ENDOCRINE RESEARCH COMMUNICATIONS, 3 ( 3 & 4 ) , 231-241 (1976)
COMPARISON OF THE ANTI-LH/FSH-RH AND ANTI-OVULATORY ACTIVITIES OF [D-Phe2, D-Leu6] -LH-RH AND [D-Phe2, D-Ala6]-LH-RH Vilchez-Martinez, D.H. Coy, E.J. Coy, A. Arimura and A.V. S c h a l l y Endocrine and P o l y p e p t i d e L a b o r a t o r i e s , Veterans A d m i n i s t r a t i o n H o s p i t a l , and Department o f Medicine, Tulane U n i v e r s i t y School o f Medicine, New Orleans, Louisiana 70146
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J.A.
Abstract The anti-LH/FSH-RH and a n t i o v u l a t o r y a c t i v i t i e s o f [D-PheZ, D-Leu6] -LH-RH and [D-Phe2, D-Ala6]-LH-RH were compared i n r a t s . Both p e p t i d e s i n h i b i t e d t h e LH and FSH r e l e a s e induced by LH-RH i n [D-Phe2, Dimmature male r a t s , b u t , 4 h r a f t e r the i n j e c t i o n , LeuGI-LH-RH s t i l l suppressed the LH and FSH r e l e a s e whereas t h e [D-Phe2, D-Ala6 ILH-RH d i d n o t . When t h e p e p t i d e s were adminis t e r e d i n equal doses on t h e a f t e r oon o f t h e day o f p r o e s t r u s i n &day c y c l i n g r a t s , [D-Phd, D-Leu2]-LH-RH more completely i n h i b i t e d t h e o v u l a t i o n o c c u r r i n g on t h e f o l l o w i n g morning than [DPhe2, D-Ala6l-LH-RH. Thus, t h e i n c o r p o r a t i o n o f D-Leucine i n t o p o s i t i o n s i x o f t h e decapeptide c h a i n g i v e s a more p o t e n t i n h i b i t o r than t h a t r e s u l t i n g from t h e i n s e r t i o n o f D-Alanine.
lntroduct ion
I n t h e search f o r b e t t e r i n h i b i t o r s o f LH-RH, we have demons t r a t e d t h a t t h e replacement o f g l y c i n e i n p o s i t i o n 6 o f t h e decap e p t i d e sequence by D-Alanine,
D-Leucine,
D-phenylalanine and D-
tryptophan, produced i n c r e a s i n g l y s t r o n g e r i n h i b i t o r s o f LHRH ( 1 ) . For instance,
[DesHis2, D-Leu6]-LH-RH
g o n i s t i c a c t i v i t y o f [DesHisZ,
showed about t w i c e t h e anta-
D-Ala6]-LH-RH
when b o t h analogs
231 Copyright 0 1976 by Marcel Dekker, Inc. All Rights Reserved. Neither thls work nor any part may be reproduced or transmitted in any form or by any means, electronlc or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher.
VILCHEZ-MARTINEZ ET AL.
232
were compared f o r i n h i b i t i o n o f LH release i n immature male r a t s (2).
I n a d d i t i o n , the i n c o r p o r a t i o n o f D-phenylalanine i n p o s i t i o n
2, r a t h e r than t h e d e l e t i o n o f H i s , increases and prolongs t h e i n h i b i t o r y e f f e c t o f many peptides.
This might be due t o increased
receptor s i t e b i n d i n g coupled w i t h increased hydrophobic p r o p e r t i e s which apparently r e s u l t i n slower r e l e a s e a f t e r
S.C.
i n j e c t i o n (2).
Making use o f these observations, we were a b l e t o b l o c k t h e phy-
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s i o l o g i c a l surge o f LH by 83% and o v u l a t i o n by 30% i n c y c l i n g hams t e r s using [D-Phe’,
D-Leu6]-LHRH (3).
t h a t t h r e e i n j e c t i o n s o f [D-Phe’,
More r e c e n t l y , we found
6 D-Leu ’J-LHRH administered to 4-
day c y c l i n g r a t s on the a f t e r n o o n o f the day o f p r o e s t r u s produced
83% blockade o f o v u l a t i o n and a s i n g l e i n j e c t i o n o f 1.5 mg o f [D6 Phe2, Phe3, D-Phe 1-LH-RH a t noon o f the proestrous day,
resulted i n
almost complete suppression o f gonadotropin release, and 86% b l o c k ade of o v u l a t i o n ( 4 ) .
Corbin and B e a t t i e (5) have a l s o demonstra-
ted a blockade o f the LH surge and o v u l a t i o n i n r a t s using[D-Phe
2
,
6
D-Ala 1-LH-RH. I n view o f t h e s i g n i f i c a n t changes i n i n h i b i t o r y potency produced by v a r i o u s m o d i f i c a t i o n s i n the s i x t h p o s i t i o n and t h e demons t r a t e d e f f e c t i v e n e s s of t h e D-Ala
6 analog, i t was o f i n t e r e s t t o
compare more e x h a u s t i v e l y t h e anti-LH-RH p r o p e r t i e s of [D-Phe’, Leu6]-LH-RH and [D-Phe2,
D-Ala6]-LHRH
D-
i n some i n v i v o systems.
M a t e r i a l s and Methods
The anti-LH-RH e f f e c t of the peptides was screened over a p e r i od o f 4 h r s u s i n g an immature male r a t method described p r e v i o u s l y
ANTI-LH/FSH-RH AND ANTI-OVULATORY ACTIVITIES (2-6).
The a n i m a l s were i n j e c t e d
w i t h the v e h i c l e alone.
w i t h 300 ug o f p e p t i d e s o r
Two hundred ng o f s y n t h e t i c LH-RH o r sa-
l i n e s o l u t i o n was i n j e c t e d times t h e r e a f t e r .
S.C.
233
S.C.
a t t h e same t i m e o r a t d i f f e r e n t
Blood was c o l l e c t e d 30 m i n a f t e r LH-RH o r sa-
l i n e administration. The a n t i o v u l a t o r y t e s t was performed u s i n g a d u l t female r a t s (SD s t r a i n , C h a r l e s R i v e r Co.) w e i g h i n g a p p r o x i m a t e l y 200 g.
They
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were k e p t i n c o n t r o l l e d t e m p e r a t u r e and l i g h t c o n d i t i o n s (14 h r l i g h t and 10 h r d a r k n e s s ) , and t h e i r v a g i n a l smears were examined d a i l y s t a r t i n g I 4 days a f t e r a r r i v a l .
Only r a t s w h i c h showed a t
l e a s t 2 s u c c e s s i v e , r e g u l a r &-day c y c l e s were used.
On t h e a f t e r -
noon o f t h e p r o e s t r u s day, t h e a n i m a l s were i n j e c t e d
S.C.
w i t h one
o r s e v e r a l doses o f t h e analogs o r w i t h t h e v e h i c l e a l o n e . was taken a t noon and 5 p.m.
o f t h e p r o e s t r o u s day.
Blood
On t h e f o l -
l o w i n g day, t h e a n i m a l s were s a c r i f i c e d and, under a d i s s e c t i n g m i croscope, t h e o v i d u c t s were i n s p e c t e d f o r ova. The s e r a were s e p a r a t e d by c e n t r i f u g a t i o n and s t o r e d a t -ZOO C u n t i l assayed f o r g o n a d o t r o p i n .
LH was d e t e r m i n e d by t h e d o u b l e a n t i -
body radioimmunoassay method o f Niswender e t a l .
( 7 ) and FSH by t h e
method o f Daane and Parlow (8) as d e s c r i b e d elsewhere ( 2 , 5 ) .
S 1 7 and NISMD-rat-FSH-RPI,
NIH-LH-
were used as LH and FSH s t a n d a r d p r e p a r a -
tions. Duncan's new m u l t i p l e range t e s t
(9) was
used f o r a n a l y z i n g t h e
s i g n i f i c a n c e s of t h e d i f f e r e n c e s i n LH and FSH l e v e l s among t h e groups.
The r e s u l t s f r o m t h e a n t i - o v u l a t o r y t e s t were expressed by
b i n o m i a l d a t a as 1 f o r o v u l a t i o n and 0 f o r n o - o v u l a t i o n ;
t h e y were
234
VILCHEZ-MARTINEZ ET AL.
f i r s t subjected t o a n a l y s i s o f variance (10-11) and then compared
(9).
by Duncan’s new m u l t i p l e range t e s t
[ D-Phe2,
D-Leu 6 1-LH-RH and [D-Phe2, D-Ala6]-LH-RH
were prepared
The p u r i t y o f
i n o u r l a b o r a t o r y by t h e s o l i d phase method (1,2).
these compounds was confirmed by TLC and amino a c i d a n a l y s i s . The i n h i b i t o r y analogs had t o be d i s s o l v e d i n a 20% propylenglycol/saline
s o l u t i o n f o r t h e i n j e c t i o n because o f t h e i r hydro-
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phobic p r o p e r t i e s .
Results
Fig. P-PheZ, rats.
1 shows a comparison o f t h e anti-LH/FSH-RH D-Leu6]-LH-RH
and
[ D-Phe2,
D-Ala6]-LH-RH
activities of
i n immature male
I t can be seen t h a t , a t t h e dose o f 300 u g / r a t ,
b o t h pep-
t i d e s i n h i b i t e d t h e LH and FSH r e l e a s e a f t e r LH-RH a d m i n i s t r a t i o n . But, a t 4 h r . ,
[D-Phe’,
D-Leu6]-LH-RH
s t i l l suppressed t h e LH and
FSH response t o LH-RH whereas [D-Phe2,
D-Ala6]-LH-RH
d i d not.
Table 1 shows t h e i n h i b i t i o n o f o v u l a t i o n by these p e p t i d e s . 1 mg/rat was administered a t noon, 3:30 and 5:OO p:m.
When
( t o t a l dose:
3 mg/rat) on p r o e s t r u s ( t r e a t m e n t No. l ) , [D-Phe2, D-Leu 6 1-LH-RH was f i v e times more p o t e n t than [D-Phe’,
6
D-Ala 1-LH-RH.
[
D-Phe’,
D-Leu6]-LH-RH
produced 62.5% i n h i b i t i o n o f o v u l a t i o n , and [D-Phe’,
D-Ala6]-LH-RH
r e s u l t e d i n o n l y 12.5% i n h i b i t i a n o f o v u l a t i o n .
When t h e p e p t i d e s were g i v e n as a s i n g l e dose of o f t h e day o f p r o e s t r u s ( t r e a t m e n t No. observed. [D-Phe2,
Z),
5
mg/rat a t noon
t h e same tendency was
The percentage o f i n h i b i t i o n o f o v u l a t i o n was 30% a f t e r D-Leu6]-LH-RH
and 11% a f t e r
[ D-Phe2,
D-Ala6]-LH-RH.
Thus,
235
ANTI-LH/FSH-RH AND ANTI-OVULATORY ACTIVITIES
a
Vehicle + b l i m (group I) Vehicle
+
LH-RH (2M) ng),(group 2)
ID-F+~.D -LH-RH - L ~goo u~ p9)I + biiM (group 3)
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Q
[E-lb2,D-Lcu61
-LH-RH + LH-RH (group 4)
FIGURE 1 Time ourse o f i n h i b i t i o n by [ D-Phe2, D-LedI-LH-RH and [D-Phe2, D-Alag]-LH-RH o f LH-RH-induced LH and FSH r e l e a s e i n immature male r a t s . The p e p t i d e s were i n j e c t e d a t time 0. The r a t s were d e c a p i t a t e d 30 min a f t e r LH-RH o r s a l i n e a d m i n i s t r a t i o n . Dose o f 300 u g / r a t . Dose o f LH-RH: 200 n g / r a t . S i x r a t s per peptide: group were used. The percentages o f i n h i b i t i o n of gonadotropin l e v e l s are shown above each bar.
t h e a d m i n i s t r a t i o n of 1 mg/rat
3
times p e r day (treatment No. 1) was
a more e f f e c t i v e schedule of treatment w i t h D-Leu6-peptide than a s i n g l e i n j e c t i o n of
5
mg.The a d m i n i s t r a t i o n o f
[ D-Phe2,
0-Ala'l-LH-
RH, e i t h e r by treatment No. 1 o r treatment No. 2 produced t h e same blockade o f o v u l a t i o n .
236
VILCHEZ-MARTINEZ ET AL. Table 1
E f f e c t o f [D-Phe’,
D-Leu6]-LH-RH
D-Ala 6 1-LH-RH on
and [D-Phe’,
O v u l a t i o n i n C y c l i n g Rats Number o f o v u l a t i n g rats t o total number o f r a t s
Group
A.
Control
Treatment No.
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B.
% of inhibition o f ovulation
Mean number o f ova/ovulating rats*
* 0.40
8/8
0%
12.0
3/8a
62.5%
9.5
0.5
7/8a *
12.5%
10.29
* 1 .l
7/1OaSb
30.0%
12.04
* 0.71
8/9
11 .O%
11.17
1.12
1**
[ D-Phe2,D-Leu6] -LH-RH
c . [ D-Phe2, D-A1 a6] -LH-RH Treatment No. 2*** D-
[ D-Phe*,D-Leu6] -LH-RH
E-
D-Phe2,D-Ala6] -LH-RH
*
;:The values a r e expressed as: means SE *“Treatment No. 1: t h r e e doses o f 1 mg/rat a t noon, 3:30 p.m. and 5:OO p.m. on p r o e s t r u s day. ***Treatment No. 2: one dose o f 5 mg administered a t noon on proe s t r u s day. Control animals were i n j e c t e d w i t h v e h i c l e (20% of propylen-glycol saline). Because no d i f f e r e n c e s were obtained i n t h e c o n t r o l s , they were combined f o r p r e s e n t a t i o n . Duncan’s multip!e range t e s t : a S i g n i f i c a n t l y d i f f e r e n t from t h e value o f c o n t r o l group; b S i g n i f i c a n t l y d i f f e r e n t from t h e value o f treatment 1 , group B .
The e f f e c t o f the peptides on t h e LH and FSH l e v e l s d u r i n g t h e a f t e r n o o n o f t h e day of proestrus, can be seen i n F i g . 2. crement o f b o t h gonadotropins a t 5 p.m.
The i n -
was s i g n i f i c a n t l y lower i n
t h e groups o f r a t s t r e a t e d w i t h t h e analogs when compared w i t h the c o n t r o l group t r e a t e d w i t h t h e v e h i c l e alone, b u t t h e h i g h e s t i n h i -
237
ANTI-LH/FSH-RH AND ANTI-OVULATORY ACTIVITIES Vehicle (group 1 )
fl
[D-Phe’,
D-Leu6J-LH-RH
(group 2 )
LD-Phe’,
D-Alo6]-LH-RH
I group
800
(52%)
-
*t
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i
600
m u
0 Control Treat. 1
Treat. 2
Control
Treat. 1
Treot
.2
FIGURE 2
E f f e c t of [D-Phe’,
D-Leu6]-LH-RH and [D-Phe*,
D-Ala 6 1-LH-RH
on
serum LH and FSH l e v e l s on t h e a f t e r n o o n o f p r o e s t r u s i n &day c y c l i n g r a t s . The values represent the means, w i t h standard e r r o r s , o f chanaes between aonadotrooin values a t noon and 5 p.m. on the proestrous day Treatment 1 :
t h r e e doses o f 1 mg/rat a t noon, 3:30 and 5 p.m.
Treatment 2: one dose o f 5 mg a t noon on proestrous day. Control animals were i n j e c t e d w i t h v e h i c l e (20% o f propylene-glycol i n saline). Because no d i f f e r e n c e s were obtained between the c o n t r o l groups o f each treatment, t h e i r values were combined. Duncan’s new m u l t i p l e range t e s t : the value of group 1.
*significantly different
from
* S i g n i f i c a n t l y d i f f e r e n t from the value o f group 2. The percent i n h i b i t i o n o f gonadotropin l e v e l s i s g i v e n i n parentheses.
VILCHEZ-MARTINEZ ET AL.
238
b i t i o n o f gonadotropin l e v e l s occurred i n r a t s i n j e c t e d w i t h the D-Leu6-analog.
T h i s t r e n d was a l s o observed when t h e peptides
were i n j e c t e d according t o e i t h e r treatments No. 1 o r No. 2.
The
lowest gonadotropin l e v e l s were found a f t e r treatment No. 1.
In
t h i s case,
i n h i b i t i o n o f LH l e v e l s by [D-Phe’,
and by [D-Phe’,
D-Ala6]-LH-RH
v e l s a f t e r [D-Phe2,
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-LH-RH was 45%. vels a f t e r -LH-RH. was
The i n h i b i t i o n o f FSH l e -
was 85% and a f t e r [D-Phe*,
D-LedI-LH-RH
was 97%
D-Ala6]
Treatment No. 2 r e s u l t e d i n 82% i n h i b i t i o n o f LH l e -
[ D-Phe,
The
o n l y 75%.
D-LedI-LH-RH
D-Leu6]-LH-RH
and 52% a f t e r [D-Phe’,
n h i b i t i o n o f FSH l e v e l s w i t h [D-Phe’,
77% and w t h [D-Phe2,
D-Ala6]-LH-RH
only
D-Ala6]
D-Leu6]-LH-RH
36%.
Discussion
From t h e r e s u l t s presented here, i t i s e v i d e n t t h a t [D-Phe D-Leu6]-LH-RH
2
,
i s a s t r o n g e r i n h i b i t o r o f LH and FSH release i n -
duced by LH-RH and t h a t i t a l s o b l o c k s t h e o v u l a t i o n t o a g r e a t e r e x t e n t than [D-Phe*,
D-Ala6]-LH-RH.
increased potency o f t h e
This i s due presumably t o the
[ D-Leu6]-peptide,
r a t h e r than a prolonga-
t i o n o f a c t i v i t y , a l t h o u g h t h e l a t t e r p o s s i b i l i t y cannot be comp l e t e l y discounted. I n p a r a l l e l s t u d i e s on t h e s t r u c t u r e - a c t i v i t y
relationship o f
LH-RH, we have found s i m i l a r e f f e c t s o f s u b s t i t u t i o n s w i t h t h e same D-amino a c i d i n p o s i t i o n s i x o f t h e c h a i n on potency of supera c t i v e a g o n i s t i c analogs. more a c t i v e than
Thus, [D-Leu6]-LH-RH
[ D-Ala6]-LH-RH,
was c o n s i d e r a b l y
when t h e i r a c t i v i t i e s were
screened over a long p e r i o d o f t i m e a f t e r i n j e c t i o n i n t o immature
ANTI-LH/FSH-RH AND ANTI-OVULATORY ACTIVITIES male rats (12).
239
However, the duration of action was essentially
identical. Corbin and Beattie (5) reported that one singe
S.C.
injection
o f 6.0 mg of [D-Phe2, D-Ala6]-LH-RH at noon o f proestrous day
brought about 40% of inhibition of ovulation in rats.
The same
total dose of this analog divided into two injections at l2:3O p.m. produced a 90% blockade of ovulation.
oon and
We were not
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able to obtain this percentage o f blockade o f ovulation w th the
[ D-A1a6] -pept i de. De la Cruz et al. (3), using 3 mg/rat of the more active [ DPhe2, D-Leu6]-LH-RH divided in 4 doses on the afternoon of the day of proestrus in hamsters, found only 30% blockade of ovulation. The reasons for this were demonstrated later when it was shown that hamsters were able to ovulate fully with approximately only 10% of the amount o f gonadotropin normally released during the afternoon o f proestrous day ( 1 3 ) . Leu6]-LH-RH
Using larger doses of [D-Phe’,
D-
in rats, 2 mg 3 times on the afternoon of proestrus, we
obtained a 82% blockade of ovulation ( 4 ) . The best results with both peptides were obtained where multiple doses were used.
This is due to the relatively short length
o f action o f these peptides at the doses used, and the fact that
they are competitive inhibitors and, hence, in moderate doses, are not able t o produce a complete inhibition of the LH and FSH surge. When higher doses a e used, the risks of producing gonadotropinrelease because o f
nherent agonist activities become greater.
Em-
ploying more potent analogs, i .e. [D-Phe*, Phe3, D-Phe6]-LH-RH at
240
VILCHEZ-MARTINEZ ET AL
.
lower doses o f 1.5 mg administered at noon o f proestrous day, we have been able to consistently produce 100% blockade o f ovulation in rats (Vi lchez-Martinez, et al., in preparation). Acknowledgements W e thank Mrs. J. Gauthier, Mrs. D. Pierson and Mr. W. Carter for their valuable technical help, Dr. G. Niswender, Dr. Ward and
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NIAMDD-rat-Pituitary Hormone Program for the gifts of materials used in radioimmunoassays and Takeda Chemical Industries, Ltd. for supplying the synthetic LH-RH. This work w a s supported by NIH Contract #NICHD-72-2741, and grants from the Veterans Administration, and the US Public Health Service AM-07467 and HD-06555. References 1.
Coy, D.H., Labrie, F., Savony, M., Coy, E.J., V., Fertil. Steril., in press, 1976.
2.
Vilchez-Martinez, J.A., Coy, D.H., Coy, E.J., Arimura, A., and Schally, A.V., Fertil. Steril., in press, 1976.
and Schally, A.
3- De la Cruz, A., Coy, D.H., Schally, A.V., Coy, E.J., d e la Cruz, K.G.,
and Arimura, A., Proc. SOC. Exp. Biol. Med.,
9:
576, 1975.
4. De la CI-UZ, A., Coy, D.H., Vilchez-Martinez, J.A., Arimura, A., and Schally, A.V., Science, 191: 195, 1976. 5.
Corbin, A. and Beattie, C.W.,
Endocrine Res. Comm.,
2:
1 , 1975.
6. Vilchet-Martinez, J.A., Schally, A.V., Coy, D.H., COY, E.J., Miller I l l , C.M., and Arimura, A., Endocrinology, 96: 1130, 1975.
7. Niswender, G.D., Midgley, A.R., Monroe, S.E. and Reichert, L. E., Proc. SOC. Exp. Biol. Med.,
128: 807,
1968.
241
ANTI-LH/FSH-RH AND ANTI-OVULATORY ACTIVITIES
8. Daane, T.A., and Parlow, A.R., Endocrinology,
88: 653, 1971.
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9. Steel, R.G.D. and Torrie, H.J., Principles and Procedures Statistics. McGraw-Hill, New York, 1960.
5:
of
515, 1969.
10.
Hsu, T., and Feldt, L . S .
11.
Seeger, P.,
12.
Vilchez-Martinez, J.A., Coy, D . H . , Arimura, A., Coy, E.J., Hirotsu, Y., and Schally, A.V., Biochem. Biophys. Res. Comm., 59: 1226, 1974.
13.
De la Cruz, A., Arimura, A., de la Cruz, K.G., Endocrinology, 98:490, 1976.
Am. Res. J.,
and Gabrielsson, A., Psychol. Bull.,
69:269, 1968.
and Schally, A.V.,
COMPARISON OF THE ANTI-LH/FSH-RH AND ANTI-OVULATORY ACTIVITIES OF [D-Phe2, D-Leu6] -LH-RH AND [D-Phe2, D-Ala6]-LH-RH Vilchez-Martinez, D.H. Coy, E.J. Coy, A. Arimura and A.V. S c h a l l y Endocrine and P o l y p e p t i d e L a b o r a t o r i e s , Veterans A d m i n i s t r a t i o n H o s p i t a l , and Department o f Medicine, Tulane U n i v e r s i t y School o f Medicine, New Orleans, Louisiana 70146
Endocr Res Downloaded from informahealthcare.com by Mcgill University on 01/06/15 For personal use only.
J.A.
Abstract The anti-LH/FSH-RH and a n t i o v u l a t o r y a c t i v i t i e s o f [D-PheZ, D-Leu6] -LH-RH and [D-Phe2, D-Ala6]-LH-RH were compared i n r a t s . Both p e p t i d e s i n h i b i t e d t h e LH and FSH r e l e a s e induced by LH-RH i n [D-Phe2, Dimmature male r a t s , b u t , 4 h r a f t e r the i n j e c t i o n , LeuGI-LH-RH s t i l l suppressed the LH and FSH r e l e a s e whereas t h e [D-Phe2, D-Ala6 ILH-RH d i d n o t . When t h e p e p t i d e s were adminis t e r e d i n equal doses on t h e a f t e r oon o f t h e day o f p r o e s t r u s i n &day c y c l i n g r a t s , [D-Phd, D-Leu2]-LH-RH more completely i n h i b i t e d t h e o v u l a t i o n o c c u r r i n g on t h e f o l l o w i n g morning than [DPhe2, D-Ala6l-LH-RH. Thus, t h e i n c o r p o r a t i o n o f D-Leucine i n t o p o s i t i o n s i x o f t h e decapeptide c h a i n g i v e s a more p o t e n t i n h i b i t o r than t h a t r e s u l t i n g from t h e i n s e r t i o n o f D-Alanine.
lntroduct ion
I n t h e search f o r b e t t e r i n h i b i t o r s o f LH-RH, we have demons t r a t e d t h a t t h e replacement o f g l y c i n e i n p o s i t i o n 6 o f t h e decap e p t i d e sequence by D-Alanine,
D-Leucine,
D-phenylalanine and D-
tryptophan, produced i n c r e a s i n g l y s t r o n g e r i n h i b i t o r s o f LHRH ( 1 ) . For instance,
[DesHis2, D-Leu6]-LH-RH
g o n i s t i c a c t i v i t y o f [DesHisZ,
showed about t w i c e t h e anta-
D-Ala6]-LH-RH
when b o t h analogs
231 Copyright 0 1976 by Marcel Dekker, Inc. All Rights Reserved. Neither thls work nor any part may be reproduced or transmitted in any form or by any means, electronlc or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher.
VILCHEZ-MARTINEZ ET AL.
232
were compared f o r i n h i b i t i o n o f LH release i n immature male r a t s (2).
I n a d d i t i o n , the i n c o r p o r a t i o n o f D-phenylalanine i n p o s i t i o n
2, r a t h e r than t h e d e l e t i o n o f H i s , increases and prolongs t h e i n h i b i t o r y e f f e c t o f many peptides.
This might be due t o increased
receptor s i t e b i n d i n g coupled w i t h increased hydrophobic p r o p e r t i e s which apparently r e s u l t i n slower r e l e a s e a f t e r
S.C.
i n j e c t i o n (2).
Making use o f these observations, we were a b l e t o b l o c k t h e phy-
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s i o l o g i c a l surge o f LH by 83% and o v u l a t i o n by 30% i n c y c l i n g hams t e r s using [D-Phe’,
D-Leu6]-LHRH (3).
t h a t t h r e e i n j e c t i o n s o f [D-Phe’,
More r e c e n t l y , we found
6 D-Leu ’J-LHRH administered to 4-
day c y c l i n g r a t s on the a f t e r n o o n o f the day o f p r o e s t r u s produced
83% blockade o f o v u l a t i o n and a s i n g l e i n j e c t i o n o f 1.5 mg o f [D6 Phe2, Phe3, D-Phe 1-LH-RH a t noon o f the proestrous day,
resulted i n
almost complete suppression o f gonadotropin release, and 86% b l o c k ade of o v u l a t i o n ( 4 ) .
Corbin and B e a t t i e (5) have a l s o demonstra-
ted a blockade o f the LH surge and o v u l a t i o n i n r a t s using[D-Phe
2
,
6
D-Ala 1-LH-RH. I n view o f t h e s i g n i f i c a n t changes i n i n h i b i t o r y potency produced by v a r i o u s m o d i f i c a t i o n s i n the s i x t h p o s i t i o n and t h e demons t r a t e d e f f e c t i v e n e s s of t h e D-Ala
6 analog, i t was o f i n t e r e s t t o
compare more e x h a u s t i v e l y t h e anti-LH-RH p r o p e r t i e s of [D-Phe’, Leu6]-LH-RH and [D-Phe2,
D-Ala6]-LHRH
D-
i n some i n v i v o systems.
M a t e r i a l s and Methods
The anti-LH-RH e f f e c t of the peptides was screened over a p e r i od o f 4 h r s u s i n g an immature male r a t method described p r e v i o u s l y
ANTI-LH/FSH-RH AND ANTI-OVULATORY ACTIVITIES (2-6).
The a n i m a l s were i n j e c t e d
w i t h the v e h i c l e alone.
w i t h 300 ug o f p e p t i d e s o r
Two hundred ng o f s y n t h e t i c LH-RH o r sa-
l i n e s o l u t i o n was i n j e c t e d times t h e r e a f t e r .
S.C.
233
S.C.
a t t h e same t i m e o r a t d i f f e r e n t
Blood was c o l l e c t e d 30 m i n a f t e r LH-RH o r sa-
l i n e administration. The a n t i o v u l a t o r y t e s t was performed u s i n g a d u l t female r a t s (SD s t r a i n , C h a r l e s R i v e r Co.) w e i g h i n g a p p r o x i m a t e l y 200 g.
They
Endocr Res Downloaded from informahealthcare.com by Mcgill University on 01/06/15 For personal use only.
were k e p t i n c o n t r o l l e d t e m p e r a t u r e and l i g h t c o n d i t i o n s (14 h r l i g h t and 10 h r d a r k n e s s ) , and t h e i r v a g i n a l smears were examined d a i l y s t a r t i n g I 4 days a f t e r a r r i v a l .
Only r a t s w h i c h showed a t
l e a s t 2 s u c c e s s i v e , r e g u l a r &-day c y c l e s were used.
On t h e a f t e r -
noon o f t h e p r o e s t r u s day, t h e a n i m a l s were i n j e c t e d
S.C.
w i t h one
o r s e v e r a l doses o f t h e analogs o r w i t h t h e v e h i c l e a l o n e . was taken a t noon and 5 p.m.
o f t h e p r o e s t r o u s day.
Blood
On t h e f o l -
l o w i n g day, t h e a n i m a l s were s a c r i f i c e d and, under a d i s s e c t i n g m i croscope, t h e o v i d u c t s were i n s p e c t e d f o r ova. The s e r a were s e p a r a t e d by c e n t r i f u g a t i o n and s t o r e d a t -ZOO C u n t i l assayed f o r g o n a d o t r o p i n .
LH was d e t e r m i n e d by t h e d o u b l e a n t i -
body radioimmunoassay method o f Niswender e t a l .
( 7 ) and FSH by t h e
method o f Daane and Parlow (8) as d e s c r i b e d elsewhere ( 2 , 5 ) .
S 1 7 and NISMD-rat-FSH-RPI,
NIH-LH-
were used as LH and FSH s t a n d a r d p r e p a r a -
tions. Duncan's new m u l t i p l e range t e s t
(9) was
used f o r a n a l y z i n g t h e
s i g n i f i c a n c e s of t h e d i f f e r e n c e s i n LH and FSH l e v e l s among t h e groups.
The r e s u l t s f r o m t h e a n t i - o v u l a t o r y t e s t were expressed by
b i n o m i a l d a t a as 1 f o r o v u l a t i o n and 0 f o r n o - o v u l a t i o n ;
t h e y were
234
VILCHEZ-MARTINEZ ET AL.
f i r s t subjected t o a n a l y s i s o f variance (10-11) and then compared
(9).
by Duncan’s new m u l t i p l e range t e s t
[ D-Phe2,
D-Leu 6 1-LH-RH and [D-Phe2, D-Ala6]-LH-RH
were prepared
The p u r i t y o f
i n o u r l a b o r a t o r y by t h e s o l i d phase method (1,2).
these compounds was confirmed by TLC and amino a c i d a n a l y s i s . The i n h i b i t o r y analogs had t o be d i s s o l v e d i n a 20% propylenglycol/saline
s o l u t i o n f o r t h e i n j e c t i o n because o f t h e i r hydro-
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phobic p r o p e r t i e s .
Results
Fig. P-PheZ, rats.
1 shows a comparison o f t h e anti-LH/FSH-RH D-Leu6]-LH-RH
and
[ D-Phe2,
D-Ala6]-LH-RH
activities of
i n immature male
I t can be seen t h a t , a t t h e dose o f 300 u g / r a t ,
b o t h pep-
t i d e s i n h i b i t e d t h e LH and FSH r e l e a s e a f t e r LH-RH a d m i n i s t r a t i o n . But, a t 4 h r . ,
[D-Phe’,
D-Leu6]-LH-RH
s t i l l suppressed t h e LH and
FSH response t o LH-RH whereas [D-Phe2,
D-Ala6]-LH-RH
d i d not.
Table 1 shows t h e i n h i b i t i o n o f o v u l a t i o n by these p e p t i d e s . 1 mg/rat was administered a t noon, 3:30 and 5:OO p:m.
When
( t o t a l dose:
3 mg/rat) on p r o e s t r u s ( t r e a t m e n t No. l ) , [D-Phe2, D-Leu 6 1-LH-RH was f i v e times more p o t e n t than [D-Phe’,
6
D-Ala 1-LH-RH.
[
D-Phe’,
D-Leu6]-LH-RH
produced 62.5% i n h i b i t i o n o f o v u l a t i o n , and [D-Phe’,
D-Ala6]-LH-RH
r e s u l t e d i n o n l y 12.5% i n h i b i t i a n o f o v u l a t i o n .
When t h e p e p t i d e s were g i v e n as a s i n g l e dose of o f t h e day o f p r o e s t r u s ( t r e a t m e n t No. observed. [D-Phe2,
Z),
5
mg/rat a t noon
t h e same tendency was
The percentage o f i n h i b i t i o n o f o v u l a t i o n was 30% a f t e r D-Leu6]-LH-RH
and 11% a f t e r
[ D-Phe2,
D-Ala6]-LH-RH.
Thus,
235
ANTI-LH/FSH-RH AND ANTI-OVULATORY ACTIVITIES
a
Vehicle + b l i m (group I) Vehicle
+
LH-RH (2M) ng),(group 2)
ID-F+~.D -LH-RH - L ~goo u~ p9)I + biiM (group 3)
Endocr Res Downloaded from informahealthcare.com by Mcgill University on 01/06/15 For personal use only.
Q
[E-lb2,D-Lcu61
-LH-RH + LH-RH (group 4)
FIGURE 1 Time ourse o f i n h i b i t i o n by [ D-Phe2, D-LedI-LH-RH and [D-Phe2, D-Alag]-LH-RH o f LH-RH-induced LH and FSH r e l e a s e i n immature male r a t s . The p e p t i d e s were i n j e c t e d a t time 0. The r a t s were d e c a p i t a t e d 30 min a f t e r LH-RH o r s a l i n e a d m i n i s t r a t i o n . Dose o f 300 u g / r a t . Dose o f LH-RH: 200 n g / r a t . S i x r a t s per peptide: group were used. The percentages o f i n h i b i t i o n of gonadotropin l e v e l s are shown above each bar.
t h e a d m i n i s t r a t i o n of 1 mg/rat
3
times p e r day (treatment No. 1) was
a more e f f e c t i v e schedule of treatment w i t h D-Leu6-peptide than a s i n g l e i n j e c t i o n of
5
mg.The a d m i n i s t r a t i o n o f
[ D-Phe2,
0-Ala'l-LH-
RH, e i t h e r by treatment No. 1 o r treatment No. 2 produced t h e same blockade o f o v u l a t i o n .
236
VILCHEZ-MARTINEZ ET AL. Table 1
E f f e c t o f [D-Phe’,
D-Leu6]-LH-RH
D-Ala 6 1-LH-RH on
and [D-Phe’,
O v u l a t i o n i n C y c l i n g Rats Number o f o v u l a t i n g rats t o total number o f r a t s
Group
A.
Control
Treatment No.
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B.
% of inhibition o f ovulation
Mean number o f ova/ovulating rats*
* 0.40
8/8
0%
12.0
3/8a
62.5%
9.5
0.5
7/8a *
12.5%
10.29
* 1 .l
7/1OaSb
30.0%
12.04
* 0.71
8/9
11 .O%
11.17
1.12
1**
[ D-Phe2,D-Leu6] -LH-RH
c . [ D-Phe2, D-A1 a6] -LH-RH Treatment No. 2*** D-
[ D-Phe*,D-Leu6] -LH-RH
E-
D-Phe2,D-Ala6] -LH-RH
*
;:The values a r e expressed as: means SE *“Treatment No. 1: t h r e e doses o f 1 mg/rat a t noon, 3:30 p.m. and 5:OO p.m. on p r o e s t r u s day. ***Treatment No. 2: one dose o f 5 mg administered a t noon on proe s t r u s day. Control animals were i n j e c t e d w i t h v e h i c l e (20% of propylen-glycol saline). Because no d i f f e r e n c e s were obtained i n t h e c o n t r o l s , they were combined f o r p r e s e n t a t i o n . Duncan’s multip!e range t e s t : a S i g n i f i c a n t l y d i f f e r e n t from t h e value o f c o n t r o l group; b S i g n i f i c a n t l y d i f f e r e n t from t h e value o f treatment 1 , group B .
The e f f e c t o f the peptides on t h e LH and FSH l e v e l s d u r i n g t h e a f t e r n o o n o f t h e day of proestrus, can be seen i n F i g . 2. crement o f b o t h gonadotropins a t 5 p.m.
The i n -
was s i g n i f i c a n t l y lower i n
t h e groups o f r a t s t r e a t e d w i t h t h e analogs when compared w i t h the c o n t r o l group t r e a t e d w i t h t h e v e h i c l e alone, b u t t h e h i g h e s t i n h i -
237
ANTI-LH/FSH-RH AND ANTI-OVULATORY ACTIVITIES Vehicle (group 1 )
fl
[D-Phe’,
D-Leu6J-LH-RH
(group 2 )
LD-Phe’,
D-Alo6]-LH-RH
I group
800
(52%)
-
*t
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i
600
m u
0 Control Treat. 1
Treat. 2
Control
Treat. 1
Treot
.2
FIGURE 2
E f f e c t of [D-Phe’,
D-Leu6]-LH-RH and [D-Phe*,
D-Ala 6 1-LH-RH
on
serum LH and FSH l e v e l s on t h e a f t e r n o o n o f p r o e s t r u s i n &day c y c l i n g r a t s . The values represent the means, w i t h standard e r r o r s , o f chanaes between aonadotrooin values a t noon and 5 p.m. on the proestrous day Treatment 1 :
t h r e e doses o f 1 mg/rat a t noon, 3:30 and 5 p.m.
Treatment 2: one dose o f 5 mg a t noon on proestrous day. Control animals were i n j e c t e d w i t h v e h i c l e (20% o f propylene-glycol i n saline). Because no d i f f e r e n c e s were obtained between the c o n t r o l groups o f each treatment, t h e i r values were combined. Duncan’s new m u l t i p l e range t e s t : the value of group 1.
*significantly different
from
* S i g n i f i c a n t l y d i f f e r e n t from the value o f group 2. The percent i n h i b i t i o n o f gonadotropin l e v e l s i s g i v e n i n parentheses.
VILCHEZ-MARTINEZ ET AL.
238
b i t i o n o f gonadotropin l e v e l s occurred i n r a t s i n j e c t e d w i t h the D-Leu6-analog.
T h i s t r e n d was a l s o observed when t h e peptides
were i n j e c t e d according t o e i t h e r treatments No. 1 o r No. 2.
The
lowest gonadotropin l e v e l s were found a f t e r treatment No. 1.
In
t h i s case,
i n h i b i t i o n o f LH l e v e l s by [D-Phe’,
and by [D-Phe’,
D-Ala6]-LH-RH
v e l s a f t e r [D-Phe2,
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-LH-RH was 45%. vels a f t e r -LH-RH. was
The i n h i b i t i o n o f FSH l e -
was 85% and a f t e r [D-Phe*,
D-LedI-LH-RH
was 97%
D-Ala6]
Treatment No. 2 r e s u l t e d i n 82% i n h i b i t i o n o f LH l e -
[ D-Phe,
The
o n l y 75%.
D-LedI-LH-RH
D-Leu6]-LH-RH
and 52% a f t e r [D-Phe’,
n h i b i t i o n o f FSH l e v e l s w i t h [D-Phe’,
77% and w t h [D-Phe2,
D-Ala6]-LH-RH
only
D-Ala6]
D-Leu6]-LH-RH
36%.
Discussion
From t h e r e s u l t s presented here, i t i s e v i d e n t t h a t [D-Phe D-Leu6]-LH-RH
2
,
i s a s t r o n g e r i n h i b i t o r o f LH and FSH release i n -
duced by LH-RH and t h a t i t a l s o b l o c k s t h e o v u l a t i o n t o a g r e a t e r e x t e n t than [D-Phe*,
D-Ala6]-LH-RH.
increased potency o f t h e
This i s due presumably t o the
[ D-Leu6]-peptide,
r a t h e r than a prolonga-
t i o n o f a c t i v i t y , a l t h o u g h t h e l a t t e r p o s s i b i l i t y cannot be comp l e t e l y discounted. I n p a r a l l e l s t u d i e s on t h e s t r u c t u r e - a c t i v i t y
relationship o f
LH-RH, we have found s i m i l a r e f f e c t s o f s u b s t i t u t i o n s w i t h t h e same D-amino a c i d i n p o s i t i o n s i x o f t h e c h a i n on potency of supera c t i v e a g o n i s t i c analogs. more a c t i v e than
Thus, [D-Leu6]-LH-RH
[ D-Ala6]-LH-RH,
was c o n s i d e r a b l y
when t h e i r a c t i v i t i e s were
screened over a long p e r i o d o f t i m e a f t e r i n j e c t i o n i n t o immature
ANTI-LH/FSH-RH AND ANTI-OVULATORY ACTIVITIES male rats (12).
239
However, the duration of action was essentially
identical. Corbin and Beattie (5) reported that one singe
S.C.
injection
o f 6.0 mg of [D-Phe2, D-Ala6]-LH-RH at noon o f proestrous day
brought about 40% of inhibition of ovulation in rats.
The same
total dose of this analog divided into two injections at l2:3O p.m. produced a 90% blockade of ovulation.
oon and
We were not
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able to obtain this percentage o f blockade o f ovulation w th the
[ D-A1a6] -pept i de. De la Cruz et al. (3), using 3 mg/rat of the more active [ DPhe2, D-Leu6]-LH-RH divided in 4 doses on the afternoon of the day of proestrus in hamsters, found only 30% blockade of ovulation. The reasons for this were demonstrated later when it was shown that hamsters were able to ovulate fully with approximately only 10% of the amount o f gonadotropin normally released during the afternoon o f proestrous day ( 1 3 ) . Leu6]-LH-RH
Using larger doses of [D-Phe’,
D-
in rats, 2 mg 3 times on the afternoon of proestrus, we
obtained a 82% blockade of ovulation ( 4 ) . The best results with both peptides were obtained where multiple doses were used.
This is due to the relatively short length
o f action o f these peptides at the doses used, and the fact that
they are competitive inhibitors and, hence, in moderate doses, are not able t o produce a complete inhibition of the LH and FSH surge. When higher doses a e used, the risks of producing gonadotropinrelease because o f
nherent agonist activities become greater.
Em-
ploying more potent analogs, i .e. [D-Phe*, Phe3, D-Phe6]-LH-RH at
240
VILCHEZ-MARTINEZ ET AL
.
lower doses o f 1.5 mg administered at noon o f proestrous day, we have been able to consistently produce 100% blockade o f ovulation in rats (Vi lchez-Martinez, et al., in preparation). Acknowledgements W e thank Mrs. J. Gauthier, Mrs. D. Pierson and Mr. W. Carter for their valuable technical help, Dr. G. Niswender, Dr. Ward and
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NIAMDD-rat-Pituitary Hormone Program for the gifts of materials used in radioimmunoassays and Takeda Chemical Industries, Ltd. for supplying the synthetic LH-RH. This work w a s supported by NIH Contract #NICHD-72-2741, and grants from the Veterans Administration, and the US Public Health Service AM-07467 and HD-06555. References 1.
Coy, D.H., Labrie, F., Savony, M., Coy, E.J., V., Fertil. Steril., in press, 1976.
2.
Vilchez-Martinez, J.A., Coy, D.H., Coy, E.J., Arimura, A., and Schally, A.V., Fertil. Steril., in press, 1976.
and Schally, A.
3- De la Cruz, A., Coy, D.H., Schally, A.V., Coy, E.J., d e la Cruz, K.G.,
and Arimura, A., Proc. SOC. Exp. Biol. Med.,
9:
576, 1975.
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