939

Pharmacological Research Communications, VoL 10, No. 10, 1978

EFFECT OF ACETYLSALICYLIC AND THEIR COMBINATION

SULFINPYRAZONE

ON COLLAGEN-INDUCED

AGGREGATION

L.T. Wong,

ACID,

PLATELET

IN GUINEA PIGS

Z. Zawidzka and B°H. Thomas

Health Protection Branch Health and Welfare Canada Tunney's Pasture Ottawa, Ontario KIA 0L2 Receivedin final form 9 November 1978

SUMMARY Acetylsaiicylic

acid

(ASA) showed a dose-dependent

tory effect on collagen-induced pigs in vitro.

Sulfinpyrazone

platelet aggregation and salicylic acid

a similar effect,

but were 14 and 57 times,

potent than ASA.

In vivo,

a dose-response

inhibi-

in guinea

(SAL)

showed

respectively, relationship

less

could

be demonstrated with ASA and sulfinpyrazone with doses up to 100mg/kg,

but SAL was ineffective

ASA and sulfinpyrazone

at that dose level.

appeared to produce a synergistic

It is likely that mutual competition result in a higher concentration finpyrazone

Combined effect.

for protein binding may

of free active ASA and sul-

interacting with plasma platelets and consequently

a synergistic

action.

0031--6989~8/100939--11/~2.00/0

©1978Theltalian ~a~acological~ciety

940

Pharmacological Research Communications, VoL ;0, No. 10, 1978

INTRODUCTION Acetylsalicy~ic

acid

(ASA) and sulfinpyrazone

both appear to be effective antithrombotic platelet 1975).

aggregation

(Didisheim et al.,

enzyme responsible

1974;

Genton et al.,

synthesis

(Smith et al.,

1975).

is less clear,

Although

1974;

a microsomal

of arachidonic

intermediates

Roth and Majerus,

1977).

effect on platelet

functions

lasting effect on platelets also inexpensive.

doses may lead to hemorrhagic action on sastric mucosa.

(Marx, 1977).

1975) and is

complications

arising from its

on the other hand is

tolerated over long periods of continuous

1977)

administration,

appears to be short-acting

(Walter

and the drug is relatively more expensive.

w o u l d appear that a combination for antithrombotic

therapy.

However treatment with such a comof the potential

that may occur resulting in the enhancement

hibition of the anticoagulant

action.

effects of ASA and sulfinpyrazone (Didisheim et al.,

1974;

It

of the two may be advantageous

bination may be hazardous without prior knowledge interactions

1977;

ingestion of large daily

Sulfinpyrazone

but its effect on platelets

like ASA,

to have a long-

(Vinazzer et al.,

However continual

on platelets

have profound

is well established

drug, ASA appears

bioRoth

(Ali and McDonald,

The fact that prostaglandins

As an antithrombotic

and Weber~

1975;

recent findings suggest that it too, activity

acid to the

of prostaglandin

the role of sulfinpyrazone

may inhibit cyclooxygenase Ali et al.,

synthetase,

for the conversion

labile cyclic endoperoxide

well

drugs which prevent

ASA has been shown to inhibit platelet release reac-

tions by inhibiting prostaglandin

et al.,

(Anturan)

or in-

Although the antiaggregatory

have been extensively

Genton et al.,

1975),

studied

their combining

Pharmacological Research Communications, Vol. 10, No. 10, 1978 a c t i o n has not been explored.

It is the i n t e n t i o n of this paper

to use the guinea pig as an e x p e r i m e n t a l the effect of ASA, lagen-induced

941

sulfinpyrazone

animal

to i n v e s t i g a t e

and their c o m b i n a t i o n

on col-

p l a t e l e t aggregation.

MATERIAL AND METHODS Platelet

aggregation

Male Hartley g u i n e a pigs 300-350

g, a c c l i m a t i z e d

environment,

solution (PRP)

were used in all studies.

syringe c o n t a i n i n g

individual

animals,

counts were

Citrated platelet

Platelets

counts w e r e r e l a t i v e l y ranging

were o b s e r v e d

from 700-1000

aggregating the PRP

5 min of i n c u b a t i o n

to the a g g r e g o m e t e r For

constant between

x I03/~i.

The f i ~ l

300 x i03/~i

properties

45-6~

up to 4 ho

stirred

In

for i0 secs and p l a c e d

the c u v e t t e ~¢as t r a n s f e r r e d

and 0.i ml of the a g g r e g a t i n g

the in vivo study,

90 min.

(0.8 ml) was a d d e d to p r e w a r m e d

(37 ° ) 0.I ml of the test solution, After

respect-

to r e a c h m a x i m u m a c t i v i t y w i t h i n

the in v i t r o experiment,

into

rich p l a s m a

All tests were p e r f o r m e d w i t h i n

and r e t a i n e x c e l l e n t

at 37°C.

animals

(PPP) w e r e o b t a i n e d by centr~-

adjusted with PPP to a p p r o x i m a t e l y

and p l a c e d at 37°C.

min

(7.2 ml) was col-

at 183 g for i0 min and 1200 g for 15 min, PRP p l a t e l e t

Canada),

0.8 ml of a 0.1M s o d i u m citrate

and p l a t e l e t poor p l a s m a

ively.

Blood

aorta of e t h e r - a n a e s t h e t i z e d

to p r e v e n t clotting.

fugation

Ontario,

for at least 7 days to the l a b o r a t o r y

lected from the dorsal a plastic

(High Oak Ranch,

0.i ml of the a g g r e g a t i n g

agent added. a g e n t was added

to 0.9 ml of PRP. Assays

of p l a t e l e t

the t u r b i d o m e t r i c

method

aggregation

were p e r f o r m e d

d e s c r i b e d by Born

(1962)

at 37 ° by using Payton

Pharmacological Research Communications, Vol. 10, No. 10, 1978

942

aggregation module connected

to a recorder to measure changes

in the optical density of the platelet suspension during aggregation.

A collagen preparation

final concentration agent.

i/i000),

(0.25% acid soluble collagen*,

was used as an aggregation-provoking

The extent of aggregation was determined by measuring

slope of the increase of the steepest portion of the curve

the

over

30 sec period. In the in vitro experiment, dissolved

in physiological

Canada Ltd., Dorva!)

ASA and sodium salicylate were

saline.

Sulfinpyrazone

was neutralized with an equal molar amount

of NaOH solution and diluted with saline. ASA was administered Sodium salicylate

(Ciba-Geigy

For in vivo studies,

orally in a gum tragacanth sulfinpyrazone

and

suspension.

(neutralized with an equal

molar amount of NaOH) were administered

orally after dissolving

in saline.

Plasma protein bindin~

was

Guinea pig plasma containing

20 ~g/ml of 14C-sulfinpyrazone

incubated at room temperature

for 2 h with sodium salicylate

in the concentration were transferred

range 0-i00 ~g/ml.

(2 ml)

to washed Visking tube bags mounted in centri-

fuged tubes and centrifuged (50 ~i) of the dialyzate for radioactivity

The incubates

for 20 min at 10000 g.

Aliquots

and undialyzed plasma were estimated

by counting

in a liquid scintillation

counter.

of drug causing a 50% inhibition

(I50) of

Statistics The concentration

Courtesy of Mr. D. Perry of McMaster Ontario,

Canada

University,

Hamilton

a

Pharmacological Research Communications, VoL 10, No. 10, 1978 platelet

aggregation

that of W a u d using

(1972)

the p r o b i t

ference values

between

and the

analysis

the effects

being

of ASA,

by collagen.

dependent

SAL was

than SAL about

(1971).

were

calculated

Significance

by S t u d e n t T s

humans

of dif-

t-test w i t h

(Seuter,

In vivo,

measured

inhibitor,

acid

(SAL)

showed

in Fig.

a 50%

is about

in rabbits

induced

formed

from

a doseASA a p p e a r e d

inhibition

(I50)

57 times m o r e p o t e n t

Sulfinpyrazone compared

These

i.

on

(I50 = 173 Bg/ml) to ASA,

results

(Rosenberg

are

but was

in close

et al.,

1971)

agreeand

1976).

levels

caused

at 2 h after

drug

SAL

relationship

(Table

The effect was

inhibition

i).

occurring

no i n h i b i t o r y

showed

(Table i, Fig. between

2).

at a dose of

effect

up to a

a similar

as A S A w i t h a 8 9 . 6 2 ± 2 . 9 6 %

in a c t i v i t y

pigs at dif-

aggregation

sulfinpyrazone

of i00 m g / k g

the d i f f e r e n c e

to g u i n e a

of p l a t e l e t

injection

showed

dose of I00 mg/kg w h i l e

of A S A

inhibition

w i t h a total

I00 mg/kg.

a dose

with

oral a d m i n i s t r a t i o n

dose-dependent

response

as d e p i c t e d

than SAL.

found

out to study

it is r a p i d l y

three drugs

less e f f e c t i v e

potent

those

and s a l i c y l i c

since

All

action

carried

of guinea pig p l a t e l e t s

of 12 pg/ml w h i c h

14 times

dose

initially

included

(I50 = 683 Bg/ml).

4 times m o r e ment with

were

of ASA.

inhibitory

concentration

that

limits

based on

significant.

sufinpyrazone

to be the m o s t potent

after

of Finney

of a g g r e g a t i o n

in vivo h y d r o l y s i s

about

95% c o n f i d e n c e

considered

experiments

the i n h i b i t i o n

ferent

by a logit p r o g r a m

AND DISCUSSION

In v i t r o

was

estimated

groups was d e t e r m i n e d

of P