrolyze at pHs 1.2 and 7.4. The discovery of new NSAID compounds with better
safety and efficiency is important for the treatment of chronic inflammatory diseases. The next step is
the pre-clinical pharmacokinetic and hepatotoxicity studies of the lactam derivative 1.

Molecules 2010, 15
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Acknowledgements
This study was sponsored by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
CAPES) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). The study protocol
(
was approved by the Brazilian Ethics Committee (no. 24/2010).
References and Notes
1
2
.
.
Harrak, Y.; Casula, G.; Basset, J.; Rosell, G.; Plescia, S.; Raffa, D.; Cusimano, M.G.; Pouplana,
R.; Pujol, M.D. Synthesis, anti-inflammatory activity, and in vitro antitumor effect of a novel
class of cyclooxygenase inhibitors: 4-(Aryloyl)phenyl methyl sulfones. J. Med. Chem. 2010, 53,
6
560-6571.
Chung, M.C.; Santos, J.L.; Oliveira, E.V.; Blau, L.; Menegon, R.F.; Peccinini, R.G. Synthesis, ex
vivo and in vitro hydrolysis study of an indoline derivative designed as an anti-inflammatory with
reduced gastric ulceration properties. Molecules 2009, 14, 3187-3197.
3
4
.
.
Silva, A.T.A.; Chung, M.C.; Castro, L.F.; Guido, R.V.; Ferreira, E.I. Advances in prodrug design.
Mini Rev. Med. Chem. 2005, 10, 893-914.
Wallace, J.L. The 1994 Merck Frosst Award. Mechanisms of nonsteroidal anti-inflammatory drug
(
NSAID) induced gastrointestinal damage—potential for development of gastrointestinal tract
safe NSAIDs. Can. J. Physiol. Pharmacol. 1994, 72, 1493-1498.
5
.
Bandhari, K.H.; Newa, M.; Yoon, S.I.; Kim, J.S.; Jang, K.Y.; Kim, J.A.; Yoo, B.K.; Woo, J.S.; Lee,
J.H.; Kim, D.D.; Choi, H.G.; Yong, C.S. Evaluation of physicochemical properties, skin permeation
and accumulation profile of ketorolac fatty ester prodrug. Biol. Pham. Bull. 2007, 11, 2211-2216.
Ranatunge, R.R.; Augustyniak, M.E.; Dhawan, V.; Ellis, J.L.; Garvey, D.S.; Janero, D.R.; Letts,
L.G.; Richardson, S.K.; Shumway, M.J.; Trocha, A.M.; Young, D.V.; Zemtseva, I.S. Synthesis
and anti-inflammatory activity of series of N-substituted naproxen glycolamides: nitric oxide-
donor naproxen prodrugs. Bioorg. Med. Chem. 2006, 14, 2589-2599.
6
.
7.
8.
9.
Winter, C.; Risley, E.; Nuss, G. Carrageenin-induced edema in hind paw of the rat as an assay for
anti-inflammatory drugs. Proc. Soc. Exp. Biol. Med. 1962, 111, 544-547.
Seigmund, E.; Cadmus, R.; Lu, G. A method for evaluating both non-narcotic and narcotic
analgesics. Proc. Soc. Exp. Biol. Med. 1957, 95, 729-733.
Cioli, V.; Putzolu, S.; Rossi, V.; Corza, B.P.; Corradino, C. The role of direct tissue contact in the
production of gastrointestinal ulcers by anti-inflammatory drugs in rats. Toxicol. Appl. Pharmacol.
1
979, 50, 283-289.
1
0. Savill, J.S.; Wyllie, A.H.; Henson, J.E.; Walport, M.J.; Henson, P.M.; Haslett, C. M. Macrophage
phagocytocis of aging neutrophils in inflammation; programmed cell death in the neutrophil leads
to its recognition by macrophages. J. Clin. Invest. 1989, 83, 865-875.
Sample Availability: Not available.
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2010 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
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