The effect of PGI2 and PGI2 analogies with increased stability on platelet cAMP content and aggregation

'rRE EFFECT OF PGI2 AHD PGI2 tibM)GXJES YlcTB INCREASED STABILXM'ON PLATELET CAMP CONTENT AXD AGGREGATION

Gy. Blask6,

E. Nemee6nszky,

G, Szab6+,

I. Stadler++

and L.A. PQlos First Department of Medioiae and Central Research Laboratories* of the Postgraduate Medical School, ia89 and Cbirroin Pharmaceutical and Chemical Works Ltd. , 1044 Budapest, Hungary

t'orm 12. Il. (Received 2S.&.19'i9; in revised Accepted by Editor bl. Versrraete)

I;)~‘:)

ABSTRACT arralogues with increased stability The effect of PGI at a pby~iologioa 2 pH on the aggregation end iatracellular CAMP coatemt of platelet6 were inveatigeted. The ethyl ester dorivrtive of PGI and its complexes with fi-oyolodertriae proved to d more stable than of adenylthe sodium salt of PO1 . Tbe stimlation elated with their inhibitory oyolaae positively oor i: effect on platelet aggregation.

INTRODUCTION Proataglaadia epoxy-APGFl) aggregation discovery (3)

(prostacyolinr

i8 tbe strongest (1) and a potent

of tbromboxaae

gate platelets Vane

12, PGI2

known

and induoe

thrombus

formation.

cellular

0AMP ooacentration

(2). After

vr6ooonatriation,

would

Like PGE

of platelet

and its ability

prestrmed there was a balance

of PG12 and Txb2 wbicb

inhibitor

vaaodilator

A2 (TXA2)

9-deoxy-6,9&-

determine

Woncada

betweeD

the to aggreand

tbe activities

the early

phase of

PGI2 elevate6 the fntra(5)s 4 . The obemical instability 1,

of PG12 wbicb

is converted

rapidly

6-keto-PGFld

at a physiological

to the stable

pH restricts

product

its therapeuti-

As

p-cyclodextrine

stabilization we expected

has proven

of different

labile fatty

that the stability

physiological

pB would

to be useful acids

in the

and volatiles,

of PGI2-derivatives

increase

when

complexed

at a

with

/3-cyclo-

dertrine, In this report

we show that PGS2, of PGI2 -etbyl

tbe stable

complex

are potent

stimulators

PGI2-ethyl

ester

and

o-cyclodertriae

of CAMP accumulation

that they are also highly

effective

ester and

in platelets

inbibitors

and

of platelet

aggregation. MATEBULS PGBl was tbe product salt and PGI2-ethyl according triae

(m.w.:

(m.u.t

were

The complex,

were

prepared

as follows:

/j-oyclodex-

of PGI2!-etbgl ester

ester

and

/3-cyclo-

382 mg of &cyclodextriae,

in 12.0 ml of distilled

and 4.0 ml of Britton-Robinson

buffer,

water

pH 8.0 at 30°C. 44 mg

of PGI -etbgl ester dissolved in 4.0 ml of otbyl-ether 2 added dropwise to the reaction mixture with continuous The

temperature

reaction

mixture

ethyl ester pby after

and

(30cyclodextrine,

in 0.5 ELM Tris-HCl

receiving

using

of blood

a Pye Unicam

of PGI: 2 by gas cbromatogra-

Model

105 gas cbroma-

substance8

from bealtby

was added

centrifuged

were dissolved

donors,

to one volume

at 200xg

and tbe platelet-rich

The PRP was then filtered column,

checked

ratio

buffer ) pH 8.3, and then stored

any drugs,

citrate,

temperature

stirring.

at

O°C

the experiment.

Xine volumes

sodium

Tbe molar

was freeze-dried.

silylation

tbrougbout

was

was kept at 30°C for 30 min and tben tbe

was 1 t 6.2. All of these

tograpb,

who were not

of 3.8 $ tri-

for 15 min at room

plasma,

PRP, separated.

a siliconized Sepbarose 4 x 20 cm, and equilibrated with Ca 2+ -free Ringer

solution,

and

Hungary.

of Cbinoin,

of PGI2-ethyl

11 $, was dissolved

humidity:

367,49)

the products

clatbrate,

was prepared

3aps.n. PGI2-sodium

of TUmClskgzi et al.(g),

1135) and the complexes

(3-cyclodextrine

dextrine

of Ono Company,

ester

to the method

ANB MBIXOBS

pH. 7.4, at room

in the void volume

tbrougb

temperature.

and collected

Platelets

in plastic

were

tubes.

2B

eluted

Platelet method

aggregation

of Born

count was corrected

/An/

We derived dextrine

complex

of

of Hayaabi

with

ceatrifugation

was measured usiag

from

an Fig.1.

the PGEl-p-cgolo-

wac)

(10) wbiob

of fl-ogolodextrine complex /B/,

1.0 ml of gel-filtered

time interval8

extractions

and PG12-

/3/ are showa

and Iabibara

PG12 or its derivatives

immediately

/AI/

by mass-spectrometry,

CAMP aesay:

various

complex

of this complex

The Calotte-modele Fi6.1. ethyl ester /A_& and tbeir

CoDtaining

to 2 x 105 per ~1.

fl-cgclodertrine

and their

the model

substantiated

to the

and 100 of I mM CaCl -3 Huoicb, or 2 I 10 n ADP.

Harm-Cbemie,

The Calo tte-models ethyl ester

according

(7) in the presence

~.&a11 collagen, The platelet

was recorded

100 pl

was incubated

100 ~1 aliqaots of ice-cold

TCA was removed

with

platelet

from

were

a CAMP-binding

to the method

protein

from

PG12-

suspe~6ion at 30°C.

removed

the sapernataat

After

by four

ethyl-ether.

oAMP

of Brow-n et al. (8),

bovine

adrenal

At

and mixed

5 $ TCA solution.

2.0 el of water-saturated

according

/AI/,

cortex+

recovery 3 of H-CAMP,

Amersham

correotions

for small

Tba

of oAKP

The doss-dependent

WI&

aorritorod

Co., USA,

by adding

a known

to the TCA solution

amsunt

making

losues.

irrhibition of platelet

by PGI2 and its derivatives

aggragatioD

ia abova ia Fig. 2,

Inhibition of platelet aggregatioD by PG12 and ita Fig. 2. derivatives: dose-response curves. These experimeDts were performed in a reaction mixture with aDd 2 phi a final volume of 2.0 ml PRP, oontainiag 1 mM CaCl compou &l 13 were ADP at a pH of 8.3. All of the investigated inoubated with platelets for 5 win at 30°C before the iaductioD of aggregation. Tbe results were expreesed ao tbe percentage inbibitioa measured by light transmission 10 miss after the PGI -sodium salt /Oa/, PG12induction of aggregation. ethyl eeter /OO/, a oomp 2 ex of 10 $ PGI -etbgl ester a/, the eamo co&plex witb 4 % wi tb /3-oyolodertrine /.APG12-ethyl ester /A-A/.

8-10 Dg per ml of the aodinm inhibited

ADP-induced

platelet

salt of PG12 Completely

aggregation.

Tbia

%LS in agree-

meat with

previously

published

ester

bad the 8ame inhibitory

20-30

ag/ml.

dextrine 200-400

The complexes

ng/ml

effeot

depeading

o:> tbe PG12 -content

of upto 800 pg per ml of

did not affect

platelet

activity

ester

aggregation

the stability

of these compounda

were

ADP-induced

f3-cvolobetween

fl-cyclodertriae

induced

aloue

by A.DP or oollagen,

of the anti-aggregating

we incubated

taken aad tested

of

of the complex.

them in PRP af 30°C

with a pH of 7.4 for 0 N 300 min. At different samples

and

at a coaceatratioae

Concentrations

To investigate

PGI24byl

at a conceatration

of PG12-ethyl

80 $ inhibition

produced

(4,6),

paper8

for these

time intervals

inbibitorg

effect

on

see Fig. 3 .

aggregation.

lo-

15

30

45

60

r20

reo M/N

240 AT 37-C

AND

Al pIi

300 7j4

The duration of the antiaggregatiag effect of Fig. 3. pGI -derivatives. PGI -8oditim salt, 10 ag/ml:o0, pGI2-ethyl e8ter, 20 ii~/mlr~0, the oomplex of PGI witb a PC1 etb$l ester and p -cyclodertriae couoeatra?iou of 80 ng/mlrA -----A, or 160 ng/mlt~--3. Tbe aotiaggregating rapidly.

activity

The full inbibitory

salt and 20 as/ml ethyl eater with is readily

of 5-10 lasted

n&l

developed

PG12-8odiUm

for 240 miu, but

bas a proloaged

at 300 min. Tbu8,

an increased

compouods

at 300 min. Tbe complex

(30oyoledextriue

complete

showed

ester

persisted

diam salt tbe oyolodextrine vative

effect

PG12-ethyl

50 $ of tbi8 effect

of these

complex

oompared

of PG12-

effect

wbicb

to the PGI -8o2 e8ter deri-

and the ethyl

lltability at a pbyeiological

PH.

Th8 antiaggregating to t&e increase measured

in 'intracellular

platelet

and compared

potsnfial

of PGK2 is closely CAMP

lev8U.

CAMP durirtg the first

t-be sffeot

of PGI2 with

2

I

3

related

We therefore

10 min of aggregation

that of PGEl (Fig. 4).

rO MIN

5

The effect of PGE aad PGI -derivative% on tbe Fig. 4. intracellular 0AM.P content tf platelgts. a, PGI -sodium, 50 ag/ml:OPGE , 50 ng/mlt ??0, PGIl-etbgl ester+~cyclodext&ne complex, 150 n&al; with 10 9 effective 8obstancetA -A. German

et al. (4) described

there wa% a rapid but transient

of PGEls

level of CAMP, witb a maximum sodium

that following

exerted

tb8 CAMP content

iucrease

effeot

PGI2-

at tbe same aoncent-

ester-/3=cgclodextrine

elovty

in tbe

level at 30 ~48~8, whereas

a more prolonged

PG12-ethyl

ration.

the addition

complex

at a conoentratioa

increased

a% bigb as 150 ng

per ml (Fig. 4). PGI2-%odinra salt, &ogclodextrine

PGI2-ethyl

complex

ester

were incubated

and PGI2-ethyl

with gel filtered

platelet8

at pH 7.4 upto 300 mine at a concentration

10 Dg/mf,

20 a&ml

and 200 ng/ml,

In the abseaoe eonteat

of platelet%

of

respactively.

of PGX2-derivatives incubated

ester-

the intracellular

at 3O*C,

300 aia was 0,7 - 1.1 p*07/7_07 plateleta,

CAMP

at a pH of 7.4 for

The effect of PGI dorlvatives of: the CAMP contel3t Fig. 5. getails: see Materiala aad Methods. of platelets. Experimental PGI -ethyl ester, 20 n&l: PGI2-eodiam, 10 ng/mlrO -0, -a, the complex of PGI -etby 2 ester with /3-cyolodextbuf? 81‘ alone: Or%e, 200 ng/mltA ---A, 0. If 10 ng/ml the amount

of PC12 -sodium

was added

of CAMP increased

to 2.8 pmol/107

eeveral

minutea

end it remained

Similar

results

were

with

obtained

PG12 -ethyl

20 ng/ml

(30cgclodextriae

to the reaction

et this

platelets

within

level for 240 min.

when platelets

eater

mixture,

were

or 200 a&11

incubated

PG12-ethyl

ester-

complex.

DISCUSSION Tbe results ite complex effect,

altbongh

ned witb

that PG12-ethyl

salt of PG12. Although

for four hours

p3. 6.keto-PGFIK

and

less than that obtaiPGI2 only bas a

of 2-3 minutes, in vitro

of CA?@ remains

ester

have an antiaggregating

tbia is 10 to 100 times

in tbe circulatioo

tory effect acouwletion

indicate

p-cgclodextrine

the sodium

half-life

logical

with

presented

it has en inbibi-

and tbe intracellular

for at least 300 min at a pbysio-

is muob

less effective

at inhibiting

aggregation

platelet

potent in raising

intracellular

in Fig, 3.

As shown the PG12-ethyl longed

in tbe current

levels

literature effect

This returned

ulia, however,

on elevating

and entrance

intracellular

the aggregation

of platelets,

inbibited

possibilities

to find

whfcb would

to the initial

and

a pro-

have

We have been unable

almost

are several

Bster

aggregation

any substance

same time in PRP, remained

less

(11).

complex

011 platelet

CAMP content*

such a proloaged

lOOO-times

and Fig. 5. PG12-ethyl

effect

the intracellular

There

CAMP

ester-fl-CyclodextriDe

inhibitory

levels.

(6) and also about

value

have CAMP

after

measured

300

at the

longer. to explain

our results:

and its derivatives might bave been bound to the regula2 tory unit of adenyl-cyclase in the membrane and become more DGI

stable

tban in solution;

active

substances

or the breakdown

products

slight have been as effective

of tbe

in raising

CAMP

as PG12 itself. Acoording tbe

complex

about 45-50 action. rapid

to our preliminary of PG12 -ethyl

minutes

ester with

in rabbits

The phareaoological

and transient

and an increase dogs after Further

deorease

a siagle

assessed

properties of blood

intravenous

pharmacological

dose

studies

to assess

the half-life

p-cyclodextrine

of

is

by its antiaggregating of the complex

pressure

of heart rate by 20040/min

these PG12 analogues therapeutic

results,

are a

by 25-35 Hgmm

in beparinized

of 75 a&g. should

bs carried

these full potential

out on

as possible

agents.

ACENOWLEDGEHEBT This work was supported by Cbinoin Ltd., grant No. 40011038/78. We are indebted to Hrs Tberese Fazskas and Mrs Susanna Orosz for excellent technical assistance and to L. Musabek Ph.D. for belpful oriticism. REFERENCES

1.

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2.

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