THRbMBOSlS

RESEARCH64;631-636,199l

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ACTION OF SOM! SALICYLATE INHIBITORY

DERIVATIVES

ON IN VITRO

AND INHIBITION

ANTA6DNISTIC

PLATELET

AtXRE6ATION.

EFFECTS

F. CASADEBAIG', J.-P. DUPIN': D. GRAVIER', G. HOU' M. BDISSEAU2 DARET2 , H. BERNARD2 , J. LARRUE2,

D. 'Faculti 2Uniti *Author

de Pharmacie,

UniversitG

ZNSERM U8, 33604 Pessac for correspondence

de Cedex

(Received 24.1 .1991; accepted

Bordeaux

11

- 33076

Bordeaux

Cedex

France

France

in revised form 11.9.1991 by Editor

J. Soria)

ABSTRACT Twenty activity gation.

salicylate derivatives were tested for their antagonistic on the inhibitory effect of aspirin on platelet aggreThe blocking effect was not limited to the salicylate some of its but also characterised substituted compounds. The substituant influence did not seem to be related to electronic or size parameters. This antagonistic activity of these derivatives decreased as concentrations increased, owing to the emergence of their own inhibitory activity: several salicylate derivatives showed dual inhibition antagonistic inhibitory and activity, with both properties present at involving dissociated activities cyclooxygenase is proposed.

the same concentration. on the two enzymatic

A

mechanism sites of

INTRODUCTION It has been established that salicylate, which has no effect on platelet functions, antagonizes the aspirin inhibition of aggregation (1,2,3). This phenomenon has been attributed to an action on cyclooxygenase It has been (2,5). suggested (6,7,8) that the enzyme possesses two sites: the first on which the binding of aspirin is necessary but not sufficient to induce its antiplatelet effect, which depends on the action of a second site. Although some other compounds with very different molecular formulae have shown a blocking effect (2,4,9), the very close chemical relation between aspirin and salicylate strengthens the hypothesis of a competitive action of the two compounds on the same site. However, few salicylate derivatives (the 6-OH derivative, diflunisal and deacetylated triflusal (l,lO,ll) have been studied for their blocking activity. Similar potential activity, as observed with salicylate in some of its derivatives, should provide better understanding concerning the structural characteristics of the binding site of aspirin on cyclooxygenase. Key

words:

Aspirin, genase.

Salicylate

derivatives, 631

Platelet

aggregation,

Cyclooxy-

molecules

Vol. 64, No. 6

ACTION OF SOME SALICYLATE DERIVATIVES...

632

We therefore studied the with different structural

effect on platelet modifications in

aggregation of their salicylate

several formula.

METHODS Platelet aggregation. Platelet-rich plasma (PRP) and platelet-poor anticoagulated plasma (PPP) were prepared by standard techniques using human blood (l/10, v/v in 3.8% sodium citrate) collected from healthy Plasubjects. The platelet count of PRP was adjusted with PPP to 3 x lOa/ml. telet aggregation was performed and measured photometrically at 37O (using a Coultronic aggregometer) with a magnetic stirrer (1200 r.p.m). To 0.430 ml PRP was added either 0.050 ml buffer as control or 0.025 ml of salicylate derivatives used at final concentration from 0.75 mmole/l to 1.5 mmole/l and 0.025 ml buffer to record inhibition curves , or 0.025 ml of salicylate derivatives and after 3 mn incubation (under stirring) 0.025 ml aspirin used at 0.15 mM or 0.5 mM final concentration (respectively when arachidonic to record antagonistic acid or collagen were used as aggregating agent) curves . Then after 3 mn incubation (under stirring), aggregation was recorded after addition of 0.020. ml arachidonic acid (AA) as sodium salt in aqueous solution at 0.4 mM final concentration (Nu Chek Prep. BP 176 Elysian Minnesota) or collagen (Coll.) (at 1.25 pg/ml final concentration: Harm). Molecules studied were obtained from Aldrich Company (EGA CHIMIE France) and used in 0.05 M Tris-HCl buffer pH 7 as sodium salt. All experiments were duplicated and performed on at least three PRP corresponding to three different donors. Results given are the mean values +standard deviation. collected from PRP Platelets were metabolism. in a 0.05 M Tris-HCl buffer ph 7.4 containing 1 mM EDTA, and resuspended in Tris-HCl buffer at 3 x lO*cells/ml. 0.44Oml either of platelet suspension was stirred and incubated for IO mn with 0.055 ml of buffer, 0.055 ml of inhibitory substance (aspirin at O.lmM or salicylate derivatives at 1.5 mM respective final concentrations), (used at 1.5 mM final concentration) or 0.030 ml of blocking agent 10 mn before 0.025 ml of aspirin (at 0.1 mM final concentration). Then concentration in 2.5% NaOH isotonic 0.005 ml of l- 14C AA at 3 uM final solution was added (l-it AA 55 mCi/mmole, Amersham International). Each tube was stirred (10 mn at 37O in obscurity), acidified with HCl N (to pH 3) and extracted with ice cold ethyl acetate. Extracts were analysed by thin layer chromatography (Silicia G plates). Chloroform, methanol, acetic acid and water (90,8,1,0.8 v/v) were used as ascending solvant prostaglandin E2 (PGE2), heptadecatrienoic with thromboxane 8 and AA as standards. (HHT) , 12-hydr$xy~T~~~~;etranoic acid (12 HETE) acid Arachidonic

centrifugation

acid

and washed twice

Rf values were 0.24, 0.36, 0.55, 0.63 was performed using a Berthold Automatic 20.

and 0.73 TLC linear

rkspectively.

analyser,

Detection

Trace

Master

RESULTS 1 .-

inhibition activity

Antagonism of some salicylic gation by aspirin

analogues

in

inhibition

of platelet

aggre-

The salicylate derivatives having a blocking effect in aspirin-induced of platelet aggregation are presented in Table I. Maximal occured for a concentration of 1.5 or 3 mmole/l, since for higher

Vol. 64, No. 6

doses of of platelet activity, acids) (Results

ACTION

salicylate aggregation

OF SOME SALICYLATE DERIVATIVES...

derivatives was less

nor di or condensed not shown).

aromatic

the intense

analogues

633

antagonistic effect in inhibition or disappeared completely. 5-NH2 derivatives had no blocking (3-OH picolinic and Z-OH nicotinic (2-OH naphtoic and pamoic acids)

TABLE I Antagonism of some salicylate of Collagen and Arachidonic Yinal

concentration

analogues acid-induced

Aspirin-induced Winpresence Collagen

(mmole/l*)

Aspirin Preincubated

in

inhibition or absence

inhibition platelet

by aspirin aggregation

of platelet ofsalicylate

aggregation derivatives

AA (0.4

(I .25 pg/ml*)

by

mM*) - --

0.15 0

0.15 1.5

0.15 3

0.15 7.5

30 + 7

9623

712 16

19+7

13A6

4Jfl5

7223

9623

752 20

492 15

5022

55210

552 12

96 f 3

91f

1826

39+11

30fll

2825

04 + 3

96 f 3

262 7

1926

68+9

7729

31 212

15k5

68 f 7

9623

I4f

28211

73 It 7

5-I

77 f 9

37 f6

3229

93 + 7

96 f 3

132 2

3326

7525

5-h

7759

45 f5

3353

95 2 2

9623

162 2

23 + 3

7125

5-F

77 f 9

40 f3

33+4

4226

9623

16f 5

925

28+7

0.5 0

0.5 1.5

0.5 3

77 +S

62216

40210

f/J f: 9

72 *5

7Jf9

72 +5

3-Cl

7729

S-Cl

compounds

0.5 7.5

(mmole/l*) R-H

3-w

3 s-CH, J

COOH OH

5

Q 3

4

R

All

compounds,

observed

tage

preincubated

at 1.5 mmole/l.

higher

than

that

-

JJf9

55 f4

53210

96f4

96 + 3

282 3

7729 ---

79+2

5325

10 f 5

96 f 3

942 2

65’115

at

0.75

mmole/l

and all compounds

observed

10

66 f 21

s-10* 5-CHO

15

at

7.5

gave

an

preincubated

percentage

inhibition at 15 mmole/l

gave

higher

an inhibition

90 f 3 35 f. 16

than

that

loercen-

mmole/l.

TABLE II Inhibitory on Collagen

effect of salicylate and some of its and Arachidonic acid-induced platelet c150

Co11.1.25

(mmole/l)

vg/ml

1 AA 0.4

derivatives aggregation C150 (mmole/l)

mM

Studied compounds

Coll. 1.25 r(g/ml

AA 0.4

ASP

0.4

0.15

5-8r SAL

5.3 20.3

5.2kO.2

5-CH3 SAL

7.4 to.6

6.7f0.8

5-I SAL

5.lkO.2

4.6kO.5

3-Cl SAL

4.5 20.3

4.2 20.4

5-IlO2 SAL

3.6 +0.4

2.0+0.3

4-Cl SAL

J.SkO.5

3.6 20.2

2-OHnaphtoic acid pamoic acid

4.3 +0.5

2.320.2

4.3 20.3

2.5kO.l

5-Cl SAL (ASP = aspirin,

5.6 20.2 SAL = salicylic

5.0f0.3

acid).

mM

1

ACTION OF SOME SALICYLATE DERIVATIVES...

634

2 .-

effect

Inhibitory

of salicylate

and some of

its

Vol. 64, No. 6

derivative?

An inhibitory effect of salicylate and some of its derivatives and halogenated salicylate deri(condensed aromatic analogues, S-NO vatives) in platelet aggregation in or AA was noted *& uced by collagen for doses about 10 or 30 times more concentrated than aspirin. (Table II). This inhibitory Some derivatives possessed effect was dose-dependent. a dual activity for the same range of concentrations: an antiplatelet activity and a blocking effect on aspirin antiplatelet action (such as Fig.l). On the other hand, 3-CH3, 4-CHy. 5-NH3’ 5-50 .t.H, 5-CHO, 5-OH and 5-OCH3 derivatives gave &ittle or no an iplate et act1 lty (for concentrations above 7.5 mmole/l).

S-Cl

5 maol/l

SAL

0.15 mmole/l

ASP

5-Cl

5. mnole/l

SAL

+ ASP 0.15 mmole/l

FIG.1 Dual activity of 5-Cl salicylic acid: inhibition of 0.4 mMAA-induced platelet aggregation and antagonism of aspirin antiplatelet action.

-3.-

-Arachidonic

acid

metabolism

modifications

The influence of the salicylate derivatives studied on prostaglandin biosynthesis was assessed by measuring the main metabolites of the cyclooxygenase pathway (TXB and HHT) and the 12 HETE from the lipoxyPGE genase pathway. Table ?I1 s$dws for aspirin the already known inhibition of cyclooxygenase and corresponding enhancement of lipoxygenase, and for 5-Cl salicycic acid the same type of dual action towards cyclooxygenase activity

as during

platelet

aggregation

assays.

TABLE III Influence

of salicylate

Derivatives mmole/l final

concentration

Control ASP 0.1 SAL 1.5 SAL 1.5

+

ASP 0.1

5-ClSAL 1.5 5-ClSAL 1.5

??

ASP 0.1

derivatives

on prostaglandin

biosynthesis

Relative quantities of main cyclooxygenase and lipoxygenase metabolites of labelled AA 3 pM %HHT %I2 HETE %PGE2 %TXB 2

18.72

3.85

24.31

43.13

4.90

3.52

12.74

67.77

19.64

4.12

23.36

44.62

13.54

2.91

16.41

56.49

15.53

3.57

17.72

54.66

15.51

3.29

18.41

52.17

Vol. 64, No. 6

ACTION OF SOME SALICYLATE DERIVATIVES...

635

3ISCLJSSION 1 .-

Structure-antagonistic

activity

relationshi.

the two functions COOH and Even in relatively ortho positions, OH of salicylic acid are not sufficient to maintain an antagonistic property in naphtalenic and pyridinic homologues of salicylic acid. The type of electronic effect and the size of the substituants do not seem to play any role, 5-Cl, 5-NO2 salicylic derivatives had nearly the since 5-CH position is of great importance since same activity. On thd’other hand, 4-substituted compounds had almost no activity, whereas 3- and 5-substituted compounds were potent blocking agents. Our results show that the blocking activity is not limited to salicylic but that it also characterises some of its substituted derivatives. acid, 2 .-

Inhibitory

activity

Values expressing antagonistic activity versus concentration reveal an unexpected decrease in this activity when the concentration increases. The loss of the antagonistic effect of a salicylic derivative was attributed to its own inhibitory activity. An antiaggregant activity has already salicylic acid) been reported for diflunisal (5-(2:4’difluorophenyl) (12) and deacetylated trifusal (2-hydroxy 4-trifluoromethyl benzoic acid) (11). Until now this phenomenon has not been mentioned in the literature, since the blocking activity concentrations lower was studied only at than those giving the inhibitory effect (1,6,7).We have new demonstrated that a compound at a concentration inducing an inhibition of platelet aggregation antagonises inhibition induced by aspirin. 3.-

Mechanism

of dual

activity

To establish that the action of salicylic derivatives has the same origin as aspirin, we studied the influence of 5-Cl salicylic acid on arachidonic acid metabolism. of compounds depending The formation on the enzymatic activity of cyclooxygenase (TXB HHT) was greatly PGE f? .$’ diminished by aspirin but much less by 5-Cl salicy lc aci at a concentration blocking the inhibitory effect of aspirin on platelet aggregation. 5-Cl salicylic acid activity seems to be cyclooxygenase-dependent. Since the inhibitory effect of aspirin and 5-Cl salicylic acid are not cumulative, it is postulated that these two drugs do not act on the same site or on two independent sites. Thereupon a mecanism involving two non-independent sites on cyclooxygenase was proposed (2,6,7) to explain the blocking activity of salicylic acid on the aspirin antiplatelet effect. One of these two sites may act as the enzymatic site, and the other one as the supplementary or binding site on which the action is necessary but not sufficient (B,9,10). Our results with ~-CL salicylic acid are in good agreement with this latter hypothesis: - the inhibitory effect should be due to an inhibition of activity of the enzymatic site of cyclooxygenase. Since the 5-Cl salicylic derivative is not a potent inhibitor, it could have a mild effect at that site (much lower than that of aspirin). Salicylic acid, which is quite ineffective as an inhibitor, should not have any activity on this site. - the blocking effect should be due to competition between aspirin and salicylate derivatives on the binding site without alteration of the enzymatic site, thus explaining the antagonistic effect of 5-Cl salicylic acid on inhibition of platelet aggregation induced by aspirin.

ACTION

636

OF SOME SALICYLATE DERIVATIVES...

Vol. 64, No. 6

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WAHLIN-BOLL,E. Salicylic BRANTMARK,B., HEDNER,U., MELANDER,A. and inhibition of antiplatelet.effect of aspirin. tancet 11, 1349, 1981.

2 VARGAFTIG,B.B. The inhibition by by aspirin is prevented

of cyclooxygenase of rabbit Platelets salicylic acid and by phenanthrolines.

European J. Pharmacol. 50, 231-241, 1978.

RAJTAR,G. and de GAETAN0.G. SalicYlate reverses 3 MERINO,J., LIvIo,M., pstaglandin in uitro aspirin inhibition of rat platelet and vascular generation. Biochem. Pharmacol. 29, 1093-l 096, 1960. 4 ALI,M. and MC DONAL0,J.W.D. Interference by sulfinpyrarone and salicylate of aspirin of inhibition of platelet cyclooxygenase activity. t'rostagtan1979. dins and Medicine 3,327-332, 5 DEJANA,E.,

CERLETTI,C., de CASTELLARNAU,C., LIVIO,M., GALLETTI,F., and de GAETANO,G. Salicylate-aspirin interaction in the rat. Evidence that salicylate accumulating during aspirin administration may protect vascular prostacyclin from aspirin-induced inhibition. J; Chin. Invest. 68, 1108-1112, 1981. LATINI,R.

6 HUMES,J.L., WINTER,C.A.,SADOWSKI,S.J. and KUEHL,Jr.F.A. Multiple sites on prostaglandin cyclooxygenase are determinants in the action of nonsteroidal antiinflammatory agents. Proc. Nat 1. Acad. Sci. LISA78 , 2053-2056, 1981. 7 CERLETTI,C.,LIVIO,M. and de GAETANO,G.Nonsteroidal antiinflammatory drugs react with two sites on platelet cyclooxygenase. Evidence from in vivo drug interaction studies in rats. Biochim., Biophys: Acta 714, 122-128 1981. 8 DE GAETANO,G.,CERLETTI,C.,DEJANA,E. inhibition in humans: implications Circulation 72, 1185-1193, 1985.

and LATINI,R. Pharmacology of platelet of the salicylate-aspirin interaction.

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ROTILIO,D.,JOSEPH,D.,HATMI,M. and VARGAFTIG,B.B. Structural requirements for preventing the aspirin-and the arachidonate-induced inactivation of platelet cyclooxygenase: additional evidence for distinct enzymatic sites. European J. Pharmacol. 97, 197-208, 1984.

11

SANCH0,M.J.. de CASTELLARNAU,C.,VILA,L.,GARCIA,J.,CARRETERO,F,.FABRA,A. and RUTLLANT,M., Ll. Effects of salicylate derivatives on cyclooxygenase. Interaction studies. BioZ. CZin. Hematot; 7, 135-743, 1985.

12 MIKHAILIDIS,D.P.,BARRADAS,M.A.,TEMPLE-SAVAGE,X.,STREETE,P.J.,HENRY,J.A., JAMES.1.M. and DAND0NA.P. The effect of diflunisal administration on platelet aggregation and cerebral blood flow. Pharmatherapeutica 4, 255-258, 1985.