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Relationship between chemical structure and plateletaggregation activity of prostaglandins
BIOCHIMICA
BBA
ET BIOPHYSICA
285
ACTA
55625
RELATIONSHIP
BETWEEN
AGGREGATION
ACTIVITY
CHEMICAL
STRUCTURE
AND PLATELET-
OF PROSTAGLANDINS
J. KLOEZE Unilever Research (Received
Laboratory,
May pth,
Vlaardingen
(The Netherlands)
1969)
SUMMARY
The finding that prostaglandin E, inhibits ADP-induced platelet aggregation in titrated platelet-rich rat-blood plasma, whereas prostaglandin E, stimulates it, has led to an investigation into the potencies of the opposing effects in relation to each other by examining mixtures of prostaglandins E, and E, at different concentrations. It has been found that the relationship between the extent of platelet aggregation, expressed in percentages of the maximum decrease in absorbance in the control (% max. d A), and the molar concentration of prostaglandins E, and E, can be written as: y’ max. dA = - 494 - 99 log [PGE,] In order and biological were examined blood plasma. group, as well at C-15.
+ 26 log [PGE,]
to gain information on the relationship between chemical structure activity, 23 prostaglandins and the methyl esters of some of them for their effects on platelet aggregation in titrated platelet-rich ratIt has been found that the C=O groups at the ring and in the carboxyl as the distance between them, are essential, as is the hydroxyl group
INTRODUCTION
Small amounts of prostaglandins are found widely distributed in different organs. Most of them have potent biological activity of some kind, as was reviewed recently by different authorsl-O. One of these activities, as I reported before’, is the effect on ADP-induced platelet aggregation in vitro. Prostaglandin E, tias found to have an inhibiting effect, whereas prostaglandin E, displayed a stimulating effect in various species. However, the stimulating effect of prostaglandin E, reached an optimum between 0.5 and I ,q/ml; at higher concentrations it decreased, probably owing to contamination of the prostaglandin E, preparation by prostaglandin E,, which on gas-chromatographic analysis appeared to be less than 1%. The inability of N. CHANDRASEKHAR to reproduce the stimulating effect of Biochim.
Biophys.
Acta,
187 (1969) z85--292
286
J. KLOEZE
prostaglandin tamination.
E,, as referred to by BERGSTROM et aL3, may be a result of this con-
The contamination is caused by the fact that the particulate enzyme fraction of sheep vesicular gland used for the biosynthesis of the prostaglandins contains very small amounts of prostaglandins E,, E, and their precursor fatty acids. As the physical and chemical properties of the prostaglandins do not differ much, it is very difficult to purify the preparations completely from the endogenous prostaglandins. For this reason prostaglandins with relative potencies lower than 1% with respect to prostaglandin E, or prostaglandin E, activity are considered inactive in the present experiments. These experiments comprise the determination of the potencies of the inhibiting and stimulating effects in relation to each other by examining mixtures of prostaglandin E, and prcstaglandin E,. Moreover, a number of prostaglandins E,- and E,related prostaglandins are examined for their effect on ADP-induced platelet aggregation in order to gain information on the relationship between chemical structure and biological
activity.
EXPERIJlENTAL
The abdominal
aorta of adult Wistar rats under ether anaesthesia is cannulated
with a polythene cannula. Blood, collected in 3.8o/o trisodium citrate solution and pooled from different animals, is centrifuged at 19oxg for 15 min at 8”. The upper layer, titrated platelet-rich plasma, is collected, stored at room temperature and used within 5 h. The prostaglandins to be investigated (Table I) and the methyl esters of some of them are dissolved in methanol, and their concentrations are determined as described by NUGTEREN et a1.8 with an accuracy of 2%. After evaporation of the methanol, they are dissolved in 0.85 “/o NaCl solution ; adenosine 5’-diphosphate (Boehringer and Sijhne) is also dissolved in 0.85% NaCl. Aggregation is induced by 0.25 ,ug ADP per ml platelet suspension (0.5 !tM) and measured by the turbidometric method of BORNEOand O’BRIEN~~, as modified by the author’. The maximum decrease in absorbance (max. AA) expressed in percentages of that in the control is taken as a criterion for the degree of platelet aggregation. The prostaglandins
are added
to the platelet
suspension
I min before
the
addition of ADP. The relationship between the actions of prostaglandins E, and E, is examined by adding different mixtures of both (see Fig. I for composition). The qualitative effects of related prostaglandins are examined by screening 0.5 and I pg/ml doses. Prostaglandins which have no activity on qualitative examination have relative potencies lower than 1% with respect to the activities of prostaglandins E, or E,. As stated in the INTRODUCTION,these are considered to be inactive. The relative potencies of the prostaglandins found to be active are determined by quantitative assay. The prostaglandins having an inhibiting effect are assayed by comparison of their dose-response curves with that of prostaglandin E,, and those Biochim. Biophys. Ada. 187
(1969) 285-292
STRUCTURE TABLE
AND
ACTIVITY
287
OF PROSTAGLANDINS
I
PROSTAGLANDINS
USED,
THEIR
Prostaglandin
PVlXUW_W
PRECURSORS
AND
MODES
Ref. No.
Mode of preparation Code * *
Name (all double bonds aye cis) E, E, a,,%dinor E, cr,p-dinor E, a-nor E, a-nor E, a-homo E, a-homo E, a-dihomo E, a-dihomo E, w-nor E, o-nor E, w-homo E, w-homo E, E, 13,14-dihydro 15.keto E, 15-keto E, F,a F,B F,a FZB A, B, B,
OF PREPARATION*
8, I I, I4-Eicosatrienoic acid 5,8,1 I,I~-Eicosatetraenoic acid 6,g,Iz-Octadecatrienoic acid 3,6,9,12-Octadecatetraenoic acid 7,1o,13-Nonadecatrienoic acid 4,7,10,13-Nonadecatetraenoic acid ~,Iz, Ig-Heneicosatrienoic acid 6,g,Iz,I5-Heneicosatetraenoic acid 10,13,x6-Docosatrienoic acid 7, IO, I 3,16-Docosatetraenoic acid 8, I I, I 4-Nonadecatrienoic acid 5,8, I I, I4-Nonadecatetraenoic acid 8, I I, I q-Heneicosatrienoic acid 5,8, I I, 14.Heneicosatetraenoic acid 5,8,11,14.17-Eicosapentaenoic acid E, Prostaglandin E, Prostaglandin E, Prostaglandin E, Prostaglandin E, Prostaglandin E, Prostaglandin E, Prostaglandin E, Prostaglandin E, Prostaglandin E, Prostaglandin E,
20:3 20:4 18:3 18:4 Ig:3 Ig:4 21:3 21:4 22:3 22:4 r9:3 19:4 21:3 21:4 20:5
06 06 06 06 06 06 w6 06 06 06 05 w5 07 w7 w3
9.10
I II,12
I
I3 1
II,12 14 1 Reduction
with NaBH,
I5 16
I Acid treatment
I7
Alkali treatment
I4
* Prepared by D. H. Nugteren, Miss C. B. Struyk, H. Vonkeman and L. van der Wolf, of Unilever Research Laboratory, Vlaardingen, The Netherlands. ** Number of C atoms; number of double bonds; position of the first double bond counted from terminal CH,-group (w-side) of the fatty acid. having
a stimulating
effect
with
that
of prostaglandin
E,*.
However,
a simple
comparison of the dose-response curves of the unknown prostaglandins with that of prostaglandin E, is impossible because an increase of the prostaglandin E, concentration very soon causes maximum platelet aggregation, which results in a poor reproducibility. Another reason is that no linear part suitable for assay is found in the dose-response
curves when maximum
aggregation
reaches
only a low value.
After the finding that no interaction occurred between the effects of prostaglandins E, and E, on platelet aggregation (see RESULTS), we overcame these difficulties by adding a constant dose of prostaglandin E, (0.008 ,ug prostaglandin E, per ml platelet suspension) to the solutions of both prostaglandin E, and the prostaglandins under assay. All prostaglandins were tested platelet-rich rat-blood plasma.
5- or 6-fold
in as many batches
of c&rated
The relative potencies of both the E,- and the E,-type are calculated by the method of parallel line bioassay. The fiducial limits are calculated as described by FIELLER~~. * A dose-response curve is obtained by plotting max. d A in percentages of the control against the prostaglandin concentration in nM. As the concentration ranges of the dose-response curves of the standard prostaglandin and the unknown prostaglandin can shift to the same extent and in the same direction depending on the batch of plasma used, both dose-response curves should be determined in the same batch of titrated platelet-rich rat-blood plasma. Biochim. Biophys. Acta, 187 (1969) 285~zgz
J. KLOEZE
288
RESULTS
The effect on platelet aggregation of different prostaglandins E, and E, mixtures added to the platelet suspension is shown in Fig. I. The horizontal plane corresponds to the control level of platelet aggregation. The relationship between platelet aggregation and the molar concentrations of prostaglandins E, and E, is illustrated by the tilted plane in Fig. I and can be satisfactorily represented by the formula “/h max. d/f = -494
- 99 log [PGE,]
+ 26 log [PGE,]
(1) This relation was obtained as a multiple regression equation after a statistical investigation showing neither a significant interaction between prostaglandin E, and E, nor any significant deviation from linearity.
nM PGE2
Fig. I. Relationship between ADP (0.5 PM)-induced platelet aggregation (in y0 max. d A) and concentrations of prostaglandin E, (PGE,) and prostaglandin E, (PGE,) (shadowed plane). The horizontal plane at the roe% level represents the extent of platelet aggregation without addition of prostaglandin.
Table II shows the relative potencies of E,-type prostaglandins. Saturation of the 13,14 double bond of prostaglandin E, decreases the activity, whereas oxidation of the hydroxyl group at c-15 inactivates prostaglandin E,. These findings are in good agreement with the results of ANGGARD~’ who tested the biological activities of prostaglandins E, and E, on smooth-muscle preparations and blood pressure. Shortening of the w-side chain by one C atom decreases the activity to about half that of prostaglandin E,, whereas elongation of this chain by one C atom increases it by a factor of about 4. Shortening or elongation of the m-side chain by one or two C atoms inactivates the prostaglandin. When prostaglandin E,, or prostaglandin E,, is added to the platelet suspension after induction of the platelet aggregation by ADP, it acts immediately without any perceptible delay’. Exactly the same phenomenon is observed for the methyl esters, which supports the assumption that these substances act as esters without preceding hydrolysis of the ester bond. Quantitatively, the methyl esters are about as active as the prostaglandins as such. Biochim.
Biophys.
Acta,
187 (1969) 285-292
STRUCTURE TABLE
AND ACTIVITY
II
RELATIVE POTENCIES OF &-TYPE IS SHOWN AT THE TOP Fiducial
PROSTAGLANDINS (E, =
131 =4
P
=
El
13,14-dihydro 15.keto w-nor w-homo n-dinor cr-nor a-homo cc-dihomo
z = = = = = =
-_2 --I +r +2
‘5
4
OH OH =o OH OH OH OH OH OH
_
Relative potency (E, = I)
as methyl ester
1.00
0.86 (o.76-o.g7)
0.64 (0.56-0.73) Inactive --I +I
0.55 (o.4g-0.61) 3.82 (3.42-4.38) 0.02 Inactive Inactive Inactive
PROSTAGLANDINS (E, =
0.53 (0.47-0.59) 2.36 (2.10-2.65)
I.OO), THE GENERAL FORMULA OF WHICH
limits are given in parentheses.
.u
Prostaglandin
P
(CH213.p COOH
15
17.18
q
Relative potency (E, assuch
EZ.
r5-keto w-nor o-homo cc-dinor m-nor cc-horn0 a-dihomo F-3
of prostaglandin
as such
III
RELATIVE POTENCIES OF EZ-~yp~ IS SHOWN AT THE TOP Fiducial
1.00). THE GENERAL FORMULA OF WHICH
limits are given in parentheses.
Prostaglandin
TABLE
289
OF PROSTAGLANDINS
-2 -I +I $2
OH =O OH OH OH OH OH OH OH
-
=
1.00
Inactive 0.09 (0.07-O. I I) 1.12 (1.03-1.22) Inactive Inactive Inactive Inactive 0.10 (0.08-0.13)
= I)
of prostaglandin
as methyl ester 1.38 (0.72-2.96) 0.15 (0.03-0.33) 1.30 (0.67-2.72)
The relative potencies of E,-type prostaglandins are represented in Table III. Oxidation of the hydroxyl group at c-15 inactivates prostaglandin E,, just as it inactivated prostaglandin E,. Shortening of the co-side chain by one C atom decreases the activity by a factor of about IO whereas elongation by one C atom has hardly any effect. Shortening or elongation of the a-side chain again inactivates the prostaglandin. Biochim. Biophys. Acta, 187 (1969) 285-2g2
J. KLOEZE
290
The methyl ester of prostaglandin E, is approximately as active as the prostaglandin as such. Prostaglandin E,, an E,-type prostaglandin with an extra double bond between C-17 and C-18, has an activity that is IO times less than that of prostaglandin
TABLE
E,.
IV
RELATIVE POTENCIES OF PRosTAGLANDINs WITH RING STRUCTURES DIFFERENT E-TYPEPRO~TAGLANDIN~, THE GENERALFORMULAOFWHICHI~ SHOWNATTHETOP The groups or bonds at the ring which indicated by heavy lines.
Prostaglandin
Ring
FROM
THOSE
OF
differfrom the structure of E-type prostaglandins are
5.6
Relative potency
(E,
= 1)
KS OH
8
F IOL
_
_--
=
Q
F 2t(
Inactive Inactive
Ok
F
18
\ ,_-0, ’
8 OH
F@
_ =
Inactive Iuactive
O;r
o. \\
a\ --
Al
@O
B,
\\ @ 1:
B,
=
0.09
Inactive Inactive
Table IV shows the relative potencies of prostaglandins with ring structures different from those of E-type prostaglandins. Substitution of the keto group at the ring by a hydroxyl group (a- and /?-isomers of prostaglandins F), inactivates the at the ring, which inprostaglandin, regardless of the stereoisomer. Dehydration troduces a double bond between the C atoms IO and II (prostaglandin A,), decreases the activity to about 0.1 of that of prostaglandin E,. When the double bond shifts to Position 8-12 (prostaglandins B1, and B,), the prostaglandin obtains a flat structure and is completely inactive. Biochint.
Biophys.
Acta,
187 (1969) 285-292
STRUCTURE
AND ACTIVITY
OF PROSTAGLANDINS
DISCUSSION
AND CONCLUSIONS
291
The potency of prostaglandin E, to inhibit platelet aggregation is shown to be many times greater than the stimulating potency of prostaglandin E,. From Formula I it follows that an increase in the prostaglandin E, concentration by a factor K needs an increase in the prostaglandin E, concentration by a factor K3es to keep the platelet aggregation at the same level. When this formula is applied to the results obtained previously’, it can be calculated that the prostaglandin E, was contaminated with about 0.3% prostaglandin E,. Extrapolation of the formula to this very low concentration is perhaps not valid, but the calculated contamination is in agreement with the gas-chromatographic data. With regard to the structural conditions being essential for the effects of prostaglandins on platelet aggregation, we can draw the following conclusions: The E-type structure of the cyclopentane ring is required for either an inhibiting or a stinlulating effect of prostaglandins on platelet aggregation. Especially the keto group at C-9 is essential, as its reduction to a hydroxyl group (F-type) inactivates the prostaglandin. Dehydration at the ring removes the hydroxyl group at C-rr and introduces a double bond between C-IO and C-II (prostaglandin A,) or between C-8 and C-12 (prostaglandins B, and B,). These structural changes at the ring, respectively, decrease the activity by about a factor of IO or inactivate the molecule. Changing the length of the a-side chain containing the carboxyl group inactivates the prostaglandin. As the methyl esters are about as active as the prostaglandins as such, the C=O group of the carboxyl seems to be the most important group.
A third essential group of the molecule is the hydroxyl group at C-15, as oxidation of this group also inactivates the prostaglandin. Changing the length of the w-side chain with one C atom causes quantitative effects in the activities of the prostaglandins. Shortening of this chain decreases, while lengthening increases the activities, although the quantitative responses of the E,- and E,-types differ somewhat in this respect. Prostaglandins stimulating platelet aggregation induced by ADP have a cisdouble bond in the a-side chain between C-5 and C-6, while those with an inhibiting effect have not. Therefore, this double bond seems responsible for the difference in effect between prostaglandins E, and E,. The &alas-double bond in the m-side chain between C-13 and C-14, which prostaglandins E, and E, have in common, is only of minor importance as saturation of this bond causes only a small quantitative effect. ACKNOWLEDGMENTS
I wish to express my thanks to Prof. Dr. D. A. van Dorp and his team for supplying the prostaglandins and for their interest in the work. Especially the critical remarks of Dr. Ir. D. H. Nugteren are greatly appreciated. Moreover, thanks are due to Messrs R. N. Lussenburg and R. van Splunter for analysis of the results and calculation of the relative potencies, and to Miss J. A. Fransen and Mr. R. Peteroff for their skillful technical assistance. Biochinz.
Biophys.
Acta,
187 (xg6g) 285~-292
J. KLOEZE
292 REFERENCES
Recent Progr. Hormone Res., 22 (1966) 153. Science. 157 (1967) 382. S.BERGSTRBM.L. A. CARLSON AND 1. R. WEEKS,Pharmacol. Rev.. 20 (1968) I. E. W. HORTON, Experientia, 21 (196;) 113. P. W. RAMWELL. T.E. SHAW. G. B. CLARKE, M. F.GROSTIC,D. G. KAISER AXD 1. E. PIKE, Progr. Chem. Faisiipzds, 9 (i968) 233. B. SAMUELSSOX, Anger. Chem. Intern. Ed. Engl., 4 (1965) 410. J, KLOEZE, Nobel Symp., 2, Prostaglandins, Stockholm, 1966, Almqvist and Wiksell, Stockholm, 1967. p 241. D.H. NUGTEREN,R.K.BEERTHUISAND D.A. VAN DORP, Rec. Trav. Chim.,85(1966)405. D. A.VAN DORP, R.K.BEERTHUIS, D.H.NUGTEREN AXDH.VOR.KEMAN, Biochim.Biophys. Acta, 90 (1964) 204. H. VONKEMAN, Natuve, 203 (1964) ICI D.A. VAN DORP,R.K.BEERTHUIS,D.H.NUGTERENAND
S. BERGSTROM,
S. BERGSTROM,
II
839. C. B.
STRUYK,R.K.BEERTHUIS,H.J.J.PABONAP~DD.
A.VAN
DoRP,K~~. Tvav.Chzm.,
X5
(1966) 1233. StockI2 C. B. STRUYK, R. K. BEERTHUIS AND D. A. VAN DORP, Nobel Symp. 2, Prostaglandins. holm, 1966, Almqvist and Wiksell, Stockholm, 1967, p. 51. BEERTHUIS,D.H.NUGTEREN,H.J.J.PABONANDD.A.VANDORP, Rec.TYav. Chim., I3 R.K. 87 (1968) 461. 238(1963) 3555. ‘4 S. BERGSTROM, R. RYHAGE, B. SAMUELSSON AND J. SJBVALL,J. Biol.Chem., AND B.~AMUELSSOX, Arkiv Kemi, 25 (1966) 293. I5 E. XNGGARD 16 (1962) 16 S. BERGSTROM, L. KRABISCH, B. SAMUELSSON AXD J. SJGVALL, Acta Chem..Scand.,
969.
A.JoHNso~T,F.P. KUPIECKI,J. Rrs. Commun., 21 (1965) 413. 18 G. \'.R. BORX, Nature, 194 (1962)927. I9 J. R. O'BRIEN, J. Clin.Pathol., 15 (1962) 446. 17 (1944) 117. 20 E. C. FIELLER, Quart. 1. Pharm. Pharmacol., 21 E. ANGGARD, Acta Physiol. Stand., 66 (1966) 509. I7
E.G.DANIELs,J.W.HINMAN,B.
Biochem.
Biochim.
Biophys.
Biophys.
Acta,
187 (1969)
285-292
W. WELSON AXD J. I<.PIKE,
BBA
ET BIOPHYSICA
285
ACTA
55625
RELATIONSHIP
BETWEEN
AGGREGATION
ACTIVITY
CHEMICAL
STRUCTURE
AND PLATELET-
OF PROSTAGLANDINS
J. KLOEZE Unilever Research (Received
Laboratory,
May pth,
Vlaardingen
(The Netherlands)
1969)
SUMMARY
The finding that prostaglandin E, inhibits ADP-induced platelet aggregation in titrated platelet-rich rat-blood plasma, whereas prostaglandin E, stimulates it, has led to an investigation into the potencies of the opposing effects in relation to each other by examining mixtures of prostaglandins E, and E, at different concentrations. It has been found that the relationship between the extent of platelet aggregation, expressed in percentages of the maximum decrease in absorbance in the control (% max. d A), and the molar concentration of prostaglandins E, and E, can be written as: y’ max. dA = - 494 - 99 log [PGE,] In order and biological were examined blood plasma. group, as well at C-15.
+ 26 log [PGE,]
to gain information on the relationship between chemical structure activity, 23 prostaglandins and the methyl esters of some of them for their effects on platelet aggregation in titrated platelet-rich ratIt has been found that the C=O groups at the ring and in the carboxyl as the distance between them, are essential, as is the hydroxyl group
INTRODUCTION
Small amounts of prostaglandins are found widely distributed in different organs. Most of them have potent biological activity of some kind, as was reviewed recently by different authorsl-O. One of these activities, as I reported before’, is the effect on ADP-induced platelet aggregation in vitro. Prostaglandin E, tias found to have an inhibiting effect, whereas prostaglandin E, displayed a stimulating effect in various species. However, the stimulating effect of prostaglandin E, reached an optimum between 0.5 and I ,q/ml; at higher concentrations it decreased, probably owing to contamination of the prostaglandin E, preparation by prostaglandin E,, which on gas-chromatographic analysis appeared to be less than 1%. The inability of N. CHANDRASEKHAR to reproduce the stimulating effect of Biochim.
Biophys.
Acta,
187 (1969) z85--292
286
J. KLOEZE
prostaglandin tamination.
E,, as referred to by BERGSTROM et aL3, may be a result of this con-
The contamination is caused by the fact that the particulate enzyme fraction of sheep vesicular gland used for the biosynthesis of the prostaglandins contains very small amounts of prostaglandins E,, E, and their precursor fatty acids. As the physical and chemical properties of the prostaglandins do not differ much, it is very difficult to purify the preparations completely from the endogenous prostaglandins. For this reason prostaglandins with relative potencies lower than 1% with respect to prostaglandin E, or prostaglandin E, activity are considered inactive in the present experiments. These experiments comprise the determination of the potencies of the inhibiting and stimulating effects in relation to each other by examining mixtures of prostaglandin E, and prcstaglandin E,. Moreover, a number of prostaglandins E,- and E,related prostaglandins are examined for their effect on ADP-induced platelet aggregation in order to gain information on the relationship between chemical structure and biological
activity.
EXPERIJlENTAL
The abdominal
aorta of adult Wistar rats under ether anaesthesia is cannulated
with a polythene cannula. Blood, collected in 3.8o/o trisodium citrate solution and pooled from different animals, is centrifuged at 19oxg for 15 min at 8”. The upper layer, titrated platelet-rich plasma, is collected, stored at room temperature and used within 5 h. The prostaglandins to be investigated (Table I) and the methyl esters of some of them are dissolved in methanol, and their concentrations are determined as described by NUGTEREN et a1.8 with an accuracy of 2%. After evaporation of the methanol, they are dissolved in 0.85 “/o NaCl solution ; adenosine 5’-diphosphate (Boehringer and Sijhne) is also dissolved in 0.85% NaCl. Aggregation is induced by 0.25 ,ug ADP per ml platelet suspension (0.5 !tM) and measured by the turbidometric method of BORNEOand O’BRIEN~~, as modified by the author’. The maximum decrease in absorbance (max. AA) expressed in percentages of that in the control is taken as a criterion for the degree of platelet aggregation. The prostaglandins
are added
to the platelet
suspension
I min before
the
addition of ADP. The relationship between the actions of prostaglandins E, and E, is examined by adding different mixtures of both (see Fig. I for composition). The qualitative effects of related prostaglandins are examined by screening 0.5 and I pg/ml doses. Prostaglandins which have no activity on qualitative examination have relative potencies lower than 1% with respect to the activities of prostaglandins E, or E,. As stated in the INTRODUCTION,these are considered to be inactive. The relative potencies of the prostaglandins found to be active are determined by quantitative assay. The prostaglandins having an inhibiting effect are assayed by comparison of their dose-response curves with that of prostaglandin E,, and those Biochim. Biophys. Ada. 187
(1969) 285-292
STRUCTURE TABLE
AND
ACTIVITY
287
OF PROSTAGLANDINS
I
PROSTAGLANDINS
USED,
THEIR
Prostaglandin
PVlXUW_W
PRECURSORS
AND
MODES
Ref. No.
Mode of preparation Code * *
Name (all double bonds aye cis) E, E, a,,%dinor E, cr,p-dinor E, a-nor E, a-nor E, a-homo E, a-homo E, a-dihomo E, a-dihomo E, w-nor E, o-nor E, w-homo E, w-homo E, E, 13,14-dihydro 15.keto E, 15-keto E, F,a F,B F,a FZB A, B, B,
OF PREPARATION*
8, I I, I4-Eicosatrienoic acid 5,8,1 I,I~-Eicosatetraenoic acid 6,g,Iz-Octadecatrienoic acid 3,6,9,12-Octadecatetraenoic acid 7,1o,13-Nonadecatrienoic acid 4,7,10,13-Nonadecatetraenoic acid ~,Iz, Ig-Heneicosatrienoic acid 6,g,Iz,I5-Heneicosatetraenoic acid 10,13,x6-Docosatrienoic acid 7, IO, I 3,16-Docosatetraenoic acid 8, I I, I 4-Nonadecatrienoic acid 5,8, I I, I4-Nonadecatetraenoic acid 8, I I, I q-Heneicosatrienoic acid 5,8, I I, 14.Heneicosatetraenoic acid 5,8,11,14.17-Eicosapentaenoic acid E, Prostaglandin E, Prostaglandin E, Prostaglandin E, Prostaglandin E, Prostaglandin E, Prostaglandin E, Prostaglandin E, Prostaglandin E, Prostaglandin E, Prostaglandin E,
20:3 20:4 18:3 18:4 Ig:3 Ig:4 21:3 21:4 22:3 22:4 r9:3 19:4 21:3 21:4 20:5
06 06 06 06 06 06 w6 06 06 06 05 w5 07 w7 w3
9.10
I II,12
I
I3 1
II,12 14 1 Reduction
with NaBH,
I5 16
I Acid treatment
I7
Alkali treatment
I4
* Prepared by D. H. Nugteren, Miss C. B. Struyk, H. Vonkeman and L. van der Wolf, of Unilever Research Laboratory, Vlaardingen, The Netherlands. ** Number of C atoms; number of double bonds; position of the first double bond counted from terminal CH,-group (w-side) of the fatty acid. having
a stimulating
effect
with
that
of prostaglandin
E,*.
However,
a simple
comparison of the dose-response curves of the unknown prostaglandins with that of prostaglandin E, is impossible because an increase of the prostaglandin E, concentration very soon causes maximum platelet aggregation, which results in a poor reproducibility. Another reason is that no linear part suitable for assay is found in the dose-response
curves when maximum
aggregation
reaches
only a low value.
After the finding that no interaction occurred between the effects of prostaglandins E, and E, on platelet aggregation (see RESULTS), we overcame these difficulties by adding a constant dose of prostaglandin E, (0.008 ,ug prostaglandin E, per ml platelet suspension) to the solutions of both prostaglandin E, and the prostaglandins under assay. All prostaglandins were tested platelet-rich rat-blood plasma.
5- or 6-fold
in as many batches
of c&rated
The relative potencies of both the E,- and the E,-type are calculated by the method of parallel line bioassay. The fiducial limits are calculated as described by FIELLER~~. * A dose-response curve is obtained by plotting max. d A in percentages of the control against the prostaglandin concentration in nM. As the concentration ranges of the dose-response curves of the standard prostaglandin and the unknown prostaglandin can shift to the same extent and in the same direction depending on the batch of plasma used, both dose-response curves should be determined in the same batch of titrated platelet-rich rat-blood plasma. Biochim. Biophys. Acta, 187 (1969) 285~zgz
J. KLOEZE
288
RESULTS
The effect on platelet aggregation of different prostaglandins E, and E, mixtures added to the platelet suspension is shown in Fig. I. The horizontal plane corresponds to the control level of platelet aggregation. The relationship between platelet aggregation and the molar concentrations of prostaglandins E, and E, is illustrated by the tilted plane in Fig. I and can be satisfactorily represented by the formula “/h max. d/f = -494
- 99 log [PGE,]
+ 26 log [PGE,]
(1) This relation was obtained as a multiple regression equation after a statistical investigation showing neither a significant interaction between prostaglandin E, and E, nor any significant deviation from linearity.
nM PGE2
Fig. I. Relationship between ADP (0.5 PM)-induced platelet aggregation (in y0 max. d A) and concentrations of prostaglandin E, (PGE,) and prostaglandin E, (PGE,) (shadowed plane). The horizontal plane at the roe% level represents the extent of platelet aggregation without addition of prostaglandin.
Table II shows the relative potencies of E,-type prostaglandins. Saturation of the 13,14 double bond of prostaglandin E, decreases the activity, whereas oxidation of the hydroxyl group at c-15 inactivates prostaglandin E,. These findings are in good agreement with the results of ANGGARD~’ who tested the biological activities of prostaglandins E, and E, on smooth-muscle preparations and blood pressure. Shortening of the w-side chain by one C atom decreases the activity to about half that of prostaglandin E,, whereas elongation of this chain by one C atom increases it by a factor of about 4. Shortening or elongation of the m-side chain by one or two C atoms inactivates the prostaglandin. When prostaglandin E,, or prostaglandin E,, is added to the platelet suspension after induction of the platelet aggregation by ADP, it acts immediately without any perceptible delay’. Exactly the same phenomenon is observed for the methyl esters, which supports the assumption that these substances act as esters without preceding hydrolysis of the ester bond. Quantitatively, the methyl esters are about as active as the prostaglandins as such. Biochim.
Biophys.
Acta,
187 (1969) 285-292
STRUCTURE TABLE
AND ACTIVITY
II
RELATIVE POTENCIES OF &-TYPE IS SHOWN AT THE TOP Fiducial
PROSTAGLANDINS (E, =
131 =4
P
=
El
13,14-dihydro 15.keto w-nor w-homo n-dinor cr-nor a-homo cc-dihomo
z = = = = = =
-_2 --I +r +2
‘5
4
OH OH =o OH OH OH OH OH OH
_
Relative potency (E, = I)
as methyl ester
1.00
0.86 (o.76-o.g7)
0.64 (0.56-0.73) Inactive --I +I
0.55 (o.4g-0.61) 3.82 (3.42-4.38) 0.02 Inactive Inactive Inactive
PROSTAGLANDINS (E, =
0.53 (0.47-0.59) 2.36 (2.10-2.65)
I.OO), THE GENERAL FORMULA OF WHICH
limits are given in parentheses.
.u
Prostaglandin
P
(CH213.p COOH
15
17.18
q
Relative potency (E, assuch
EZ.
r5-keto w-nor o-homo cc-dinor m-nor cc-horn0 a-dihomo F-3
of prostaglandin
as such
III
RELATIVE POTENCIES OF EZ-~yp~ IS SHOWN AT THE TOP Fiducial
1.00). THE GENERAL FORMULA OF WHICH
limits are given in parentheses.
Prostaglandin
TABLE
289
OF PROSTAGLANDINS
-2 -I +I $2
OH =O OH OH OH OH OH OH OH
-
=
1.00
Inactive 0.09 (0.07-O. I I) 1.12 (1.03-1.22) Inactive Inactive Inactive Inactive 0.10 (0.08-0.13)
= I)
of prostaglandin
as methyl ester 1.38 (0.72-2.96) 0.15 (0.03-0.33) 1.30 (0.67-2.72)
The relative potencies of E,-type prostaglandins are represented in Table III. Oxidation of the hydroxyl group at c-15 inactivates prostaglandin E,, just as it inactivated prostaglandin E,. Shortening of the co-side chain by one C atom decreases the activity by a factor of about IO whereas elongation by one C atom has hardly any effect. Shortening or elongation of the a-side chain again inactivates the prostaglandin. Biochim. Biophys. Acta, 187 (1969) 285-2g2
J. KLOEZE
290
The methyl ester of prostaglandin E, is approximately as active as the prostaglandin as such. Prostaglandin E,, an E,-type prostaglandin with an extra double bond between C-17 and C-18, has an activity that is IO times less than that of prostaglandin
TABLE
E,.
IV
RELATIVE POTENCIES OF PRosTAGLANDINs WITH RING STRUCTURES DIFFERENT E-TYPEPRO~TAGLANDIN~, THE GENERALFORMULAOFWHICHI~ SHOWNATTHETOP The groups or bonds at the ring which indicated by heavy lines.
Prostaglandin
Ring
FROM
THOSE
OF
differfrom the structure of E-type prostaglandins are
5.6
Relative potency
(E,
= 1)
KS OH
8
F IOL
_
_--
=
Q
F 2t(
Inactive Inactive
Ok
F
18
\ ,_-0, ’
8 OH
F@
_ =
Inactive Iuactive
O;r
o. \\
a\ --
Al
@O
B,
\\ @ 1:
B,
=
0.09
Inactive Inactive
Table IV shows the relative potencies of prostaglandins with ring structures different from those of E-type prostaglandins. Substitution of the keto group at the ring by a hydroxyl group (a- and /?-isomers of prostaglandins F), inactivates the at the ring, which inprostaglandin, regardless of the stereoisomer. Dehydration troduces a double bond between the C atoms IO and II (prostaglandin A,), decreases the activity to about 0.1 of that of prostaglandin E,. When the double bond shifts to Position 8-12 (prostaglandins B1, and B,), the prostaglandin obtains a flat structure and is completely inactive. Biochint.
Biophys.
Acta,
187 (1969) 285-292
STRUCTURE
AND ACTIVITY
OF PROSTAGLANDINS
DISCUSSION
AND CONCLUSIONS
291
The potency of prostaglandin E, to inhibit platelet aggregation is shown to be many times greater than the stimulating potency of prostaglandin E,. From Formula I it follows that an increase in the prostaglandin E, concentration by a factor K needs an increase in the prostaglandin E, concentration by a factor K3es to keep the platelet aggregation at the same level. When this formula is applied to the results obtained previously’, it can be calculated that the prostaglandin E, was contaminated with about 0.3% prostaglandin E,. Extrapolation of the formula to this very low concentration is perhaps not valid, but the calculated contamination is in agreement with the gas-chromatographic data. With regard to the structural conditions being essential for the effects of prostaglandins on platelet aggregation, we can draw the following conclusions: The E-type structure of the cyclopentane ring is required for either an inhibiting or a stinlulating effect of prostaglandins on platelet aggregation. Especially the keto group at C-9 is essential, as its reduction to a hydroxyl group (F-type) inactivates the prostaglandin. Dehydration at the ring removes the hydroxyl group at C-rr and introduces a double bond between C-IO and C-II (prostaglandin A,) or between C-8 and C-12 (prostaglandins B, and B,). These structural changes at the ring, respectively, decrease the activity by about a factor of IO or inactivate the molecule. Changing the length of the a-side chain containing the carboxyl group inactivates the prostaglandin. As the methyl esters are about as active as the prostaglandins as such, the C=O group of the carboxyl seems to be the most important group.
A third essential group of the molecule is the hydroxyl group at C-15, as oxidation of this group also inactivates the prostaglandin. Changing the length of the w-side chain with one C atom causes quantitative effects in the activities of the prostaglandins. Shortening of this chain decreases, while lengthening increases the activities, although the quantitative responses of the E,- and E,-types differ somewhat in this respect. Prostaglandins stimulating platelet aggregation induced by ADP have a cisdouble bond in the a-side chain between C-5 and C-6, while those with an inhibiting effect have not. Therefore, this double bond seems responsible for the difference in effect between prostaglandins E, and E,. The &alas-double bond in the m-side chain between C-13 and C-14, which prostaglandins E, and E, have in common, is only of minor importance as saturation of this bond causes only a small quantitative effect. ACKNOWLEDGMENTS
I wish to express my thanks to Prof. Dr. D. A. van Dorp and his team for supplying the prostaglandins and for their interest in the work. Especially the critical remarks of Dr. Ir. D. H. Nugteren are greatly appreciated. Moreover, thanks are due to Messrs R. N. Lussenburg and R. van Splunter for analysis of the results and calculation of the relative potencies, and to Miss J. A. Fransen and Mr. R. Peteroff for their skillful technical assistance. Biochinz.
Biophys.
Acta,
187 (xg6g) 285~-292
J. KLOEZE
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Biophys.
Acta,
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