- Email: [email protected]
Anti-platelet aggregating and disaggregating activities of 6,9-methano PGI2
Life Sciences, Vol. 27, pp. 695-701 Printed in the U.S.A.
Pergamon Press
ANTI-PLATELET AGGREGATING AND DISAGGREGATING ACTIVITIES OF 6,9METHANO PGI 2 A. Morita, M. Mori, K. Hasegawa,
K. Kojima and S. Kobayashi
Central Research Laboratories, Sankyo Co., Ltd. Shinagawa-ku, Tokyo 140, Japan.
(Received in final form June 16, 1980)
Summary Anti-platelet aggregating and disaggregating activities of the chemically stable 6,9-methano prostaglandin I2 (6,9-methano PGI 2) were investigated. 6,9-Methano PGI 2 inhibited ADP-induced platelet aggregation in PRP from humans, rabbits and rats. 6,9-Methano PGI 2 also inhibited rabbit platelet aggregation induced by ADP, collagen, thrombin, arachidonic acid and ll,9-epoxymethano PGH 2. Antiaggregating activities of 6,9-methano PGI 2 were 0.3 to 2.0 times greater than those of PGE I. 6,9-Methano PGI 2 facilitated platelet disaggregation in a dose related manner. Antiaggregating and disaggregating activities of 6,9-methano PGI 2 were markedly enhanced by incubation with the phosphodiesterase inhibitor, theophylline. It is well known that prostaglandin 12 (PGI2), an intermediate in the metabolic pathway of arachidonic acid, is a potent inhibitor of human platelet aggregation in vitro (30 times more potent than PGEI) (i). It is also known that PGI 2 reverses aggregated platelets to their non-aggregated state in vitro(2) and in vivo(3). However, PGI 2 is chemically unstable in aqueous medium at pH 7.6 and will lose its ability to inhibit platelet aggregation in i0 minutes at 37°C(4). Therefore several attempts were made to modify its chemical structure in order to increase its stability. Recently four types of stable PGI 2 analogs were reported, viz. 6,9-thia PGI2(5), 5,6-dihydro PGI2(6,7), 6,9-nitrilo PGII(8) and 6,9methano PGI2(9,10,11). This paper describes the antiaggregatory and disaggregatory action of 6,9-methano PGI 2 in comparison with those of prostaglandin EI(PGEI). Materials and methods 6,9~-Methylene-lle,15e-dihydroxyprost-5(E),13(E)-dienoic acid (6,9-methano PGI2) (Fig. I) was synthesized by Kojima et al (9). 6,9-Methano PGI 2 was dissolved in a small amount of 1.3% NaHCO 3 followed by addition of 0.9% NaCI(pH 8.0). The solution was stable for more than one week at room temperature. PGEl(Sigma, St. louis, Mo., U.S.A.) was dissolved in a same manner as 6,9-methano PGI 2.
0024-3205/80/340695-07502.00/0 Copyright (c) 1980 Pergamon Press Ltd
696
6,9-Methano PGI 2 on Platelet Aggregation
Vol. 27, No. 8, 1980
COOH
x
-o-
PGI2
-CH2- 6,9-Methano PGI2 OH
OH Fig.
1
Structure of PGI 2 and 6 , 9 - m e t h a n o PGI 2
Inhlbition of p l a t e l e t a g g r e g a t i o n P l a t e l e t rich plasma(PRP) was p r e p a r e d by c o l l e c t l n g human, rabblt and rat blood into 0.i v o l u m e of 3.8% t r i s o d i u m citrate followed by c e n t r i f u g a t i o n at 95 x g for 15 m l n u t e s at room temperature. The number of platelets was adjusted to 300,000 p l a t e l e t s / ~ l (human) or 600,000 p l a t e l e t s / ~ l ( r a b b i t and rat). Suspensions of w a s h e d rabbit p l a t e l e t (WP) were p r e p a r e d as described b y H a m b e r g et al(12). In brief, PRP was c e n t r i f u g e d at 650 x g for 15 min. The p l a t e l e t p e l l e t was w a s h e d twice w i t h 0.15 M NaCi-0.15 M Tris-HCl buffer (pH 7.4)-0.077 M sodium E D T A (90;8:2, v/v/v) and subsequently recentrifuged. The p e l l e t of washed platelets was suspended in K r e b s - H e n s e l e i t m e d i u m not c o n t a i n i n g calcium. The number of p l a t e l e t s was a d j u s t e d to 1,000,000 platelets/~l. P l a t e l e t a g g r e g a t i o n was m o n i t o r e d by contlnuous r e c o r d l n g of light t r a n s m i s s i o n in a Born aggregometer(13). Ten ~M(human) or 5 ~M(rabbit and rat) a d e n o s i n e - 5 ' - d i p h o s p h a t e ( A D P ; Sigma) and 4 ~g/ml c o l l a g e n ( H o r m o n Chemie, M~nchen, W e s t Germany) were used as a g g r e g a t i n g agents in PRP. One ~g/ml a r a c h i d o n i c acid(Sigma), 0.5 U/ml thromb~n(Sigma) and 1 ~g/ml d l - l l , 9 - e p o x y m e t h a n o PGH 2 were used in w a s h e d rabbit platelets. Drugs were added 2 m i n u t e s prior to the a d d i t i o n of the a g g r e g a t i n g inducers. In some experiments, theophylline and drugs were s i m u l t a n e o u s l y added 2 m i n u t e s p r i o r to the a d d i t i o n of ADP. The IC50 was c a l c u l a t e d as the c o n c e n t r a t i o n required to reduce the a g g r e g a t i o n by 50% of its control value. Facilitation
of p l a t e l e t d i s a @ g r e g a t l o n
Rabbit PRP was p r e p a r e d as d e s c r i b e d before. The a g g r e g a t i o n inducer was 30 or 60 ~M ADP. A f t e r m a x i m a l a g g r e g a t i o n was observed, drugs were added. The time r e q u i r e d to cause 50% disa g g r e g a t i o n and the degree of d i s a g g r e g a t i o n in 20 m i n u t e s were calculated. In some experiments, t h e o p h y l l i n e was added simultaneously w i t h 6,9-methano PGI 2 or PGE I. Results Inhibition
of p l a t e l e t a g g r e g a t i o n
6 , 9 - M e t h a n o PGI 2 inhibited A D P - i n d u c e d
platelet aggregation •
Vol. 27, No. 8, 1 9 8 0
6,9-MethanoPGI2 on Platelet Aggregation
697
TABLE I Inhibition of ADP-induced platelet aggregation in PRP from several species by 6,9-methano PGI 2 and PGE I. Values given are the results of 6 to 8 experiments and are expressed as the concentration causing 50% inhibition of aggregation. IC50 Human 6,9-Methano PGI 2 PGE 1
(ng/ml) Rabbit
Rat
50 (43-57)*
73
(61-87)
40 (31-51)
29 (25-33)
36
(32-42)
39 (35-43)
*95% confidence limits
TABLE II Inhibition of rabbit platelet aggregation induced by several aggregation inducers by 6,9-methano PGI 2 and PGE I. Values given are the results of 6 to 8 experiments and are expressed as the concentration causing 50% inhibition of aggregation. Aggregation inducer
Platelets
Collagen ADP Arachidonic acld Thrombin ll,9-Epoxymethano PGH 2
PRP PRP WP WP WP
IC50 (ng/ml) 6,9-Methano PGI 2 PGE 1 49 73 15 21 20
(46-52)* (61-87) (13-17) (16-28) (15-26)
15 36 29 37 30
(14-16) (32-42) (25-33) (30-45) (26-35)
*95% confidence limits
in the PRP of the various species tested (Table I). 6,9-Methano PGI 2 was 2 times less potent than PGE 1 in human and rabbit PRP and equipotent to PGE 1 in rat PRP. 6,9-Methano PGI? also inhibited rabblt platelet aggregation induced by ADP, colIagen, arachidonic acid, thrombin and ll,9-epoxymethano PGH2(Tabl e II). 6,9-Methano PGI2 was two to three times less potent than PGE 1 in collagen- and ADP-induced aggregation of rabbit PRP. On the other hand, 6,9methano PGI 2 was 1.5 to 1.9 times more potent than PGE 1 in arachido. nlc acid-, thrombin- and ll,9-epoxymethano PGH2-induce d aggregation of washed rabbit platelets. Effect of theophylline Incubation of PRP with 50 ~g/ml theophylline, which itself had no effect on ADP-induced platelet aggregation, and with 200 ~g/ml theophylline, whlch itself inhibited ADP-induced platelet aggregation by 15.6 ± 1.2% (N=8), makedly augmented the effects of 6,9-methano PGI2. A dose-related shift of the dose-inh~bltion curve for 6,9-methano PGI 2 by theophylllne is shown an Fig. 2 (left). Relative increases in antiaggregatory activity of 6,9methano PGI 2 incubated with theophylllne (50 and 200 ~g/ml) are 2.3 and 9.7 respectively. Similar effects were seen when PRP was incubated with theophylline and PGE 1 (Fig. 2 right).
698
6,9-MethanoPGI2 on Platelet Aggregation
Vol. 27, No. 8, 1980
6,9-Methano PGI 2 100,
PGE1 100
,//,/ 0
~_ 80~ , . 60
q~
=
.a
80'
so,
"'40
..
40
/
o
-
20
z~
~2o
+/+//
#
L"
1o
loo
"
Concentration ( n g / m l ) Fig. 2
0
1 fo lo% Concentration ( n g / m l )
Potentiation of 6,9-methano PGI 2 (left)- and PGE 1 (right)induced inhibltlon of rabbit platelet aggregation by theophylline. Aggregation was carried out in the presence of 50 pg/ml theophylllne ( D , • ) , 200 ~g/ml theophylline ( O, • ) or in the absence of theophylline (~, •). Results are mean ± S.E. of 6 to 8 experiments.
ADP #
..~
_-
....
_ 30 0
t 6,9-Methano PGI2 Fig. 3
n,/ml
5 min
Superimposed disaggregation showing dose-dependent facil~tation of platelet disaggregation induced by 6,9-methano PGI 2
Vol. 27, No. 8, 1980
6,9-Methano PGI 2 on Platelet Aggregation
699
T A B L E III F a c i l i t a t i o n of p l a t e l e t d i s a g g r e g a t i o n b y 6 , 9 - m e t h a n o PGI 2 and PGE I. P l a t e l e t a g g r e g a t i o n w a s i n d u c e d b y 30 o r 60 ~M ADP. R e s u l t s are m e a n ±S.E. of 6 e x p e r i m e n t s .
Dose (ng/ml)
Drugs
Control 6,9-Methano
T i m e r e q u i r e d for 50% d i s a g g r e g a t i o n (min) 18.0 17.1 13.6 5.1 i.I 13.5 10.0 2.2 0.6
i0 30 i00 300 10 30 100 300
PGI 2
PGE 1
* P < 0.05
+± -+ -+ +-+ ± -+ -+
1.0 1.4 1.3" 1.0"* 0.i** 1.6 1.8"* 0.7** 0.i**
] 4
± ± -+ + _+ -+ +_ ± +
2.6 3.1 5.5 2.4** 2.1"* 1.9 2.7** 3.3** 2.4**
PGE1
] lb
1do
Concentration ( ng/mi ) Fig.
57.5 58.0 69.3 75.3 91.7 61.9 72.5 78.9 89.9
** P < 0.01
6,9-Methano PGi 2
i
P e r c e n t of final disaggregatlon
\\,\-.. i Concentration ( n g / m l )
P o t e n t i a t i o n of 6 , 9 - m e t h a n o P G I 2 ( l e f t )- and P G E l ( r i g h t ) i n d u c e d f a c l l i t a t i o n of r a b b i t p l a t e l e t d i s a g g r e g a t i o n b y t h e o p h y l l i n e , w h i c h was a d d e d w i t h 6 , 9 - m e t h a n o PGI 2 o r PGE 1 s i m u l t a n e o u s l y . The o r d i n a t e shows the time r e q u i r e d for 50% d i s a g g r e g a t i o n . Theophylline concentr a t i o n s were: 0 ~g/ml (~,A) : 50 pg/ml ( D , m ) and 200 ~g/ml ( 0 , 0 ) . E a c h p o i n t r e p r e s e n t s the m e a n ± S.E. of 6 to 8 e x p e r i m e n t s .
700
6,9-Methano PGI2 on Platelet Aggregation
Facilitatlon
Vol. 27, No. 8, 1980
of p l a t e l e t d i s a g g r e @ a t i o n
6,9-Methano PGI 2 p r o d u c e d a d o s e - r e l a t e d f a c i l i t a t i o n of p l a t e l e t d i s a g g r e g a t i o n and almost complete d l s a g g r e g a t i o n was o b s e r v e d for a 6,9-methano PGI2 c o n c e n t r a t i o n of 0.3 ~g/ml (Fig. 3). The time required to cause 50% d i s a g g r e g a t i o n and the degree of d i s a g g r e g a t i o n are shown in Table III. Incubation of PRP with 50 or 200 ~g/ml of theophylline, w h i c h themselves had no effects on p l a t e l e t disaggregation, augmented the disa g g r e g a t i n g action of 6 , 9 - m e t h a n o PGI 2 in a dose related m a n n e r (Fig. 4). DISCUSS~On 6,9-Methano PGI~ inhibited p l a t e l e t a g g r e g a t i o n by v a r l o u s a g g r e g a t i o n i n d u c e r s - i n various species. Our results are in a g r e e m e n t w i t h the r e c e n t l y p u b l i s h e d report of M o r t o n and B r o k a w (ii), in w h i c h 6 , 9 - m e t h a n o PGI 2 Is shown to be as potent as PGE 1 in inhibition of A D P - i n d u c e d human p l a t e l e t aggregatlon. The relative p o t e n c y of 6~9-methano PGI 2 to PGI 2 in i n h i b i t l n g i) p l a t e l e t a g g r e g a t i o n in PRP was 0.012 (human), I) 0.085 (rabblt) and 0.19 (rat) ±) . C o m p a r e d w i t h o t h e r k n o w n analogs of PGI 2, 6,9m e t h a n o PGI 2 was 2 tlmes more potent than 6,9-thla PGI2(5), e q u i p o t e n t to 5,6-dihydro PGI2(6,7) and 100 tlmes less p o t e n t than 6,9-nltrllo PGII(8). A n t i - p l a t e l e t a g g r e g a t o r y action of PGI 2 is c l o s e l y related to the s t i m u l a t i o n of p l a t e l e t adenylate cyclase(14,15,16) and is e n h a n c e d by p h o s p h o d i e s t e r a s e inhibltor(17). Theophylline m a r k e d l y e n h a n c e d the a n t i a g g r e g a t o r y action of 6 , 9 - m e t h a n o PGI 2, as well as PGE 1 w h l c h also stimulates p l a t e l e t adenylate cyclase (18). These results suggest that 6,9-methano PGI 2 inhibits p l a t e l e t a g g r e g a t i o n by e l e v a t i n g p l a t e l e t c-AMP level. 6 , 9 - M e t h a n o PGI 2 showed m a r k e d and d o s e - r e l a t e d f a c i l i t a t i o n of p l a t e l e t d i s a g g r e g a t ~ o n w h i c h was e n h a n c e d by incubation w l t h theophylllne. D i s a g g r e g a t ~ n g a c t l v i t y of 6 , 9 - m e t h a n o PGI 2 was equal to that of PGE I. O h t s u et al reported that PGI 2 was about ten times more active than PGE 1 in r e v e r s l n g a r a c h l d o n i c acidinduced r a b b i t p l a t e l e t aggregatlon(19). So d i s a g g r e g a t o r y a c t i v l t y of 6,9-methano PGI 2 m l g h t be about i0 times smaller than that of PGI2. The p r e s e n t study indicates that 6 , 9 - m e t h a n o PGI 2 has a p r o s t a c y c l i n e - l i k e a c t i v i t y on b l o o d platelets, though 5 to 80 tlmes less potent. A proper c o n s i d e r a t i o n of p o t e n t i a l clinlcal use of 6 , 9 - m e t h a n o PGI 2 m u s t await a more general survey of Its b i o l o g i c a l actlvities, p a r t i c u l a r l y on the c a r d i o v a s c u l a r and g a s t r o i n t e s t l n a l systems. Studies of this nature are now in progress in our labolatorles. Acknowledgements We w i s h to thank Dr. K. Murayama, D i r e c t o r of Central R e s e a r c h L a b o r a t o r i e s and Dr. H. Takagl, D i r e c t o r of P h a r m a c o l o g l c a l Research Laboratory, Sankyo Co., Ltd., for thelr c o n s t a n t encourage. menh and p e r t i n e n t suggestions, and also Miss K. Kohata for her skilled technical assistance. i) as c a l c u l a t e d
from reference
(15).
Vol. 27, No. 8, 1980
6,9-Methano PGI 2 on Platelet Aggregation
701
References
7.
8.
9. i0. ii. 12. 13. 14. 15. 16. 17. 18. 19.
S. MONCADA, R.J. GRYGLEWSKI, S. B U N T I N G and J.R. VANE, Nature 263 663-665 (1976). S. MONCADA, R.J. GRYGLEWSKI, S. B U N T I N G and J.R. VANE, P r o s t a g l a n d i n s i__22715-737 (1976). R.J. GRYGLEWSKI, R. K O R B U T and A. OCETKIEWICZ, P r o s t a g l a n d i n s 15 637-644 (1978). R.J. G R Y G E L E W S K I , S. BUNTING, S. MONCADA, R.J. F L O W E R and J.R. VANE, P r o s t a g l a n d i n s 1 2 6 8 5 - 7 1 3 (1976). R.J. G R Y G L E W S K I and K.C. NICOLAOU, E x p e r i e n t i a 3 4 1 3 3 6 - 1 3 3 8 (1978). G. TOGNA, C. GANDOLFI, A. ANDREONI, A. FUMAGALLI, C. PASSAROTTI, F. F A U S T I N I and C. PATRONO, Pharmacol. Res. Comm. ~ 909-916 (1979). B.H. CRANE, T.L. MAISH, Y.T. MADDOX, E.J. COREY, I. S Z E K E L Y and P.W. RAMWELL, J. Pharmacol. Exp. Ther. 2 0 6 1 3 2 - 1 3 8 (1978). G.L. B U N D Y and J.M. BALDWIN, T e t r a h e d r o n Lett. 1371-1374 (1978). K. K O J I M A and K. SAKAI, T e t r a h e d r o n Lett. 3743-3746 (1978). K.C. NICOLAOU, W.J. SIPIO, R.L. MAGOLDA, S. SEITZ and W.E. BARNETTE, J.C.S. Chem. Comm. 1067-1068 (1978). D.R. M O R T O N and F.C. BROKAW, J. Org. Chem. 4 4 2 8 8 0 - 2 8 8 7 (1979). M. H~MBERG, J. SVENSSON, T. W A K A B A Y A S H I and B. SAMUELSSON, Proc. Nat. Acad. Sci. U.S.A. 7 1 3 4 5 - 3 4 9 (1974). G.V.R. BORN, Nature 1 9 4 9 2 7 - 9 2 9 (1962). J.E. TATESON, S. M O N C A D A and J.R. VANE, P r o s t a g l a n d l n s 13 389-397 (1977). R. GORMAN, S. B U N T I N G and O.V. MILLER, P r o s t a g l a n d i n s 1_3 377-388 (1977). L.C. BEST, T.J. MARTIN, R.G.G. R U S S E L L and F.E. PRESTON, Nature 2 6 7 8 5 0 - 8 5 2 (1977). B.J.R. WHITTLE, S. M O N C A D A and J.R. VANE, P r o s t a g l a n d l n s 16 373-388 (1978). G. BALL, G.G. BRERETON, M. FULWOOD, D.M. IRELAND and P. YATES, B1ochem. J. 12___0709-718 (1970). A. OHTSU, K. NOGUCHI and K.C. NICOLAOU, A C T A HAEM. JAP. 42 659-666 (1979).
Pergamon Press
ANTI-PLATELET AGGREGATING AND DISAGGREGATING ACTIVITIES OF 6,9METHANO PGI 2 A. Morita, M. Mori, K. Hasegawa,
K. Kojima and S. Kobayashi
Central Research Laboratories, Sankyo Co., Ltd. Shinagawa-ku, Tokyo 140, Japan.
(Received in final form June 16, 1980)
Summary Anti-platelet aggregating and disaggregating activities of the chemically stable 6,9-methano prostaglandin I2 (6,9-methano PGI 2) were investigated. 6,9-Methano PGI 2 inhibited ADP-induced platelet aggregation in PRP from humans, rabbits and rats. 6,9-Methano PGI 2 also inhibited rabbit platelet aggregation induced by ADP, collagen, thrombin, arachidonic acid and ll,9-epoxymethano PGH 2. Antiaggregating activities of 6,9-methano PGI 2 were 0.3 to 2.0 times greater than those of PGE I. 6,9-Methano PGI 2 facilitated platelet disaggregation in a dose related manner. Antiaggregating and disaggregating activities of 6,9-methano PGI 2 were markedly enhanced by incubation with the phosphodiesterase inhibitor, theophylline. It is well known that prostaglandin 12 (PGI2), an intermediate in the metabolic pathway of arachidonic acid, is a potent inhibitor of human platelet aggregation in vitro (30 times more potent than PGEI) (i). It is also known that PGI 2 reverses aggregated platelets to their non-aggregated state in vitro(2) and in vivo(3). However, PGI 2 is chemically unstable in aqueous medium at pH 7.6 and will lose its ability to inhibit platelet aggregation in i0 minutes at 37°C(4). Therefore several attempts were made to modify its chemical structure in order to increase its stability. Recently four types of stable PGI 2 analogs were reported, viz. 6,9-thia PGI2(5), 5,6-dihydro PGI2(6,7), 6,9-nitrilo PGII(8) and 6,9methano PGI2(9,10,11). This paper describes the antiaggregatory and disaggregatory action of 6,9-methano PGI 2 in comparison with those of prostaglandin EI(PGEI). Materials and methods 6,9~-Methylene-lle,15e-dihydroxyprost-5(E),13(E)-dienoic acid (6,9-methano PGI2) (Fig. I) was synthesized by Kojima et al (9). 6,9-Methano PGI 2 was dissolved in a small amount of 1.3% NaHCO 3 followed by addition of 0.9% NaCI(pH 8.0). The solution was stable for more than one week at room temperature. PGEl(Sigma, St. louis, Mo., U.S.A.) was dissolved in a same manner as 6,9-methano PGI 2.
0024-3205/80/340695-07502.00/0 Copyright (c) 1980 Pergamon Press Ltd
696
6,9-Methano PGI 2 on Platelet Aggregation
Vol. 27, No. 8, 1980
COOH
x
-o-
PGI2
-CH2- 6,9-Methano PGI2 OH
OH Fig.
1
Structure of PGI 2 and 6 , 9 - m e t h a n o PGI 2
Inhlbition of p l a t e l e t a g g r e g a t i o n P l a t e l e t rich plasma(PRP) was p r e p a r e d by c o l l e c t l n g human, rabblt and rat blood into 0.i v o l u m e of 3.8% t r i s o d i u m citrate followed by c e n t r i f u g a t i o n at 95 x g for 15 m l n u t e s at room temperature. The number of platelets was adjusted to 300,000 p l a t e l e t s / ~ l (human) or 600,000 p l a t e l e t s / ~ l ( r a b b i t and rat). Suspensions of w a s h e d rabbit p l a t e l e t (WP) were p r e p a r e d as described b y H a m b e r g et al(12). In brief, PRP was c e n t r i f u g e d at 650 x g for 15 min. The p l a t e l e t p e l l e t was w a s h e d twice w i t h 0.15 M NaCi-0.15 M Tris-HCl buffer (pH 7.4)-0.077 M sodium E D T A (90;8:2, v/v/v) and subsequently recentrifuged. The p e l l e t of washed platelets was suspended in K r e b s - H e n s e l e i t m e d i u m not c o n t a i n i n g calcium. The number of p l a t e l e t s was a d j u s t e d to 1,000,000 platelets/~l. P l a t e l e t a g g r e g a t i o n was m o n i t o r e d by contlnuous r e c o r d l n g of light t r a n s m i s s i o n in a Born aggregometer(13). Ten ~M(human) or 5 ~M(rabbit and rat) a d e n o s i n e - 5 ' - d i p h o s p h a t e ( A D P ; Sigma) and 4 ~g/ml c o l l a g e n ( H o r m o n Chemie, M~nchen, W e s t Germany) were used as a g g r e g a t i n g agents in PRP. One ~g/ml a r a c h i d o n i c acid(Sigma), 0.5 U/ml thromb~n(Sigma) and 1 ~g/ml d l - l l , 9 - e p o x y m e t h a n o PGH 2 were used in w a s h e d rabbit platelets. Drugs were added 2 m i n u t e s prior to the a d d i t i o n of the a g g r e g a t i n g inducers. In some experiments, theophylline and drugs were s i m u l t a n e o u s l y added 2 m i n u t e s p r i o r to the a d d i t i o n of ADP. The IC50 was c a l c u l a t e d as the c o n c e n t r a t i o n required to reduce the a g g r e g a t i o n by 50% of its control value. Facilitation
of p l a t e l e t d i s a @ g r e g a t l o n
Rabbit PRP was p r e p a r e d as d e s c r i b e d before. The a g g r e g a t i o n inducer was 30 or 60 ~M ADP. A f t e r m a x i m a l a g g r e g a t i o n was observed, drugs were added. The time r e q u i r e d to cause 50% disa g g r e g a t i o n and the degree of d i s a g g r e g a t i o n in 20 m i n u t e s were calculated. In some experiments, t h e o p h y l l i n e was added simultaneously w i t h 6,9-methano PGI 2 or PGE I. Results Inhibition
of p l a t e l e t a g g r e g a t i o n
6 , 9 - M e t h a n o PGI 2 inhibited A D P - i n d u c e d
platelet aggregation •
Vol. 27, No. 8, 1 9 8 0
6,9-MethanoPGI2 on Platelet Aggregation
697
TABLE I Inhibition of ADP-induced platelet aggregation in PRP from several species by 6,9-methano PGI 2 and PGE I. Values given are the results of 6 to 8 experiments and are expressed as the concentration causing 50% inhibition of aggregation. IC50 Human 6,9-Methano PGI 2 PGE 1
(ng/ml) Rabbit
Rat
50 (43-57)*
73
(61-87)
40 (31-51)
29 (25-33)
36
(32-42)
39 (35-43)
*95% confidence limits
TABLE II Inhibition of rabbit platelet aggregation induced by several aggregation inducers by 6,9-methano PGI 2 and PGE I. Values given are the results of 6 to 8 experiments and are expressed as the concentration causing 50% inhibition of aggregation. Aggregation inducer
Platelets
Collagen ADP Arachidonic acld Thrombin ll,9-Epoxymethano PGH 2
PRP PRP WP WP WP
IC50 (ng/ml) 6,9-Methano PGI 2 PGE 1 49 73 15 21 20
(46-52)* (61-87) (13-17) (16-28) (15-26)
15 36 29 37 30
(14-16) (32-42) (25-33) (30-45) (26-35)
*95% confidence limits
in the PRP of the various species tested (Table I). 6,9-Methano PGI 2 was 2 times less potent than PGE 1 in human and rabbit PRP and equipotent to PGE 1 in rat PRP. 6,9-Methano PGI? also inhibited rabblt platelet aggregation induced by ADP, colIagen, arachidonic acid, thrombin and ll,9-epoxymethano PGH2(Tabl e II). 6,9-Methano PGI2 was two to three times less potent than PGE 1 in collagen- and ADP-induced aggregation of rabbit PRP. On the other hand, 6,9methano PGI 2 was 1.5 to 1.9 times more potent than PGE 1 in arachido. nlc acid-, thrombin- and ll,9-epoxymethano PGH2-induce d aggregation of washed rabbit platelets. Effect of theophylline Incubation of PRP with 50 ~g/ml theophylline, which itself had no effect on ADP-induced platelet aggregation, and with 200 ~g/ml theophylline, whlch itself inhibited ADP-induced platelet aggregation by 15.6 ± 1.2% (N=8), makedly augmented the effects of 6,9-methano PGI2. A dose-related shift of the dose-inh~bltion curve for 6,9-methano PGI 2 by theophylllne is shown an Fig. 2 (left). Relative increases in antiaggregatory activity of 6,9methano PGI 2 incubated with theophylllne (50 and 200 ~g/ml) are 2.3 and 9.7 respectively. Similar effects were seen when PRP was incubated with theophylline and PGE 1 (Fig. 2 right).
698
6,9-MethanoPGI2 on Platelet Aggregation
Vol. 27, No. 8, 1980
6,9-Methano PGI 2 100,
PGE1 100
,//,/ 0
~_ 80~ , . 60
q~
=
.a
80'
so,
"'40
..
40
/
o
-
20
z~
~2o
+/+//
#
L"
1o
loo
"
Concentration ( n g / m l ) Fig. 2
0
1 fo lo% Concentration ( n g / m l )
Potentiation of 6,9-methano PGI 2 (left)- and PGE 1 (right)induced inhibltlon of rabbit platelet aggregation by theophylline. Aggregation was carried out in the presence of 50 pg/ml theophylllne ( D , • ) , 200 ~g/ml theophylline ( O, • ) or in the absence of theophylline (~, •). Results are mean ± S.E. of 6 to 8 experiments.
ADP #
..~
_-
....
_ 30 0
t 6,9-Methano PGI2 Fig. 3
n,/ml
5 min
Superimposed disaggregation showing dose-dependent facil~tation of platelet disaggregation induced by 6,9-methano PGI 2
Vol. 27, No. 8, 1980
6,9-Methano PGI 2 on Platelet Aggregation
699
T A B L E III F a c i l i t a t i o n of p l a t e l e t d i s a g g r e g a t i o n b y 6 , 9 - m e t h a n o PGI 2 and PGE I. P l a t e l e t a g g r e g a t i o n w a s i n d u c e d b y 30 o r 60 ~M ADP. R e s u l t s are m e a n ±S.E. of 6 e x p e r i m e n t s .
Dose (ng/ml)
Drugs
Control 6,9-Methano
T i m e r e q u i r e d for 50% d i s a g g r e g a t i o n (min) 18.0 17.1 13.6 5.1 i.I 13.5 10.0 2.2 0.6
i0 30 i00 300 10 30 100 300
PGI 2
PGE 1
* P < 0.05
+± -+ -+ +-+ ± -+ -+
1.0 1.4 1.3" 1.0"* 0.i** 1.6 1.8"* 0.7** 0.i**
] 4
± ± -+ + _+ -+ +_ ± +
2.6 3.1 5.5 2.4** 2.1"* 1.9 2.7** 3.3** 2.4**
PGE1
] lb
1do
Concentration ( ng/mi ) Fig.
57.5 58.0 69.3 75.3 91.7 61.9 72.5 78.9 89.9
** P < 0.01
6,9-Methano PGi 2
i
P e r c e n t of final disaggregatlon
\\,\-.. i Concentration ( n g / m l )
P o t e n t i a t i o n of 6 , 9 - m e t h a n o P G I 2 ( l e f t )- and P G E l ( r i g h t ) i n d u c e d f a c l l i t a t i o n of r a b b i t p l a t e l e t d i s a g g r e g a t i o n b y t h e o p h y l l i n e , w h i c h was a d d e d w i t h 6 , 9 - m e t h a n o PGI 2 o r PGE 1 s i m u l t a n e o u s l y . The o r d i n a t e shows the time r e q u i r e d for 50% d i s a g g r e g a t i o n . Theophylline concentr a t i o n s were: 0 ~g/ml (~,A) : 50 pg/ml ( D , m ) and 200 ~g/ml ( 0 , 0 ) . E a c h p o i n t r e p r e s e n t s the m e a n ± S.E. of 6 to 8 e x p e r i m e n t s .
700
6,9-Methano PGI2 on Platelet Aggregation
Facilitatlon
Vol. 27, No. 8, 1980
of p l a t e l e t d i s a g g r e @ a t i o n
6,9-Methano PGI 2 p r o d u c e d a d o s e - r e l a t e d f a c i l i t a t i o n of p l a t e l e t d i s a g g r e g a t i o n and almost complete d l s a g g r e g a t i o n was o b s e r v e d for a 6,9-methano PGI2 c o n c e n t r a t i o n of 0.3 ~g/ml (Fig. 3). The time required to cause 50% d i s a g g r e g a t i o n and the degree of d i s a g g r e g a t i o n are shown in Table III. Incubation of PRP with 50 or 200 ~g/ml of theophylline, w h i c h themselves had no effects on p l a t e l e t disaggregation, augmented the disa g g r e g a t i n g action of 6 , 9 - m e t h a n o PGI 2 in a dose related m a n n e r (Fig. 4). DISCUSS~On 6,9-Methano PGI~ inhibited p l a t e l e t a g g r e g a t i o n by v a r l o u s a g g r e g a t i o n i n d u c e r s - i n various species. Our results are in a g r e e m e n t w i t h the r e c e n t l y p u b l i s h e d report of M o r t o n and B r o k a w (ii), in w h i c h 6 , 9 - m e t h a n o PGI 2 Is shown to be as potent as PGE 1 in inhibition of A D P - i n d u c e d human p l a t e l e t aggregatlon. The relative p o t e n c y of 6~9-methano PGI 2 to PGI 2 in i n h i b i t l n g i) p l a t e l e t a g g r e g a t i o n in PRP was 0.012 (human), I) 0.085 (rabblt) and 0.19 (rat) ±) . C o m p a r e d w i t h o t h e r k n o w n analogs of PGI 2, 6,9m e t h a n o PGI 2 was 2 tlmes more potent than 6,9-thla PGI2(5), e q u i p o t e n t to 5,6-dihydro PGI2(6,7) and 100 tlmes less p o t e n t than 6,9-nltrllo PGII(8). A n t i - p l a t e l e t a g g r e g a t o r y action of PGI 2 is c l o s e l y related to the s t i m u l a t i o n of p l a t e l e t adenylate cyclase(14,15,16) and is e n h a n c e d by p h o s p h o d i e s t e r a s e inhibltor(17). Theophylline m a r k e d l y e n h a n c e d the a n t i a g g r e g a t o r y action of 6 , 9 - m e t h a n o PGI 2, as well as PGE 1 w h l c h also stimulates p l a t e l e t adenylate cyclase (18). These results suggest that 6,9-methano PGI 2 inhibits p l a t e l e t a g g r e g a t i o n by e l e v a t i n g p l a t e l e t c-AMP level. 6 , 9 - M e t h a n o PGI 2 showed m a r k e d and d o s e - r e l a t e d f a c i l i t a t i o n of p l a t e l e t d i s a g g r e g a t ~ o n w h i c h was e n h a n c e d by incubation w l t h theophylllne. D i s a g g r e g a t ~ n g a c t l v i t y of 6 , 9 - m e t h a n o PGI 2 was equal to that of PGE I. O h t s u et al reported that PGI 2 was about ten times more active than PGE 1 in r e v e r s l n g a r a c h l d o n i c acidinduced r a b b i t p l a t e l e t aggregatlon(19). So d i s a g g r e g a t o r y a c t i v l t y of 6,9-methano PGI 2 m l g h t be about i0 times smaller than that of PGI2. The p r e s e n t study indicates that 6 , 9 - m e t h a n o PGI 2 has a p r o s t a c y c l i n e - l i k e a c t i v i t y on b l o o d platelets, though 5 to 80 tlmes less potent. A proper c o n s i d e r a t i o n of p o t e n t i a l clinlcal use of 6 , 9 - m e t h a n o PGI 2 m u s t await a more general survey of Its b i o l o g i c a l actlvities, p a r t i c u l a r l y on the c a r d i o v a s c u l a r and g a s t r o i n t e s t l n a l systems. Studies of this nature are now in progress in our labolatorles. Acknowledgements We w i s h to thank Dr. K. Murayama, D i r e c t o r of Central R e s e a r c h L a b o r a t o r i e s and Dr. H. Takagl, D i r e c t o r of P h a r m a c o l o g l c a l Research Laboratory, Sankyo Co., Ltd., for thelr c o n s t a n t encourage. menh and p e r t i n e n t suggestions, and also Miss K. Kohata for her skilled technical assistance. i) as c a l c u l a t e d
from reference
(15).
Vol. 27, No. 8, 1980
6,9-Methano PGI 2 on Platelet Aggregation
701
References
7.
8.
9. i0. ii. 12. 13. 14. 15. 16. 17. 18. 19.
S. MONCADA, R.J. GRYGLEWSKI, S. B U N T I N G and J.R. VANE, Nature 263 663-665 (1976). S. MONCADA, R.J. GRYGLEWSKI, S. B U N T I N G and J.R. VANE, P r o s t a g l a n d i n s i__22715-737 (1976). R.J. GRYGLEWSKI, R. K O R B U T and A. OCETKIEWICZ, P r o s t a g l a n d i n s 15 637-644 (1978). R.J. G R Y G E L E W S K I , S. BUNTING, S. MONCADA, R.J. F L O W E R and J.R. VANE, P r o s t a g l a n d i n s 1 2 6 8 5 - 7 1 3 (1976). R.J. G R Y G L E W S K I and K.C. NICOLAOU, E x p e r i e n t i a 3 4 1 3 3 6 - 1 3 3 8 (1978). G. TOGNA, C. GANDOLFI, A. ANDREONI, A. FUMAGALLI, C. PASSAROTTI, F. F A U S T I N I and C. PATRONO, Pharmacol. Res. Comm. ~ 909-916 (1979). B.H. CRANE, T.L. MAISH, Y.T. MADDOX, E.J. COREY, I. S Z E K E L Y and P.W. RAMWELL, J. Pharmacol. Exp. Ther. 2 0 6 1 3 2 - 1 3 8 (1978). G.L. B U N D Y and J.M. BALDWIN, T e t r a h e d r o n Lett. 1371-1374 (1978). K. K O J I M A and K. SAKAI, T e t r a h e d r o n Lett. 3743-3746 (1978). K.C. NICOLAOU, W.J. SIPIO, R.L. MAGOLDA, S. SEITZ and W.E. BARNETTE, J.C.S. Chem. Comm. 1067-1068 (1978). D.R. M O R T O N and F.C. BROKAW, J. Org. Chem. 4 4 2 8 8 0 - 2 8 8 7 (1979). M. H~MBERG, J. SVENSSON, T. W A K A B A Y A S H I and B. SAMUELSSON, Proc. Nat. Acad. Sci. U.S.A. 7 1 3 4 5 - 3 4 9 (1974). G.V.R. BORN, Nature 1 9 4 9 2 7 - 9 2 9 (1962). J.E. TATESON, S. M O N C A D A and J.R. VANE, P r o s t a g l a n d l n s 13 389-397 (1977). R. GORMAN, S. B U N T I N G and O.V. MILLER, P r o s t a g l a n d i n s 1_3 377-388 (1977). L.C. BEST, T.J. MARTIN, R.G.G. R U S S E L L and F.E. PRESTON, Nature 2 6 7 8 5 0 - 8 5 2 (1977). B.J.R. WHITTLE, S. M O N C A D A and J.R. VANE, P r o s t a g l a n d l n s 16 373-388 (1978). G. BALL, G.G. BRERETON, M. FULWOOD, D.M. IRELAND and P. YATES, B1ochem. J. 12___0709-718 (1970). A. OHTSU, K. NOGUCHI and K.C. NICOLAOU, A C T A HAEM. JAP. 42 659-666 (1979).