Synthesis and biological testing of Acyl-CoA-ketoprofen conjugates as selective irreversible inhibitors of COX-2

Article abstract of DOI:10.1016/S0968-0896(01)00330-3

Ketoprofenoyl-CoA thioester 3 was synthesized by coupling ketoprofen to coenzyme A using the mixed anhydride method. Diastereoisomeric compounds 3a and 3b corresponding to the enantiomers of ketoprofen, were obtained in optically pure form by preparative

Full text of DOI:10.1016/S0968-0896(01)00330-3

Bioorganic & Medicinal Chemistry 10 (2002) 753–757
Synthesis and Biological Testing of Acyl-CoA–Ketoprofen
Conjugates as Selective Irreversible Inhibitors of COX-2
Nicolas Levoin,b Francoise Chretien,a Francoise Lapicqueb,* and Yves Chapleura,*
a
´
´
´
Groupe SUCRES, Unite Mixte 7565 CNRS—Universite Henri Poincare-Nancy 1, BP 239, F-54506 Nancy, Vandoeuvre, France
b
´
Unite Mixte 7561CNRS—Universite Henri Poincare-Nancy 1, BP 187, F-54506 Nancy, Vandoeuvre, France
´
´
Received 11 July 2001; accepted 17 September 2001
Abstract—Ketoprofenoyl-CoA thioester 3 was synthesized by coupling ketoprofen to coenzyme A using the mixed anhydride
method. Diastereoisomeric compounds 3a and 3b corresponding to the enantiomers of ketoprofen, were obtained in optically pure
form by preparative HPLC. A non-acylating analogue, rac-3-(3-benzoylphenyl)-2-oxo-butanoyl-CoA (7) was also prepared. The
biological evaluation suggested that 3a and 3b are reversible inhibitors of COX-1 and irreversible inhibitors of COX-2. Compound
7 appears to be a poor but selective inhibitor of COX-1. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
molecules. For most drugs, these metabolic pathways are
Prostaglandins are active mediators of inflammatory
responses and also provide cytoprotection in the sto-
mach and intestine. The key enzyme of their biosynth-
esis is prostaglandin H2 synthase (PGHS or
cyclooxygenase, COX), which converts arachidonic acid
to PGH2, further metabolised by specific synthases and
isomerases to give various prostanoids. COX exists as
two isoforms: in most tissues, COX-1 is expressed con-
stitutively and COX-2 is transiently up-regulated by
pro-inflammatory mediators and down-regulated by
corticosteroids. Numerous works in this area suggest
that constitutive COX-1 protects the GI tract, whereas
inducible COX-2 mediates inflammation. However,
COX-2 is also involved in ulcer healing, renal physiol-
ogy, female reproductive process,1 and even in the
resolution of the inflammatory response.2,3
hydroxylation and glucuronic acid conjugation. Con-
jugation to glycine,4 lipids incorporation,5 and inversion
of configuration6 are observed in vivo for certain
NSAIDs, and point out a particular conjugation to coen-
zyme A, acting as an intermediate for these reactions.
Acyl-CoA from diverse drugs have been shown to inhibit
several enzymes like acetyl-CoA carboxylase,7 glu-
8
tathione S transferase and COX.9 Since acyl-CoA are
chemically activated carboxylic acids, their effects may
be due to their acylating properties and occur by cova-
lent binding. Adduct formation has been actually
demonstrated for fibrate derivatives which acylate
unknown proteins.10,11 This ability of adduct formation
is particularly important in the case of carboxylic acid-
containing xenobiotics, since their acyl chain retains the
pharmacological activity and carries the reactive mole-
cule to definite pharmacological targets.
Non-steroidal anti-inflammatory drugs (NSAIDs) are
widely used for the treatment of inflammation and
articular diseases. Their pharmacological activity is
mainly attributed to a competitive inhibition of COX.
As many other xenobiotics, NSAIDs undergo two
metabolic phases following their absorption: functiona-
lisation, for example oxidative phase, and conjugation,
consisting of the addition of various small hydrophilic
The aim of this study was to prepare ketoprofenoyl-
CoA 3 and its analogue 7 in order to study their effects
on COX. Pharmacological activity often depends on the
absolute configuration of chiral centres in the molecule,
particularly for NSAIDs for which (S) enantiomer is a
more potent inhibitor of COX than its (R) anti-
pode.12,13 Therefore, we describe in this paper the
synthesis of 3 starting from ketoprofen, and the separa-
tion step giving pure (S) and (R) diastereoisomers. In
order to highlight the inactivation mechanism of COX
by acyl-CoA, an analogue of ketoprofenoyl-CoA was
designed. Thus the replacement of the thioester group
*Corresponding authors. Tel.: +33-383-59-2619; fax: +33-383-59-2621;
e-mail: francoise.lapicque@medicine.uhp-nancy.fr (F. Lapicque); Tel.
+33-383-91-2355 fax: +33-383-91-2479; e-mail: yves.chapleur@meseb.
uhp-nancy.fr (Y. Chapleur)
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00330-3

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N. Levoin et al. / Bioorg. Med. Chem. 10 (2002) 753–757
Scheme 1. Reagents: (i), 2,6-lutidine, CH2Cl2 then ClCO2Et, rt, 1 h;
(ii), THF, CoASH, H2O, Ph=6.6, rt, 2 h.
by a thiomethylene keto group would provide a non-
acylating species. It was assumed that such a slight
modification would not affect the recognition of the
inhibitor by COX. The compound selected was rac-3-(3-
benzoylphenyl)-2-oxo-butanoyl-CoA (7).
Scheme 2. Reagents: (i), ClCO2Cl, rt, 1 h; (ii), diazald, 0 ꢀC, 2 h; (iii),
anhydrous HCl in Et2O; (iv), CoASH, H2O/DMF, CsCO3, rt, 24 h.
suggesting an epimerisation, due to epimerase activity.15
It cannot, therefore, be concluded that compound 3b is
(R) ketoprofenoyl-CoA. Crystallization of each com-
pound 3a and 3b is currently under investigation to
provide a definitive answer to this issue.
Results
The synthesis of compound 3 was performed by cou-
pling rac-2-(3-benzoylphenyl)propionic acid (ketopro-
fen [KPF] 1) to coenzyme A (CoASH) using the mixed
anhydride method14 (Scheme 1). Formation of the rather
unstable compound 2 was almost achieved quantita-
tively within 1 h. When at most 10% of the starting
compound 1 was detectable by HPLC monitoring,
CoASH was added to the reaction mixture. The forma-
tion of compound 3 was monitored by HPLC. After 2 h
of reaction, compound 2 completely disappeared. The
organic solvent was evaporated under reduced pressure
and unreacted 1 was extracted from the aqueous layer
using hexane. Diastereoisomeric compounds 3a and 3b
were obtained in optically pure form after two pre-
parative HPLC steps. The first one allowed the separa-
tion of the mixture of diastereoisomers 3 from residual
reactants; the second one gave pure 3a and 3b. In order
to assign the absolute configuration to compounds 3a
and 3b, the same synthesis was carried out starting
from optically pure R-KPF. However both diastereo-
isomers were obtained, and the same result was obtained
starting from optically pure S-KPF.
The synthesis of compound 7 was based on the final
alkylation of coenzyme A using the chloromethylketone
analogue 6 of KPF. The synthesis of 7 is outlined in
Scheme 2. Ketoprofenyl chloride 4, obtained by reac-
tion of oxalyl chloride with 1, was treated with diazo-
methane to give rac 3(-3-benzoylphenyl)-2-oxo-
diazobutane (5). Conversion of the crude resulting diazo
group to the corresponding chloride by reaction of an
anhydrous ethereal solution of hydrogen chloride, gave
the compound 6 in 95% overall yield. Alkylation of
coenzyme A by the compound 6 was performed in a
water–dimethylformamid mixture in the presence of
caesium carbonate. An optimised H2O/DMF ratio of
3:14 gave the best result. Contrary to 3, neither analytic
nor semi-preparative HPLC allowed diastereoisomeric
purification of 7.
Biological Evaluation
The biological specificity of the two isoforms of COX
reported before can be used to obtain more efficient
NSAIDs without gastro-intestinal side effects. Most
classical NSAIDs are reversible or time-dependent
reversible inhibitors, except aspirin which acetylates
Ser530of COX-1 (Ser516 of COX-2). They generally
inhibit both COX-1 and COX-2 to different extents; for
this reason, extensive studies were conducted in recent
years for the development of new molecules that
selectively inhibits COX-2.17 These studies were based
on crystal structure determinations of the enzymes:12,18
Therefore, an epimerisation reaction occurred during
the overall process, as reported by Carabaza.15 As
roughly the same diastereoisomeric mixture was
obtained whatever the KPF enantiomer used as the
starting material, assignment of one configuration to
one chromatographic eluate of 3 could not be done at
this stage. Microsomial biosynthesis with rac-1 using R-
16
AINS-specific Acyl-CoA synthetase
produced only
compound 3b, but incubation of 3a or 3b with the
microsomes resulted in the presence of the only 3b,

N. Levoin et al. / Bioorg. Med. Chem. 10 (2002) 753–757
755
Figure 2. Kinetics of KPF adducts formation. COX-2 was incubated
for 1, 3, 5, 7, 9, and 11 h with 3 before Western blotting.
Figure 1. Detection of KPF adducts after 1.5 h incubation of COX-2
with 3. Th-KPF: chemical adduct of KPF on Thyreoglobulin (positive
control), B: COX-2+KPF, 3: COX-2 incubated with compound 3.
incubation time at room temperature from 1 to 11 h
(Fig. 2). This confirms that acylation is certainly at the
origin of the irreversible inhibition of COX-2 by keto-
profenoyl-CoA.
Table 1. IC50(mM) values of ketoprofen derivatives for COX-1 and
COX-2a
Compd
COX-1b,c
Rec COX-1b,d
COX-2b,c
Rec COX-2b,d
(R) 1
(S) 1
3a
3b
7
0.695
0.003
0.043
0.083
0.317
>10,000
>10,000
900
140
>10,000
80
5
71
55
>10,000
>10,000
107
108
>10,000
Conclusion
The present work reports the synthesis and the enantio-
meric purification of ketoprofenoyl-CoA (3a, 3b) and its
non-acylating analogue 7. The biological evaluation of
compound 3 showed that it is an irreversible selective
inhibitor of COX-2. This result contributes to better
understand the mechanism of action of NSAIDs and
their anti-inflammatory properties. It is also meaningful
for the research in articular therapeutics, and the devel-
opment of more efficient drugs without side effects.
5000
aCOX inhibitions were performed as described in biological assays.
bAverage of four determinations.
cImmediate inhibition.
dRemaining inhibition after recovery.
the most striking difference between the isoforms is that
the active site of COX-1 is more stable and less bulky
than COX-2. 19,20 Therefore, most selective inhibitors of
COX-2 display steric groups that hinder their binding
on the active site of COX-1.
Besides, acyl-CoA are key intermediates in many bio-
chemical pathways, acting as cofactors of enzymes and
as intermediates of protein modification, for example
palmitoylation. A convenient way to provide these
intermediates and their non-acylating analogues is of
large interest to elucidate biological processes.21,22
The results reported in Table 1 show that acyl-CoA 3a
and 3b are good inhibitors of both COX-1 and COX-2
with an IC50 one order of magnitude higher than the S
enantiomer of the parent drug. It is worth noting that
they do not present the marked enantioselectivity of
KPF, probably due to epimerisation. Reversibility of
the inhibition was tested by recovery experiments, where
inhibitors were carefully removed before measuring the
enzymatic activity. COX incubated with (R) or (S) 1
recovered thus, almost all their catalytic capacity
(IC50>10,000), as expected from reversible inhibitors.
In contrast, COX incubated with 3a or 3b were still
inhibited: very weakly for COX-1, since IC50 values
were higher in recovery experiment than during
immediate inhibition, but strongly for COX-2, with
comparable values of IC50 in both conditions. Thus, 3a
and 3b appear essentially reversible inhibitors of COX-1
and completely irreversible inhibitors of COX-2.
Experimental
General indications
FTIR spectra were recorded on Perkin-Elmer Spectrum
1
1000 on NaCl windows or KBr pellets. H NMR and
13C spectra were recorded on a Bruker AC 250or on a
Bruker DRX 400 spectrometer. Unless otherwise stated,
all spectra were recorded in D2O. Attribution of 13C
signals are based on the J-modulated spin-echo
sequence and/or heteronuclear two-dimensional techni-
ques. Mass spectra were recorded on a Trio 1000
Thermo Quest spectrometer in the electron impact
mode or a Platform Micromass in the electro spray
mode. Elemental analyses were obtained from the Ser-
vice Central de Microanalyse du CNRS, Vernaison
(France). Analytical thin-layer chromatography was
performed on Merck 60F 254 pre-coated silica gel
plates. Preparative chromatography was performed on
silica gel 60(23–040mesh ASTM). Analytic reverse
phase HPLC was performed on a C18 column (Waters
RadialPak 8ꢁ10mm in Waters RCM module), using at
flow rate of 1.5 mL minꢂ1 and a mobile phase of MeOH
(45%), 9 mM phosphate buffer pH=5.5 (55%). Elution
was monitored at 254 nm, using Waters 996 photodiode
Compound 7 was shown to be a slight inhibitor of both
COX and to be COX-1 selective. As expected, it is a
reversible inhibitor and this argues for an acylation
mechanism responsible for the irreversible inhibition of
COX-2 by KPF acyl-CoA. In fact, incubation of COX-
2 with 3 resulted in the formation of KPF adducts, as
revealed by Western blot experiments, using anti-KPF
antibody (Fig. 1). The maximum rate of acylation was
achieved within 1 h and adducts were stable since no
clear difference in labelling was observed by varying the

756
N. Levoin et al. / Bioorg. Med. Chem. 10 (2002) 753–757
array detector and Waters 510pump. Semi-preparative
reverse phase HPLC was performed on a Merck
Lichrosorb RP-18 column, (7 mm, 250ꢁ10mm), at a
flow rate of 2.5 mL min-1, and a mobile phase of
MeOH (35%), 9 mM phosphate buffer pH=5.5 (65%)
by injections of 700 mL and monitoring at 290nm.
prepared diazomethane (generated from 7.2 g diazald in
diethyl ether at 0 ꢀC). The resulting solution was
allowed to stand for 2 h at 0 ꢀC. The reaction mixture
was quenched with heptane and the solution was con-
centrated to dryness, and led to the crude compound 5,
which was used without purification in the next step. To
a solution of the crude compound 5 in anhydrous die-
thyl ether was added dropwise a saturated etheral solu-
tion of hydrogen chloride (8 mL of a solution obtained
by extraction of 12 M HCl with diethyl ether and drying
over magnesium sulphate). The resulting solution was
stirred for 1 h at room temperature and the solvent was
concentrated under reduced pressure. The residue was
purified by column chromatography on silica gel using
hexane/diethyl ether 3:2 (v:v) to give 1.35 g of com-
pound 6. An analytical sample was recrystallised from
ethyl ether. Rf 0.17 (hexane/diethyl ether 3:2); IR:
2-(3-Benzoylphenyl)propionyl-CoA (3). To a solution of
rac-2-(3-benzoylphenyl)propionic acid [ketoprofen
(KPF)] (1) (122 mg, 0.48 mmol) in anhydrous dichloro-
methane (10mL) was added 2,6 lutidine (56 mL, 0.48
mmol) and ethyl chloroformate (46 mL, 0.48 mmol) in
anhydrous dichloromethane (3 mL). The reaction mix-
ture was stirred at room temperature under a nitrogen
atmosphere. The reaction was monitored by analytical
HPLC. After 1 h of reaction compound 1 (retention
time 9.5 min) was no longer detectable and only com-
pound 2 (retention time 6.4 min) was present. The
solution was concentrated to dryness and the crude
residue was diluted in anhydrous THF (9 mL). A solu-
tion of coenzyme A (36.8 mg, 0.048 mmol) in water (9
mL) was then added. The pH of the resulting solution
was adjusted to 6.2 using NaOH (5M) and stirred under
a nitrogen atmosphere at room temperature. The reac-
tion was monitored by analytical HPLC. After 2 h,
CoASH (retention time 1.3 min) disappeared and two
peaks corresponding to the diastereoisomers of ketoyl-
CoA 3 appeared (retention time 4.7 for 3a and 5.2 min
for 3b). The mixture was then concentrated under
reduced pressure to give a milky liquid. The aqueous
layer was extracted with hexane (10ꢁ10mL) and con-
centrated. The residue was purified by semi-preparative
HPLC to yield the compounds 3a and 3b as amorphous
white powder. The compound obtained after chroma-
tography was washed ten times with MeOH in order to
1
1734,1657 cmꢂ1 ; H NMR (250MHz, CDCl 3): d 1.48
(d, 3H, J=7 Hz, CH3), 4.05 (d, 1H, Jgem=15 Hz,
CH2Cl), 4.12 (d, 1H, CH2Cl), 4.15 (q, 1H, CH), 7.63 (m,
9H, Ph); 13C NMR (62.9 MHz, CDCl3): d 17.81 (CH3),
47.34 (CH2Cl), 49.69 (CH), 128.43, 129.16, 129.52,
130.07, 131.69, 132.73 (9C, CH Ph), 137.26, 138.45,
139.69 (3C, C Ph), 196.21, 201.78 (2C, CO); EIMS (m/
z): 288 (7%) (M+2)+, 286 (19%) (M)+. Anal. calcd for
C17H15ClO2: C,71.21; H,5.27; Cl,12.36. Found: C,71.06;
H,5.17; Cl, 12.56.
3-(3-Benzoylphenyl)-2-oxo-butanoyl-CoA (7). Co-enzyme
A disodium salt (90mg, 0.12 mmol) was dissolved in
water (300 mL) then diluted with DMF (1.4 mL). The
resulting solution was added dropwise to a suspension
of compound 6 (40mg, 0.14 mmol) and Cs 2CO3 (52 mg,
0.16 mmol) in DMF (1 mL). The reaction mixture was
stirred for 24 h at room temperature. The reaction was
monitored by analytic HPLC. The retention time for 7
was 3.7 min. The solvent was evaporated under reduced
pressure and the crude residue was purified by semi-
1
remove inorganic phosphate. Compound 3a: H NMR
(400 MHz): d 0.55 (s, 3H, CH3), 0.69 (d, 3H, J=2.5Hz,
CH3 ), 1.32 (d, 1H, J=7 Hz, CH3 ), 2.11 (br m, 2H),
2.79 (m, 1H), 2.86 (m, 1H), 3.15, (br m, 4H), 3.37 (dd,
1
preparative HPLC. H NMR: d 0.53 (s, 3H, CH3), 0.72
(s, 3H CH3), 1.06 (d, 1H, J=7 Hz, CH3), 2.22 (br m,
3
1H, Jgem=9.5 Hz, JHP=5 Hz, CH2OP), 3.66 (dd, 1H,
CH2OP), 3.84 (s, 1H, CHOH), 3.90(m, 1H, C HCH3),
4.09 (br m, 2H, H-5rib, H50rib), 4.43 (br m, 1H, H-4rib),
5.07 and 5.20 (br m, 2H, H-2rib, H-3rib), 6.08 (d, 1H,
J=6 Hz, H-1rib), 7.40(m, 9H, Ph), 7.99 (s, 1H, H-2ad),
4H), 2.96 (m, 2H), 3.23 (br m, 2H), 3.39 (dd, 1H,
3
Jgem=9.5 Hz, JHP=5 Hz, CH2OP), 3.71 (dd, 1H,
CH2OP), 3.87 (m, 2H), 4.12 (br m, 2H, H-5rib, H50rib),
4.44 (br m, 1H, H-4rib), 4.66 (br m, 2H, H-2rib, H-
3rib), 5.97 (d, 1H, J=6 Hz, H-1rib), 7.18 (m, 9H, Ph),
7.96 (s, 1H, H-2ad), 8.35 (s, 1H, H-8ad); 13C NMR: d
19.66 (CH3keto), 20.80, 23.73 (2C, CH3CoA), 33.44,
38.08, 38.17, 40.7 (5C, CH2), 41.16 (C(CH3)2), 51.10
2
8.21 (s, 1H, H-8ad), 31P NMR: d ꢂ11 (d, 1P, J=20
Hz),ꢂ10.5 (d, 1P), 2.73 (s, 1P). Compound 3b: 1H NMR
(400 MHz): d 0.46 (s, 3H, CH3), 0.65 (s, 3H, CH3), 1.00
(d, 1H, J=7 Hz, CH3), 2.07 (br m, 2H), 2.58 (br m, 2H),
2.95, (br m, 2H), 3.34 (br m, 1H, CH2OP), 3.53 (m, 1H,
CHCH3), 3.64 (br m, 1H, CH2OP), 3.81 (s, 1H,
CHOH), 4.06 (br m, 2H, H-5rib, H50rib), 4.37 (br m,
1H, H-4rib), 4.80(br m, 2H, H-2rib, H-3rib), 5.97 (d,
1H, J=6 Hz, H-1rib), 7.10(m, 9H, Ph), 7.91 (s, 1H, H-
2ad), 8.27 (s, 1H, H-8ad). 31P NMR: d ꢂ10.8 (d, 1P,
2J=20Hz), ꢂ10.4 (d, 1P), 2.68 (s, 1P).
2
(CHOH), 53.10 (CHCH3), 68.37 (d, J=4 Hz, C-5rib),
2
2
74.68 (d, J=6 Hz, CH2OP), 76.58 (d, J=3.5 Hz, C-
3
3rib), 77.03 (d, J=3.5 Hz, C-2rib), (m, C-4rib), 89.37
(C-1rib), 121.18 (C-5ad), 131.20, 131.88, 132.21, 132.73,
135.73, 135.45, 136.15 (9C, CHPh), 139.00, 139.89 (2C),
142.43 (C-8ad), 142.68 (C-6ad), 151.85 (C-2ad), 155.44,
158.08 (2C), 176.17, 177.41 (2C, COamide), 201.26,
2
211.67 (2C, CO); 31P NMR: d ꢂ11 (d, 1P, J=20Hz),
3-(3-Benzoylphenyl)-2-oxo-chlorobutane (6). KPF (1)
(1.27 g, 5 mmol) was dissolved in oxalyl chloride (3
mL). The solution was stirred at room temperature for 1
h, and then concentrated to dryness. The crude residue
of rac-2-(3-benzoylphenyl)propionyl-chloride (4) was
coevaporated twice with toluene. The dried residue was
dissolved in diethyl ether and added dropwise to freshly
ꢂ10.5 (d, 1P), 2.73 (s, 1P); ESMS negative mode (m/z):
1016.2 (MꢂH)ꢂ.
Biological assays
COX assays were conducted using intact cells expressing
preferentially COX-1 or COX-2: BPAEC cells for COX-1

N. Levoin et al. / Bioorg. Med. Chem. 10 (2002) 753–757
757
and LPS stimulated J774.2 for COX-2 (1mg/mL for 14
References and Notes
h).23 We checked for reversibility of inhibition by
recovery experiments.24 All tests were performed in 12
wells plates (27ꢁ104 cells per well). After 30min pre-
incubation of cells and tested compounds, arachidonic
acid 30 mM was added for 15 min, then supernatants
analysed for 6-keto-PGF 1 a (BPAEC) and PGE2
(J774.2) contents. Cells were then washed twice for 40
min, and arachidonic acid 30 mM was added for 15 min
to test COX activity recovery, quantifying pros-
taglandins as previously. 6-keto-PGF 1 a and PGE2
concentrations in supernatants were determined by EIA
from Assay Designs, Ann Arbor, MI, USA.
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Analysis of COX-2adducts by SDS-PAGE and Western
blot
Purified COX-2 (6ꢁ10ꢂ11 mol) (Cayman Chemicals,
Ann Arbor, MI) in 1 mM hematine, 300 mM diethyl
dithiocarbamate and Tween 200.1% 50mM pH=7.4
phosphate buffer were incubated for various times at
20 ꢀC with 3 (2ꢁ10ꢂ10 mol). Samples were mixed with
Laemmli reagent (final concentration 2% SDS, 10%
glycerol, 5% 2-mercaptoethanol, 0.002% bromophenol
blue in Tris buffer). SDS-PAGE was performed using
9% acrylamide for separating gel and 4% stacking gel.
Proteins were transferred onto Immobilon P membrane
(Millipore, Bedford, MA, USA) by semi-dry electro-
blotting, using a 0.75 M glycine, 0.1 M Tris, 10%
MeOH buffer adjusted to pH=7.4, for 1 h at 12 V.
Blots were saturated with 3% BSA in 0.04% Tween 20
in PBS. Immunodetection was performed using rabbit
anti-KPF polyclonal antibody diluted 1/6000 and sec-
ondary goat anti-rabbit IgG alkaline phosphatase con-
jugated 1/5000. Anti-KPF antibody and KPF-
thyreoglobuline were kind gift of Pr. E. Benoit (Ecole
Nationale Veterinaire, Lyon, France).
16. Brugger, R.; Garcia Alia, B.; Reichel, C.; Waibel, R.;
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Acknowledgements
N.L. and F.L. wish to thank the Association de Recherche
sur la Polyarthrite (ARP 1999-2000) and the Contrat de
Programme en Recherche Clinique du CHU de Nancy
(CPRC 1999) for financial support. Warmly dedicated
to Pr Gerard Descotes on occasion of his retirement.
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