Photodegradation and in vitro phototoxicity of aceclofenac

Article abstract of DOI:10.1691/ph.2007.5.5139

Aceclofenac (Airtal) (1) is a photoallergy and phototoxic anti-inflammatory and analgesic agent. This drug is photolabile under aerobic and anaerobic conditions. Irradiation of an ethanol-solution of aceclofenac under oxygen or argon at 290-320 nm affords

Full text of DOI:10.1691/ph.2007.5.5139

ORIGINAL ARTICLES
Laboratorio de Fotoqu ´ı mica, Centro de Qu ´ı mica, Instituto Venezolano de Investigaciones Cient ´ı ficas I.V.I.C., Caracas,
Venezuela
Photodegradation and in vitro phototoxicity of aceclofenac
F. Vargas, C. Rivas, T. Zoltan, A. Fuentes, L. Padr o´ n, Y. D ´ı az, C. Izzo
Received August 9, 2005, accepted December 8, 2005
Dr. Franklin Vargas, Laboratorio de Fotoqu ´ı mica, Centro de qu ´ı mica,
Instituto Venezolano de Investigaciones Cient ´ı ficas I.V.I.C., Carretera Panamericana Klm. 11,
Apartado 21827, Caracas 1020-A, Venezuela
fvargas@ivic.ivic.ve
Pharmazie 62: 337–341 (2007)
doi: 10.1691/ph.2007.5.5139
1
Aceclofenac (Airtal ) (1) is a photoallergic and phototoxic anti-inflammatory and analgesic agent. This
drug is photolabile under aerobic and anaerobic conditions. Irradiation of an ethanol-solution of aceclo-
fenac under oxygen or argon at 290–320 nm affords via decarboxlation compound 2 as the main
isolated and spectroscopically identified photoproduct, besides hydroxylamine derivates 3 and 4. A
radical intermediate was evidenced spectrophotometrically with GSH and DTNB, as well as by the
dimerization of cysteine. Red blood cell lysis photosensitized by 1–4 was investigated. Furthermore, in
a lipid-photoperoxidation test with linoleic acid the in vitro phototoxicity of aceclofenac was also veri-
fied. The photoinduced generation of peroxide by compound 1 was determined during the irradiation in
presence of NADPH by chemiluminescence. In relation to the photoallergic activity of this drug, the
interaction of aceclofenac with human serum albumin (HSA) has been studied through fluorescence
spectroscopy. No photoinduced binding was observed after irradiation of compounds 1 in the pre-
sence of human serum albumin.
1. Introduction
2. Investigations and results
0
0
Aceclofenac
[[2-(2 ,6 -dichlorophenyl)amino]phenylace-
The photolysis of 1 (absorption maxima at 205 and
284 nm in methanol) was carried out under UV-B light
(290–320 nm) and a decrease was followed by monitoring
the disappearance of these bands, at 20 min intervals. The
results are shown, for a methanolic solution (1 ꢀ 10 M)
of 1, in Fig. 1. No difference in the photoproduct yields or
in the velocity of the reaction was found in the photolysis
under either O2 or argon.
The photolysis reaction of 1 is shown in the Scheme. The
major photoproduct of 1 was 2 (yield ¼ 89%). The forma-
tion of photoproduct 2 is compatible with an initial excita-
tion of aceclofenac after light absorption, followed by a
toxyacetic acid] (1), is a non-steroidal anti-inflammatory
drug with analgesic properties, which is widely used in
tablets and intramuscular injections (Grau 1991; Brogden
ꢁ4
1
996; Legrand 2004). This compound is capable of initiat-
ing adverse light-induced biological effects. Some cases of
photodermatitis from this drug have been reported (Lud-
wig 2003). Recently, cases of photoallergic reactions trig-
gered by aceclofenac have also been reported (Goday Bu-
jan 2001). The possibility that photoproducts of the drug
are involved in the photosensitivity, photoallergic or
phototoxic reactions is now being studied by us. It is pre-
sumed that there must be a relationship between photoche-
mical behavior and phototoxicity. In this context, very lit-
tle is known as yet about the interrelation between
photochemistry and its phototoxicity. This fact has
prompted us to examine the photolysis of 1. These studies
have led to the isolation of the photoproducts of 1 and
their characterization by spectroscopic means. The forma-
tion of 2 probably involves the generation of a radical
intermediate. The latter could be detected when the irra-
diation was carried out in presence of either reduced glu-
tathione or cysteine used as radical scavengers (Costanzo
1
989). The present work also reports the phototoxic ef-
fects of 1 and its photolysis products on human erythro-
cytes, out of which compounds 2–4 (Scheme) turned out
to be less effective than 1 in their photohemolytic proper-
ties (Costanzo 1989) and the in vitro study of the photo-
oxidant properties of aceclofenac by chemiluminescence
Fig. 1: UV monitoring of the photolysis of aceclofenac by irradiation with
UV-B light at intervals every 2 min.
(
Mouithys-Mickalad 2000).
Pharmazie 62 (2007) 5
337

ORIGINAL ARTICLES
Scheme
cleavage of the CH2ꢁCOOH bond (decarboxylation)
which would give a radical intermediate that via hydrogen
abstraction could give rise to this compound. The methyl
ester derivate 2 formed in the photolysis of 1 was com-
pared with the compound described in the literature, used
as a compound for determination by gas liquid chromato-
graphy of this drug and their metabolites, as well as in the
synthesis of new prodrugs of its degradation product, di-
clofenac (Sastry 1988; Poiger 2001; Sharma 2003).
The formation of compounds 3 and 4 (yield ¼ 7 and 4%
respectively) could come from the hydroxylation of the
amino group and subsequent hydrolysis of the resulting
molecule. Similar compounds arising from the hydroxyla-
tion of the diclofenac have been previously reported in the
literature (Bort 1999).
An indication of the free radical formation was given by
the capacity of 1 to induce dimerization of reduced glu-
tathione (GSH) under the same conditions of irradiation,
as detected by UV-Vis spectrophotometry (Figure 3) by
titration with 5,5 dithiobis(2-nitrobenzoic acid) (DTNB) at
412 nm, and the presence of cysteine dimers as detected
by mass spectroscopy.
It may be inferred that the phototoxicity mechanism for
aceclofenac most probably involves reactions of a free ra-
dical intermediate and stable photoproducts with cellular
components. Future studies will include development of
devices to discriminate between effects due either to stable
photoproducts or to short-lived intermediates as well as
the use of cultures and co-cultures of different human
cells mimicking the human skin.
0
Our investigation has indicated that 1 undergoes photohe-
molysis after 20 min irradiation (l ¼ 290–320 nm) at any
of the concentrations tested under either aerobic or anaero-
bic conditions. Nevertheless, oxygen markedly enhances
lysis. Typical experiments are illustrated in Fig. 2. No
lysis was observed either when cells were irradiated for
We are currently investigating the toxic effects of the iso-
lated photoproducts using in vitro as well as in vivo mod-
els. In the near future patients photosensitive to aceclofe-
nac will be patch-tested with these photoproducts.
The observed photohemolysis induced by aceclofenac
might reflect extensive photoperoxidation of the mem-
brane lipids (Fig. 4). When phosphate buffer saline (PBS)
solutions of linoleic acid were irradiated in the presence
of 1 significant amounts of dienic hydroperoxides were
formed, as evidenced spectrophotometrically by the appear-
ance of a new UV-absorption band at 233 nm (Recknagel
and Glenden 1984).
The chemiluminescence (CL) observed both in the pro-
cesses induced by H2O2 as well as by ferrous ion in lumi-
nol was used to evaluate the generation of radical hydro-
xyl (OH) of 1. In the former, a quantitative increase was
observed when 1 was irradiated in the presence of nicotin-
amide adenine dinucleotide phosphate (NADPH). The CL
2
0 min in the absence of drugs or photoproducts, or when
ꢂ
they were incubated for 2 h in the dark at 37 C with
100 mg/ml of the drug.
The possibility that singlet oxygen was produced during
the irradiation of 1 in the red blood cell (RBC) solution
was examined. In fact, this compound did not produce
singlet oxygen as was shown when the photolysis of 1
was carried out in the presence of the usual singlet oxygen
and superoxide anion scavengers such as 2,5-dimethyl-
furan (DMF), sodium azide (NaN3) and of superoxide dis-
mutase (SOD) respectively used during the photohemolysis
test. No changes were observed during the photohemoly-
sis test of 1 in the presence of the reactive oxygen species
activity generated by luminol reflects the release of reac-
1
.ꢁ
.
(
ROS) scavengers as O2 and O2.
tive oxygen species (ROS) specially OH (Lundqvist and
0
0
0
0
0
0
0
,7
,6
,5
,4
,3
,2
,1
0
0,4
0,35
0,3
0
0
0
,25
,2
,15
,1
0
0
,05
0
0
10
20
30
40
50
Time (min)
6
Fig. 2: Photohemolysis of RBC (3.3 ꢀ 10 cells/ml): & photosensitized
with UV-B and aceclofenac (1); ~ in the presence of radical sca-
ꢁ
4
vengers [GSH] ¼ 1.0 ꢀ 10 M and * control in darkness. Each
point represents the mean ꢃ SEM (less than 5%) derived from four
observations
Fig. 3: Photoinduced dimerization of GSH (DTNB titration at 412 nm) in
presence of aceclofenac (1)
338
Pharmazie 62 (2007) 5

ORIGINAL ARTICLES
photohemolysis occurring via a radical-chain mechanism.
0
0
0
0
,8
,6
,4
,2
0
The photohemolysis assay, as well as an in vitro phototoxi-
city test, has evidenced the involvement of radical-
mediated cellular membrane damage in the skin photosen-
sitization by 1. Lipid photoperoxidation certainly correlates
with the damage produced in cell membranes and thus
with the observed photohemolysis. It was also possible to
prove the generation of hydroxyl radicals during the irra-
diation of the 1 by means of chemiluminescence methods.
The results reported in the present paper may be very use-
ful from the medical standpoint. The methods used here
may not only help to elucidate the photobiological action
of 1 but also that of other photoreactive pharmaceutical
drugs. That these compounds are not bound to HSA could
be of physiological significance as this protein could be the
initial target of these drugs in the organism. This result can
be incongruous with the reported cases of photoallergy.
0
10
20
30
40
50
60
Time (min)
Linoleic acid + 1 (hv) Linoleic acid (hv) Linoleic acid + 1 (dark)
ꢁ3
Fig. 4: Photoperoxidation of linoleic acid (10 M) sensitized by aceclofe-
4. Experimental
nac (1)
4
.1. Chemicals
Aceclofenac (1) was extracted from Airtal1 (Leti laboratory) by means of
a Soxhlet extractor with ethanol and recrystalized from the same solvent.
2
2
1
1
0
0
,5
,0
,5
,0
,5
,0
1
The purity was 99.2% as determined by H NMR-spectroscopy and UV-Vis
0
spectrometry. 5,5 -Dithiobis(2-nitrobenzoic acid) (DTNB), reduced gluta-
thione (GSH), sodium azide (NaN3), lysis buffer (NH4Cl, KHCO3, EDTA),
histidine, nicotinamide adenine dinucleotide (NADH) and cysteine were
commercially obtained from Aldrich (Steinheim, Germany), while superox-
ide dismutase (SOD) was purchased from Sigma (St. Louis, MO, USA).
Human serum albumin (HSA) was purchased from Sigma. All analytical
or HPLC grade solvents were obtained from Merck (Darmstadt, Germany).
1
1
/ UV-B
(darknes)
4
.2. Photolysis
Photolysis of 1 was carried out in methanol solution (1.50 mmol in 50 ml)
ꢂ
0
2
4
6
8
10
at 20 C during 6 h in a Rayonet photochemical chamber reactor (model
T im e(s)
RPR-100, Southern New England Ultraviolet Company-USA) equipped
with 16 phosphorus lamps with an emission maximum in UV-A between
3
20–400 nm and UV-B 290–320 nm (23 mW/cm2 of irradiance as meas-
Fig. 5: Chemiluminescence generated by H2O2 and luminol after the irra-
diation of aceclofenac in the presence of NADPH. Data are the
mean and SEM, (n ¼ 4, p < 0.05 vs. control; analysis of variance)
ured with a UVX Digital Radiometer, Melles Griot, USA). The distance
between the light sources to the test aliquots was 10 cm. The temperatures
detected in the cuvette during a standard 1 h irradiation were no higher
ꢂ
than 28 C. In the determination of quantum yields the photolysis was
Dahlgren 1996). Hydroxyl radical was also generated by
the addition of a freshly prepared FeSO4 solution to a
mixture containing luminol as measured by chemilumines-
cence. These results are in agreement with previous obser-
vations with other drugs (Yildiz and Demiry u¨ rek 1998)
where the addition of a ferrous ion salt to buffered solu-
tions generates the hydroxyl radical-mediated oxidative re-
actions. The increase of the production of CL of aceclofe-
nac is shown in Fig. 5.
limited to less than 10% to minimize light absorption and reaction of
photoproducts. The photon flux incident on 3 ml of solution in quartz cuv-
ettes of 1 cm optical path was measured by means of a ferric oxalate actin-
ometer and was of the order of 10 –10 quanta s (Vargas 2002).
Either oxygen or argon was bubbled through the reaction mixture through-
out the whole irradiation process. The photodegradation reaction was
followed by means a Perkin Elmer 559 UV-visible spectrophotometer, a
15
16
ꢁ1
Milton-Roy Spectronic 3000 array instrument (Milton Roy Company-
USA), 1H NMR as well as by TLC and HPLC (Water Delta Prep 4000
equipped with a 3.9 ꢀ 300 mm, 10 mm Bondapak C18 column using a
CH2Cl2/MeOH binary solvent system) (El Kousy 1999). After the irradia-
tion was stopped the solvent was evaporated under reduced pressure
No photoinduced binding was observed after irradiation of
(
(
14 Torr) and the residue was purified by chromatography on a silica gel
230 mesh) column. The elution was carried out by means of solvent mix-
1
in the presence of human serum albumin.
tures (dichloromethane/methanol) (3 : 1 vol/vol). The structures of the iso-
lated products were elucidated by 1H NMR and C NMR (Bruker Aspect
13
3. Discussion
3
000, 300 and 100 MHZ respectively), IR (Nicolet DX V 5.07) and mass
spectra (Varian Saturn 2000) in connection with a Varian chromatograph
equipped with a 30-m capillary (CP-Sil, 8CB-MS). Elemental analyses
were performed by the “Laboratorio Nacional de An a´ lisis Qu ´ı mico” –
IVIC – Centro de Qu ´ı mica, Caracas, Venezuela in a Fisions Instrument
EA-1108. The results were in an acceptable range.
Irradiation of a methanol solution of 1 produces three
photoproducts, one major product, compound 2, is gener-
ated by photodecarboxylation and the two minor products
3
and 4 by photooxidation of the amino group. 1 under-
Photoproduct 2 (yield: 88%): IR (KBr): 3050, 2960, 1770, 1735, 1580,
goes photodecarboxylation by homolytic rupture of the
COOH-CH2O bond from its singlet state.
ꢁ1
1
1
490, 1454, 1383, 1230, 1205, 1183, 1090, 810, 750, 735 cm . H NMR
(CDCl , 300 MHz): d ¼ 8.74 (m, 2 H, aromatic-H), 8.03 (s, 1H, NꢁH),
3
After establishing the photochemistry of 1 in solution, it
appeared interesting to evaluate its in vitro phototoxicity
and to extend this study to its photoproducts 2, 3 and 4.
The photohemolysis of red blood cells was chosen as a
suitable test for this purpose. Our results showed that only
7.49 (m, 1 H, aromatic-H), 7.19 (m, 1 H, aromatic-H), 6.93 (m, 1 H, aro-
matic-H), 6.85 (m, 1 H, aromatic-H), 6.83 (m, 1 H, aromatic-H), 3.94 (s,
1
3
2
H, CꢁCH2ꢁCOOꢁ), 3.67 (s, 3 H, CH3). C NMR (CDCl3, 100 MHz):
d ¼ 178.00 (s, C¼O), 142.60 (s, aromatic-C), 141.20 (s, aromatic-C),
129.70 (d, aromatics-2 CH), 128.80 (d, aromatic-CH), 127.89 (d, aromatic-
CH), 127.30 (s, aromatics-2 CꢁCl), 124.20 (d, aromatic-CH), 118.00 (d,
aromatic-CH), 116.10 (s, aromatic-C), 108.79 (d, aromatic-CH), 51.70 (q,
CH3), 36.50 (t, CH2). MS: m/z (%) ¼ 313 (7), 311 (56), 309 (100), 276
1
was able to photosensitize the lysis of red bood cells.
The addition of GSH as a radical scavenger resulted in an
enhanced lifetime of erythrocytes, which is in favor of
(25), 274 (82), 242 (44), 214 (55), 178 (20).
C H Cl NO
2
15
13
2
Pharmazie 62 (2007) 5
339

ORIGINAL ARTICLES
Photoproduct 3 (yield: 7%): IR (KBr): 3450, 3055, 3000, 2700, 2500,
4.6. Chemiluminescence experiments
ꢁ1 1
1
745, 1617, 1530, 1495, 1454, 1090, 930, 810, 743, 732 cm
. H NMR
Chemiluminescence (CL) was generated in cell-free systems; H2O2-in-
duced CL (as a blank): H2O2 (3.5 mM) was added to phosphate buffered
saline solution (PBS, 10 mM KH2PO4 and 150 mM NaCl, pH 7.2) and
luminol (250 mM, prepared daily in 2 M NaOH and diluted with PBS).
The aceclofenac-induced CL at different concentrations was dispense after
(
CDCl3, 300 MHz): d ¼ 8.72 (m, 2 H, aromatic-H), 8.48 (br.s, 2 H, ꢁCOOH
and NꢁOH), 7.70 (m, 1 H, aromatic-H), 7.23 (m, 1 H, aromatic-H), 6.99
(
4
1
m, 1 H, aromatic-H), 6.92 (m, 1 H, aromatic-H), 6.89 (m, 1 H, aromatic-H),
13
.13 (s, 2 H, ꢁCH2ꢁCOOH), 3.77 (s, 2 H, CH2ꢁCO). C NMR (CDCl3,
00 MHz): d ¼ 170.50 (s, C¼O), 168.10 (s, C¼O), 144.80 (s, aromatic-
irradiation with 2 phosphorus lamps with a emission maximum in UV-B
CꢁN), 138.90 (s, aromatic-CꢁN), 132.30 (s, aromatic-2 CꢁCl), 128.40 (d,
ꢂ
2
90–320 nm in presence of NADH. The generated CL at 37 C was meas-
aromatics-2 CH), 127.50 (d, aromatic-2 CH), 127.47 (d, aromatic-CH), 122
ured continuously for 10 min in
a Luminoskan Ascent luminometer
(
(
3
s, aromatic-C), 118.10 (d, aromatic-CH), 110.40 (d, aromatic-CH), 69.40
(ThermoLabsystems, Finland) in a 96-well Thermo Labsystems Microtiter
t, CH2), 36.00 (t, CH2). MS: m/z (%) ¼ 373 (13), 371 (68), 369 (100),
plate. (Lundqvist 1996; Vargas 2003; Yildiz 1998).
55 (12), 279 (22), 215 (47), 214 (90), 213 (38), 150 (12), 78 (10), 51
(
10). 42 (4).
C16H13Cl2NO5
4.7. Titration of the aceclofenac solutions with HSA
Photoproduct 4 (yield: 5%): IR (KBr): 3500, 3050, 3000, 2690, 2500,
Titration of the aceclofenac solutions (1.0 ꢀ 10ꢁ M) with HSA was per-
4
ꢁ1 1
1
740, 1094, 1430, 930, 810, 752, 733 cm
d ¼ 9.50 (br.s, 2 H, –COOH and NꢁOH), 8.72 (m, 2 H, aromatic-H), 7.90
m, 1 H, aromatic-H), 7.40 (m, 1 H, aromatic-H), 7.10 (m, 1 H, aromatic-H),
. H NMR (CDCl3, 300 MHz):
formed by addition of appropriate aliquots of an aqueous-buffered PBS
10 mM KH2PO4 and 150 mM NaCl) HSA stock solution at 1.0 mM
pH 7.4) concentration directly to the absorbance or fluorescence cell so
(
(
(
6
.95 (m, 1 H, aromatic-H), 6.90 (m, 1 H, aromatic-H), 3.40 (s, 2 H,
CH2ꢁCOO). C NMR (CDCl3, 100 MHz): d ¼ 175.00 (s, C¼O), 145.00
ꢁ
4
13
that the final protein concentration was in the range from 0 to 5.0 ꢀ 10
ꢁ
M. The solutions were allowed to incubate in the dark for 20 min. Then,
samples were irradiated in 1-cm2 Suprasil quartz cells under the above
mentioned conditions for various time periods. Control included drug pro-
tein mixtures kept in the dark and HSA solutions irradiated for the same
periods of time. The drug was separated from the protein using a Sepha-
dex G-25 column equilibrated with PBS. The photobinding was monitored
by fluorescence spectroscopy (Moreno 1999).
(
s, aromatic-CꢁN), 137.20 (s, aromatic-CꢁN), 133.00 (s, aromatic-
2
CꢁCl), 127.52 (d, aromatics-2 CH), 128.40 (d, aromatic-CH), 127.50 (d,
aromatic-2 CH), 126.20 (d, aromatic-CH), 123.50 (s, aromatic-C), 117.00
(
(
2
d, aromatic-CH), 109.00 (d, aromatic-CH), 35.00 (t, CH2). MS: m/z
%) ¼ 315 (8), 313 (59), 311 (100), 278 (40), 277 (35), 276 (80), 241 (5),
13 (56), 177 (24), 150 (15), 78 (4), 64 (8).
C14H11Cl2NO3
4
.8. Statistical treatment of results
4
.3. Photoinduced hemolysis of RBC by aceclofenac
At least three independent experiments were performed except where indi-
cated otherwise. The results are expressed as a mean ꢃ S.E.M. derived
from 3–4 observations. The level of significance accepted was p ꢄ 0.05.
A red blood cells (RBC) suspension from three different samples of
freshly obtained human erythrocytes was prepared by washing them four
times with tenfold volume of a phosphate-buffered saline solution (PBS)
pH 7.4 (0.01 M phosphate buffer and 0.135 M NaCl), centrifuging the cells
each time at 2500 ꢀ g for 15 min and carefully removing the supernatant.
For the photohemolysis experiments RBC were diluted in PBS containing
the compounds 1, 2 or 3 so that the resultant suspension had an optical
density (OD) of 0.4–0.8 at 650 nm. An OD value of 0.5 corresponded to
Acknowledgements: This research was supported by a grant from “Consejo
Nacional de Investigaciones Cient ´ı ficas y Tecnol o´ gicas” CONICIT-Vene-
zuela (S1-2502 and S1-960011724), Laboratorio Nacional de An a´ lisis Qu ´ı -
o
mico, proyecto N Lab. 1998003690 and Fundaci o´ n Polar.
6
ꢁ1
3
.3 ꢀ 10 cells ml . The photon flux incident on the cuvettes (measured
16 ꢁ1
as before) was 2 ꢀ 10 photon s . Hence samples received, on the aver-
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5
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ꢁ
4
1
.0 ꢀ 10 M GSH as radical scavengers. The hemolysis rate was deter-
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4
.4. Photosensitized oxidation of glutathione and cysteine by aceclofenac
To determine the photoinduced oxidation of glutathione in the presence of
aceclofenac, it was irradiated in presence of compound 1. Detection of
glutathione depletion was carried out with 5,5 -dithiobis(2-nitrobenzoic
0
acid) (DTNB) and performed by means of similar assays as those de-
scribed in Beutler’s text book (Beutler 1984). However, the red cells used
as a blank were hemolyzed with a “lysis buffer” (NH4Cl, KHCO3, EDTA,
pH 7.5). The same experiment was used to determine the photoinduced
oxidation of cysteine in the presence of aceclofenac. Similar control ex-
periments were carried out without irradiation of the samples.
In a separate experiment, in order to detect the probable formation of a
radical intermediate, aceclofenac (1) (0.5 mmol in 50 ml H2O) was irra-
diated under the conditions described above, in the presence of equimo-
lar quantities of reduced glutathione (GSH) or cysteine as radical scaven-
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4
.5. Photosensitized peroxidation of linoleic acid
ꢁ
3
Linoleic acid 10 M in PBS phosphate buffered saline solution PBS
(10 mM KH2PO4 and 150 mM NaCl) in water, pH 7.2, with Tween 20
(
0.1%), was irradiated in the presence of compound 1 and in a pre-irra-
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ꢁ5
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sive increase of a new band at l ¼ 233 nm (Recknagel and Glenden 1984).
340
Pharmazie 62 (2007) 5

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