Inhibitory effects of aspirin and indomethacin on the biosynthesis of PGE2 and PGF2α

Vol. 57, No. 2,1974

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

INHIBITORY EFFECTS OF ASPIRIN AND INDOMETHACIN ON THE BIOSYNTHESIS OF PGE2 AND PGF2o Joseph

W. Horodniak,

Received

Marc Julius, John E. Zarembo and A. Douglas Research and Development Division Smith Kline and French Laboratories Philadelphia, Pa. 19101

February

Bender

4,1974 SUMMARY

A gas-liquid chromatography system has been used to study the effects of indomethacin and aspirin on the biosynthesis of PGE2 and PGFZs by the prostaglandin synthetase system of bovine seminal vesicle. Both compounds were found to inhibit the production of PGE2 and PGFZs. However, based on statistical analyses, the inhibitory effect of indomethacin was found to be non-selective while aspirin produced statistically significant preferential inhibition of PGE2 over PGF20.

INTRODUCTION Several taglandin

groups

formation

agents.

These

while

amount

suggested

a variety

PG-like

activity.

of prostaglandin the

Recognizing

the differential selective

conceivably

could

be of potential

alter

have been formed

simultaneous

could

have effects

inhibition the balance therapeutic

study important

0 I Y74 by Accrdemic Press, of reproduction in my form

Inc. re.rerved.

including

lung6,

the measurement measure

employed

of the products

of the synthesis in a particular

of pros-

only

interest.

To observe

PGE22 formation,

acid3.

Flower

of the action

of

implications.

of a specific tissue

and

to simultaneously

from arachidonic

physiological

bovine1*233

human skin7,

of PGEZ and PGFZe on various

539 Copyright All rights

for

pig

of pros-

antiinflammatory

of tissues

some of which

techniques

synthetase

could

of methods

employed,

prostaglandin

systems,

a variety

have been

that

organ

employed

inhibition

by non-steroidal

, dog spleen3s5, guinea

quantitative

the

et a1.3

arachidonic

have

estimate

Recently, estimate

acid

Also,

produced others

from

vesicles4

brainS¶S.

taglandin

have demonstrated

studies

and ram seminal rabbit

of investigators

tissues

prostaglandin

and such a result selective

and

inhibition

Vol. 57, No. 2, 1974

demands

accurate

analytical

chromatography synthesized

BIOCHEMICAL

system

arachidonic

The inhibitory

effects

PGE2 and PGF2o have been

results

of PGE2 and PGF2, than

to measure by bovine

of aspirin

and radiochemical of the current

extent

acid

reported

the

amounts

seminal

vesicle

and indomethacin

previously

study

suggest

that

of PGE2 and PGF2, microsomes

analytical

(BSVM). of

bioassay

methods3.

inhibits

aspirin

a gas-liquid

employing

indomethacin

while

to the same extent,

study

on the production

in studies

and spectrophotometric

inhibits

The

the synthesis PGE2 to a greater

PGF2,.

MATERIALS 1)

In the present

procedures.

was developed

from

systems6,

test

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

AND METHODS

Biological A.

B.

BSVM were

prepared

as frozen

suspensions

The biological

as described instead

reaction

of being

conditions

The concentration

et al.g.

by Takeguchi

continue

for

production with

initial different

were

also

of components

studies

six

minutes.

under inhibitor

times

acid

with

in order

were

The inhibitory

effects

preincubated

of these

of conditions. at 25'C

was stopped

the reaction

First, for

five

540

from

Takeguchi

pH 8.3

was permitted

a time

and immersion

and indomethacin

stored

shown below:

the reaction

The reaction

to a pH of 3.0-3.5

sets

are

to construct

aspirin

two different

adapted

0.05 M Tris 0.33 mM 2.0 mM 4-6 mglml 0.04% 10 ml 6 min 37.5" c

of experiments

of PGE2 and PGF2e.

25% formic

the

series

but were

lyophilized.

Buffer Arachidonic Acid L-epinephrine Bitartrate BSVM Cutscum Reaction Volume Reaction Time Reaction Temperature In the

et a1.9,

curve

for

the

by acidification in an ice time

two compounds

bath.

before

In

was uniformly were

studied

the enzyme preparation minutes

to

substrate

and was

Vol. 57, No. 2, 1974

added.

Second,

directly cate

BIOCHEMICAL

the

inhibitor

to the reaction samples

incubated

were

was not

mixture

run

AND BIOPHYSICAL

in all

preincubated

containing

with

both

experiments.

All

RESEARCH COMMUNICATIONS

enzyme,

enzyme data

but

was added

and substrate.

presented

are

Dupli-

for

pre-

samples.

2) Analytical A.

The acidified washed

reaction

with

distilled

8.0 phosphate

to pH 3-3.5

with

distilled

Excess

and ether

was then

treated

The pyridine

standard

graphic B.

at 6O'C.

with

2% methoxamine was removed

an internal

authentic

acid

to react

Diazomethane for

15 minutes.

of N2 at R.T.

in pyridine under

was added

The

at 60°C for

a N2 stream,

and bis

and 1% trimethylchlorosilane) Chemical

Co.)

The resulting

prostaglandins

in pyridine solution

standard

were

was added

was used

for

and reacted gas chromato-

analyzed

by gas chromatography

Standard

curves

internal

standard.

method.

PGF2d and PGE2 containing G.C.

The PGE2 and PGF2d were

obtained

Chemicals

other

All

prepared

Perkin Elmer 900 3% OV 225 100/120 Gas Chrom Q glass 6 ft x 2 mm 210°c 20 cc/min He F.I.D. 225" (glass)

Column Temperature Flow Detector Injector Port

(Japan).

were

Conditions

Instrument Column

C.

25% formic

to dryness.

a stream

pH

analysis.

The derivatized using

under

with

was backwashed

cholanic

and allowed

removed

and was back-

with

The ether

were

solvent

(BSTFA and TMCS) (Pierce one hour

adidified

was evaporated

residue

ether

was reextracted

ether.

(trimethylsilyl)trifluoroacetamide

for

layer

The GLC internal

to the

with

was then

with

and the solution

reagent

4 hour.

The ether

and extracted

was added

residue

was extracted

The buffer

H20.

point

in ether

H20.

buffer.

acid

at this

mixture

from chemicals

541

the Hormel were

Institute

reagent

grade

or Ono or the

with

Vol. 57, No. 2,1974

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FIGURE FORMATION

240-

OF

I.

PROSTAGLANOIN

. TOTAL

WITH

TIME

PG

220210 200 180 5 F

160-

5: 2

140-

2

9

I20 100 -

60 40 -

( .:f ..

, , , , , , ,

0

4

2

6

8 TIME

highest

purity

commercially

10

12

14

16

(MIN.)

obtainable,

and were

used without

further

purification. RESULT AND DISCUSSION Figure

I shows a typical

a function usually

of time. 15-20%.

ratios

for

The percentage

Under

typically

curve

are

of PGE2 and PGF2, as

conversion

of substrate

conditions

described

but vary

from

to 70:30

are

studies

the reaction 60:40

the formation

50:50

at six above,

for

minutes

is

the PGE2:PGF2,

any given

BSVM

preparation. The data incubated the added

with

synthesis

vation

II

BSVM.

Under

of both

directly

inhibitory

in Figure

is

these

consistent

who have noted

that

mixture

of indomethacin with

aspirin

the

inhibitor

was pre-

and indomethacin

inhibited

In studies

in which

containing

enzyme and substrate,

and aspirin

the work

incubation

in which

conditions

PGE2 and PGF2,.

to the reaction effects

from

of Flower

conditions

are

542

were

not

the inhibitor

apparent.

et al. 3 and Smith critical

in studies

This

was the obser-

and Lands 10 of this

type.

A,

0

0

inhibition

and 0

show

0

0

from

Molar)

10-6

is an average

1.3

PGF2,

1.8

:

1

1 43

60 1.4

TOTAL

1 :

42

Ratio

in reaction

55

1.3

10-5

/

of duplicates.

Aspirin

lndomethacin

Activity

concentrations

I

8

:

PGE2

point

l(

/

d A

01

0

TOTAL

controls

0

INDOMETHACIN

:

and

Each

A

AND

INOOMETHACIN

BY ASPIRIN

59

Aspirin 100

I PGE2

experiments.

Cone.

lo-4

I

ASPIRIN

PG SYNTHESIS

the sum of PGF2,

different from

(PM)

three

1.7

ID56 lndomethacin

is calculated

“Gb PGE2

prostaglandin

points

(Log

INHIBITOR

I

lo-5

I

0

PGE2

TOTAL

10-6

11

AND

INDOMETHACIN

PGF2,

I

0

0

A

OF PGE2,

10-4

the experimental

A

0

0

ASPIRIN

INHIBITION

of total

I

10-5

I

10-6

b0

The perwnt with inhibitor.

The

A

0

PGF2,

II.

0

INDOMETHACIN

FIGURE

and

10-4

0

V

ASPIRIN

v

reactlons

lo- -3

BIOCHEMICAL

Vol. 57, No. 2, 1974

AND 5lOPHYSlCAL

RESEARCH COMMUNICATIONS

TABLE I ID50 Values

Published

And Indomethacin

Method of Analysis

Tissue*

Reference

For Aspirin

ID50

Activity Ratio**

(UN

Indomethacin

Aspirin

BSVM

GLC

1.4

60

(1)

BSVM

Adrenochrome

2.0

820

(2)

BSVM

Radiochemical

7.0

15000

RSVM

Oxygen

9.0

9000

This

work

(10)

*BSVM = Bovine **Activity

Seminal

Ratio

electrode

Vesicle

by the method

indicate duces

that

indomethacin

a greater

(0.8-2.3)

and 1.8

activity

recently

These

greater

authors

also

than

effect

in PGF2o.

indomethacin

and aspirin

Selective

1OOO:l

Vesicle

Microsomes

that

found

two other

that

were

obtained

while

aspirin

was in

and

to be 1.3

reported

synthetase preferential intermediate.

was 4.5

inhibiting

times

as effective

the production

reduced

of the biosynthetic

pro-

ratios

a prostaglandin-like

phenylbutazone

products

data

found

et a1.12

benzydamine

of PGF2, as it

analyzed

of prostaglandins

Wlodawer

to PGFle with

were

The respective

inhibition

by others.

formation

11 , the

bioassay

a non-selective

et al. 3 reported

Flower

extent

line

and aspirin

in PGE2 than

(1.1-3.2).

the

indomethacin

of parallel

of PGEl as opposed

in inhibiting PGE2.

for

has been reported

inhibition More

limits

for

produces

reduction

95% confidence

2143:l

= 1ndomethacin:Aspirin

When the PGF2,. 'PGE2 ratios statistically

410: 1

RSVM = Ram Seminal

Microsomes,

43:l

of

PGF2, and PGE2 to a process,

namely,

PGD2

and malondialdehyde. The potency was found Table

ratio

to be 43:l

I this

value

between (using

the

is compared

indomethacin ID50 for to the

and aspirin total

ratios

544

in the

prostaglandin reported

by other

current

study

synthesis).

In

workers.

1,2,10

Vol. 57, No. 2, 1974

Reviewing

BIOCHEMICAL

these

different

analytical

between

ID50 values vary

however, the

data

(see Table

systems reported

for

methods

in the reaction

sequence.

thacin

by each of these

observed

an early

step

step.

This

and aspirin

is

the degree

ID50 ratios. other

that

again

in Table cofactor

the

good agreement for

aspirin,

may be due in large which

measure

consistent

while it

that

of activity

steps

inhibits

wide

at

variation

at more than

proposed

for

observation

to

of indomeit

the very

may inhibit

part

different

activity

suggests

our earlier

despite

one

indomethacin

of a difference

in

selectivity.

of Tomlinson Here

values

indicates

with

that

that,

The ID50 values

differences

pathway,

in the site

consistent

apparent

is reasonably

techniques

indicates

of inhibitory

The data

there

The relatively

of aspirin

difference

is

employed,

in the biosynthetic

in the potency

it

indomethacin.

These

analytical

I)

employed,

considerably.

different

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I,

et al.*

indicate

that

the

ID50 of indomethacin

but

the

may also

ID50 of aspirin influence

the

cofactor is

may also

in agreement

is much larger. activity

influence with This

of an inhibitor.

Acknowledgements We are indebted to Mr. R. Gifford and Ms. L. Lathan with the BSVM preparations and G.C. analysis, and to Mr. statistical evaluation of the data.

for their J. Pauls

help for the

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Takeguchi, C., and Sih, C.J., Prostaglandins (1972) 2 169-184. Tomlinson, R.V., Ringold, H.J., Quershi, M.C., and Forchielli, E., Biochem. Biophys. Res. Comm. (1972) 46 552-559. Flower, R.J., Cheung, H.S., and Cushman, D.W., Prostaglandins (1973) 4 325-341. xaz, A., Stern, H., and Kenig-Wakshal, R., Prostaglandins (1973) 2 337-352. Flower, R. Gryglewski, R., Herbaczynska-Cedro, K., and Vane, J.R., Nature New Biol. (1972) -238 104-106. Vane, J.R., Nature New Biol. (1971) -231 232-235. W., Br. J. Pharm. (1972) 46 172-175. Greaves, M.W., and McDonald-Gibson, Flower, R.J., and Vane, J.R., Nature (1972) -240 410-411. Takeguchi, C., Kohno, E., and Sih, C.J., Biochemistry (1971) 10 2372-2376 Smith, W.L., and Lands, W.E.M., J. Biol. Chem. (1971) 246 6700-6702. Finney, D.J., "Statistical Methods in Biological Assay"(1952), Hafner Publishing, N.Y. Wlodawer, P., Samuelson, B., Albonico, S.M., and Corey, E.J., J. Am. Chem. Sot. (1971) -93 2815-2816.

545