Different in vitro activity of flurbiprofen and its enantiomers on human articular cartilage

Article abstract of DOI:10.1016/S0014-827X(03)00196-4

The 2-arylpropionic acid derivatives or 'profens' are an important group of non-steroidal anti-inflammatory drugs that have been used for the symptomatic treatment of various forms of arthritis. These compounds are chiral and the majority of them are stil

Full text of DOI:10.1016/S0014-827X(03)00196-4

Il Farmaco 58 (2003) 1339Á1344
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www.elsevier.com/locate/farmac
Different in vitro activity of flurbiprofen and its enantiomers on
human articular cartilage
A.M. Panico a,*, V. Cardile b, F. Vittorio a, G. Ronsisvalle a, G.M. Scoto c, C. Parenti c,
B. Gentile c, R. Morrone d, G. Nicolosi d
a Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Catania, V.le A. Doria 6, 95125 Catania, Italy
b Department of Physiological Sciences, University of Catania, V.le A. Doria 6, 95125 Catania, Italy
c Department of Pharmaceutical Sciences, Pharmacology section, Faculty of Pharmacy, University of Catania, V.le A. Doria 6, 95125 Catania, Italy
d CNR Á
/
Istituto di Chimica Biomolecolare, Sezione Catania Via del Santuario 110, 95028 Valverde Catania, Italy
Received 4 April 2003; accepted 19 July 2003
Abstract
The 2-arylpropionic acid derivatives or ‘profens’ are an important group of non-steroidal anti-inflammatory drugs that have been
used for the symptomatic treatment of various forms of arthritis. These compounds are chiral and the majority of them are still
marketed as racemate although it is known that the (S)- form is the principal effective in the cyclooxygenase inhibition. However,
recent findings suggest that certain pharmacological effect of 2-arylpropionic acids cannot be attributed exclusively to the (S)-(ꢀ)
/
enantiomer. To obtain further insights into the pharmacological effect of profens, the present study investigated the influence of
racemic and pure enantiomers of flurbiprofen on the production of nitric oxide and glycosaminoglycans, key molecules involved in
cartilage destruction. The culture of human articular cartilage stimulated by interleukin-1b (IL-1b), which plays an important role in
the degradation of cartilage, has been established, as a profit experimental model, for reproducing the mechanisms involved in the
pathophysiology of arthritic diseases. Our results show that mainly (S)-(ꢀ)-flurbiprofen decreases, at therapeutically concentra-
/
tions, the IL-1b induced cartilage destruction.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: (S)-(ꢀ)-Flurbiprofen; Arthritis; Cartilage; NO; GAGs
/
1. Introduction
enzymes, called NO synthases [4Á6]. Two NO synthases
/
are constitutively expressed (eNOS and nNOS), the
third one (iNOS) is expressed in response to cytokines
or lipolysaccarides in smooth muscle cells, macrophages
and hepatocytes as well as in chondrocytes and it could
play a role in inflammatory and immunological host
defence reactions [7,8]. NO has been shown to mediate
some of the catabolic responses elicited by IL-1b in
chondrocytes such as suppression of proteoglycan
synthesis, induction of apoptosis, activation of collage-
nase gene expression and inhibition of cell proliferation
The destruction of connective tissue matrix compo-
nents in rheumatoid arthritis (RA) and osteoarthritis
(OA) is a serious condition that causes the progressive
destruction of joint. It is characterized by loss of
articular cartilage and it has been suggested that
interleukin-1b (IL-1b) plays the major role in the
pathophysiology of these diseases [1Á3]. In articular
/
chondrocytes and synovial cells, IL-1b induces the net
loss of cartilage proteoglycan by both stimulating the
proteoglycan degradation and inhibiting the proteogly-
can synthesis [3]. Moreover elevation of IL-1b induces
the production of nitric oxide (NO) in fibroblasts and
chondrocytes. NO is a free radical produced by three
[9Á14].
/
Non-steroidal anti-inflammatory drugs (NSAIDs) are
one of the widely used forms of therapy for OA. The
traditional rationale for NSAIDs use is their ability to
inhibit cyclooxygenase (COX) and hence suppress the
inflammatory process and the pain [15,16]. Recently, it
has been suggested that some NSAIDs might protect
* Corresponding author.
E-mail address: panico@unict.it (A.M. Panico).
´
0014-827X/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0014-827X(03)00196-4

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A.M. Panico et al. / Il Farmaco 58 (2003) 1339Á1344
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against cartilage and bone destruction in OA. Whereas
other, such as indomethacin, might accelerate joint
damage, possibly as a result of inhibitory effects on
cartilage proteoglycan synthesis or by enhancing pro-
2. Materials and methods
2.1. Preparation of enantiopure (R)- and (S)-
flurbiprofen
duction of cartilage bone-destructive IL-1b or both [17Á
/
To a solution of racemic flurbiprofen, (9)-1 (1 g;
/
24]. There are several reports that certain NSAIDs may,
in addition to inhibiting COX, modify cytokine produc-
tion [25]. The inhibition of these cytokines by NSAIDs
may be an alternative mechanism of action [26].
4.098 mmol) in CH3CN (100 ml) containing tripropyl
orthoformate (2.65 ml; 12.294 mmol), 10 g of lipase
from Candida antarctica (Novozyme 435†) were added
(Scheme 1). The mixture was incubated under shaking at
45 8C (300 rpm,) and the reaction progress was moni-
tored by HPLC equipped with a chiral column (S,S)-
The 2-arylpropionic acid derivatives, or ‘profens’,
constitute an important group of NSAIDs. Chemically
they are weak acids and by virtue of a chiral carbon
atom on the propionic acid side chain exist as enantio-
mer pairs, with (S)-isomers primarily responsible for
inhibition of prostaglandin production and of inflam-
Whelk-O1, 5 mm, 250ꢁ
/
4 mm; hexane/2-propanol/acetic
acid 98:2:0.5 as the eluent at a flow rate of 0.9 ml/min;
for the enantiomers of 1 the selectivity factor (a) and the
resolution factor (Rs) were 1.21 and 1.38, respectively).
The ee value of flurbiprofen propyl ester 2 was
matory events [27Á29]. The majority of these acids is
/
still employed as racemate, this is the case for the
flurbiprofen [(R,S)-2-(2-fluoro-4-biphenylyl)propanoic
acid]. For our studies we have realised to separation of
both the enantiomers resorting to two complementary
enzymatic processes on the racemate as reported in
Scheme 1.
In this paper we have report the different properties of
rac-, (R)- and (S)-flurbiprofen on the anti-inflamma-
tory action, focusing our investigation on the produc-
tion of NO and glycosaminoglycans (GAGs), key
molecules involved in cartilage destruction. The enan-
tiopure forms of flurbiprofen have been obtained
through enantiomeric separation of the racemate by a
modified biocatalysed procedure reported in literature
[30,31]. The culture of human articular cartilage stimu-
lated by IL-1b has been established as a profitable
experimental model for reproducing the mechanisms
involved in arthritic diseases [32,33].
calculated from cꢂ
/
ees/(eesꢀeep) according to Ref.
/
[34]. After 120 h the reaction was stopped and the
enzyme removed by filtration. Removal of the solvent in
vacuo left a residue that was portioned between hexane
and NaHCO3 solution. The organic phase, made
anhydrous with Na2SO4, subjected to evaporation
furnished 670 mg of (R)-2 with 66% ee. Acidification
of the aqueous phase, followed by extraction with
hexane, gave 390 mg of S-1 (yield, 39%) with ee ꢀ98%.
/
The enantioenriched ester (R)-2 (500 mg) was dis-
solved in CH3CN (50 ml) containing 500 ml of water and
lipase from C. antarctica (1 g) was added. The mixture
was incubated under shaking at 45 8C (300 rpm) until
HPLC analysis indicated a conversion of 30% and ee
value ꢀ
of the reaction mixture furnished 150 mg of (R)-1.
[a]Dꢂ 42.78 (c 1 CHCl3), Literature [30] [a]Dꢂ
41.48 for (S)-1 with ee ꢀ95%.
/
98 for the (R)-acid produced. Workup as above
/
ꢃ
/
/
ꢀ
/
/
Scheme 1.

A.M. Panico et al. / Il Farmaco 58 (2003) 1339Á
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1344
1341
2.2. Drugs
3. Results
The test drugs were dissolved in dimethyl sulfoxide,
appropriately diluted in Dulbecco’s Modify Medium
(DMEM) and dispensed to the wells in 20 ml volumes to
give the desired final concentrations.
3.1. Effect of rac-, (R)- and (S)-flurbiprofen on the IL-
1b induced NO production
As showed in Fig. 1, NO production in cartilage
under basal condition (control condition) is very low
16.7091.1 mainly due to activity of the (cNOS). When
human articular cartilage was treated with IL-1b 10 ng/
ml for 120 h a substantial increase in NO production to
/
2.3. Culture of human articular cartilage
Human articular cartilage was obtained at replace-
ment surgery from patients with femoral neck fractures.
Cartilage was reduced in fragments of small dimensions,
and washed in 25 ml of Hank’s balanced salt solution/
antibiotic (penicillin 100 U/ml, streptomycin 100 mg/ml)
and shaken vigorously for 30 s; such operation mini-
mizes the risk of infection during the cartilage culture.
The single fragments of cartilage were set in a sterile
plate to 24 wells each containing 1 ml of DMEM phenol
red free, 10% v/v foetal calf serum, 10 mM glutamine,
100 U/ml penicillin, 100 mg/ml streptomycin, 50 mg/ml
gentamycin and 2.5 mg/ml of amphotericin B and it was
kept in an incubator to 37 8C with 5% of CO2 and 95%
of air humidified for 24 h. After 24 h the culture media
were removed and cartilage samples were treated. After
120 h the supernatants of cartilage culture were collected
for different assays.
62.7991.6 mM was observed probably due to up-
/
regulation of iNOS.
Rac-, (R)- and (S)-flurbiprofen at 10 mM in combina-
tion with IL-1b reduced the production of nitrite
induced to 37.619
respectively.
/
1.6, 39.439
/
0.7, 34.9390.6 mM,
/
3.2. Effect of rac-, (R)- and (S)-flurbiprofen on the IL-
1b induced GAGs release
In Fig. 2 the levels of GAGs are reported. The
untreated control samples released, in the culture
medium, a GAGs amount of 51.02 mg/ml. When the
samples were treated with IL-1b at the dose of 10 ng/ml
a release of 180 mg/ml was observed.
Rac-, (R)- and (S)-flurbiprofen at 10 mM in combina-
tion with IL-1b reduced the IL-1b induced GAGs
release from matrix cartilage to 86.189
/
1.3, 106.009
/
2.4. Determination of nitrite
2.5, 61.5691.9 mg/ml.
/
Nitrite was determined by adding 100 ml of Griess
reagent (1% sulfanilamide and 0.1% naphthyl-ethylene-
diamine dihydrochloride in 5% of chloridric acid) to
100 ml of samples [35]. The optical density at 570 nm was
measured using a microtitrer plate reader. Nitrite
concentrations were calculated by comparison with
respective optical densities of standard solutions of
sodium nitrite prepared in medium.
4. Discussion
Despite their therapeutic efficacy for pain relief in the
treatment of OA, the role of NSAIDs in cartilage
metabolism is still debatable. Several in vitro and in
vivo studies have suggested that some NSAIDs promote
degeneration of articular cartilage by inhibiting the
biosynthesis of proteoglycans, which are crucial for
maintaining the viscoelastic function of this tissue [38].
Flurbiprofen belongs to the 2-arylpropionic acid
group of NSAIDs, and is available as racemate. In our
work the esterification in organic solvent of rac-1
catalysed by lipase from C. antarctica, using tripropyl
orthoformate as nuclephyilic donor, furnished the
enantiopure (S)-acid, (S)-1, as unreacted substrate
and the enantioenriched (R)-ester, (R)-2. The corres-
ponding hydrolysis of the (R)-2 in the presence of the
same lipase gave the enantiopure acid (R)-1 (Scheme 1).
The (R)-flurbiprofen is before primarily considered to
be the inactive isomer because it does not inhibit COX
activity [28]. However, previous studies have revealed
that it has antinociceptive and anti-tumor effects
whereas (S)-isomer is primarily responsible for inhibi-
tion of prostaglandin production and of inflammatory
events [29].
2.5. Determination of the GAGs
The level of GAGs was measured by spectrophoto-
metry with a solution of 1,9-dimethylmethylene blue by
the method of Farndale et al. [36,37].
The amount of GAGs was calculated from a
standard curve obtained for shark chondroitin
sulfate.
2.6. Statistical analysis
All the results presented are means9SEM of three
/
experiments performed on quadruplicate samples. The
Student’s t-test was used to evaluate the differences
between the means of each group. P B
/0.05 was
considered to be statistically significant.

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A.M. Panico et al. / Il Farmaco 58 (2003) 1339Á1344
/
Fig. 1. NO production (means9
flurbiprofen with IL-1b. Values are expressed as mM. *, significantly different from IL-1b treated samples (P B
/
SEM) in the culture medium from articular chondrocytes 120 h after the addition of (10 mM) of rac, S, R
/
0.05).
In this study we have investigated the effects of
flurbiprofen as racemate and (R)- and (S)-enantioforms
on production of NO and GAGs release in human
articular cartilage stimulated with IL-1b. The potential
role of NO in the pathophysiology of chronic inflam-
mation has been extensively explored. Studies have
demonstrated that sustained high NO levels can acce-
lerate cartilage catabolism and reduce its anabolism
and found that at concentration of 10 mM, which is
within the range of plasma concentration, (R) and (S)
enantiomers and racemate inhibited of 37.2, 44.3 and
40.10%, respectively with respect to IL-1b treated
samples IL-1b induced nitrite accumulation in cartilage
culture supernatants.
Next we examined the effect of rac-, (R)-, and (S)-
flurbiprofen on the release of sulfated GAGs, which is
likely to reflect proteoglycan release from chondrocytes.
In general the erosion of cartilage in OA is initially
caused by a decrease in the proteoglycan content,
followed by destruction of collagen fibers. Therefore,
we focused in the release proteoglycan in the matrix of
[10Á15] hence, interest in mechanism underlying the
/
regulation of nitrite level as well as pharmacological
approaches has substantially increased in recent years.
Therefore we have examined the effect of rac-, (R)-, and
(S)-flurbiprofen on the IL-1b induced production of NO
Fig. 2. GAGs release (means9
expressed as mg /ml. *, significantly different from IL-1b treated samples (P B
/
SEM) from articular chondrocytes 120 h after the addition of (10 mM) rac, S, R flurbiprofen, with IL-1b. Values are
/0.05).

A.M. Panico et al. / Il Farmaco 58 (2003) 1339Á
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