In vitro and in vivo inhibition of prostaglandin synthesis by fenoprofen, a non steroid anti-inflammatory drug

Pharmacological Research Communications, Vo/. 6, No. 5., 1974

509

IN VITRO AND IN ~;[IVO ~ I B I T I O N CF PROSTAGIANDIN SYNTHESIS BY FENOPRCFEN, A NON STEROID ANTI-Ib~IAMMATORY D~JG.

C. Patrono,

G. C i a b a t t o n i

and D. G r o s s i - B e l l o n i

Institute of Pharmacology and Centro di ShJ~io per la Fisicpatologia delIo Shock C.N.R., Universit~ Cattolica del S. O/ore, 00168 Rome, Italy. Received 5 June 1974

~JMMARY:

The in v i t r o

the superfused

uterine

by f e n o p r o f e n

thacin. i.p.

in v i v o

horns

Fenoprofen

sma v a l u e s

rats,

or d i s s o l v e d

is likely

to be r e f e r r e d shows

on the u t e r i n e

PG-synthetase.

with

some d e g r e e

indomethacin. activity

fenoprofen

and a n t i f l o g i s t i c

the c o r r e s p o n d i n g

The e f f e c t

follow

activity

pla-

of p l a t e l e t with

effects

the e f f e c t of b o t h

a linear r e l a t i o n

slightly more potent

discrepancy

in

on serum PGF2~,

of c o r r e l a t i o n

The i n h i b i t o r y

being

The r e l a t i v e

either

that of indome-

to the i n h i b i t i o n

on serum PGF20~concentrations the dose,

acid),

PGF2¢&serum c o n c e n t r a t i o n s

extent.

PG-synthetase,

drugs

propionic

is i n h i b i t e d

in the i n c u b a t i o n medium.

not a f f e c t i n g

to a s i g n i f i c a n t

rats

comparable with

also reduces while

F20~(PGF20~) from

of non p r e g n a n t

is q u a n t i t a t i v e l y

treated

which

of P r o s t a g l a n d i n

(d!-2-(3-phenoxyphenyl)

administered The e f f e c t

release

between

than

PG-inhibitory

of these d r u g s

is b r i e f l y

discussed. INTRODUCTION: during

A considerable

the last few years,

tory a c t i v i t y ability Vane,

1972).

It seems

of a s p i r i n - l i k e

This has b e e n fractions

therefore

that

pharmacological

in many organs,

an i m p o r t a n t

reported

of F e n o p r o f e n

in v i t r o

shown

to w h o l e

evaluation

We had p r e v i o u s l y ability

up

spontaneous

has

accumulated

the a n t i - i n f l a m m a -

drugs m a y be a t t r i b u t e d

that the a s s e s s m e n t

should r e p r e s e n t

systems,

from s e v e r a l

data,

of

agent.

to i n h i b i t

as w e l l as e s t r a d i o l - s t i m u l a t e d

1973).

acti-

demonstrating

1971)

during

from

species.

of P G - i n h i b i t o r y

et al.,

rat u t e r u s

(see

ranging

step in the p r o c e s s

preliminary

(Patrono and C i a b a t t o n i ,

to their

and r e l e a s e

of a new a n t i f l o g i s t i c

(Nickander

lease from. the non p r e g n a n t fusion

indicating

to i n h i b i t Pros tag land in s y n t h e s i s

subcellular

vity

body of e v i d e n c e

the

PGF re-

continuous

The p r e s e n t

the

super-

studies

Pharmacological Research Communications, Vol. 6, No. 5, 1974

510

were u n d e r t a k e n bitory

in order

properties

to c h a r a c t e r i z e

of this drug both

MATERIALS AND METHODS: F e n o p r o f e n (3-phenoxyphenyl) generously

supplied

(Merck-Sharpe substance, tivity.

propionic

se from the rat uterus

superfusion

was

as the r e f e r e n c e PG-inhibitory

ac-

PGF relea-

of PGF d u r i n g

continuous

in vitro.

uterine

horns

by Serra and M i d g l e y

single rat anterior

611 69 : dl-2-

to stimulate

obtained

studied by a m o d i f i c a t i o n

nique d e s c r i b e d schematically

was used

in vitro.

Indomethacin

chosen

confirmed

the in vitro release

of isolated

g n a n t rats was

(Sigma)

the PG-inhi-

salt, dihydrate)

Italia.

was arbitrarly

in view of the largely

17~-estradiol

(compound

sodium

by the Eli Lilly

& Dohme)

Superfusicn system:

in v i v o and

sodium

acid,

further

pituitary

represented

glands.

in Fig.

from non pre-

of the original

(1970)

tech-

for the study of

The whole

apparatus

is

I.

O2 C 0~,

U

U

Fig.1 Incubation design: (1,2) buffer containers with 95% oxygen and 5% carbon d i o x i d e c o n t i n u o u s l y bubbled; (3) peristaltic pump; (4) t h e r m o s t a t i c bath; (5,6) incubation chambers; (7) test tubes. It consists cm wide)

containing

and attached were used

of two identical a porous

glass

chambers

glass

sept in the

to i n l e t and outlet

simultaneously

tubing.

to a c c o m o d a t e

(6 cm high and 5 lower half,

The two chambers

the separated

horns

of

Pharmacological Research Communications, Vol. 6, No. 5, 1874 the same uterus. sept,

Right

the c h a m b e r s

static tion

bath

were

at 32°C.

- was

after

from

by s u p e r f u s i n g

The

the m e d i u m

were

into

changed

frozen

tion For

diol

action

was d i s s o l v e d

0.5 ~g/ml.

Both

continuous

gas

In vivo studies: rats

red w i t h animals

and

phase

serum PGF

treated

were

the

used

for

values

the second

Approximately

treatment

the a n i m a l s

by

test

subto

to the sti-

transferring were

were

the

estraof

kept u n d e r

and 5% CO 2.

were measured

in m a l e

or i n d o m e t h a c i n , animals.

of b o t h drugs. before

and

and c o m p a -

Groups

These

two and a h a l f were

solu-

of e t h a n o l

horns

95% 02

the e v e n i n g

anti-

at a c o n c e n t r a t i o n

in c o n t r o l

each dose

were

exposed

amount

container

fenoprofen

intraperitoneally

collection.

solution

with

the

Tubes

The

the

container

concentrations

with

with

of both

effected

other

by b u b b l i n g

corresponding

nistered sample

this

from

per minute.

and b e i n g

a separate

in De Jalon'

horns,

of the s u p e r f u s i o n .

superfused

Exposure

into

pump

in De Jalon'

or to the same

was

same

of the assay.

was d i s s o l v e d

of e s t r a d i o l tubing

time

there-

a constant

and the s u p e r f u s a t e s

as a c o n t r o l

alone

the

(LKB

chamber,

the u t e r i n e

of 0.1 ml

the

one horn was

the d r u g .

inlet

by

the b e g i n n i n g

serving

to d i s s o l v e

Wistar

from

solution

of the

at a rate

tested

present

the other

mulatory end

to be

experiment

physiologic

aspirated

at -20°C u n t i l

agent

each

tubes

bathing

solu-

pump

while maintaining

at 30 rain. intervals,

and was

stance,

used

test

- De Jalon'

into the c l o s e d

After

on each

in a t h e r m o -

by a p e r i s t a l t i c

tissues

1.5 ml.

horns

and p l a c e d

medium

transferred

continuously

and kept

flogistic

superfusion

the e x p o s e d

was

the u t e r i n e

closed

its c o n t a i n e r

level of a p p r o x i m a t e l y

chambers

tightly

continuously

Multiperpex)

placing

511

were

the d a y

hours

decapitated

of 10 admiof

after

and

the b l o o d

t

collected. the

time

in the

Ser~n

of the assay.

same

the in v i t r o previous

study,

were

frozen

Occasionally,

in order

PGF r e l e a s e

and k e p t female

to o b t a i n

from

at -20°C u n t i l

rats

a parallel

the uterus,

were

included

evaluation

as d e s c r i b e d

of

in the

section.

~io~oassay in p l a s m a

samples

~rocedures: PGF c o n c e n t r a t i o n s a n d serum

The d e t a i l s

samples

of our m e t h o d

were measured

for P G F ~ h a v e

in s u p e r f u s a t e s ,

and

by r a d i o i m m u n o a s s a y .

been

previously

descri-

Pharmacologica/ Research Communications, Vo/. 6, No. 5, 1974

51 2

bed

(Patrono,

by means

1973).

Serum samples were also assayed

of a recently developed

1974 a). 3H-PGF1~(specific Nuclear Corp.

were purchased

Standard Prostaglandins

of the Upjohn Company. for PGF1~were

Incubation

identical

(Patrono et al.,

73 Ci/mM)

activity:

120 Ci/mM)

(specific activity:

antiserum

were

and 3H-PGF20% from New England

the generous

and separation

to those described

RESULTS AND DISCUSSION:

The superfused

Essentially, during

the first 30-60 minutes,

the m e c h a n i c a l

uterine horns

considerably

lower values.

superfusion medium

elicits

and release

A virtually

at

can be obserto the

increase of PGF 1973).

is generally

of a single uterus,

obtai-

simultaneously

one horn can be taken

of confidence

the effects

of fenoprofen

to represent

culated percentage fen as compared

of equimolar

a relia-

concentrations

and indomethacin

and estradiol-stimulated

PGF synthesis

inhibition

in terms of PGF,

The concentration

in superfusates

since the assay for P G F ~ a l s o (Patrono,

in our laboratory

1973).

PGFl~Concentrations

both compounds

are immeasurably

that its contribution

measures However,

PGF10 (

the

antiserum

1974 a) has ena-

in the same superfusates. low in most samples,

to the m e a s u r e m e n t

to be negligible.

of

is expressed

of a guinea-pig

with high affinity for PGFI~((Patrono et al., bled us to quantitate

The cal-

is 84% in the case of fenopro-

present

at I/5 of its concentration recent production

on the spontaneous

and release.

to 54% for indomethacin.

immunoreactive material

assumed

of

of the other.

2 depicts

(0.033 mM)

are quite high

and drop within

release

a dose-dipendent

Thus,

that

1973).

The addition of estradiol

to the same stimulus.

ble control

pattern

constant plateau

identical pattern of response

with a reasonable degree

Fig.

A continuous

of non

as a consequence

(Patrono and Ciabattoni,

ned from the two horns exposed

probably

to reach a relatively

ved for as long as 8 hours.

synthesis

in superfusates

trauma suffered by the tissue,

the first two hours

procedures

(Patrono and Ciabattoni,

PGF concentrations

gift

for P G F ~

pregnant rats release PGF with a characteristic we have already described

for PGFI~

so

of PGF2~can be safely

513

Pharmaco/ogica/ Research Communications, Vol. 6, No. 5, 1974 RAT UTERI Oestradlol

0.5

,,glrnl

ii

15 -1

/

L-'L_

I0

3 7 6

..q

__I

1

!

5 C

i

4

L. 3

2

o. 3 cn C

FENOPROFEN ~..

%._,j7"'1

-%.

! ! t

c O

o!9 E u c 0 u u.

0.033

mM

i

0.8 0.7

L..

0.6 0,5'

i i t i

~-'q

I

1

,,~..J L 2"-

0.4

I

I

I

I

I

I

i

I

I

I

e,

Time

!

0

2

3

4

5

hrs

6

----treated

~ ~

Oestradlol

~0

0.5

horn

l=glml

9 8

~

6

f

s

~

,5

J

! ,,

2 ~ •"3

2

[--

~ ~ .,~

L----

I

g ~

~ E u c 0 u

O o.

INDOMETHACIN

i t

0.9

0.033

I I

0.8 0.7

mM

0.6

0"5 0.4 Time

I

-

0

'1

1

1

2

., •

..,

!

I

I

3

4

5

6

hrs

Fig.2 C o n c e n t r a t i o n of PGF in s u p e r f u s a t e s f r o m the two horns of a s i n g l e r a t uterus, f o l l o w i n g e x p o s u r e to e s t r a d i o l (0.5 ~g/ml) i n c u b a t i o n medium: De Jalon' s o l u t i o n i n c u b a t i o n medium: De Jalon' s o l u t i o n c o n t a i n i n g e q u i m o l a r c o n c e n t r a t i o n s of f e n o p r o f e n or indcamethacin. Although

fenoprofen

in the same r a n g e repeated results

and

of c o n c e n t r a t i o n s

experiments was

indomethacin

observed,

a considerable affecting

both

were

found

to be active

(0.5 - 10 ~g/ml), degree

on

of v a r i a t i o n

the e x t e n t

in

of s t i m u l a -

Pharmacological Research Communications, Vo/. G, No. 5, 1974

51 4

tion by e s t r a d i o l g i s t i c drugs.

inhibition

This m i g h t be t e m p t a t i v e i y

in s e n s i t i v i t y of p r e c u r s o r s Indirect

and the p e r c e n t a g e

of the P G - s y n t h e t a s e as a f u n c t i o n

support

to this

the d e m o n s t r a t i o n , tion follows

ascribed

to changes

s y s t e m and/or

availability

of the time of the estrous

interpretation

provided

in other

a cyclic pattern,

of o v a r i a n h o r m o n e s

by a n t i f l o -

(Caldwell

most

can be d e r i v e d

species,

1972;

from

that PGF p r o d u c -

likely under

et al.,

cycle.

the i n f l u e n c e

Patrono

et al.,

1 974 b). Inhibition

of PGF s y n t h e s i s

and r e l e a s e

by a s p i r i n

and i n d o m e t h a c i n

has been

spontaneous However,

contractions

In order

by p a p a v e r i n e

to c h a r a c t e r i z e

fenoprofen,

experiments

in a p r e v i o u s

section.

compared

the effect

toneally

injected d r u g

immunoreactive found

in b o t h

controls,

does

were

significant.

Table

I - Fenoprofen in vivo.

out in vivo,

In a p r e l i m i n a r y

series

in Table

as c o m p a r e d were

Number

of samples

Number

of samples

Number

immeasurably

114

9

Serum PGF (pg/ml) M e a n + SEM

1488

low

P r o b a b i l i t y of s t a t i s t i c a l S t u d e n t ' s t test.

+

were

to

and r e l e a s e

Fenoprofen treated rats

+ 34.5

of

not s t a t i s t i -

of PGF s y n t h e s i s

177.8

we

of i n t r a p e r i -

I, lower v a l u e s

animals

of

as d e s c r i b e d

concentrations

Control rats P l a s m a PGF (pg/ml) M e a n + SEM

activity

of studies,

(10 mg/kg)

on s e r u m and p l a s m a

inhibition

1972).

of u t e r i n e

the P G - i n h i b i t o r y

the p l a s m a d i f f e r e n c e s

cally

inhibited

1972; Aiken,

the a b o l i s h m e n t

carried

of treated

with

not p r e v e n t per se PGF release.

further

As shown

fluids

although

(1972),

of a single dose

PGF.

associated

(Vane and W i l l i a m s ,

as shown by A i k e n

contractions

from p r e g n a n t rat uteri

+ 16.6

P

N.S.

9

134

132

+ 52.5

10

10

0

3

significance

was

calculated

<0.01

with

Pharmacologica/ Research Communications, Vo/. 6, No. 5, 1974 The r e a s o n

for m u c h

in c o n t r o l s during

lies

observed

concentrations to the h i g h l y sites

uterus

between

(Silver

serum

in m a l e

to the i n h i b i t o r y

is

likely

n o t be

activity

PGs

1972).

can be taken

of r e d u c t i o n

of p l a s m a

rats m a y

et al.,

PG-synthetase.

the d e g r e e s

origin

to plasma,

of p l a t e l e t

concentration

and in p l a s m a

eterogeneous

as c o m p a r e d

and r e l e a s e

of the p l a t e l e t

in serum

of s y n t h e s i s

accessible

levels,

clotting

90% of the

the a c t i v i t y

discrepancy

The of PGF

to be r e f e r r e d

PGF,

whose

main

as s e n s i t i v e

of f e n o p r o f e n

as

and/or the

or the platelets.

In an a t t e m p t kg)

of blood

approximately

to r e f l e c t

serum

in the s y n t h e s i s

the p r o c e s s

Thus,

higher

51 5

to c o m p a r e

of i n t r a p e r i t o n e a l l y

systems, fusates

correlation

cases.

administered

fenoprofen

horns

PGF

the e f f e c t

Fig.

were measured

of treated

was g e n e r a l l y

of serum

although

of a single

PGF c o n c e n t r a t i o n s of u t e r i n e

bition

the effects

observed

levels

and

on s e r u m

3 depicts

and c o n t r o l

PGF r e l e a s e

PGF was m o r e

(10 rag/

on those

in s e r u m

between

one of these

dose

and

rats.

of inhi-

the uterus,

pronounced

experiments,

superA good

the d e g r e e s from

two

in m o s t

in w h i c h

to

D

~

10

-------

control

rat

- -

Fenoprofen

--

treated

rat

¢.-

C --.

5

--

4

-

Oestrad

iol 0.5..g~ml

i

_

am

D t

.._.J

C~ 1

--

I--,- l I

C l

C O

|

0-5-

I

m

. . . .

0"4-L

0-3-C O

0-2--

U.

0-1

-i

I

,

0

1

2

-

i-

i

I

i

3

4

5

6

""

hrs

Time

F i g . 3 C o n c e n t r a t i o n of PGF in s u p e r f u s a t e s from rat uteri, f o l l o w i n g e x p o s u r e to e s t r a d i o l (0.5 ~g/ml).

Pharmacological Research Communications, Vo/. 6, No. 5, 1974

51 6

a 60% i n h i b i t i o n rat vs.

of PGF r e l e a s e

the c o n t r o l

animal,

serum PGF c o n c e n t r a t i o n We then studied

of a second

corresponded

(145 p g / m l vs.

the effects

and i n d o m e t h a c i n

f r o m the u t e r u s

an 85% d e c r e a s e

doses

on serum PGF c o n c e n t r a t i o n s . system

us to c a l c u l a t e

the a b s o l u t e

PGF2~,

of s i m u l t a n e o u s

equations.

reported scale

in Fig.

against

centrations.

4, where

the dose

the p e r c e n t a g e

The a v a i l a b i l i t y

concentrations

The r e s u l t s

of d r u g

inhibition

Each point r e p r e s e n t s

of f e n o p r o f e n

for PGF,IG((Patrono et al.,

1974 a) e n a b l e d by m e a n s

of

940 pg/ml).

of c o m p a r a b l e

radioimmunoassay

of the treated

is p l o t t e d

of c o n t r o l

the p e r c e n t a g e

of

are on a log

PGF2~concalculated

100

90

& 80

70

-

60 c 0

7A

.c

50

E

40

c

..'.

o n 30

20

10 -

0

I

0.1

I

tt

1

I

It

I i I ii

i

!

I

I 1 I III

1

I

10 Dose

I

II I

I I I I I ill

100

(mg/kg)

Fig.4 I n h i b i t i o n of PGF2~%serum c o n c e n t r a t i o n s and indomethacin, A .

by fenoprofen, • ,

Pharmacological Research Communications, Vol. 6, No. 5, 1974 on the m e a n c o n c e n t r a t i o n control group. are linear Several

The

log dose r e s p o n s e

and p a r a l l e l w i t h i n

investigators

activities

anti P G - s y n t h e t a s e

In our

laboratory,

being

almost

tions.

equally

This r e l a t i v e

encountered

tests

The p o s s i b l e

factors

review

active

responsible

that the v a s t m a j o r i t y

of a r a c h i d o n i c

Tomlinson

et al.

(1972)

no q u a n t i t a t i v e We have found

lower

that f e n o p r o f e n

extent

studies,

technical gifts

too,

refers

out on the to the

of the s'ynthesis although

Should

This

that the d i f f e r e n t

aspect

cell s y s t e m

We w i s h

assistance,

of P r o s t a g l a n d i n s .

by f u r t h e r

toward

Alternatively, to d i f f e r e n c e s precursors being

of

the

this appain a v a i l a b i -

and/or

essen-

investigated

in vitro.

to thank Mr.

and Dr.

reduction

PG-synthetases

sensitivities

is p r e s e n t l y

and

to a c o n s i d e r a b l y

this be c o n f i r m e d

of the v a r i o u s

fashion,

a significant

although

m i g h t be a s c r i b e d

omogeneous

of a F e l l o w s h i p

drugs

in a p r e l i m i n a r y

a c t i o n of the same drug.

ACKNOWLEDGEMENTS:

thus far c o l l e c t e d

is able to induce

suggest

lity or c o n c e n t r a t i o n

in a m o r e

be p o i n t e d

inhibition

the same cell s y s t e m have d i s t i n c t

tial cofactors.

in a r e c e n t

acid into PGE 2 and PGF2~.

this p r o b l e m

than PGF2~.

rent discrepancy

1972).

in r e l a t i v e

acid by the same drugs,

concentrations

it m i g h t

inhibitory

anti-

d a t a were reported.

investigated

of P G F 1 ~ s e r u m

(see Vane,

by Vane

of a s p i r i n - l i k e

found a l s o

of PGE I from e i c o s a t r i e n o i c

the d i f f e r e n t

it should p e r h a p s

reduced

while

not i n f r e q u e n t l y

upon

discussed

activity

of carra-

serum P G F 2 ~ c o n c e n t r a -

for v a r i a t i o n s

anti P G - s y n t h e t a s e conversion

to be three

employed

of i n f o r m a t i o n

and

in p r e p a r a t i o n ) ,

is however

and P G - s y n t h e t a s e s

between

drugs

as an i n h i b i t o r

depending

error.

1972).

has been found

discrepancy

In addition,

correlation

(see Vane,

in s u p p r e s s i n g

have b e e n t h o r o u g h l y

(1972).

of e x p e r i m e n t a l

a good

et al.,

the

vs.

of the two drugs

of a s p i r i n - l i k e

(Fischetti

w i t h other drugs,

inflammatory

potencies

limits

than f e n o p r o f e n

oedema

of I0 animals

curves

activities

indomethacin

times more potent geenin-induced

the

have r e p o r t e d

the a n t i - i n f l a m m a t o r y their

of each g r o u p

51 7

J.E.

Pike

G. R i p a n t i

for s e v e r a l g e n e r o u s

Dr. D. G r o s s i - B e l l o n i

from the Eli L i l l y

for skilled

Italia.

is the r e c i p i e n t

518

Pharmacological Research Communications, Vol. 6, No. 5, 1974

REFE~KIES

A i k e n J.W. (1972): N a t u r e 240, 21. C a l d w e l l B.V., T i l l s o n S.A., Brock W.A. and Speroff L. (1972) : P r o s t a g l a n d i n s 1, 21 7. N i c k a n d e r R.C., Kraay R.J. and M a r s h a l l W.S. (I 971) : Fed. Proc. 3_..9.0, 2 (Abst. no 2059). P a t r o n o C. (1973) : J.Nucl. Biol.Med. 17, 25. P a t r o n o C. and C i a b a t t o n i G. (1973) : in S y m p o s i u m on R a d i o i m m u n o a s s a y and R e l a t e d P r o c e d u r e s in C l i n i c a l M e d i c i n e and Research, I n t e r n a t i o n a l A t o m i c Energy Agency, I A E A - S M - 1 7 7 / 9 2 , in press. P a t r o n o C., G r o s s i - B e l l o n i D. and C i a b a t t o n i G. (1974 a) : s u b m i t t e d to the N a t i o n a l M e e t i n g of the ~/nerican F e d e r a t i o n for C l i n i c a l Research, A t l a n t i c City, M a y 4-5. P a t r o n o C., G r o s s i - B e l l o n i D., C i a b a t t o n i G., Serra G.B. and D e l l ' A c q u a S. (1974 b): S u b m i t t e d to the F o u r t h I n t e r n a t i o nal C o n g r e s s on H o r m o n a l Steroids, Mexico, S e p t e m b e r 2-7. Serra G.B. and M i d g l e y A.R.Jr. (1970) : Proc. S o c . E x p . B i o l . M e d . 13__~3, 1370. Silver M.J., Smith J.B., I n g e r m a n C. and Kocsis J.J. (1972) : P r o s t a g l a n d i n s 2, 75. T o m l i n s o n R.V., Ringold H.J., Q u r e s h i M.C. and F o r c h i e l l i E. (1972) : Biochem. B i o p h y s ° R e s . C o m m . 46, 552. V a n e J.R. (1972) : in I n f l a m m a t i o n - M e c h a n i s m and control, I.H. L e p o w & P.A. Ward Eds., A c a d e m i c Press, p. 261. V a n e J.R. and W i l l i a m s K.I. (1972) : B r i t . J o P h a r m a c o ! ° 45, 146P.