Hormone antagonists 9783111506753, 9783110086133

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Hormone Antagonists

Hormone Antagonists Editor M. K. Agarwal

W G_ Walter de Gruyter • Berlin • New York 1982 DE

Editor M. K. Agarwal, B. Sc.; M. Sc.; Ph. D„ M. D. Maître de Recherche au CNRS Scientific Director: Laboratoire de Physio-Hormono-Réceptéroiogie Faculté de Médecine Broussais Hôtel-Dieu Université Pierre et Marie Curie 15, rue de l'Ecole de Médecine 75270 Paris Cédex 06, France.

CIP-Kurztitelaufnahme der Deutschen Bibliothek Hoimom antagonists/ ed. M. K. Agarwal. Bertin; New York: de Gruyter, 1982. ISBN 3-11-008613-1 NE: Agarwal, Manjul K. [Hrsg.]

Library of Congress Cataloging in Publication Data Hormone antagonists. Includes indexes. 1. Hormone antagonists. 2. Hormone antagonistsTesting. I. Agarwal, M. K. [DNLM: 1. Hormone antagonists. WK102 H8117] QP571.5.H67 615'.74 81-19424 ISBN 3-11-008613-1 AACR2

Copyright © 1982 by Walter de G ruyter & Co., Berlin 30. All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced in any form - by photoprint, microfilm or any other means nor transmitted nor translated into a machine language without written permission from the publisher. Printing: Druckerei Hildebrand, Berlin. - Binding: Dieter Mikolai, Berlin. Printed In Germany.

PREFACE Antagonists for hormones are of importance not only for their clinical potential but especially as tools to probe mechanisms of hormone action at the cellular and the molecular levels. The present book aims to group under one single cover, for the first time, all hormones for which specific antagonists have been established. The first section of the volume deals with steroid hormone antagonism, and the second with antagonists for peptide hormones. The clinical use as well as the conceptual challenge posed by various systems have been presented in all possible cases. The last section groups together substances already well familiar, e.g. antihistamines, as well as areas that are just beginning to emerge, e.g. anti-pheromones, but which represent tremendous potential for basic and industrial research. The influence of a hormone can be antagonized at various levels such as synthesis, degradation, biological half life, peripheral physiological effects, to mention only a few. The most effective antagonism, however, is possible at the level of the cellular receptor specific for the hormone. The widespread interest in this regard is evident from the explosion of articles in contemporary journals of endocrinology, biochemistry, pharmacology, and neurosciences. Specialized symposia, monographs, reviews, and workshops, too, compete for attention thereby cutting down the time available for work at the bench. Due to the large spectrum of biological responses, laboratory techniques, and experimental approaches developed in one field are often not consulted by workers in other, related fields. Bringing all these models together under one cover should lead to cross fertilization of ideas and stimulate further research. The photo-offset method of production assures that the book will reach the reader before specialized reports make it obsolete. It is expected that the present volume will remain a major reference source to advanced graduate students, research scientists, and clinicians with

VI

a f l a i r for f u n d a m e n t a l s ,

for s o m e t i m e to

come.

In a n y u n d e r t a k i n g o f t h i s s i z e , o n e c a n be

criticized

for t h a t w h i c h w a s n o t i n c l u d e d a n d c o u l d o r s h o u l d h a v e integrated.

been

It is h o w e v e r i m p o s s i b l e to p r o v i d e a t i m e l y

c o v e r a g e f r o m a l l l e a d e r s in d i f f e r e n t f i e l d s w i t h v a r i o u s degrees of commitments.

I n d u l g e n c e is a s k e d o f all

concerned.

T h e o r e t i c a l c r i t i c i s m is v a l i d o n l y if t e m p e r e d w i t h

prac-

tical experience. Another, later edition may cover that

which

w a s left o u t f r o m t h e p r e s e n t o n e . N e v e r t h e l e s s ,

been

it h a s

a p r i v i l e g e a n d p l e a s u r e to e d i t t h i s v o l u m e a n d t h a n k s d u e to all c o n t r i b u t o r s w h o r e s p o n d e d t i m e l y to m a k e work

possible.

September

1981,

Paris

M . K. A g a r w a l

are

this

CONTENTS SECTION 1: STEROID HORMONE ANTAGONISTS Steroid Agonists and Antagonists: Molecular Conformation, Receptor Binding and Activity W.L. Duax, J.F. Griffin, D.C. Rohrer, C.M. Weeks

3

Antihormonal Activities of Steroids with Modified Rings A or B L. Starka, R. Hampl

25

Antagonism of Steroid Hormones by Specific Antibodies P. Vecsei, D. Haack, K.G. Gless

39

Antagonism of Steroid Hormone Action by Interference of Receptor Function with Low Molecular Weight Inhibitors B. Sato, Y. Nishizawa, Y. Maeda, K. Noma, K. Matsumoto, Y. Yamamura

61

Anti-Steroids and Animal Behaviour: A Review C. Fabre-Nys

77

Agonistic and Antagonistic Properties of Clomiphene J.H. Clark, S.C. Guthrie, S.A. McCormack, B.M. Markavarich

89

Hydroxylated Antioestrogens: New Pharmacological Probes to Investigate Oestrogen and Antioestrogen Action V.C. Jordan

109

Biological Actions and Binding Properties of a New Estrogen Antagonist, LY117018 L.J. Black

129

Binding Properties and Ligand Specificity of an Intracellular Binding Site with Specificity for Synthetic Oestrogen Antagonists of the Triphenyl ethylene Series R.L. Sutherland, C.K.W. Watta, L.C. Murphy

147

Estrogen Receptor Binding Parameters of the High Affinity Antiestrogen 3 H-H1285 T.S. Ruh, J.L. Keene, P. Ross

163

Potential Antiestrogenic and Antitumour Mechanisms of Tamoxifen Action in Breast Cancer R.I. Nicholson, P. Daniel, J.S. Syne, P. Davies

179

Anti-Tumor Action of Non-steroidal Antiestrogens S. Sekiya, N. Inaba and H. Takamizawa

203

Progesterone Inhibition of Nuclear Estrogen Receptor Retention W.W. Leavitt, R.W. Evans, W.C. Okulicz, R.G. McDonald, W.J. Hendry, W.F. Robidoux Jr

213

VIII Advances in the Study of Antiprogestational Agents K.E. Kendle

233

Pharmacological and Clinical Effects of Antiandrogens R. Neri, N. Kassem

247

Mechanism of Action of a Steroidal Anti-androgen TSAA-291, 16ß-ethyl-17ß-hydroxyestr-4-en-3-one R. Nakayama, K. Hiraga, T. Miki

269

Structure-Affinity Relationship of Antimineralocorticoid Active Steroids G. Wambach, A. Helber

293

Progestin Antagonism of Aldosterone Action in the Kidney M. K. Agarwal

307

Clinical Pharmacology and Therapeutic Use of Aldosterone Antagonists L.E. Ramsay 335 Antiglucocorticoids: Differential Antagonism of in vivo Responses to Dexamethasone P. H. Naylor, F. Rosen

365

Antagonism of Glucocorticoid Action in vivo M. K. Agarwal

381

Discrimination between Glucocorticoid Agonists and Antagonists by Means of Receptor Binding Studies P. A. Bell, T.R. Jones

391

Antagonism of Glucocorticoid Action by Insulin M.H. Cake, K.K.W. Ho, G.C.T. Yeoh

407

Inhibition of Adrenal Steroid Biosynthesis by Metyrapone N. Sonino

419

SECTION 2: PEPTIDE HORMONE ANTAGONISTS Structural, Conformational and Dynamic Considerations in the Development of Peptide Hormone Antagonists V.J. Hruby, H.I. Mosberg

433

Inhibitors of the Renin-Angiotensin System K. Yamamoto, K. Miura, Y. Abe, T. Komori, S. Yoshimoto

475

Anti-Prolactin and Anti-Growth Hormone Receptor Antibodies, and their use as Anti-Hormonal Agents M.J. Waters, H.G. Friesen

487

Clinical Applications of Prolactin Lowering Drugs C. Ferrari, R. Benco, P. Rampini

503

IX Receptor Antagonists of the Actions of G a s t r o i n t e s t i n a l on Pancreatic Acinar C e l l s J.D. Gardner, R.T. Jensen

Peptides 527

G a s t r i n Antagonism B.H. H i r s t

549

Antigonadal Effects of Gonadotropin-Releasing Hormone and Agonist Analogs J.P. Harwood, K.J. Catt

571

Antagonists of Hypothalamic Regulatory Peptides and Dopamine C. Denef, M. Andries

589

Lutenizing Hormone Releasing Hormone Analogs as Antihormones F. Bex, A. Corbin

609

Antigonadotropic, A n t i s t e r o i d o g e n i c and A n t i s t e r o i d a l A c t i v i t i e s of Agonist Analogs of Lutenizing Hormone Releasing Hormone as Revealed by t h e i r Antireproductive A c t i v i t i e s B.H. Vickery

623

SECTION 3: MISCELLANEOUS HORMONES AND ANTAGONISTS Hormonal I n h i b i t i o n of Adenylate Cyclase: A P o s s i b l e Mechanism f o r P h y s i o l o g i c a l Antagonism L. E. Limbird

661

Naloxone and Naltrexone: Endorphin Antihormones M.S. Gold, A.C. Pottash

671

Antihistamines J. Dry, A. Pradalier

679

X-Ray C r y s t a l l o g r a p h i c Studies of Thyroid Hormone, Analogue and Competitor Binding I n t e r a c t i o n s with Human Serum Prealbumin S . J . Oatley, J.M. Burridge, C.C.F. Blake

705

Antipheromones? J. Bai 11 e t , J. Pail l a r d

717

AUTHOR INDEX

725

SUBJECT INDEX

727

SECTION 1: STEROID HORMONE ANTAGONISTS

STEROID AGONISTS AND ANTAGONISTS:

MOLECULAR

CONFORMATION,

RECEPTOR BINDING AND ACTIVITY

William L. Duax, Jane F. Griffin, Douglas C. Rohrer and Charles M. Weeks Medical Foundation of Buffalo, Inc. 73 High Street, Buffalo, New York 14203, USA

Introduction Many characteristic hormonal responses of steroids are contingent upon their binding to specific receptors in target tissue.

While

it is known that response depends upon

interaction

of the receptor and nuclear chromatin, the precise details of this interaction and the role played by the steroid in this process remains undetermined.

Structural details

undoubtedly

have a direct bearing upon receptor affinity and will directly or indirectly influence receptor activation, transport and nuclear interaction.

The existence of antagonists that com-

pete for the steroid binding site of the receptor with high affinity demonstrate that the phenomena of binding and activity are at least partially independent. major topics:

This review will cover two

Structural requirements

gestin receptor binding and the

for estrogen and pro-

structural differences

between

estrogen, androgen, glucocorticoid and mineralocorticoid agonists and antagonists.

Structural Features of Estrogens Compounds that bind to the estrogen receptor and exhibit ity have an amazing variety of structures. these compounds are illustrated in Figure 1.

© 1982 Walter de Gruyter & Co., Berlin • New York Mormone Antagonists, Editor M. K. Agarwal

activ-

Just a sample of Structures w i t h

4

a'

OH

o

c)

HO

HO'

HO

d:

HO

OH O

¿

H 3

Figure 1. Compounds that bind to the estrogen receptor and have varying degrees of estrogenic activity include (a) estradiol, (b) 8a-D-homo-estradiol, (c) ll-keto-9$-estrone, (d) mirestrol, (e) diethylstilbestrol and (f) trans zearalenone. relatively high affinity

for the receptor almost without

exception contain a phenolic ring.

Lven the most weakly

binding compounds have at least a benzene ring.

On the basis

of a comparison of the chemical structures of estradiol diethylstilbestrol

Keasling and Schueler

the structural requirements

(1) proposed

and

that

for estrogenic activity are that

the molecule be flat, that there be hydrophilic groups

(such

as the hydroxyls on estradiol and diethylstilbestrol, DISS) at either end of the molecule, that the middle of the molecule be hydrophobic and that the distance between terminal yls be highly specific.

The crystallographically

hydrox-

observed

structure of estradiol and DES are compared in Figure 2. fect agreement in the overall shape of these and other genic compounds

is not possible.

Per-

estro-

In Figure 2, when the best

overlap of the A-ring of the estradiol and one of the phenol rings of DES is achieved by a least-squares process, the differences at the other e«d of the molecules are maximized. The corresponding distances between the hydroxyl groups in estradiol and DES are not and cannot be identical. tances in estradiol and DES are 10.9% and 12.1$ Although there is some flexibility

The dis-

respectively.

in the two molecules,

5

Figure 2. Comparison of the crystallographically observed structures of estradiol and DES viewed (a) perpendicular and (b) parallel to the phenol ring. Dark = common structure, shaded = estradiol, open = DES. neither of these distances can be altered by more than 0.28 without disrupting their chemical integrity.

The phenyl rings

in DES may be rotated relative to each other and the ethyl groups take up different conformations in four different crystal forms

(2) but the distances between the hydroxyls is fixed

by the rigidity of the phenyl rings and the central double bond.

Similarly the flexibility in the overall shape of

estradiol illustrated by its minor variation in three forms

crystal

(hemihydrate, propanol and urea complexes) does not

greatly alter the 0(3)-0(17) distance which is 10.938, and 11.038 in the three independent structure (3,4,5).

10.998,

determinations

The fact that the distances between the terminal

hydroxyl groups in these and other estrogenic compounds are not equal could mean one or more of the following botli hydroxyls arc not necessary

things:

for binding and/or

activity,

the receptor is flexible, or solvent may act as a link between the hormone and the receptor

(6).

A comparison of the conformation of estradiol with four other estrogenic compounds 8a-D-homo-estradiol, mirestrol, and zearalenone

ll-keto-93-estrone,

(Figure 3) further illustrates

molecular planarity is not a requirement for activity. the phenol rings, a common element in all of these

that

When

structures,

are superimposed, the degree of structural variation that is compatible with receptor binding and activity is illustrated. The fact that 8a-D-homo-estradiol and ll-keto-93-estrone

are

6

Figure 3. Comparison of the crystallographically observed conformation of estradiol with those of (a) 8a-D-homo-estradiol, (b) ll-keto-96-estrone, (c) mirestrol, and (d) zearalenone. Shaded molecule is estradiol is each figure. more estrogenic than the natural 83 and 9a configurations respectively, prompted X-ray crystallographic

analysis of

both compounds in order to unambiguously demonstrate that the less planar unnatural configuration analogue was indeed more active form

(7,8).

Zearalenone

(Figure If), a mycotoxin

graminearum

causes estrogenic syndrome in swine.

produced by

analogues and naturally occurring derivatives of have been tested for estrogenic activity. C1 ' -C 2 * eis

the

The

Fusarium A number of zearalenone

synthetic

zearalenone is more active than Cl'-C2'

trans

zearalenone and the 8'-hydroxy derivative of trans

zearale-

none is inactive.

zearale-

Because the chemical formula of

none bears little resemblence to that of estradiol striking similarity

the

in overall shape of the molecules

3d) is somewhat surprising.

The

(Figure

crystallographically

observed conformation of ais and trans

zearalenone and the

7 8-hydroxy derivative of trans pared in Figure 4.

zearalenone

Despite differences

(10,11) are com-

in the 14-member

ring

conformation in individual structures the overall shape remains very nearly identical.

Since the 8-hydroxy derivative

is inactive it is clear that similar shape and the presence of a phenol ring will not assure activity. The 8-hydroxy substituent may inhibit binding by direct steric interference with the receptor or by electronic

repulsion.

In order to achieve this good agreement in overall

space

filling properties between estradiol and zearalenone, it is necessary to match the C(3) atom of the phenol ring of estradiol with the atom between the two hydroxy substituted bons

in zearalenone

(Figure 5).

If hydrogen bonding

carinvolv-

ing the A ring is important for receptor binding and if zearalenone binds to the site in the orientation shown in Figure 5 relative to the binding orientation of estradiol, then there must be some flexibility in the specific of the hydroxyl oxygen.

position

Exactly where the hydroxy1 is may

be less important than its ability to form a hydrogen bond to the appropriate hydrophilic site on the receptor. be achieved

This could

if either 0(2) or 0(4) of zearalenone can hydrogen

,(b) :0(C)

06 Figure 4. Stereo comparison of (a) ais zearalenone, zearalenone and (c) 8-hydroxy trans zearalenone.

(b) trans

8

\

\ \

017

F i g u r e 5. S u p e r p o s i t i o n d r a w i n g of e s t r a d i o l a n d trans zeara l e n o n e i l l u s t r a t i n g s i m i l a r i t i e s in o v e r a l l c o n f o r m a t i o n a n d p o s s i b l e h y d r o g e n b o n d i n g to t h e s a m e r e c e p t o r s i t e (R). b o n d to the s a m e

s i t e as 0 ( 3 )

are o r i e n t e d r e l a t i v e if t h e r e c e p t o r donation

0(3)

zearalenone of

0(4)

zearalenone

site.

forms

p o s i t i o n R1 s h o w n at position R2.

to e a c h o t h e r

is f l e x i b l e

from either

estradiol,

donates

acts

for e s t r o g e n i c of b o u n d

has d e m o n s t r a t e d

binding

of e s t r a d i o l

displace

demonstrate either

end

prebound

for

these

oid surface

(13,14).

and

estradiol

on

that

for

of atom

0(4) is

is, supports

our choice

in

estradiol.

that 0(2) of the

not this

relative

zearalenone.

prevents

and p-sec-amyl

(12) .

These

phenol

results

flat molecule with hydroxy

assumed

receptors

receptor

structure

that t e t r a h y d r o n a p h t h o l

is n o t e s s e n t i a l

of s u b s t r a t e

(9)

but 0(4)

supports

of

to a n a c c e p t o r

of 0(3)

the r e c e p t o r

that a large

It h a s b e e n c o m m o n l y oids

to

structures

to a w a t e r m o l e c u l e

and Mirocha

estradiol

Mueller will

bond

reason we have proposed

activity

suggestion and indirectly orientation

5 or

In the c r y s t a l

the a n a l o g u e

molecules

in F i g u r e

a hydrogen bond

this as

5.

the

to a c c e p t h y d r o g e n

In the c r y s t a l

The d e m o n s t r a t i o n by Pathre essential

as s h o w n

enough

a hydrogen bond

in Figure For

of e s t r a d i o l w h e n

clearly

groups

at

binding.

that

the

high affinity

can only be r a t i o n a l i z e d

contact

over

Because

the

the m a j o r i t y 3 - h y d r o x y l of

of

ster-

on the basis

of t h e

ster-

estradiol

9 c a n act as a h y d r o g e n b o n d a c c e p t o r and d o n o r at the

same

t i m e , it a l o n e * c a n a c c o u n t for as m u c h as 6 k c a l / m o l e binding energy

.

It is not h a r d to i m a g i n e

that the

of this e n e r g y w i t h that g a i n e d from s t a c k i n g involving

of coupling

interactions

the p h e n o l ring a n d o t h e r h y d r o p h o b i c

interactions

w i t h the A a n d B rings m i g h t be s u f f i c i e n t to a c c o u n t m o s t of the e n e r g y of b i n d i n g .

The p o t e n t i a l

of a

h y d r o x y l g r o u p w i l l , of c o u r s e , be d e t e r m i n e d by the m a k e u p of the m o l e c u l e

to w h i c h

it is a t t a c h e d .

these o b s e r v a t i o n s we h a v e p r o p o s e d a m o d e l

for

specific chemical

In light

for e s t r o g e n bind-

ing to the r e c e p t o r that i n v o l v e s c l o s e a s s o c i a t i o n of s t e r o i d A - r i n g or a n a l o g o u s p a r t of a n o n - s t e r o i d a l w i t h the r e c e p t o r probably 6).

(15).

In this

the

estrogen

interaction a hydroxyl

acts as a h y d r o g e n b o n d d o n o r and a c c e p t o r

A l t h o u g h the m o l e c u l e s

oxygen

(Figure

are n o t all flat and there is no

s p e c i f i c o x y g e n o x y g e n d i s t a n c e , m o s t of the a c t i v e

estrogens

d i s c u s s e d h e r e have an o x y g e n at the e n d of the m o l e c u l e is a n a l o g o u s

of

that

to the s t e r o i d D r i n g .

C h e r n a y a e v et al h a v e o b s e r v e d that r e m o v a l of the 17 h y d r o x y l from e s t r a d i o l almost

significantly decreases receptor binding

totally abolishes estrogenic activity

(16) .

The

and activ-

F i g u r e 6. M o d e l for e s t r o g e n r e c e p t o r b i n d i n g in w h i c h the p h e n o l i c ring lias an i n t i m a t e a s s o c i a t i o n w i t h the r e c e p t o r a n d d i f f e r e n c e s in v a r i a b l e D - r i n g r e g i o n w i l l c o n t r o l e v e n t s s u b s e q u e n t to b i n d i n g that g o v e r n a c t i v i t y . * E s t i m a t e s of the s t r e n g t h of a s i n g l e h y d r o g e n b o n d f r o m 1 to 5 k c a l / m o l e .

vary

ity of each of the steroids is greater than would be expected on the basis of the binding retained (Table I). the removal of the 3-hydroxyl almost eliminates

In contrast, receptor

binding while retaining activity in proportion to the reduced amount of binding.

These results demonstrate that 0(3) is

more important to binding and 0(17) is more important to activity.

The reduced binding of the steroid without a 17-

hydroxyl group illustrates that our A-ring model fies the requirement for binding.

oversimpli-

Possible explanations

this 0(17) dependence are long range conformational mission effects

for

trans-

(17), or 17-hydroxy participation in stabiliz-

ing a conformational change in the receptor after binding. In accordance w i t h the principal of conformational

transmis-

sion the removal of the 17-hydroxy may alter the electronic balance in the structure reducing the strength of hydrogen bonds involving 0(3) and the stability of other A ring amino acid interactions.

The observation that the difference

in

binding affinity between estrone and estradiol is due to the slower off-rate of estradiol

(18) is compatible w i t h the

suggestion that the D-ring hydroxyl may stabilize a conformational change in the receptor thereby impeding facile substrate

dissociation.

Table I. Affinity to cytoreceptor of rabbit uterus cytosol (in percent) and estrogenic activity (in the Allen Doisy test) of monohydroxylated estratrienes relative to estradiol.

Estradiol 1,3,5(10)Estratriene-17-ol 1,3,5(10)Estratriene-3-ol

Relative Binding

Relative Activity

[100] 1.7±0.4 14.4±2.3

[100] 1 .2

Ratio of Binding to Activity [1.0] 1.7 72

Structural Features of Progestins A number of structural variations are compatible with high

11

affinity binding to the progesterone receptor.

The

diagrams of 19 steroids that were observed to have binding affinity than progesterone

chemical greater

for the progestin

receptor

in rabbit or human uterus are shown in Figure 7 (19-22). Examination of these diagrams

indicates that

gesterone side chain is not essential substituent

is not essential

(a) the

(norgestrel);

(progesterone);

the 19-methyl group enhances binding; and

(c) removal of

(d) additional

hydrophobic bulk at the 10a, 16a, and 18 positions essential but can enhance binding.

176-pro(b) a 17a

is not

Finally, the most con-

spicuous common feature of all the high-affinity binders

in

•these studies is the 4-ene-3-one composition.

F 13) DU 41164

14) W Y 4 3 5 5

15)R=OH 16) R= F1

17)

Figure 7. Chemical diagrams of 19 steroids observed to have equal or greater binding affinity than progesterone for the progestin receptor in rabbit and human uterus. X-Rav studies have been completed for compounds 1-8 and 10.

12 The e n h a n c e m e n t of b i n d i n g b r o u g h t a b o u t b y h y d r o p h o b i c s t i t u e n t s at C(16ci), C ( 1 7 a ) , C ( 1 7 3 ) , and C ( 1 8 ) , by s u b s t i t u e n t s at C(113)

sub-

hydrophilic

and C(21) , and by the t r a n s f e r of a

m e t h y l g r o u p from 3 face to a face in r e t r o p r o g e s t e r o n e g e n e r a l l y b e e n a s s u m e d to r e s u l t hydrophilic

i n t e r a c t i o n b e t w e e n the s u b s t i t u e n t and the

tein s u r f a c e

(14) .

This s u g g e s t s

at b e s t a v e r y

s p e c i f i c fit of m o s t of these t i g h t l y b i n d i n g the a and 3 faces of the B, C, and D r i n g s sequently particular

there is little j u s t i f i c a t i o n o r i e n t a t i o n of the D ring

e l e m e n t for c o m p a r i n g

steroids

(Figure

high-affinity binding. substituents

across Con-

reference

o v e r a l l c o n f o r m a t i o n of p r o g e s t i n s The p r i m a r y s i g n i f i c a n c e

or

in

of b u l k y they

s t r a i n that is t r a n s m i t t e d to the

A ring, the only c o m m o n e l e m e n t

non-

that a

in the C and D ring m a y in fact be that

introduce molecular

pro-

8).

for a s s u m i n g

is a v a l i d

or

loose or

that this e n d of the s t e r o i d p l a y s a m a j o r d i r e c t role

Because

has

from d i r e c t h y d r o p h o b i c

in s t r o n g l y b i n d i n g

flexible

hormones.

the A r i n g , the D ring, and the 173 side c h a i n of p r o -

g e s t e r o n e are the p r i n c i p a l conformational

areas of m o l e c u l a r

t r a n s m i s s i o n c o u l d be p l a y i n g

role in p r o g e s t i n b i n d i n g as it does

flexibility,

as s i g n i f i c a n t

in c h e m i c a l

a

reactivity.

A l t h o u g h the f e a t u r e c o m m o n to all of the p r o g e s t i n s

in

Figure

F i g u r e 8. C o m p o s i t e d r a w i n g i l l u s t r a t i n g sites on the p r o g e s t e r o n e m o l e c u l e w h e n b i n d i n g is e n h a n c e d by e i t h e r subs t i t u t i o n (A) or s u b s t i t u e n t r e m o v a l (B). The c o m p l e m e n t a r i t y of fit b e t w e e n the s t e r o i d and the r e c e p t o r site c a n n o t be t i g h t over the a or 3 faces of the B, C, and D r i n g s .

13 7 is the 4 - e n e - 3 - o n e A r i n g , m a n y o t h e r s t e r o i d s h a v i n g structural

feature

the p r o g e s t e r o n e Close

(i.e., t e s t o s t e r o n e )

receptor

do not b i n d w e l l

m a t i o n of the m o s t p o t e n t p r o g e s t i n s steroids having a flexibility

reveals

4-ene-3-one A rings.

observed that

confor-

s e v e r a l of

The A r i n g s of

natural

4 - e n e - 3 - o n e c o m p o s i t i o n are o b s e r v e d to h a v e

ranging

from the la,26 half c h a i r

(Figure 9a) to the l a - s o f a c o n f o r m a t i o n

conformation

(Figure 9b)

(23).

s h o u l d be n o t e d that in e i t h e r case the 2 6 - h y d r o g e n a t o m in the a x i a l o r i e n t a t i o n p e r p e n d i c u l a r ring.

(24)

is

position

( 1 7 , 2 1 - d i m e t h y l - 1 9 - nor - 4 , 9 - p r e g n a d i e n e - 3 , 2 0 -

dione)

and o t h e r p o t e n t p r o g e s t i n s

change

in the A ring in w h i c h the 2 6 - h y d r o g e n takes up

equatorial

It

to the p l a n e of the A

A n a d d i t i o n a l d o u b l e b o n d at the C ( 9 ) - C ( 1 0 )

in R 5 0 2 0

to

(19) .

i n s p e c t i o n of the c r y s t a l l o g r a p h i c a l l y

them have unusual

this

orientation

b e e n c a l l e d the 1 3 , 2 a

induces a

(Figure 9c).

conformational

This c o n f o r m a t i o n

(inverted) h a l f c h a i r

an has

conformation.

R e m o v a l of the 1 9 - m e t h y l g r o u p , a f e a t u r e c o m m o n to m a n y the h i g h e s t a f f i n i t y b i n d e r s , has also b e e n s h o w n to

of

contrib-

ute to the d e s t a b i l i z a t i o n of the n o r m a l c o n f o r m a t i o n

in

f a v o r of the i n v e r t e d form

changes

at C(9)

a n d C(10)

(25) .

The c o n f i g u r a t i o n a l

in r e t r o p r o g e s t e r o n e

inversion

(26).

Finally,

6a-methyl

substitution

also

lead to A

ring

the c o m b i n a t i o n of 1 7 a - a c e t o x y

appears

to be r e s p o n s i b l e

for

and

in-

F i g u r e 9. Of 94 s t e r o i d s h a v i n g 4 - e n e - 3 - o n e c o m p o s i t i o n , the A r i n g s of 88 range b e t w e e n two c o n f o r m a t i o n s : (a) the la,26 h a l f - c h a i r a n d (b) .the la sofa. The u n u s u a l 16,2a (inverted) h a l f - c h a i r c o n f o r m a t i o n (c) is o b s e r v e d in six 4 - e n e - 3 - o n e s t r u c t u r e s and in ten 4 , 9 ( 1 0 ) - d i e n e - 3 - one s t r u c t u r e s for which X - r a y a n a l y s i s has b e e n c o m p l e t e d .

14

verting the A ring in medroxyprogesterone acetate

(MPA)

(27) ,

a synthetic steroid that binds to the human progestin receptor with twice the affinity of progesterone

(22).

The fact that

the 4-ene-3-one A ring is the only common feature of the progestins w i t h highest affinity for the uterine receptor and the fact that the unusual inverted A-ring conformation is common to many of them, has led us to propose that the high affinity of binding is due to a tight binding of the A ring to the receptor

(27)

inverted

(Figure 10).

A specific A-rijig conformation has also been proposed as a requirement for the activity of vitamin D (28) and the molting hormone ecdysone

(29).

A conformational

insect

requirement

for activity implies that this is also a requirement for binding since binding is required but not sufficient for activity.

In the case of androgens several

investigators

have concluded that the 178-hydroxylated D ring is the structural feature of primary importance to receptor binding 31,32).

Because a structure having a phenyl-pyrazole

(30,

moiety

Figure 10. The superposition of the nearly identical A rings of high-affinity binders (a) R5020, (b) R24S3, and (c) medroxyprogesterone acetate suggests a receptor site for progestins that provides intimate contact over the 3 face of atoms C-2 through C-6 and the a face of the conjugated system and far less specific binding on the remainder of the steroid.

15 f u s e d to the 2-3 p o s i t i o n of the b a s i c g l u c o c o r t i c o i d is 17 times m o r e e f f e c t i v e

than d e x a m e t h a s o n e

with dexamethasone

for g l u c o c o r t i c o i d

it seems u n l i k e l y

that there is a close

A ring a n d the g l u c o c o r t i c o i d

Agonists and Antagonists

in

nucleus

competing

receptor binding fit of the

receptor.

Antagonists of h o r m o n e a c t i o n m a y e x e r t their e f f e c t by

fering w i t h the s y n t h e s i s or m e t a b o l i s m of a s p e c i f i c or any of the p r o t e i n s

that are e s s e n t i a l

that s t e r o i d ' s p h y s i o l o g i c a l suggest

response.

that some a n t a g o n i s t s

the h o r m o n e b i n d i n g s h o u l d be p o s s i b l e responsible

steroid

to the e x p r e s s i o n of

act by d i r e c t c o m p e t i t i o n

for

features

to a c o m m o n site and w h i c h

it are

structural

responses.

is further c o m p l i c a t e d by u n c e r t a i n t y

to

In these c a s e s

to i d e n t i f y w h i c h s t r u c t u r a l

for b i n d i n g

inter-

T h e r e is e v i d e n c e

site on the r e c e p t o r .

f e a t u r e s c o n t r o l a g o n i s t and a n t a g o n i s t cess

(33) ,

steroid

This

pro-

as to w h e t h e r

re-

s p o n s e is due to the p r e s u m m e d a n t a g o n i s t or a m e t a b o l i t e the reo f. T a m o x i f e n and n a f o x i d i n e antiestrogenic

are p r e s u m e d to e x e r t m o s t of

a c t i v i t y as a r e s u l t of c o m p e t i t i v e b i n d i n g

the e s t r o g e n r e c e p t o r .

A l t h o u g h b o t h s t r u c t u r e s have

r i n g s , t a m o x i f e n has no h y d r o x y l s u b s t i t u e n t a n d has a m e t h o x y g r o u p . ascertain which, as an A - r i n g been

For this r e a s o n it is d i f f i c u l t

analogue

in b i n d i n g

to the r e c e p t o r .

tive s h o w n in F i g u r e

lib

(34).

(Figure

11c)

It has

of t a m o x i f e n in r a t ,

Similarly,

trans-tamoxifen

is m e t a b o l i z e d in vivo (35).

to

function

m o u s e , r h e s u s m o n k e y a n d dog is the m o n o h y d r o x y l a t e d has s h o w n that the a n t i e s t r o g e n U 2 3 , 4 6 9

to

phenyl

nafoxidine

if any, of the p h e n y l rings m i g h t

found that the p r i m a r y m e t a b o l i t e

nafoxidine)

their

deriva-

Katzenellenbogen

(an a n a l o g u e

to a p h e n o l i c

of

metabolite

These monohydroxylated metabolites

and U 2 3 ,469 have b e e n s h o w n to m a i n t a i n

of their

16

Figure 11. Chemical drawings of (a) n a f o x i d i n e and the m o n o h y d r o x y l a t e d m e t a b o l i t e s of the potent a n t i e s t r o g e n s , (b) trans-tamoxifen and (c) U 2 3 , 4 6 9 . a n t i e s t r o g e n i c character while having higher affinity for estrogen receptor

(35,36).

It is reasonable to suppose

the phenol ring of m o n o h y d r o x y l a t e d imposed on the A ring of estradiol in Figure 12.

tamoxifen can be in two ways as

the

that

super-

illustrated

In either o r i e n t a t i o n the absence of o x y g e n

Figure 12. C o m p a r i s o n of the c r y s t a l l o g r a p h i c a l l y o b s e r v e d c o n f o r m a t i o n and hydrogen bonding of estradiol w i t h that of tamoxifen. For purposes of the comparison, the A ring of estradiol is superimposed on the phenyl ring of tamoxifen that is h y d r o x y l a t e d in animal m e t a b o l i s m . The two p o s s i b l e superpositions of these rings are illustrated in (a) and (b). C o m m o n structural features are dark, agonist structure is shaded and antagonist is light.

substitution comparable

to 0(17)

for the i n a c t i v i t y / a n t a g o n i s m

(0(20)

in DES) m a y

of the c o m p o u n d .

account

In a d d i t i o n

to this

the a m i n o s u b s t i t u t e d ring m a y p r e s e n t s t e r i c

ference

to a m o l e c u l a r

binding

in the r e c e p t o r or g e n o m i c hormone action.

(i.e., c o n f o r m a t i o n a l

interaction)

two p o s s i b l e p h e n o l

(Figure

change

that is e s s e n t i a l

W h e n the c o n f o r m a t i o n of n a f o x i d i n e

c o m p a r e d to that of e s t r a d i o l

interto

(37)

is

13), only one of the

ring o r i e n t a t i o n s w i l l p e r m i t

maximum

o v e r l a p of the n a f o x i d i n e fused rings on the A and B r i n g s estradiol. appear

Once a g a i n the a n t i e s t r o g e n i c p r o p e r t i e s

to be a result of the a b s e n c e of a h v d r o g e n b o n d

in a p o s i t i o n a n a l o g o u s ference

in the D - r i n g

W h e n the s t r u c t u r e s ticoid

to 0 ( 1 7 )

on e s t r a d i o l

of

would donor

or s t e r i c

inter-

region.

of a l d o s t e r o n e

spironolactone

a n d the

are c o m p a r e d ,

antimineralocor-

the o n l y c o m m o n

is their 4 - e n e - 3 - o n e c o n f o r m a t i o n s .

There

ity in the s t r u c t u r e s

and c a n r e n o n e , a p r i n c i -

pal metabolite

of s p i r o n o l a c t o n e

corticoid activity. spironolactone

of a l d o s t e r o n e

is g r e a t e r

feature

that m a i n t a i n s

antimineralo-

The A and B rings of a l d o s t e r o n e

are s u p e r i m p o s e d

is s e e n to be w e l l b e l o w

in Figure

14.

similar-

and

The s p i r o

the plane of b o t h m o l e c u l e s .

ring

The

F i g u r e 15. C o m p a r i s o n of o b s e r v e d c o n f o r m a t i o n s and h y d r o g e n Common strucb o n d i n g p o t e n t i a l of e s t r a d i o l a n d n a f o x i d i n e . ture d a r k , a g o n i s t s h a d e d , a n t a g o n i st u n s h a d e d .

18

Figure 14. Comparison of the conformation and hydrogen bonding potential of aldosterone and the antimineralocorticoid canrenone. Common structure dark, agonist shaded, antagonist unshaded.

antagonist behavior of spironolactone and canrenone may be due in part to steric interference caused by the spiro ring placement but it is more likely to be due to the absence of hydrogen bond donating groups capable of mimicing the tion of the aldosterone 0(20) and 0(21) hydroxyl

func-

groups.

These groups on aldosterone can act as hydrogen bond donors and/or acceptors while

those on canrenone and

could only act as hydrogen bond

spironolactone

acceptors.

Dexamethasone oxetanone was found to have significant glucocorticoid activity

(38).

When

anti-

its X-ray crystal

ture is compared with the potent glucocorticoid

struc-

dexamethasone

the A, B, and C rings of the agonist and antagonist are

found

to have nearly identical conformations but the differences the D-ring are appreciable

(Figure IS).

in

In this case there

does not appear to be a steric impediment to receptor

inter-

19

Figure 15. Comparison of the conformation and hydrogen bonding potential of dexamethasone and the antiglucocorticoid dexamethasone oxetanone. Common structure dark, agonist shaded, antagonist unshaded. action since the bulk fit in the D-ring region is good.

The

primary difference between the structures is in the chemical character of the D-ring region of the molecule. obvious difference is in the hydrogen bonding of the D-ring substituents.

The most

capabilities

While both agonist and antagonist

can accept a hydrogen bond to 0(20) only the agonist can also donate two hydrogen bonds to effect or stabilize a receptor or macromolecular

interaction.

Cyproterone acetate

(CPA) and chlormadinone acetate

(CMA)

have identical chemical constitution except for a la, 2otmethylene bridge in CPA (Figure 16). gestins

Both are potent pro-

(39) but CPA is also a very potent antiandrogen

which binds to a dihydrotestosterone

(40)

(DMT) receptor in the

20

Figure

16.

Chemical

rat v e n t r a l p r o s t a t e

f o r m u l a for (41).

(a) CPA a n d

(b)

CMA.

The u n i q u e b i n d i n g p r o p e r t i e s

C P A m u s t be due to the e f f e c t of the m e t h y l e n e b r i d g e . prerequisites

for e f f e c t i v e b i n d i n g

to the A - r i n g

DHT site are not c l e a r

from the c o m p a r i s o n

DHT

However,

(43)

(Figure

participation

17).

of C P A

the r e q u i r e m e n t

the

(42)

and

of D - r i n g

in the e x p r e s s i o n of s t e r o i d h o r m o n e

w o u l d e x p l a i n the p r o g e s t a t i o n a l

at

activity

a c t i v i t y of C P A and CMA

the lack of a n d r o g e n a g o n i s t a c t i v i t y

of

The

and

in C P A .

Summary We h a v e p r o p o s e d that the s t e r o i d A ring sible

for i n i t i a t i n g

and m a i n t a i n i n g

e s t r o g e n and p r o g e s t i n r e c e p t o r s . agonists

and a n t a g o n i s t s

is p r i m a r i l y

hormone binding

W h e n the s t r u c t u r e s

of s p e c i f i c s t e r o i d h o r m o n e s

c o m p a r e d , they g e n e r a l l y e x h i b i t s i m i l a r i t i e s region and dissimilarities gesting

for r e c e p t o r b i n d i n g w h i l e activity. receptor

"dimer",

the r e c e p t o r , or (15,26).

(a) i n f l u e n c i n g

(b) i n d u c i n g

sug-

expression

in g e n o m i c

of

rings

the s t a b i l i t y of

a conformational

that c o m p e t e

A-ring

responsibility

the D - r i n g c o n t r o l s

(c) p a r t i c i p a t i n g

Antihormones

are

in t h e i r

The p o s s i b l e m e a n s by w h i c h the s t e r o i d D include

the of

in the D - r i n g r e g i o n f u r t h e r

that the s t e r o i d A - r i n g b e a r s p r i m a r y

control activity

responto

change

the

in

interaction

for the r e c e p t o r

a s t e r o i d h o r m o n e m a y be e x p e c t e d to h a v e s t r u c t u r a l

site

of

features

21

l i g u r e 17. C o m p a r i s o n of the c o n f o r m a t i o n and h y d r o g e n b o n d i n g p o t e n t i a l of d i h y d r o t e s t o s t e r o n e and the a n t i a n d r o g e n cyproterone acetate. Common structure dark, agonist shaded, antagonist unshaded. appropriate formation)

for r e c e p t o r b i n d i n g and lack s t r u c t u r a l

lize s u b s e q u e n t features a n d

receptor

functional

(A-ring c o m p o s i t i o n a n d

features

functions

that induce or

(D-ring

con-

stabi-

conformational

groups).

Acknowledgements R e s e a r c h s u p p o r t e d in part by N I A M D D G r a n t No. A M - 2 6 5 4 6 , G r a n t No. L M - 0 2 5 5 5 a n d DRR G r a n t No. R R - 0 S 7 1 6 . w i t h to e x p r e s s

their a p p r e c i a t i o n

The

to M r s . Q u e e n i e

M i s s G l o r i a Del Bel, M r s . C a t h e r i n e D e V i n e , M r s .

authors Bright,

Jean

Gallmeyer, Mrs. Brenda Giacchi, Miss Deanna Hefner,

NLM

Mrs.

22 Kathleen McCormick, Miss Phyllis for a s s i s t a n c e manuscript.

Strong

and M i s s M e l d a

in the o r g a n i z a t i o n and p r e p a r a t i o n of

A l l f i g u r e s e x c e p t 1 a n d 7 w e r e d r a w n on

an N I H s p o n s o r e d b i o m e d i c a l

computer

Tugac this PROPHET,

network.

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K e a s l i n g , H. H., S c h u e l e r , 39, 87-90 (1950).

F. W.:

J. A m e r . P h a r m .

2.

H o s p i t a l , M . , B u s e t t a , B., C o u r s e i l l e , C., P r e c i g o u x , J. S t e r o i d B i o c h e m . 6, 2 2 1 - 2 2 5 (1975).

3.

B u s e t t a , B., H o s p i t a l , M.: 567 (1972).

4.

B u s e t t a , B., C o u r s e i l l e , C., G e o f f r e , S., H o s p i t a l , M. : A c t a C r y s t a l l o g r . B2_8, 1349- 1351 (1972).

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D u a x , W. L.,: A c t a C r y s t a l l o g r .

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7.

D u a x , W. L., S m i t h , G. D . , S w e n s o n , D. C . , S t r o n g , P. W e e k s , C. M . , A n a n c h e n k o , S. N., E g o r o v a , V. V.: J. S t e r o i d B i o c h e m . U , 1-7 (1981).

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S e g a l o f f , A . , G a b b a r d , R. B . F l o r e s , A . , B o r n e , R. F., B a k e r , J. K., D u a x , W. L., S t r o n g , P. D. , R o h r e r , D. C . : S t e r o i d s 35, 1 3 3 5 - 1 3 4 9 (1980).

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P a t h r e , S. V . , M i r o c h a , C. J.: in " E s t r o g e n s in the E n v i r o n m e n t " , J. A. M c L a c h l a n (Ed.), E l s e v i e r / N o r t h H o l l a n d , New Y o r k , 265-279 (1980). Acta

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T a y l o r , I. F., Jr., W a t s o n , W. H.: B 3 2 , 710-714 (1976).

11.

G r i f f i n , J. F., D u a x , W. L., S t r o n g , P. D.: Amer. Cryst. Assoc. Meeting, College Station, Texas, Abst. No. PB18, 35 (1981).

12.

M u e l l e r , G., Kim, U . - H . : (1978).

13.

Z e e l e n , F J.: Bological Activity and Chemical tures, E l s e v i e r S c i e n t i f i c , A m s t e r d a m , 1 4 7 - 1 6 0

14.

Lee, D. L., K o l l m a n , P. A . , M a r s h , F. H . , W o l f f , M. J. Med. C h e m . 20, 1 1 3 9 - 1 1 4 9 (1977).

15.

D u a x , W. L., W e e k s , C. M.: in " E s t r o g e n s in the E n v i r o n m e n t " , J. A. M c L a c h l a n (Ed.), E l s e v i e r / N o r t h - H o l l a n d , New Y o r k , 11-31 (1980).

Endocrinology

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102,

1429-1435 Struc(1977). E.:

23 16.

C h e r n a y a e v , G. A . , B a r k o v a , T. I., E g o r o v a , V. V . , S o r o k i n a , I. B., A n a n c h e n k o , S. N . , M a t a r o d z e , G. D . , S o k o l o v a , N. A . , R o z e n , V. B.: J. S t e r o i d B i o c h e m . 6, 1 4 8 3 - 1 4 8 8 (1975).

17.

B a r t o n , D. H. R.:

18.

W e i c h m a n , B. M . , N o t i d e s , A. C.: 439 (1980).

19.

S m i t h , II. E., S m i t h , R. G . , T o f t , D. 0 . , N e e r g a a r d , J. R., B u r r o w s , E. P., O ' M a l l e y , B. W.: J. B i o l . C h e m . 249 , 5 9 2 4 - 5 9 3 2 (1974).

20.

R a y n a u d , J. P., P h i l i b e r t , D . , A z a d i a n - B o u l a n g e r , G.: in " P h y s i o l o g y and G e n e t i c s of R e p r o d u c t i o n " , E. C o u l t i n k a and F. F u c h s (lid.), P l e n u m P r e s s , N e w Y o r k , 1 4 3 - 1 6 0 (1973) .

21.

T e r e n i u s , L.:

22.

K o n t u l a , K., J a n n e , 0 . , V i h k o , R., de J a g e r , E . , de V i s s e r , .J., Z e e l e n , F.: A c t a E n d o c r i n o l . 7_8, 5 7 4 - 5 9 2 (1975) .

23.

D u a x , W. L., W e e k s , C. M . , R o h r e r , D. C.: in " T o p i c s in S t e r e o c h e m i s t r y " , E. L. E l i e l and N. A l l i n g e r (Eds.), Wiley I n t e r s c i c n c e , New Y o r k , 9, 2 7 1 - 3 8 3 (1976).

24.

B u s e t t a , B., Coniberton, G., C o u r s e i l l e , C . , H o s p i t a l , A c t a C r y s t a l l o g r . B30, 2 7 5 9 - 2 7 6 1 (1974).

25.

P r e c i g o u x , P. G., B u s e t t a , B., C o u r s e i l l e , C . , M.: A c t a C r y s t a l l o g r . B 3 1 , 1 5 2 7 - 1 5 3 2 (1975).

26.

B r a u n , P. B., Ilornstra, Ii., L e e n h o u t s , J. I.: Res. Rep. 24, 4 4 5 - 4 4 9 (1969).

27.

D u a x , IV. L., C o d y , V . , G r i f f i n , J. F., R o h r e r , D. C . , W e e k s , C. M . : .J. T o x i c o l . E n v i r o n . H e a l t h 4 , 2 0 4 - 2 2 7 (1978) .

28.

O k a m u r a , W. II., N o r m a n , A. W . , W i n » , R. M . : A c a d . Sei. USA 7^, 4 1 9 4 - 4 1 9 7 (1974).

29.

lihright, R. A.:

30.

L i a o , S., L i a n g , T . , Fang, S., C a s t a ñ e d a , E . , S h a o , T. C.: J. Biol. C h e m . 2£8 ,' 6154 - 6162 (1973).

31.

L o b l , T. J . , C a m p e l l , J. A . , T i n d a l l , D. J . , C u n n i n g h a m , G. R., M e a n s , A . R.: in " T e s t i c u l a r D e v e l o p m e n t , S t r u c ture a n d F u n c t i o n " , A. S t e i n b e r g e r and E. S t e i n b e r g e r (Eds.), R a v e n P r e s s , New Y o r k , 323-330 (1980).

32.

S c h m i t , J . - P . , Q u i v y , J. I., R o u s s e a u , G. G.: o i d B i o c h e m . , 1^5, 1 3 8 7 - 1 3 9 4 (1980).

33.

H a r m o n , J. M . , S c h m i d t , T. J . , T h o m p s o n , E. C.: S t e r o i d B i o c h e m . 14_, 2 7 3 - 2 7 9 (1981).

Experientia Suppl.

Endocrinology

Steroids ^3, 909-918

Private

II, 121-137

(1955).

106,

434-

(1974).

M.:

Hospital, Phillips

Proc.

Natl.

communication.

J.

SterJ.

24

34.

F r o m s o n , J. M . , P e a r s o n , S., B r a m a k X e n o b i o t i c a 3, 693-709 (1973).

35.

H a y e s , J. R., R o r k e , 1:. A . , R o b e r t s o n , D. W . , K a t z e n e l l e n b o g e n , B. S., K a t z e n e l l e n b o g e n , J. A . : Endocrin o l o g y 10_8, 164-172 (1981).

36.

B i n a r t , N . , C a t e l l i , M. G . , G e y n e t , C . , P u r i , V . , H a h n e l , R., M e s t e r , J . , B a u l i e u , E. E.: Biochem. Biophys. Res. C o m m . 9_1 , 812- 818 (1979) .

37.

C a m e r m a n , N . , C h a n . L. Y. Y . , C a m e r m a n , A . : C h e m . 2_3 , 9 4 1 - 9 4 5 (1980) .

38.

D u a x , W. L., S i m o n s , S. S.:

39.

B r e n n a n , D. M . , K r o a y , R. J.: 367-379 (1963).

40.

H a m a d a , H . , N e u m a n n , F., J u n k m a n n , C o p e n h . , £ 4 , 3 8 0 - 3 8 8 (1963).

41.

J o h a n s s o n , R.:

42.

C h a n d r o s s , R. J . , B o r d n e r , J.: 1 5 8 1 - 1 5 8 5 (1974).

43.

C o u r s e i l l e , C., P r e c i g o u x , G., L e r o y , I:. , B u s e t t a , C r y s t . S t r u c t . C o m m . 3, 4 4 1 - 4 4 6 (1973).

Acta Endocr.

(ne6 M e e k ) ,

Unpublished

S.:

J. M e d .

data.

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Acta

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(1976).

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44,

A N T I H O R M O N A L A C T I V I T I E S O F S T E R O I D S W I T H M O D I F I E D RINGS A O R B

L u b o S S t ^ r k a , R i c h a r d Harapl Research Institute of Endocrinology CS-116 94 Praha, Czechoslovakia

Introduction T h e s e a r c h for h o r m o n a l a n d a n t i h o r m o n a l a g e n t s led to the investigation of steroid compounds with modified

cvclopenta-

n o p e r h y d r o p h e n a n t h r e n e n u c l e u s . E s p e c i a l l y a m o n g the

antian-

d r o g e n s r e p r e s e n t a t i v e s of these t y p e s o f s t r u c t u r e are the m o s t p o t e n t d r u g s . A n t i a n d r o g e n i c a c t i o n of

A-norprogesterone

(1,2) h a s b e e n u s e d for the h o r m o n a l t h e r a p y o f a c n e , mo-17B-hydroxy-17a-nethyl-4-oxa-5a-androstan-3-one

6a-bro-

(BOMT) e x -

e r t s its a n t i h o r m o n a l a c t i o n in all a n d r o g e n - d e p e n d e n t w i t h the e x c e p t i o n of the c e n t r a l n e r v o u s s y s t e m -methyl-3-nortestosterone

tissues

(3,4),

17a-

r e p r e s e n t s o n e o f the e a r l i e s t c l i -

nically useful antiandrogens

(5,6), a n d

6a-chloro-17o-hydroxy-

-la,2ot-methylene-4 , 6 - p r e g n a d i e n e - 3 ,20-dione 1 7 - a c e t a t e , t e r o n e a c e t a t e is the c l i n i c a l l y a n d c o m m e r c i a l l y m o s t sful a n t i a n d r o g e n

cyprosucces-

(7-11).

In spite of these facts the s y s t e m a t i c i n v e s t i g a t i o n s o f the effects of steroid nucleus modifications on the hormonal

and

a n t i h o r m o n a l a c t i v i t y is m i s s i n g a n d the i n f o r m a t i o n o n b i o c h e m i c a l p r o p e r t i e s c o n c e r n i n g the b i o l o g i c a l a c t i v i t y are

scan-

ty. In t h i s p a p e r w e t r y to g a t h e r i n f o r m a t i o n o n t h e

influence

o f ring A o r B m o d i f i c a t i o n s , e s p e c i a l l y o f the e x p a n s i o n

and

the c o n t r a c t i o n of the rings o r t h e i r c l e a v a g e , o n the b i n d i n g p r o p e r t i e s to the r e c e p t o r s a n d c o n s e q u e n t l y o n t h e b i o l o g i c a l a c t i v i t y o f some

steroids.

© 1982 Walter de Gruyter & Co., Berlin • New York Hormone Antagonists, Editor M. K. Agarwal

26

Heteroatom in Ring A Ring A oxa-steroids 6a-Bromo-17 6-hydroxy-17a-raethyl-4-oxa-5a-androstan-3-one (BOMT) selectively suppresses the nuclear binding of dihydrotestosterone in androgen dependent tissues in vitro. In addition BOMT competes effectively for the specific, high affinity binding sites for dihydrotestosterone in cytoplasm of the rat prostate gland and reduces the transfer of dihydrotestosterone into chromatin in a reconstituted, cell-free system. BOMT also antagonises the stimulation of RNA polymerase activity in the prostate gland after the administration of testosterone in vivo. Conversly, BOMT had no effect on the rate of formation of dihydrotestosterone in androgen-dependent tissues. On the basis of binding studies it was suggested that the antiandrogenic properties of BOMT are best explained by its competition for dihydrotestosterone-binding sites. The relative binding to these sites was 51% at a 50-fold BOMT excess which is close to the value for cyproterone acetate 44% (3). The high competing efficiency for androgen receptors in accessory sexual glands contrasts with the low affinity of BOMT for the central nervous system (4). Among the steroids with high inhibitory effects on the bin3 ding of H-dihvdrotestosterone in dialysed rat prostate cytosol and in the purified nuclear component of the prostate cells were 17B-hydroxy-2-oxa-4,9-estradien-3-one and 17e-hydroxy-17o-methyl-2-oxa-4,9-estradien-3-one (12). The relative inhibition of dihydrotestosterone binding was 90.5% and 88.1% resp. in cytosol (cyproterone acetate 77.1%) and 9 4.4% and 93.5%, resp., in nuclear fraction (cyproterone acetate 80.2%). The steroids that achieved the highest degree of inhibition were those compounds which exhibited a general planar geometric shape. They showed also a distinct androgenic activity (12).

Similar to progesterone in respect to binding to glucocorti-

27

cold cytosolic receptors in hepatoma tissue culture cells was l78-hydroxy-17a-methyl-2-oxa-4,9,ll-estratrien-3-one (13). This compound as well as progesterone have decreasing relative binding activities on increasing incubation time and seem to have both antiglucocorticoid and antimineralocorticoid properties. They are also able to counteract the induction of tyrosinaminotransferase by potent glucocorticoids in hepatoma cell culture. Selenasteroids For 2-selena-A-nor-5a-androstan-17B-ol the ability to bind selectively with the specific receptors of 5a-dihydrotestosterone in all androgen dependent tissues in the cytosol and to be retained in the nuclei in an unaltered form was demonstrated (14).

Norsteroids A-Norsteroids According to the observations of Raynaud et al.(l3) a certain class of antiestrogens appears to exert their antagonistic activity by virtue of the fast dissociation of complex they form with the estrogen receptor compared to that of the natural hormone. The relative binding activity of such compounds is high but it declines with prolonged incubation. One of the compounds of this type was shown to be 2-ethynyl-5a-A-norestrane-2,176-diol (RU 26143) which forms fast-dissociating complexes with estrogen receptors, similarly as cyproterone acetate does with the androgen receptors. Antifertility effectiveness in adult cycling female baboons was investigated using 2a,l7a-dlethynyl-A-nor-5a-androstane-28,17B-diol (Anordrin) and 2a,17a-diethynyl-A-nor-5a-estrane-

28

-2g,17g-diol (Dinordrin I) or its 2 g-ethynyl-isomer (DInordrin II). Dinordrin I was the most potent compound of these in rat uterotrophic activity bioassay and it was parallel with antifertility effect (15). B-Norsteroids B-Noranalogues of testosterone, dehydroepiandrosterone, 17a-methyltestosterone, 4-androstene-3,17-dione, 5-androstene-3g, 17 6-diol, 3a-hydroxy-5a-androstan-17-one and 17e-hydroxy-5a-androstan-3-one were either extremely weak androgens in chick comb test or fully inactive in biological assay. These findings indicate a considerable decrease in androgenicity as a result of elimination of one carbon atom from ring B. However, the B-nor form of 17a-methyltestosterone was active as antiandrogen and marginally active antiandrogens were free B-nor-deoxycorticosterone and its acetate (16). Biological test for antiestrogenic activity was positive in a series of B-norsteroidss 3g-hydroxy-B-nor-5-pregnen-20-one, B-norprogesterone, 17a-ethenyl-B-nortestosterone,B-nor-deoxycorticosterone and 3b,21-dihydroxy-B-nor-5-pregnen-20-one (16). Potential hormonal activity of various B-norsteroids, analogues of corticosteroids, estrogens and androgens, in terms of displacement ability of appropriate radioligand from receptor binding was searched (17). The binding properties were compared with parent hormones with normal steroid nucleus. Glucocorticoids B-norcortisol and B-nor-11-deoxycortisol, if compared in terms of their binding to rat liver cytosol, show a decrease in binding ability of approximately three orders of magnitude relative to corticoids without contraction of ring B. The binding of llg-hydroxy- and 11-oxo-B-norprogesterone was quite negligible in contrast with a relatively high binding of 11(3-hydroxyproges terone. Similar situation was found for estrone and estradiol-170 and their noranalogues. In 3-norestrogens a lower ability to

29

Fig.l

Structures of some steroids with modified ring A or B

m M

n

K

i 0

OH

M XI

m

M OH

YTTT

XX

XXI

YM

xxm XXIV

XXz

31

displace ^l-estradiol-17$ from uterine cytosol receptors was found than in C,„-estrogens (17). 1o Only 17a-methyl-B-nortestosterone was bound in an extent comparable to the binding of 17a-methy1testosterone to rat prostate cytosol (17) which is in agreement with its distinct antiandrogenic activity (5,6). Five 4a,5-cyclo-A-homo-B-nor-androstane derivatives (Fig.l, Table 1, compounds I-V) were assayed in vivo on mice for their anti- resp. syn-androgenic activity and the effect was compared with that of cyproterone acetate. The inhibition of dihydrotestosterone binding to rat prostate cytosol by the compound correlated with the in vivo effects. The antiandrogenic activity of 17B-acetoxy-4a,5-cyclo-A-homo-B-nor-5a-androst-l-en-3-one was found to be comparable with that of cyproterone acetate. It was also antirenotrophic but unlike cyproterone acetate it was lacking antianabolic activity and corticoid-like action on relative weight of spleen. A lower antiandrogenic activity was found for l7f5-hydroxy-17a-methyl-4a,5-cyclo-A-homo-B-nor-5a-androst-l-en-3-one, the remaining three compounds (I,II,II) with saturated ring A were only very weak competitors for dihydrotestosterone binding in prostate and for testosterone action in vivo in accessory sex organs (18).

A- or B-homosteroids In contrast to several extensive investigations on D-homoestrogens and their potential hormonal and binding activities (19-21) with respect to their conformation, there is only one report (22) on a series of A-homo- and 3-hono-androstane and pregnane derivatives (Fig.l, Table 1., compounds X-XIX). The steroids with expanded rings A or B were tested for potential synandrogenic or antiandrogenic activity by binding assay on rat prostate cytosol receptors. The binding to sex hormone binding 6-globulin and the inhibition of 5a-reductase was maesured, too. The results of the in vitro screening

TABLE 1 THE CORRELATION OF HORMONAL AND ANTIHORMONAL BIOLOGICAL ACTIVITY WITH THE BINDING PROPERTIES OF STEROIDS WITH MODIFIED RINGS A OR B

Compound

Relative binding to rat prostate cytosol %

a)

Relative binding to Te3G

b)

Inhibition of 5 a-reductase

%

c)

Biological activity

d)

%

I II III IV V

0.1 no no 75.6 3.46

VI VII VIII IX X

3.98 12.0 1.47 1.10 no

MR2 < GR < MR^ which could be resolved only partially due to superimposition of various peaks. This ruled out competition between steroids for the same vector and further established that an antagonist R-5020 may bind to one subspecies of the MR while leaving another subspecies intact for concurrent attachment of an agonist aldosterone. Receptor separation based both on molecular weight and charge was attempted on Sephadex-DEAE-A-25 columns. Data in Fig. 7a

reveal 4 peaks of radioactivity when aldosterone was used

to label renal cytosol but only three were present with pro14 gesterone under these conditions (7b). Serum bound C-progesterone coeluted as a single peak (Fig. 7b). Thus, MR agonist and agonist inhibition may proceed through a difference in the relative abundance of the various components that constitute MR. It has previously been pointed out that KC1, required for elution from A-25 resin, leads to aggregation of MR in low salt concentrations and disaggregation as the ion strength is increased (2). Nevertheless, MR multiplicity was a function of the steroid (aldosterone vs progesterone in the kidney) as well as of the tissue since progesterone does not label the same entities in the kidney (Fig. 7b) vs heart (not shown).

325

Fig. 7. Fractionation of renal MR on Sephadex A-25 columns. 4 ml renal cytosol was incubated with either -^-aldosterone or 3h- progesterone, both 10"^ M, for figures a and b respectively. For 7b, 2 ml serum was incubated with progesterone. Cochromatography was performed as in the text and described earlier (3). It is to be noted that KC1 gradients were used in these experiments, in place of the Na phosphate buffer usually employed with DE-52 resin.

326 Molecular Filtration of the Renal Mineralocorticoid Receptor. Data in Fig. 8a show two peaks of bound aldosterone in the 67,000 and 122,000 ranges. Under these conditions, bound progesterone could be resolved into four peaks in the kidney (Fig. 8b) but only three could be observed in the heart (not shown here; see 3). The profile was still different when R-5020 was used in place of progesterone bound progesterone eluted

(Fig. 8c). Serum

primarily as a single peak

(Fig.

8b) and this is also true of serum corticosterone binders (6). These further support the argument that renal MR is very heterogeneous. The relative abundance of these multiple peaks is indeed required for the expression of agonist or antagonist activity, depending upon the steroid and the tissue. This view is different from the classical stand that the receptor protein is a single, homogeneous, unitary entity capable of accepting all agonists and antagonists, albeit with different affinities. A word may be added as to the choice of the resin. As explained in this chapter, DEAE-cellulose-52 seems to give the best resolution when used with 0.001-0.2 M phosphate gradient. The resolution deteriorates rapidly with NaCl in place of PO4 and is the worst with KC1. High salt required with Sephadex A-25, therefore, hinders receptor separation

(Fig. 7). Since

the various MR subspecies appear to have similar molecular weights, Ultrogel columns, too, are of limited value (Fig. 8). These results with MR are supported by our studies on liver GR.

A future challenge would therefore reside in the identi-

fication of steroids that selectively bind a given subpopulation of the MR or the GR. Studies with animal models in vitro are conducted in the hope that they have physiological relevance in vivo. What is the relationship between receptor activity, as described here, and physiological status of the host in vivo? This is rather difficult to assess at the moment given the fact that a good model of hormone activity in vitro is currently unavailable. We therefore turned our attention to patients where unilateral

nephrectomy produced a clinically defined diseased tissue. It was hoped that the relative abundance of diverse MR subspecies could be related to a given pathology. As controls were taken normal part of the diseased human kidney as well as rat kidney as described above.

328 C

K

¿00 200

E ¿0 b * 20 u

k • f l ^ r t

FRACTION NUMBER

50

TOO

0

Fig. 8. Molecular filtration of kidney MR on Ultrogel ACA-44. 3 2 ml renal cytosol was incubated with either H-aldoste3 rone (a), 3H-progesterone (b), or H-R-5020 (c), all at 10~7 M. For 8b, 1 ml serum was incubated with 14 C-corticosterone and charcoal treated separately in order to perform double labelled cochromatography. Further details are given in the text and have been published (3). Analysis of the Mineralocorticoid Receptor in Human Kidney. Data in Fig. 9 show that in the normal tissue, associated with the pathological part of the human kidney, aldosterone —8 binding proteins were saturated by 10 M aldosterone, in keeping with KD (37°C) of 5 x 10~ 1 0 M for rat MR. Even at -7 10 M, tissue from patients with nephrolithiasis, hydronephrosis, and one adenocarcinoma, did not show as —8much bound aldosterone as the normal tissue with only 10 M. This suggests a correlation between cortical atrophy and lack of hormone receptor. In two neoplasms, however, normal levels of bound steroid were observed with 10 ^ M aldosterone alth—8 ough the quantity bound with 10 M corticoid was far less than in the non-diseased part of the same kidney. Histological examinations did not reveal morphological differences between the three types of neoplasms. It is not clear, therefore, why 10 fold greater concentrations were required to maximally saturate MR in two neoplastic renal tissues.

329

C

1CT9 5x10"91CF8

5x10~8107?

CONCENTRATION

(M)

Fig. 9. Saturation characterstics of human renal MR. Normal kidney pool (O 0), lithiasis (0), hydronephrosis (X), and three different adenocarcinoma (•, A, A) were studied with different concentrations of ^H-aldosterone and the quantity bound calculated per rrg protein. For other details see text and (17, 18). Data in Fig. 10

show that progesterone was more effective

in revealing the MR^ and the MR^ components in human kidney than

the natural steroid - aldosterone. This is surprising

when compared to studies on rat kidney where aldosterone reveals MR£ that is apparently absent in the human kidney. The possibility can not be ruled out, however, that MR2 was inactivated during surgery and subsequent freezing of human kidney. As in the rat, MR^ eluted with serum bound corticosterone or progesterone

(Figs. 10a and 10b, respectively).

The difference between human vs rat kidney was also noted with R-5020. Whereas rat kidney binds great quantities of this progestagen, human kidney does not bind at all (not shown here). Interspecific extrapolations would therefore require great caution. Pathological human kidney did not show any new MR peaks and both MR^ and MR^ could be observed as in the normal portion of human kidney

(17, 18).

330

FRACTION NUMBER Fig. 10. Resolution of human kidney MR on DEAE-52 columns. Renal cytosol (normal pool) was equilibrated with 10~7 M of either 3n-aldosterone (a) or 3n-progesterone (b). 2 ml human serum was incubated with the 14C-isotope of the corresponding steroid for cochromatography. For further details see text and (17,18).

331 Persepectives It seems clear from the foregoing that multiplicity appears to be an inherent property of MR and of receptors for other classes of steroid hormones (3). Future challenge would require identification of molecules exhibiting specificity for a defined chromatographic peak. In this manner one can expect to trigger a

desired

agonist or antagonist action

a lot more precisely than is hitherto attempted with all or none techniques based entirely on binding studies. What is the chemical explanation of this sort of multiplicity? One can either visualize different proteins, for the various agonists and antagonists of a given class of hormone, or one

single protein with either one or multiple binding

sites. The former can not be supported in view of the fact that the organism does not come in contact with synthetic steroids either during ontogeny or phylogeny. As a corollary of this hypothesis it would have to be assumed that cell cytoplasm already contains receptors for all conceivable analogs of any given hormone class. Certainly this would represent useless redundance of genetic information at a great cost to the host. Furthermore, one single mutation in cultured cells in vitro is known to alter receptivity for all analogs of the hormone in question (2). Post-transcriptional modification of proteins is a well known phenomenon. As examples can be cited the genesis of hormones or enzymes from propeptides (19). In earlier studies it has been established that endogenous proteolysis does not appear to be responsible for the apparent multiplicity of the receptor protein (20-22). Thus, one is left with the alternative of the Multipolar model presented earlier (2,21). In its simplest form this model envisions an uncommitted, nascent proreceptor protein in the cytoplasm or on the ribosomes. The binding site(s) for the ligand would then be endowed with a flexible stereospecificity. As soon as the steroid

332

interacts with the vector, a definite, fixed, three-dimensional conformation would result. The nature of the hormone would dictate the eventual charge density of the receptor thus formed from a precursor and thus also its elution pattern from the ion-exchange resins. One of the most irksome problems in recepterology today is the fact that the receptor can only be quantified after it has interacted with the steroid in vitro. Thus, the in vivo nature of the receptor, prior to its occupation by the hormone, remains in dark. The high lability of MR further undermines efforts for detailed characterization in vitro. Purification of the receptor protein would obviously be of great help in resolving these and other related problems. Acknowledgements Over the past years the experiments reviewed in this chapter have been aided by grants from: Institut National de la Santé et de la Recherche Médicale, Délégation Générale à la Recherche Scientifique et Téchnique, Centre National de la Recherche Scientifique, and UER Broussais Hôtel Dieu. Some financial assistance was also provided by pharmaceutical industry. Some of the radioactive steroids were procured from Roussel UCLAF since they are not available commercially. Drs. G. Lazar (Hungary) and S. Sekiya benefitted from the INSERM Post-doctoral fellowships which allowed them various periods of time in this laboratory in Paris. Dr.

J. Paillard

is a Research Associate supported by the Department of Physiology of this University. Dr. Baviera has provided much help with histological analysis. Surgical specimens were obtained through the courtesy of Service of Surgery, Hôpital Broussais and Hôpital St. Joseph, Paris. Administrative help was kindly extended by Pr. J. Baillet of the Department of Physiology. For technical assistance and some other experiments, thanks are due to Ms. M. Philippe, F. Coupry, D. Blondel.

333 References 1. Landau, R.: Progesterone vs Aldosterone. In: "Antihormone^' Ed. Agarwal, M. K., Elsevier/North Holland, pp 153-166 (1979). 2. Agarwal, M.K.: Physical Characterisation of Cytoplasmic Gluco- and Mineralo- Steroid Receptors, FEBS Letters 85, 1-8 (1978). 3. Agarwal, M.K.: Paradoxical Nature of Mineralocorticoid Receptor Antagonism by Progestins, Biocehm. Biophys. Res. Comm. 8j3: 77-84 (1979) . 4. Agarwal, M.K.: Ed. Multiple Molecular Forms of Steroid Hormone Receptors, Elsevier/North Holland, (1977). 5. Agarwal, M.K.: Chromatographic Demonstration of Mineralocorticoid-specific Receptors in Rat Kidney, Nature 254, 623-625 (1975). 6. Agarwal, M.K.: Identification and Properties of Renal Mineralocorticoid Receptors in Relation to Glucocorticoid Binders in Rat Liver and Kidney, Biochem. J., 154, 567575 (1976). 7. Agarwal, M.K.: Differential Binding to Renal Receptor Subpopulations as an Explanation of Mineralocorticoid Agonist Action, FEBS Letters 67, 260-263 (1976). 8. Agarwal, M.K., Coupry, F., Philippe, M.: Physiological Activity and Receptor Binding of 9-a-fluorohydrocortisone, Biochem. Biophys. Res. Comm. 78, 747-753 (1977). 9. Agarwal, M.K.: Chromatographic Conditions in the Expression of Corticosteroid Receptor Specificity, Experientia 32, 531-532 (1976). 10. Agarwal, M.K.: Demonstration of Steroid Specific Hormone Receptors by Chromatography, FEBS Letters 6^, 25-29 (1976). 11. Agarwal, M.K.: Further Characterization and Subunit Nature of Mineralo- and Gluco- Steroid Receptors from Rat Liver and Kidney, Int. J. Biochem. 8, 877-881 (1977). 12. Agarwal, M.K.: Analysis of Rat Serum Transcortin-Steroid Hormone Association by Column Chromatography, Arch. Biochem. & Biophys. 180, 140-145 (1977). 13. Agarwal, M.K., Berger, L.: Polymorphism of Blood Serum Transcortin during Column Chromatography, Int. J. Biolog. Macromolecules _1, 211-214 (1979). 14. Agarwal, M.K., Philippe, M: Physical Characterization of Corticosteroid Binders in Adult Rat Heart, J. Molecular & Cellular Cardiol. 11, 115-126 (1979).

334

15. Wambach, G. Higgins, J.R.: Antimineralocorticoid action of progesterone. In "Antihormones" Ed. Agarwal, M.K., Elsevier/North Holland, pp 167-180 (1979). 16. Agarwal, M.K., Paillard, J., Philippe, M.: Evidence for an unusual, multifunctional protoreceptor in hormone action, Experientia 3j>, 1010-1012 (1980) . 17. Paillard, J., Baviera, E., Agarwal, M.K.: Physicochemical analysis of gluco- and mineralo- corticoid receptors from human liver and kidney. In "Hormones in Normal and Abnormal Human Tissues", Ed. Fotherby, K. and Pal, S.P., Walter de Gruyter, pp 145-166 (1981). 18. Paillard, J., Baviera, E., Agarwal, M.K.: Gluco- and mineralo- corticoid receptors in human liver and kidney, Biochem. Med. 24, 201-209 (1980) . 19. Hales, C.N., Docerty, K.: Lysosomal proteases and polypeptides: evolutionary precursors of polypeptide hormones? In "Proteases and Hormones" Ed. Agarwal, M.K., Elsevier/ North Holland, pp 19-46 (1979) . 20. Agarwal, M.K., Philippe, M.: Can gluco- and mineralocorticoid hormone receptor multiplicity be an expression of limited proteolysis? In "Proteases and Hormones" Ed. Agarwal, M.K., Elsevier/North Holland, pp 93-118 (1979). 21. Agarwal, M.K., proteolysis on "Proteases and North Holland,

Paillard, J.: The influence of partial sex steroid binders in rat liver. In Hormones" Ed. Agarwal, M.K., Elsevier/ pp 119-140 (1979) .

22. Agarwal, M.K., Philippe, M.: The influence of various proteases and inhibitors on steroid hormone receptors in rat liver and kidney, Biochem. Med. (in press).

CLINICAL PHARMACOLOGY AND THERAPEUTIC USE OF ALDOSTERONE ANTAGONISTS

Lawrence E. Ramsay Department of Therapeutics, Royal Hallamshire Hospital, Sheffield, U.K.

Abstract

Spironolactone undergoes complex metabolism, and its therapeutic properties are probably attributable to the metabolite canrenone.

Plasma

canrenone concentrations vary little in health or in hypertensive

patients,

but pharmacokinetic studies in heart failure or chronic liver disease are conflicting.

Formulations of spironolactone now marketed have reliable

bioavailability.

The drug should be taken after food, and the daily dose

need not be divided.

W i t h the exception of certain endocrine effects the

action of spironolactone can be related to mineralocorticoid

antagonism.

The importance of inhibition of aldosterone biosynthesis remains to be explored.

Spironolactone is a drug of first choice in treating

primary

aldosteronism, uncommon forms of hypertension associated w i t h m i n e r a l o corticoid excess, and fluid retention due to chronic liver disease.

In

essential hypertension it is a useful alternative w h e n thiazide diuretics are contraindicated, and it has an important place in treating hypertension.

resistant

It is a valuable adjunct to loop diuretics in resistant

cardiac failure.

Spironolactone is more convenient and reliable than

potassium supplements in correcting diuretic-induced hypokalaemia, and may have an added hypotensive action.

There is no consistent evidence for

important drug interactions involving spironolactone. most dangerous adverse effect, is generally avoidable.

Hyperkalaemia, the Endocrine

side-

effects such as gynaecomastia and menstrual irregularity are common w i t h high doses, and the m e c h a n i s m is incompletely understood. spironolactone should exceed 100 m g daily only in special

© 1982 Walter de Gruyter & Co., Berlin • New York Hormone Antagonists, Editor M. K. Agarwal

The dose of circumstances.

336 Introduction

This review is concerned largely w i t h spironolactone, w h i c h satisfies the criteria for specific reversible competitive antagonism of aldosterone and other mineralcorticoids years.

(1), and has been in clinical use for almost

twenty

Potassium canrenoate is a water soluble spirolactone derivative

used in some parts of the world.

The therapeutic action of b o t h drugs is

probably attributable to their common metabolite, canrenone, w h i c h has itself found limited clinical use.

A new spirolactone derivative,

prorenoate, has a potency threefold higher than spironolactone yet to be evaluated in the clinic.

potassium

(2-4) but has

The structures of these compounds are

shown in Figure 1.

Pharmacology

Cellular action.

Aldosterone and other mineralocorticoids produce

their

physiological effects by a process which involves binding to stereospecific receptor proteins in the cytoplasm

of target cells;

translocation

of the aldosterone-receptor complex to chromatin acceptors in the nucleus; induction of RNA;

new protein synthesis;

changes in electrolyte transport.

and finally the physiological

The spirolactones compete w i t h

aldosterone for binding to the cytoplasmic receptors, but the resulting spirolactone-receptor complexes are unable to translocate to the nuclear acceptors, and thus the train of events leading to altered electrolyte transport is interrupted

(5).

The affinity of a series of

spirolactones

for aldosterone receptors has b e e n studied in vitro to elucidate their structure-activity relationships

(6).

At the cellular level B ring

unsaturation at C-6/C-7 (eg canrenone, Fig.1) reduced affinity, whereas C-7 esterification or thioesterification (eg spironolactone, Fig.1) affinity.

increased

Opening of the gamma-lactone ring to form water soluble

(eg potassium canrenoate or prorenoate, Fig.1) resulted in almost

salts complete

loss of binding, probably because these compounds are largely ionized at physiological pH and cannot cross lipid cell membranes.

These data provide

valuable insight on the mode of action of the spirolactones, but they cannot be extrapolated directly to intact animals or man.

337

Q'y

-SCOCH3

SPIRONOLACTONE

CANRENONE COOK

POTASSIUM CANRENOATE COOK OH

POTASSIUM Fig. 1.

PRORENOATE

Structures of some spirolactones referred to in the text.

338 Pharmacological action.

The spirolactones satisfy the criteria for

reversible specific competitive antagonism of aldosterone and other mineralocorticoids

(7).

Thus they have no important

pharmacological

action in the absence of mineralocorticoids, they reverse all the k n o w n effects of mineralocorticoids, and the responses to different doses of agonist and antagonist vary in accordance w i t h the Law of Mass A c t i o n

(7).

Mineralocorticoids influence electrolyte transport in several organs other than the kidney, for example the gut, sweat glands and salivary glands, and the spirolactones are active at all of these sites (8,9).

However, the

kidney, and in particular the distal renal tubule (8), is the important target for aldosterone and its antagonists.

Here the

spirolactones

increase urinary sodium excretion, decrease renal p o t a s s i u m loss, diminish titratable acidity

(9) and impair maximal urinary acidification (10).

The

ratio of urinary sodium to potassium (Na/K) has long been used as a n index of mineralocorticoid and antimineralocorticoid activity, and logarithmic transformation of this metameter renders its distribution normal, stabilizes its variance, and increases the precision of bioassay procedures

(7).

It was mentioned above that the presence of a m i n e r a l o -

corticoid agonist is necessary for the spirolactones to have pharmacological activity, but it is important to recognize that m i n e r a l o corticoids need not be present in excess.

Thus the spirolactones have w e a k

but measurable natriuretic activity in healthy m a n even w h e n endogenous mineralocorticoids have been suppressed by sodium loading

Bioassay procedures.

(8).

Simple but sensitive bioassay methods in laboratory

animals (rat, dog, rhesus monkey) and in healthy m e n have allowed accurate quantitative and qualitative comparison of different spirolactone

compounds.

The animal method used most extensively has b e e n the Kagawa test, w h i c h measures the urine Na/K ratio in response to spirolactones in adrenalectomized rats pre-treated w i t h DOCA (7).

K a g a w a examined the

structure-

activity of a large number of spirolactones in this experimental m o d e l , but unfortunately the effect of structural modifications proved inconsistent and unpredictable

(7).

somewhat

In healthy m a n the renal

electrolyte

effects of spirolactones can be measured readily if a state of m i n e r a l o corticoid excess is induced either by stimulating endogenous

aldosterone,

339 or by administering exogenous mineralocorticoid.

Secondary hyperaldoster-

onism can be produced by dietary salt restriction (11) or by diuretic treatment (12).

Exogenous mineralocorticoid may be given as an

aldosterone infusion (13) or, more conveniently, by pretreatment with oral doses of the synthetic mineralocorticoid fludrocortisone(3 ,4,14-16).

The

latter method has been used to examine the structure-activity of fourteen spirolactones in healthy men (17) and in general the results showed only a modest correlation with those from in vitro binding studies (6) or animal bioassays (7).

This was not unexpected, as some spirolactones undergo

extensive biotransformation and there are major differences between species in pharmacokinetics.

It would seem wise to examine the activity of

any new spirolactone in healthy man at the earliest possible stage of development.

Clinical pharmacology Pharmacokinetics.

Spironolactone is metabolized extensively when

administered to healthy man.

Although Abshagen et al (18) reported

"considerable amounts" of unaltered spironolactone in plasma 30 minutes after dosing, this is at variance with other studies (19,20) which suggest that the drug is metabolized completly on first pass through the liver, 80% of the dose being converted to canrenone (Fig.1) by removal of the 7tf\acetylthio substituent (29) . The remaining 20% undergoes partial metabolism at the 7 0( position, resulting in a large number of sulphurcontaining metabolites found mainly in the urine (19,21,22).

The only

sulphur-containing compound identified in plasma was the 6 p - 0 H , 1 0 e

(1)

, ip values for a left or right handed ahelix: y -51°, IJJ ^ -47° (78). Thus, in view of the high activity of the 8-a-methylphenylalanine analogue, it would appear that one of these sets of ,i|j values complements the receptor geometry in the region of the carboxy terminus. The apparent requirement of a fixed relative orientation between the phenylalanine aromatic and carboxylate moieties suggests a binding role for the anionic acid group. This inference p is further strenghtened by the weak binding of the [desPhe ]-AII heptapeptide (106), the low activity of [Pheo NH_ ]-All (109), which has a neutral carboxamide terminus, and 1 5 8 the very weak antagonist activity of [Asn , Val , BAla ]-AII (116) compared with [Asn1, Val 5 , Gly 8 ]-AII (129).

In the

latter case the introduction of an extra methylene group

456

between the aromatic ring and carboxylic acid function reduces the potency approximately 100 fold. Further correlations between structural features of the carboxy-terminal side chain and antagonist potency are apparent from the data in Table 3. For example, for amino acids with unbranched aliphatic side chains, increased side chain length leads to increased potency: Gly 8 < Ala 8 < Abu 8 < Nva 8 < Nle 8. The increased chain length (and increased lipophilicity) is also correlated with increased residual pressor activity (106). Position 8 substituted analogues containing aliphatic side chains that are branched at the 6 or y position (Leu, lie, Val) are approximately equipotent antagonists with the corresponding unbranched side chains in the in vitro assays reported in Table 3. However, these branched analogues are more potent antagonists of the in vivo pressor response (9, 106) .

The most potent All antagonists are substituted in position 1 as well as in position 8. By far the majority of these contain secondary amino acids at the N-terminus (N-methylglycine (sarcosine), proline, N-methylleucine, etc.) and thus part of their increased potencies may be attributed to increased resistance to enzymatic degradation. The Sar^ derivatives have several interesting properties, including slow rates of association and dissociation with the receptor (77, 133), and increased tendencies to induce tachyphylaxis (8). This latter property has been associated with the presence of a positively charged amino group on the N-terminal amino acid (93, 94). Thus, for example, 1-pyroglutamic acid and 1-succinic acid substituted All analogues do not induce tachphylaxis nor does All itself at alkaline pH (129). The greater tendency of Sar1 derivatives to induce tachyphylaxis at physiological pH may be due to a higher pKa of the methylated amino group. This same property may also result in the observed slow kinetics of association/dissociation. Khosla and Bumpus and co-workers have examined the effects of

457

Table 3.

Inhibiting Activities of Angiotensin II Analogues

Analogue

110.

[p-F-Phe4]-AII [ (ctMe)DOPA8]-AII [Sar1, (aMe)DOPA8]-All [Cha8]-All [Sar1, H e 8 , Pro9]-AII [Asn1, Ind8]-AII [Pgl8]-All [Asn1, Pgl8]-All [Z-Asn1, Pgl8]-All [Sar1, Pgl8]-All

111.

[ (H2N-CO(CH2)2CO)1,

101. 102. 103. 104. 105. 106. 107. 108. 109.

Lit. Ref. weak

136

weak weak weak

1

T

strong strong strong strong strong strong

1 57 56 48 141 141 141 141 141

8

112. 113. 114. 115. 116. 117. 118. 119. 120. 121. 122. 123. 124. 125. 126. 127. 128. 129. 130.

Pgl ] - A H [2-Pyrrolidone-N-acetyl1, strong 8 Pgl ] - A H [Prolyl-Sar1, lie8, Pro9]2.49* AII [Chi1]-All 5.0**5 [Sue1, Phe4, Tyr8]-AII 5.0*§ 1 5 8 [Asn , Val , gAla ]-AII 5.08* [D-Phe8] - A H 5 . 37 4 8 [Phe , Ala ]-AH 5. 5* [Acpc8]-All 5.69 [Sar1, Nip 4 , Nip8 ] - A H 6.12* 1 8 [Sar , Car ]-AH 6.12* [35%

Effective blocking of prolactin

of specific binding) was also shown with one

antiserum (at 1:50) in membrane preparations from a number of species (rabbit, mouse, rat, man).

In the case of the GH

receptor, where binding is largely restricted to the liver, an examination of the efficacy of four antisera In inhibiting binding to the liver of five species (rabbit, mouse, rat, sheep, man) showed differences in the relative abilities of different antisera to inhibit

125

I-ovine GH binding (7).

Perhaps more importantly, these antisera were able to inhibit binding to rabbit liver GH receptors to a considerably greater

490 extent than binding to GH receptors from other species.

This

species restriction may be responsible for the relative ineffectiveness of these antibodies in blocking GH stimulated 3-0-methyl glucose transport in rat diaphragm (J. Schwartz, personal communication).

For this reason, efforts have been

made to purify rat liver GH receptors, but without owing to the low content of these receptors.

success,

It is likely

that these antisera will be of greatest use in defining the physiological role of GH in the rabbit, and that in other species, higher dose levels of gamma globulins will be needed in order to block GH action; in some cases control gamma globulins are inhibitory at these levels (vide infra). However, it is likely that a variety of other antibodies are also present in these antisera, directed against located away from the hormone binding site.

determinants

These may act in

conjunction with hormone binding site directed antibodies to enhance down regulation of receptors and tissue desensitization to growth hormone.

Use of Anti-Prolactin Receptor Antibodies as Anti-Hormones Shiu and Priesen (17) were able to use these antibodies to demonstrate that the prolactin binding glycoprotein that they had isolated was indeed the prolactin receptor.

Thus, using

cultured explants derived from pseudo-pregnant rabbit mammary tissue, they were able to show that

prolactin-stimulated

amino-acid uptake and casein synthesis could be blocked by pretreatment in vitro only with anti-prolactin antibodies.

receptor

Specificity was demonstrated by the lack of

effect of both antisera and control (non immune) sera on insulin and Cortisol stimulated amino-acid uptake and glucose oxidation in these explants.

Of interest was the slight

stimulation of amino acid uptake and glucose oxidation by anti-receptor antisera alone, which may indicate a short term

491

hormone-like effect analogous to that seen with insulin receptor antibodies in vitro (5,18).

Similar phenomena have

recently been observed in the Noble lymphoma line, which replicates in response to exquisitely low concentrations of prolactin (19, Shiu and Friesen, unpublished).

Addition of

anti-PrlR Abs to these cells blocked their response to added prolactin, while antisera alone mimicked in part the actions of prolactin. These antisera are also of value in establishing the

identity

of a prolactin binding protein or the involvement of a prolactin receptor mediated event.

Thus Waters et al.

(20)

were able to show that the prolactin binding component of rabbit milk fat globule membranes was antigenically to the receptor in the mammary gland.

identical

Leontic et al.

(21)

used these antisera to assess the role played by prolactin receptors in the decrease in water permeability of human term amnion membranes seen when prolactin is added to the fetal side of the membrane.

Preincubation of membranes for 15 mins

with antiserum at 1:100 effectively blocked any effect of prolactin in the system, even at high doses (10 pg/ml). At the present time, the in vivo action of PrlR Abs have been the subject of only one study, performed in the female rat (16).

In these experiments, antisera (0.15 ml/day)

administered to normal cycling rats from diestrus 2 to the following diestrus 1 resulted in an approximate doubling of corpora lutea number and an increase in ovarian weight by the second diestrus 2.

At this time serum progesterone

were unaffected, while those of prolactin were elevated in two of three studies.

levels

significantly

No attempt was made to

confirm ovulation by detection of ova in the oviducts.

The

authors interpreted these findings to be the result of blocking of the luteolytic action of prolactin (22) and desensitization of the hypothalamic feedback sensor

regulating

492 prolactin secretion.

However, in view of the recent report by

Wang et al. (23) that prolactin inhibits estrogen production in preantral follicle granulosa cells in culture, it is possible that the antibody has allowed multiple ovulations to occur by preventing the inhibitory effect of prolactin on estrogen-induced follicular maturation (vide infra).

The

inhibitory effect of prolactin on ovulation was recently demonstrated in the rabbit (24).

The lack of effect on

diestrus 2 serum progesterone levels is in accord with the autonomous nature of progesterone secretion during the estrus cycle, following the proestrus trigger phase (25).

In any

case, the diestrus 2 corpus luteum is essentially quiescent. The luteotrophic role of prolactin would best be examined in the pseudo-pregnant rat model, where diurnal prolactin surges maintain the corpus luteum until mid pregnancy (25). A second study was carried out by Bohnet et al. (16) with lactating rats.

Here, injection of the mothers with 0.2 ml

antiserum per day for 5 days significantly decreased total litter weights relative to normal serum injected controls, indicating an inhibition of milk production resulting from blockade of prolactin's galactopoietic action.

Again, serum

prolactin levels were elevated, although serum progesterone was unaffected.

Comparison of the Use of Anti-Prolactin Receptor and AntiProlactin Antibodies as Anti-Hormonal Agents and Likely Targets of Anti-Prl Receptor Action Anti-hormone antibodies have several potential disadvantages when compared with anti-receptor antibodies, and these relate to their different modes of action.

Thus, with large antigens

such as prolactin or gonadotrophins, binding of antibody may not block binding to the receptor; furthermore, the prolonged half life of antibody-hormone complexes may lead to enhanced

493 biological potency of the hormone

(26,27).

Approximately

equivalent affinities of receptor and antibody for hormone may also lead to exchange of hormone onto the receptor unless excessive amounts of antibodies are present.

In addition,

unless damage to the secretory cells occurs as a consequence of antibody action, blockade of negative feedback loops controlling secretion may result in hypersecretion of hormone, with correspondingly enhanced requirements for antisera.

By

contrast, anti-receptor antibodies produce an extended state of desensitization of target tissues in addition to their direct steric effects.

However, the requirement for blocking

concentrations of antibody will vary with "spare content.

There are also some physiological

receptor"

circumstances

where purified hormonal antigen is not available (such as rat placental lactogen, which binds to prolactin receptors in the rat) or where the hormone is present in great excess (such as maternal plasma placental lactogen levels or fetal somatomedin like activity

plasma

(28) in the sheep) which are not

amenable to anti-hormonal probes.

Nevertheless, antibodies to

hormones are of great use, and have been responsible for the discovery of human placental lactogen and prolactin and nonsuppressible insulin-like

activity.

Anti-prolactin antibodies have been used to demonstrate the role of prolactin in maintaining the corpus luteum of pseudopregnancy in the rat (29) and in lactating rats

(30).

Tomogane et al. (31) examined litter weight gain and progesterone secretion on day 10 of lactation in the rat and found highly significant decreases in both parameters response to rat prolactin antisera.

in

An analysis of this data

indicates that the lack of effect on progesterone seen by Bohnet et al. (16) may have been the result of administration of insufficient antiserum to block ovarian

progesterone

secretion, since effects on litter weight gain were not great.

Tomogane et al.'s antiserum was unusually

effective

494 when compared to similar studies on weight gain in pups alone (32,33).

Purandare et al. (3*0 found that anti-prolactin

antisera blocked ovulation in cyclic mice, and no newly formed corpora lutea were detected, in contrast to the findings with anti-PrlR Ab.

Pseudo-pregnancy was also shortened

significantly, as expected.

In contrast, Wuttke and Meites

(36) found ergocornine suppression of serum prolactin in rats did not prevent cycling and resulted in an increased number of corpora lutea, attributed to a blockade of prolactin's luteolytic action along similar lines to Bohnet et al. (16). Other workers have reported similar findings in rats (37) and mice (35) using ergocornine or bromocryptine, and the former group (37) provided additional evidence that prolactin induces luteolysis rather than preventing multiple ovulations in hypophysectomized, gonadotrophin ovulated rats (22).

Evidence

for a physiological role of prolactin as a luteolytic agent in intact rats still appears to be inadequate.

The complexity of

the role played by prolactin in inhibiting progesterone production in immature follicles while promoting it in estrogen matured follicles and corpora lutea (38,39) precludes a simple statement on the luteotrophic and luteolytic roles of prolactin in the normal cycle.

Varying follicular

concentrations of prolactin are probably a factor, as are the direct actions of prolactin in enhancing steroid precursor supply and blocking further metabolism of progesterone. However, the work of Richards' group (40,^1) demonstrating the role of diurnal prolactin surges in maintaining estradiol and LH receptors in the corpus luteum of pregnancy or pseudopregnancy may hold the key.

Curiously, this mechanism depends

on the state of differentiation of the follicle cells, since estrogen production by rat preantral granulosa cells is inhibited by prolactin (23), a mechanism which would be counter productive to luteinized cells reliant on estrogen as well as prolactin for luteal support (MO).

Functional

prolactin receptors are apparently induced in very immature

495 follicles by FSH. These findings seem highly relevant to clinical conditions of anovulatory amenorrhea resulting from hyperprolactlnemia, or less severe luteal phase defects which may result from inadequate estrogen priming of granulosa cells as well as defects in hypothalamic LHRH release mechanisms (42).

It is

possible that anti-prolactin receptor antibodies are present in some of these patients, particularly those without galactorrhea which cannot be normalized with bromocryptine, since low levels of prolactin are apparently necessary for progesterone production by advanced follicles (38,43) and for ovulation (42).

Hence, prolactin receptors of human corpora

lutea are mostly present in early luteal tissue samples (44). No studies have been Undertaken with anti-Prl Abs in the male, and results obtained with other blockers of prolactin action are not definitive.

Prolactin potentiates the steroidogenic

actions of LH on the Leydig cell In some species (particularly rodents), in part by increasing LH receptor content and in part by increasing precursor supply (45,46).

Prolactin

receptors are present on Leydig cells, and can be blocked by anti-PrlR Abs (47).

Hyperprolactinemia in man results in

hypogonadism and loss of libido as a result of dopaminergic suppression of LHRH release, rather than by a direct effect on testicular function.

On the other hand, suppression of

prolactin with anti-prolactin or bromocryptine leads to a fall in plasma testosterone in a number of species, as well as decreases in the size of the accessory reproductive glands, partly by blocking direct actions of prolactin

(48,45).

Dysfunction of the accessory glands would appear to be the most likely markers for the presence of anti-PrlR Abs in man. Adrenal steroidogenesis is also regulated in part by prolactin receptors in the cortex (49); these too can be blocked by

496 anti-PrlR Abs, but as yet no functional studies have been performed.

Prolactin acts to enhance corticosterone secretion

in the rat by decreasing its disposal by the 5-alpha-reductase (50), although in man the elevated levels of dehydroepiandrosterone sulphate of hyperprolactinemia would suggest a block at the 3-beta-hydroxysteroid dehydrogenase

(42).

This review would be incomplete without a comment on the use of the dopamine agonist bromocryptine (CB154, parlodel, ergocryptine) as a blocker of prolactin secretion.

This

compound is of great importance in many studies, yet suffers several limitations.

Because of its mode of action, it is not

totally specific, producing a lowering of plasma catecholamines in addition to plasma prolactin, and small increases in plasma ACTH and Cortisol (51).

Furthermore,

doses sufficient to decrease circulating prolactin levels to undetectable levels may produce unpleasant side effects (52a).

It is also unsuitable in circumstances where lactogen

secretion is not under dopaminergic control, such as human amniotic fluid prolactin secretion (42) or placental lactogen secretion (52b).

In both of these cases, anti-PrlR Abs would

be useful anti-hormonal agents.

Nevertheless, in the majority

of cases, bromocryptine is of great value.

Anti-Growth Hormone Receptor and Anti-GH Antibodies as AntiHormones These antibodies may prove to be a particularly valuable tool, since somatostatin is considerably less specific in its actions than bromocryptine, and has a very short life.

Thus

somatostatin lowers plasma insulin and glucagon levels and impairs TSH responses to TRH (53). Anti-GH antisera have been used to block the actions of administered GH in hypophysectomized

rats (54), as well as to

497 produce a protracted retardation of growth after injection into neonatal rats (55,56,57).

Interestingly, basal plasma GH

concentrations were normal in these animals after sexual maturation, despite depleted pituitary reserves.

Since this

antiserum did not affect the growth of young (less than 20 day old) animals, a role of pituitary-dependent plasma GH surges in the growth process after 20 days of age is implied. Neutralization of endogenous GH is associated with a decrease in liver, kidney and brain ornithine decarboxylase, a marker of cellular growth (58).

Anti-rat GH antiserum administered

to rats was able to produce a significant inhibition of protein synthesis in diaphragms removed 3 hours after injection, again implying a role for endogenous GH.

This was

accompanied by loss of the GH dependent "refractoriness" or desensitization phenomenon (59).

Atrophy of thymic and

peripheral lymphatic tissue resembling that seen in dwarf mice has also been reported in response to GH antiserum treatment in mice (57). Studies of the metabolic role of GH using anti-GH antisera have been limited until recently.

Only one study has examined

the lipolytic effects of endogenous GH, in this case in the pigeon, and found no effect with uncharacterized

antisera.

Recently, careful studies by Schwartz (60) have shown that the insulin sensitivity of adipose tissue from antiserum treated rats (measured as stimulation of glucose oxidation) was enhanced, although insulin tolerance curves were not significantly different.

Adipose tissue from these animals

was sensitive to added GH (stimulation of glucose oxidation), unlike tissue from control animals, implying an in vivo role of endogenous GH in desensitization of target tissues to GH action.

Other in vitro studies of this phenomenon with GH

antisera have used GH stimulated sugar transport as a parameter, and have concluded that 90 mins contact of tissue with GH is necessary to produce delayed refractoriness, even

498 though the stimulatory effects of .GH in vitro can be produced after only 10 mins exposure.

These important studies provide

a basis for a number of useful studies which could be carried out with anti-GHR Abs. The only study performed to date with anti-GHR Abs in vivo, involving the administration of 80 pg or 320 yg of gamma globulins daily for 5 days to hypox HGH treated rats, has unfortunately shown equivalent inhibition of growth (33%) with both the high dose of control and anti-GHR gamma globulins (Waters and Friesen, unpublished observations).

However, the

lower dose anti-GHR Abs produced the same level of inhibition of HGH stimulated growth, so it is possible that an effect was indeed produced.

Future studies should be undertaken in

intact, 30 day old rats without HGH administration, since exogenous GH may have overcome the blocking effects of the antibodies if a sizeable pool of "spare receptors" was present.

Alternatively, as mentioned, for reasons of species

specificity it may not be appropriate to undertake these studies in the rat.

These studies, along with others aimed at

the production of monoclonal antibodies to the receptors, are currently in progress

Summary Specific anti-prolactin receptor and anti-GH receptor antibodies have been raised against purified receptor preparations, and characterized.

These are capable of

blocking the binding of prolactin and GH to their respective receptors.

Limited in vitro studies have indicated the

utility of anti-PrlR Abs in blocking stimulation of mammary gland function by prolactin and the effects of prolactin on water transport in amniotic membranes, as well as the replication of a prolactin dependent lymphoma line.

499 Administration of anti-PrlR antlsera to cycling rats resulted In an apparent block of luteolysis, and reduced milk yield In lactating rats.

No satisfactory data on the blocking actions

of GHR Abs is available at present.

Comparisons with the

effects of anti-prolactin and anti-GH antisera are made, and likely targets for receptor antibody action suggested.

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8 7 7 - 8 8 3 (1980). Reagan,

CLINICAL APPLICATIONS OF PROLACTIN LOWERING DRUGS

Carlo Ferrari, Rosanna Benco, Pietro Rampini Second Department of Medicine, Fatebenefrateili Hospital, 23 Corso di Porta Nuova, 20121, Milano, Italy

Introduction Ten years have now elapsed since Daughaday and his associates (1) first reported that prolactin (PRL) hypersecretion may be pharmacologically inhibited. Although L-dopa, the drug used in that study, was found to be of little clinical benefit because of its short duration of action, in the next few years several clinically useful PRL-lowering drugs were developed, including bromocriptine (2-4), lergotrile (5,6), metergoline (7-10), lisuride (11,12) and pergolide (13,14). All of these drugs are ergoline derivatives and most of them are believed to inhibit PRL secretion by stimulation of dopamine receptors, both in the pituitary lactotrophs and in the hypothalamus; the mechanism of action of metergoline, which is mainly a serotonin antagonist, is still uncertain, and both an anti-serotoninergic and an indirect dopaminergic mechanism have been suggested (15-17). Another ergoline derivative with antiserotoninergic and dopaminergic properties is methysergide (7,8) but its clinical results in hyperprolactinemic states have been relatively poor (9,18). Nonergolinic PRL-lowering drugs include dopamine itself (19,20), which has however to be infused intravenously, and the dopamine agonists apomorphine (21), piribedil (22) and nomifensine (23, 24), but their therapeutic employment is hampered either by the need of parenteral administration or by their very weak PRL-lowering potency in hyperprolactinemic states. Cyproheptadine, an antiserotoninergic, anticholinergic and antidopaminergic drug which does not affect PRL secretion in healthy subjects (25 ) ,

© 1982 Walter de Gruyter & Co., Berlin • New York Hormone Antagonlsts, Editor M. K. Agarwal

504

has also been tried in hyperprolactinemic amenorrhea, but PRL levels were not at all (9) or only mildly (26) decreased. One of the most effective PRL-lowering agents, lergotrile, had yet to be abandoned because of liver toxicity (27), so that the drugs currently available for therapeutic purposes include bromocriptine, metergoline, lisuride and pergolide.

Therapeutic applications of PRL-lowering drugs Prevention and suppression of puerperal lactation. The important role played by the physiological PRL hypersecretion in puerperal lactogenesis and galactopoiesis is well established (28). Therefore, the availability of potent and safe PRL-lowering drugs has resulted in a new and more rational approach to the inhibition of puerperal lactation. The high effectiveness of bromocriptine both in preventing and in suppressing already established lactation has been shown in many trials since 1971 (29 for review). The recommended dosage schedules advise a treatment period of at least 14 days with 2.5 mg twice daily, preferably followed by a 7 days period with 2.5 mg daily to lower the rate of rebound lactation. Surprisingly, it has subsequently been shown that metergoline administration at the dose of 4 mg thrice daily for only 5-7 days is associated with even better results, virtually eliminating the rebound of lactation on withdrawal (16,30), in spite of less complete inhibition of PRL secretion. Good results have also been obtained with lisuride administration (0.1-0.2 mg thrice daily for 7 days); although the rate of rebound lactation was high (50% and 30% respectively) , a persistent suppression was obtained by a further one week treatment (31). Interestingly, administration of ergolinic drugs in postpartum women is not associated with the usual side effects observed (vide infra). Controlled studies are presently needed to establish the best drug and regimen for the inhibition of puerperal lactation.

505

Treatment of pathological hyperprolactinemlc states. Non-physiological hyperprolactinemia, i.e. elevation of serum PRL levels above 15-20 ng/ml in the absence of pregnancy or puerperal lactation, is a relatively common condition, which accounts for a consistent percentage of various reproductive disorders in females and, at a lower extent, in males (28,32,33). Although the causes of hyperprolactinemia are numerous, the most common include administration of drugs ( dopamine antagonists, estrogens, progestogens, antiandrogens, opiates, i^-histamine receptor antagonists), PRL-secreting pituitary micro-and macroadenomas, acromegaly, hypothalamic lesions, hypothyroidism and socalled idiopathic hyperprolactinemia, i.e. hyperprolactinemia with normal sella turcica on tomography and CT scan, in the absence of any known cause of this disorder (28,32). Hyperprolactinemia is frequent in uremic patients (34,35) and may occur in severe liver cirrhosis (36). By far the most frequent types of pathological hyperprolactinemia observed in clinical practice are pituitary adenomas ( especially microprolactinomas ) and idiopathic hyperprolactinemia. Both of these conditions are amenable to successful medical therapy with PRL-lowering drugs. The most widely used agent has so far been bromocriptine. Numerous trials since 1972 have shown that this drug is able to suppress PRL secretion and restore gonadal function in most patients with hyperprolactinemia, irrespective of the underlying etiology (2, 4,28,32). Available data indicate that normalization of PRL lev^ els may be obtained with bromocriptine therapy in about 70-80% of cases without major differences between patients with idiopathic disease, micro- or macroprolactinomas, and that a significant PRL decrease ( i.e. a decrease to below 50% of pre-treatment values ) occurs in most of the remainders (32, 37) . Complete resistance to bromocriptine therapy is rather uncommon, accounting for less than 10% of treated patients, and may be overcome in about half of the cases by increasing the drug doses (37). The clinical results of bromocriptine therapy are even more satisfactory, since some patients show a clinical benefit in spite of only partial PRL lowering. In females, resumption of menses

506

occurs in 80-90% of the subjects with hyperprolactinemic secondary amenorrhea, usually within 1 to 3 months, and ovulation is induced in most of them, as well as in the high majority of women with hyperprolactinemic anovulation (32,37). Galactorrhea ceases or markedly decreases promptly after institution of

treat-

ment (32). Luteal insufficiency due to hyperprolactinemia is also easily corrected by bromocriptine treatment in many cases (38). Completion of puberty with occurrence of menarche and ovulatory menses may be induced in patients with hyperprolactinemic primary amenorrhea by PRL-lowering therapy with either bromocriptine or metergoline (39,40), although the treatment period required may be as long as 2 years. Restoration of ovulation by bromocriptine therapy obviously results in a high pregnancy rate. While it was formerly believed that pregnancy might carry a serious risk to hyperprolactinemic women, recent evidence indicates that clinically significant pituitary enlargement occurs during gestation in less than 5% of hyperprolactinemic women with idiopathic disease or microprolactinoma (41), and that some patients even show clinical and biochemical amelioration of their disorder after pregnancy (42). These data suggest that prophylactic adenomectomy or radiation therapy must be performed before induction of pregnancy only in patients with large pituitary adenomas. The outcome of gestation in mothers given bromocriptine is also uneventful, since there is no increased risk of abortion, multiple pregnancy or occurrence of malformations in the infants (43). In hyperprolactinemic males, bromocriptine treatment is associated with restoration of sexual potency in most cases; normal serum testosterone levels and sperm counts are also restored in many instances (44-46). The bromocriptine dose usually employed in the therapy of the hyperprolactinemic disorders has been 2.5 mg twice ( or sometime thrice ) daily, which is sufficient to normalize PRL levels in most cases. Recent evidence shows that increasing of the drug dose up to 20 mg per day may result in adequate PRL suppression in many patients resistant to the usual regimens (37).

507

Treatment has to be initiated in slowly increasing doses to avoid side effects ( vide infra ). As anticipated in the introduction, other ergoline derivatives have been successfully used in the treatment of hyperprolactinemic states. Lergotrile gave initially promising results (6), but is no longer in use due to liver toxicity. Experience with lisuride, another dopamine agonist, is rapidly accumulating and suggests that this drug in doses ranging between 0.4 and 2 mg per day divided in two to four administrations is nearly as effective as bromocriptine both in lowering PRL levels and in restoring ovarian and testicular function (11,12,37,47). A newly introduced dopamine agonist is pergolide, which may be administered once a day due to its long duration of action. A very preliminary study showed that this drug at doses of 0.10.2 mg per day may suppress PRL levels and restore menses in hyperprolactinemic women (14). The 5 year experience of the authors with metergoline indicates that this drug may be used effectively and safely in the treatment of the hyperprolactinemic disorders (10,16,17). Among 82 women treated with 8-24 mg per day in 3 divided doses, 50% had PRL levels normalized and 25% reduced to below 50% of basal; ovarian function was restored in 74% of the patients. These figures are somewhat lower than those obtained with bromocriptine, but the difference may be dose-rather than drug-related since many of the treatment failures occurred in subjects given 8 mg metergoline per day (9) and since increasing of the drug doses was associated with better clinical and biochemical results (17). On the other hand, bromocriptine is probably a more potent PRL-suppressing drug than metergoline, lisuride and lergotrile, since it completely suppresses the PRL responses to TRH and sulpiride (4,15,17) while the latter drugs either do not affect or only partially inhibit these responses (15,17,25,48,49). Many reports have now appeared showing that PRL-lowering therapy with bromocriptine or lisuride may result in size reduction of some prolactinomas, especially of the suprasellar portion of large adenomas (50-53). These favorable results have generally

508 been obtained after long-term drug administration ( 1 to 6 years), but in some cases a few days or weeks have been sufficient (50, 53). The proportion of tumors which may shrunk on medical treatment has been estimated to be about 20% (54) or 60% (53) in the two investigations performed in sufficiently large series of patients to make such a calculation meaningful. The mechanism of this effect is still unknown,but it is observed only in patients with secreting adenomas showing PRL suppression by the drug, suggesting a link between inhibition of PRL secretion and tumour shrinkage. In contrast with the impressive results reported for macroprolactinomas, evidence for size reduction of microadenomas during bromocriptine therapy has yet to be provided. Whether this depends on the limitations of the presently available radiological technics or on a different behavior of these adenomas remains to be established. However, we were unable to observe evidence of tumor regression in 30 patients with microprolactinomas who were treated for at least 1 year with either bromocriptine or metergoline; on the contrary, a marked enlargement of one such tumor with destruction of the sellar floor occurred in a patient after 1 year of apparently successful bromocriptine therapy as suggested by persistently normalized PRL levels during treatment (17). Further growth of a macroprolactinoma during bromocriptine therapy has also been recently described in a case (52). Another approach to evaluate the possible effect of medical treatment on the natural history of the hyperprolactinemic states has relied on measurement of serum PRL concentration after drug withdrawal. Significantly lower PRL levels 1 or more months after stopping bromocriptine treatment have been found by some authors in patients with macroprolactinomas (52,55) and idiopathic disease (56) but not microprolactinomas (55). However, some patients with pituitary tumors had also had radiotherapy, and it is to be pointed out that convincing evidence of pharmacological cure of hyperprolactinemia has not yet been provided. In the author's experience only a minority of patients treated with either bromocriptine or metergoline have some PRL suppression persist-

509 ing after 2 months of drug withdrawal,and none of them shows normal basal PRL levels and normal PRL secretory dynamics (17). In spite of the rapid rebound in PRL levels, cyclic ovarian function may persist on drug withdrawal for many months, sometimes with evidence of ovulation, but the reproductive disorder eventually recurs in most cases (17,57). Thus, the presently available data suggest that ergolinic drugs, though very effective agents for the treatment of hyperprolactinemic states, are unable to yield permanent cure. A particular kind of hyperprolactlnemia probably due to pituitary dopamine receptor dysfunction is present in most uremic patients (35,58). In these subjects the lowering of PRL levels which is associated with chronic, but not acute, bromocriptine treatment (58) only seldomly results in restoration of gonadal function suggesting that other factors ( probably impairment of the positive estradiol feed-back effect ) are involved in the anovulation of chronic renal failure (35). Treatment of acromegaly. Medical treatment of acromegaly became possible after the important observation by Liuzzi and coworkers that dopamine agonist drugs are able to reduce growth hormone (GH) secretion in about 50% of acromegalics (59). Acutely effective drugs include L-dopa, dopamine, apomorphine, bromocriptine, lergotrile, lisuride, piribedil, methysergide and metergoline, which may act as a dopamine agonist in acromegaly, but the only drugs so far shown to be effective during chronic treatment are bromocriptine, lergotrile and lisuride (47,49,60,61). Bromocriptine has been used in most trials, and in about 50% of treated patients serum GH levels have been lowered to below 50% of basal, near normal levels having been attained in about 30% of the cases (60). Although escape phenomena have been occasionally reported, the GH-lowering effect of bromocriptine is almost always maintained during long-term treatment. The drug doses used are generally 10-20 mg per day; higher doses ( up to 60 mg per day ) have been found to increase the percentage of responsive patients by some authors, but this has not been confirmed by others (60,

510

61). The clinical results of bromocriptine treatment have generally been satisfactory even in patients showing poor GH suppression, with reduced soft tissue swelling, sweating, headache and blood pressure values and improved libido and potency or restoration of menses in most cases; improvement of glucose tolerance and normalization of hydroxyprolinuria generally occur (60,61). Recent trials with lisuride at doses ranging between 0.6-2 mg per day showed that this drug is as effective as bromocriptine in the treatment of acromegaly (47,60). That neither bromocriptine nor lisuride administration results in cure of the disease is however indicated by the rapid rebound of GH to the pretreatment levels (47,60). The possible antitumor effect of dopaminergic drugs in acromegaly has recently been investigated, but the results have been less encouraging than those obtained in prolactinomas, with only 4 instances of tumor shrinkage documented by X-ray or CT scan of the skull during bromocriptine or lisuride treatment (54,60,61,62). The most recent data

by Liuzzi and

coworkers indicate clear-cut signs of reduced tumor size in only 2 of 20 acromegalics with large adenomas treated with dopamine agonists ( personal communication ). However, the real incidence of this phenomenon in acromegalic patients responsive to dopaminergic therapy has yet to be determined, since only 4 subjects of this series ( including the 2 showing signs of tumor shrinkage ) were GH responders. Treatment

of parkinsonism. Another well established clinical

application of PRL-lowering drugs is treatment of parkinsonism. This syndrome is associated with decreased dopaminergic transmission in the basal ganglia due to a somewhat selective degeneration of the dopaminergic nigrostriatal tract and depletion of dopamine with however intact striatal dopamine receptors. Cotrias and colleagues in 1967 were able to show that the administration of L-dopa, the dopamine precursor, is associated with dramatic improvement of parkinsonism (63). The addition of extracerebral L-aromatic aminoacid decarboxylase inhibitors as carbidoca or benserazide allowed then continued theraDeutic ef-

511

ficacy of L-dopa up to 2-6 years of' treatment with reduced side effects such as anorexia, nausea and vomiting ( 50 for review ). However, increasing disability develops in more than 50% of the patients at this time in spite of L-dopa administration, probably due to further degeneration of dopaminergic nigrostriatal neurons. Since these patients should theoretically benefit from drugs that stimulate the striatum directly, not requiring conversion to dopamine by the degenerating nigrostriatal neurons, trials with direct dopamine agonists were undertaken. Present knowledge (50,64) indicates that such drugs possess in fact an antiparkinsonian effect, but not in all patients and only at high doses, much higher than those used in PRL-lowering therapy. Bromocriptine has been the most widely used agent, and may now be considered an useful adjunct to L-dopa therapy; it is usually advisable to give it in combination with submaximal doses of Ldopa. Combined L-dopa-bromocriptine treatment results in significant improvement in rigidity, tremor, bradykinesia and gait disturbance in secondary L-dopa f ailures, with at least onestage improvement in about 40% of individual patients (64). The optimal dose range of bromocriptine is 40 to 80 mg per day, which is unfortunately associated with frequent and severe side effects, especially psychiatric reactions and involuntary movements, so that only about

50% of patients may remain on bromo-

criptine treatment for prolonged periods. The results of lergotrile are comparable with those of bromocriptine (64), but the use of this drug is hampered by its hepatotoxicity. Lisuride and pergolide are now undergoing clinical trials, and preliminary results suggest that both drugs may be effective in advanced parkinsonism, even in some patients with secondary Ldopa and bromocriptine failures, at dose levels of 1-5 mg per day combined with L-dopa (65) . Bromocriptine has been tried in other extrapyramidal disorders, and some amelioration of Huntinqton's chorea and tardive dyskinesia has been reported, but it is unlikely that these findings will have any major clinical impact because of the narrow therapeutic dose range in these conditions (50).

512

Possible therapeutic application of PRL-lowering drugs PRL-lowering drugs have revolutionized the treatment of some endocrine and neurologic diseases, as above reviewed. The dramatic results obtained in the management of hyperprolactinemic disorders, acromegaly and parkinsonism plus some theoretical considerations on the possible roles played by PRL and by dopamine in certain physiological or pathophysiological conditions led to attempts to use PRL-lowering drugs in many other disorders, including Cushing's disease,normoprolactinemic infertility,impotence, breast diseases, premenstrual syndrome,hepatic encephalopathy, diabetes mellitus and hypertension,but their therapeutic value in these conditions has not yet been established. In the following section the results obtained with the administration of PRL-lowering agents in these disorders will be briefly reviewed. Treatment of Cushing's disease and Nelson's syndrome. Attempts to treat Cushing's disease with neuroactive drugs were unsuccessful until 1975, when Krieger and her associates reported that administration of cyproheptadine at doses of 24 mg per day. for 3 to 6 months was associated with biochemical and clinical evidence of amelioration of the disease in 3 patients (66). There has been a considerable debate on the effectiveness of cyproheptadine in the management of Cushing's disease and Nelson's syndrome in the following years. In her most recent revision, Krieger reported an about 50% remission rate among over 100 patients with Cushing's disease treated with cyproheptadine by herself or by others (67). Clinical remission is characterized by resumption of menses, decrease of hirsutism, disappearance of facial redness, decrease of hypertension and loss of weight, is associated with normalization of urinary corticosteroid excretion and a return of normal ACTH and Cortisol periodicity and dexamethasone suppressibility within 6-12 months, and is generally sustained throughout treatment. Relapse is the rule on discontinuance of medication, but 2 cases of sustained remission on withdrawal have been described (68,69). Cyproheptadine has also

513

been used as an adjunct to pituitary irradiation to achieve a more rapid remission of Cushing's disease, and reports have appeared suggesting that the drug may induce clinical and biochemical amelioration of Nelson's syndrome in some cases, which may on occasion be sustained after stopping treatment (67,70). It is however to be pointed out that many investigators were unable to show any beneficial effect of cyproheptadine treatment in most patients with Cushing's disease (71). The mechanism of action of cyproheptadine in decreasing ACTH secretion is unknown, but may be related to its antiserotonin properties. There have been two reports of effective treatment of childhood Cushing's disease with metergoline at the dose of 12-16 mg per day (72,73), but favourable results have not been obtained in 4 adult patients (71). Bromocriptine has also been tried in Cushing's disease, since it had been shown in acute studies that the drug lowers plasma ACTH and Cortisol levels in several cases of Cushing's disease and Nelson's syndrome (74). Some of the few patients treated chronically with bromocriptine 5-20 mg per day for up tó 1 year showed initially a good suppression of corticosteroid secretion, but escape phenomena were frequently observed, which could be overcome only for short periods by increasing the drug doses (74). Thus although there are certain patients with Cushing's disease or Nelson's syndrome who respond

to serotonin antago-

nists or dopamine agonists, the role of neuroactive drugs in the management of these disorders is still under investigation. Treatment of normoprolactinemic amenorrhea, infertility, impotence, galactorrhea, and gynecomastia. It has been claimed by some authors that bromocriptine administration leads to resumption of menses in several women with normoprolactinemic amenorrhea or infertility. However, it has been shown by controlled trials that bromocriptine treatment is not superior to placebo in both of these conditions (75,76). Similarly, bromocriptine as well as metergoline and methysergide have been shown to be no

more effective than placebo in sexually impotent men (lit

78).Normoprolactinemic oligospermia is also unresponsive to

514

bromocriptine treatment (79). On the other hand, galactorrhea ceases or decreases on administration of PRL-lowering drugs also in normoprolactinemic subjects, suggesting that lactation is PRL-dependent also in these cases (6,28). While gynecomastia related to hyperprolactinemia subsides during PRL-lowering treatment (44), most patients with this disorder are normoprolactinemic and do not benefit from this therapy. Treatment of premenstrual syndrome. Conflicting results have been reported on the effects of bromocriptine administration in women suffering from the premenstrual syndrome ( 80 for review ). However, controlled trials have shown that mastodynia and possible bloating and depression are the only symptoms of this syndrome which are improved by the use of bromocriptine

(81,82).

Treatment of breast diseases. Mastalgia associated with fibrocystic disease or fibroadenosis of the breast is relieved by bromocriptine therapy according to several investigators; adenomatous or cystic nodules often become smaller and softer, and the smaller ones may even disappear (83,84). On the other hand, the inhibitory effect of bromocriptine treatment on the progression of breast cancer is only minimal (84 for review ). However, it has been reported that clinical responses of metastatic disease to antiestrogen therapy are less likely in the presence of raised serum PRL levels (85) . The recent finding that PRL levels are frequently elevated after mastectomy (86) should therefore prompt further investigations on the possible value of PRL-lowering agents in conjunction with other drugs in the treatment of breast cancer. Treatment of hepatic encephalopathy. Eleven years after the first attempts to use neurotransmitter therapy with L-dopa in fulminant liver failure, the efficacy of dopamine agonists in the treatment of hepatic encephalopathy remains controversial (87). The apparent benefit of L-dopa in both acute and chronic encephalopathy reported in many studies was not Confirmed by a

515

controlled trial. A double blind controlled trial of bromocriptine therapy gave also disappointing râsults, but other studies, one of which a controlled trial, showed benefical effects of the drug at slowly increasing doses up to 15 mg per day in pafc tients with chronic encephalopathy unresponsive to conventional therapy (88 for review). It was suggested that the subjects more likely to benefit from bromocriptine treatment are those with the hepatocerebral (non-Wilsonian) form of hepatic encephalopathy . Treatment of miscellaneous conditions. Preliminary studies suggest that ergoline derivatives-might be of value in the management of mild diabetes mellitus, since improved glucose tolerance was found in puerperal women during suppression of lactation with bromocriptine (89) as well as in patients with chemical diabetes during a short course of metergoline administration (90). However, trials of prolonged treatment have not been performed. Dopamine agonists and metergoline as well may induce postural hypotension after acute administration. The mechanism of this response is thought to depend on drug effects on central nervous system, probably stimulation of dopamine receptors. A few trials of bromocriptine administration in hypertensive patients indeed confirmed that the drug decreases blood pressure in some subjects and that it potentiates the antihypertensive effect of methyldopa (50, 91). Ergoline derivatives, particularly those endowed with antiserotoninergic properties such as methysergide, metergoline and lisuride, have been used with some success in the chronic preventive treatment of migraine (16,92,93). Methysergide and cyproheptadine are frequently effective in controlling the diarrhea and malabsorption of the carcinoid syndrome, probably due to their antiserotoninergic action (92). Central dopaminergic drugs as nomifensine proved to have a good antidepressant activity in depressive illness (94).

516

Side effects of PRL-lowering drugs PRL-lowering drugs are not devoid of side effects. Two major groups of adverse reactions have been described,the first group occurring with the initiation of therapy, and the second during long-term administration. Both groups of side effects also occur during treatment with L-dopa and probably depend on stimulation of dopamine receptors in the central nervous system and in peripheral tissues. The side effects of bromocriptine, the most widely used drug, have been studied more extensively (50 for review), but it appears that many of them also occur with the other PRL-lowering ergoline derivatives. Adverse reactions with initiation of bromocriptine therapy include nausea, vomiting and postural hypotension. The true incidence of these effects has not been established, but all are frequently observed except in puerperal women. However, these reactions are minimized or prevented in most subjects by giving the drug with food and by initiating therapy with small doses, such as 1.25 mg per day in two divided administrations. The dose is then gradually increased by 1.25 or 2.5 mg per day every 2-4 days to attain the optimal dose usually within 1 to 3 weeks. If side effects occur, the dose can be maintained or reduced as required. In some patients the adverse reactions cannot be avoided, but tolerance then develops with continuation of therapy. Adverse reactions with chronic bromocriptine therapy include headache and nasal stuffiness, which are generally mild and often transitory; constipation, which is common with high dose therapy and may be treated symptomatically; dyspepsia, which may occur occasionally with high drug doses; cold-sensitive digital vasospasm,whichisrelatively common with high dose therapy and can be reverted by lowering the dose; alcohol intolerance, increased arousal and leg cramps, which may sometimes occur but do not constitute a major problem. Gastrointestinal bleeding from peptic ulcer has been described in 6 of 96 acromegalics treated with bromocriptine and may be related to the drug's ability to increase gastric acid secretion (95). Other adverse reactions have

517

been reported only in parkinsonism

patients treated with very

high doses of bromocriptine and include dyskinesia, which is probably a manifestation of drug overdosage, psychiatric reactions such as depression, hallucinations, delusions and dementia, which are frequently encountered and necessitate drug withdrawal in most cases, and erythromelalgia, which develops in about 5-10% of subjects. Recently it has been reported that pleuropulmonary changes occur in some parkinsonian patients during long-term treatment (96). Discontinuation of bromocriptine, results in disappearance of side effects usually in a few days,but psychiatric reactions in parkinsonian patients may persist for several weeks. Presently available data suggest that bromocriptine is not teratogenic (43,50). Bromocriptine administration throughout pregnancy also appears not to adversely affect fetal growth (97). Lergotrile, lisuride and pergolide have similar side effects as bromocriptine both on acute and chronic administration, but experience is still limited (6,14,37,47). Metergoline treatment seems to be associated with somewhat different and perhaps less severe side effects which include dizziness, insomnia, nausea and postural hypotension with initiation of therapy, and constipation or, rarely, rhinitis, asthma or postural hypotension on chronic treatment (16,37). This drug, which decreases gastric acid secretion (98), might be preferred to bromocriptine in patients with gastric or duodenal disorders. The availability of different PRL-lowering drugs is important, because some patients who do not tolerate a drug may be treated with another without side effects. In a study with bromocriptine, metergoline and lisuride treatment in 193 hyperprolactinemic women, the initially used agent had to be withdrawn in 12 cases, but changing of the drug allowed PRL-lowering treatment to be continued in 11 of them (37). As reported in previous sections, lergotrile is hepatotoxic (27) and is no longer in clinical use. Another drug with severe adverse reactions is methysergide, which may induce retroperitoneal fibrosis, cardiac and pulmonary fibrosis and pleural effusions (92). The more frequent side effects of cyproheptadine

518

are hyperphagia and somnolence, but they usually subside on chronic treatment (67). Although PRL-lowering drugs may interfere with the secretion of pituitary hormones other than PRL, clinically significant alterations do not occur during chronic treatment (16, 37, 50 for review). It is finally worth of mention the fact that administration of PRL-lowering drugs may result in PRL oversuppression with consequently inadequate luteal phase (99).

Diagnostic and prognostic applications of PRL-lowering drugs Many studies have been performed to evaluate the possible application of acutely administered PRL-lowering drugs in the differential diagnosis of the hyperprolactinemic states and other endocrine disorders. Unfortunately, both pituitary acting dopamine agonists or precursors such as dopamine itself (4-5 ug/Kg/min infused intravenously for 120-240 min), L-dopa (500 mg po) and bromocriptine (2.5 mg po), and selective activation of central nervous system dopaminergic pathways with either L-dopa (100 mg) plus carbidopa (35 mg) administration after pretreatment with carbidopa (50 mg po every 6 hours for 1 day) or nomifensine (200 mg po) failed to discriminate between adenomatous and idiopathic disease (6,20,24,58,100-102). The observation of impaired PRL suppression by direct dopamine agonists in some cases and by brain dopaminergic activation in most cases of both adenomatous and idiopathic hyperprolactinemia indicates the existence of common defects in dopaminergic inhibition of PRL secretion in these conditions and indeed suggests that many patients with hyperprolactinemia of unknown etiology may harbor a pituitary microadenoma too small to be radiologically evident (102). The serotonin antagonists metergoline and methysergide have also been tried as possible tools in the differential diagnosis of hyperprolactinemia, but the results have been disappointing (8, 101). As reported in the section on treatment of acromegaly, direct dopamine agonists, which usually elevate serum GH levels in

519

healthy subjects, are able to inhibit GH release in about half of the patients. Since this effect is confined to acromegalics, the finding of GH suppression by acutely administered dopaminergic drugs is of value in confirming the diagnosis of acromegaly (60). Moreover, the results of acute testing are of prognostic value in this condition, since there is a highly significant correlation between the GH suppression induced by acute and chronic administration of bromocriptine or lisuride (47,60). On the other hand, the acute PRL response to these drugs as well as to metergoline has less predictive value in hyperprolactinemic patients, who sometimes show acute inhibition with escape phenomena on chronic treatment or, less uncommonly, PRL lowering on chronic but not acute drug administration (47, and unpublished data). Acute bromocriptine testing has no predictive value in Cushing's disease due to the high incidence of escape phenomena during chronic treatment (74).

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Leading article : Br. Med. J. 282, 171-172 (1981).

88.

Schenker, S., Desmond, P. V. , Speeg, K.V., Hoyumpa, A. M.: Gastroenterology 7_8, 1 094-1 097 (1 980).

89.

Peters, F. D., Roemer, V. M.: Br. J. Obstet. Gynaecol. 84, 531-533 (1977).

90.

Ferrari, C., Barbieri, C., Caldara, R., Magnoni, V., Testori, G. P., Romussi, M. : Eur. J. Clin. Pharmacol. 1_5, 395-399 (1979) .

91.

Lewis, M. J., Henderson, A. H.: Br. J. Clin. Pharmacol. 9, 57-60 (1980).

92.

Grahame-Smith, D. G.: In: Endocrinology, Eds. De Groot, L. J., Cahill, G. F., Odell, W.D., Martini, L., Potts, J. T., Nelson, D. H., Steinberger, E., Winegrade, A. I., Grüne and Stratton, New York, vol. 3, pp. 1721-1731 (1979).

93.

Herrmann, W. M., Horowski, R., Dannehl, K., Kramer, U., Lurati, K.: Headache 1_7, 54-60 (1977).

94.

Brodgen, R. N., Heel, R. C., Speight, T. M., Avery, G. S.: Drugs 1_8, 1-24 (1 979) .

95.

Caldara, R., Ferrari, C., Romussi, M., Paracchi, A.: 'J. Endocrinol. Invest. 2, 45-49 (1979) .

96.

Rinne, U. K., Krupp, P., Le Witt, P. A., Calne, D. B.: Lancet 1_, 44-45 (1 981) .

97.

Bigazzi, M., Ronga, R., Lacranjian, I., Branconi, F., Buzzoni, P., Martorana, G., Scarselli, G. F., Del Pozo, E.: J. Clin. Endocrinol. Metab. 48, 9-12 (1979).

98.

Caldara, R., Ferrari, C., Barbieri, C-, Romussi, M., Rampini, P., Telloli, P.: Eur. J. Clin. Pharmacol. 1_7' 13-18 (1980). Schultz, K. -D., Geiger, W., Del Pozo, E., Künzig, H. J.: Am. J. Obstet. Gynecol. 132, 561-566 (1978).

99.

525 100. 101.

102.

Gomez, F., Reyes, F. I., Faiman, C.: Am. J. Med. ¿2, 648660 (1977) . Ferrari, C., Travaglini, P., Mattei, A., Caldara, R., Moriondo, P., Romussi, M., Crosignani, P. G.: In: Pituitary Microadenomas, Eds. Faglia, G., Giovanelli, M. A., MacLeod, R. M., Academic Press, London and New York, pp, 399-406 (1 980) . Crosignani, P. G., Ferrari, C., Malinverni, A., Barbieri, C., Mattei, A. M., Caldara, R. , Rocchetti, M.: J. Clin. Endocrinol. Metab. 51, 1068-1073 (1980).

RECEPTOR ANTAGONISTS OF THE ACTIONS OF GASTROINTESTINAL PEPTIDES ON PANCREATIC ACINAR CELLS

Jerry D. Gardner, Robert T. Jensen Digestive Diseases Branch, National Institute of Arthritis, Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20205, USA

Introduction The initial step in the action of a gastrointestinal peptide on its target tissue is reversible binding of the peptide to receptors on the outer surface of the plasma membrane of the target cell.

Interaction of the peptide with its receptors

initiates a series of changes in various cellular biochemical reactions, the last of which is usually referred to as the "response" of the target cell.

This paper reviews what is

known of the agents that will antagonize the interactions of various gastrointestinal peptides with their cell surface receptors on pancreatic acinar cells and by so doing inhibit the peptide-induced stimulation of enzyme secretion.

All agents that have been found to be capable of stimulating pancreatic enzyme secretion appear to act by initiating one of the two functionally distinct sequences of biochemical changes or coupling mechanisms illustrated in Figure 1 (for review see reference 1).

Some secretagogues interact with specific recep-

tors to cause release of cellular calcium and, after a series of undefined steps, stimulation of enzyme secretion.

Other

secretagogues interact with specific receptors to cause activation of adenylate cyclase, increased cellular cyclic AMP, activation of cyclic AMP-dependent protein kinase and, after a series of unknown steps, stimulation of enzyme secretion.

© 1982 Walter de Gruyter & Co., Berlin - New York Hormone Antagonists, Editor M. K. Agarwal

528

SEC RET AGOG UES

PANCREATIC ACINAR CELL

Acetylcholine

CCK

Bombesin

Physalaemin

Cholera Toxin

VlP-Preferring

Secretin- Preferring

Figure 1 Mechanisms of action of secretagogues on pancreatic acinar cells. Secretagogues, by interacting with their membrane receptors, can cause one of two functionally distinct sequences of changes that produce stimulation of enzyme secretion. One sequence is characterized by mobilization and release of cellular calcium; the other involves activation of adenylate cyclase and increased cellular cyclic AMP. There are four classes of receptors that cause mobilization of cellular calcium and three classes of receptors that activate adenylate cyclase. Of the receptors that activate adenylate cyclase, only two produce stimulation of enzyme secretion.

Those secretagogues that act by causing release of cellular calcium do not alter cellular cyclic AMP or the increase in cyclic AMP caused by other secretagogues.

Although the two

different mechanisms for coupling the stimulus to secretion have initial steps that are functionally distinct, these two mechanisms do interact at some presently unknown step.

One

consequence of this interaction is potentiation of enzyme secretion (1,2), that is, the increase in enzyme secretion caused by a secretagogue that increases cyclic AMP plus a

529 secretagogue that increases calcium release is substantially greater than the sum of the effect of each secretagogue acting alone. With the development of techniques for preparing radiolabeled secretagogues of high specific activity, it has become possible to measure directly the interaction of secretagogues with receptors on pancreatic acinar cells and to explore the quantitative relation between the number of receptors occupied and the corresponding changes in cellular function.

In general,

binding of the radiolabeled ligand is reversible, temperaturedependent and to a finite number of sites located on the plasma membrane.

Because there are a finite number of receptors, one

can monitor the interaction of a particular agent with the receptors by measuring that agent's ability to compete with the radiolabeled ligand - the higher the affinity of the secretagogue for the receptor, the lower the secretagogue concentration required to inhibit binding of radioactivity.

From the

standpoint of classifying different pancreatic secretagogues in terms of the repeptors with which they interact, those secretagogues that inhibit binding of a given radiolabeled ligand interact with the same class of receptors, whereas those secretagogues that do not inhibit binding of the ligand do not interact with the same receptors as the ligand.

The availa-

bility of a radiolabeled ligand, however, is not essential for distinguishing different classes of receptors (for detailed discussion see reference 3).

For example, competitive anta-

gonists can be used to distinguish different classes of receptors in that those secretagogues whose actions can be inhibited competitively by a particular antagonist interact with the same class of receptors, Whereas those secretagogues whose actions are not inhibited by the antagonist interact with other receptors.

The classification of receptors that mediate the

actions of adrenergic agents (a and 6), cholinergic agents (muscarinic and nicotinic) and histamine (H^ and Hj) developed primarily from studies using selective, competitive antagonists.

530 By comparing the ability of a secretagogue to inhibit binding of a radiolabeled ligand with the accompanying secretagogueinduced changes in acinar cell function, one can investigate the relation between receptor occupation and the secretagogue-induced response.

In some instances, there is a one-to-one cor-

relation between receptor occupation and secretagogue-induced changes in cell function.

In other instances, however, there

are "spare receptors" that occupation of only a fraction of the receptors is sufficient to produce a maximal secretagogueinduced change in function and occupation of the remaining receptors by the secretagogue is not accompanied by a secretagogue-induced change in cell function. There are 4 classes of receptors that mediate the actions of secretagogues that stimulate pancreatic enzyme secretion by causing mobilization of cellular calcium (Fig. 1).

One class

interacts with muscarinic cholinergic agonists such as acetylcholine and can be antagonized by muscarinic antagonists such as atropine (Table 1).

This class of receptors will not be

considered further because the present paper deals with receptor antagonists of the actions of peptides.

A second class of

receptors interacts with cholecystokinin (CCK), gastrin and caerulein, a decapeptide isolated from the skin of Hyla caerulea (Table 1).

A third class of receptors interacts with

bombesin, a peptide isolated from frog skin that probably has a structurally related counterpart in animals, as well as other naturally occurring, structurally related peptides such as litorin, ranatensin and alytesin (Table 1).

A fourth class

of receptors interacts with physalaemin, substance P, eledoisin and kassinin (Table 1). There are 3 classes of receptors that interact with peptides that increase cellular cyclic AMP in pancreatic acinar cells (Fig. 1).

One class interacts with cholera toxin, whereas the

other two classes both interact with vasoactive intestinal pep-

531 TABLE 1

CLASSES OF RECEPTORS

PANCREATIC SECRETAGOGUES

CLASSES OF RECEPTORS

NATURALLY OCCURRING

CCK

CCK (2 nM) GASTRIN (2 uM) CAERULEIN (0.2 nM)

BOMBESIN

BOMBESIN (4 nM)

AGONISTS (EC50)

LITORIN (40 nM) RANATENSIN (12 nM) ALYTESIN (12 nM) PHYSALAEMIN

PHYSALAEMIN (2 nM) SUBSTITUTE P (5 nM) ELEDOISIN (100 nM) KASSININ (250 nM)

CHOLINERGIC

MUSCARINIC CHOLINERGIC AGONISTS

VIP-PREFERRING

VIP (1 nM) SECRETIN (7 pM) PHI (25 nM)

SECRETIN-PREFERRING

SECRETIN (300 pM) VIP (80 nM) PHI (100 nM)

CHOLERA TOXIN

CHOLERA TOXIN (3 nM)

EC50 refers to the concentration required to occupy fifty percent of the receptors.

532

OH

Bt,cGMP IN'. 0*'-dibutyry1 Qusnotint 3': 5'-mqnophosph*t«i O

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J H

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. C-0

BENZOTRIPT IN p chloro b . n i o v

L Uyptoph.n;

Structures of Bt^cGMP, proglumide and benzotript.

tide (VIP), secretin and other naturally occurring, structurally related peptides such as PHI (Table 1). Of the two classes of receptors that interact with VIP and secretin, one is "VIP-preferring" and has a high affinity for VIP and a low affinity for secretin; the other is "secretin-preferring" and has a high affinity for secretin and a low affinity for VIP (Table 1). Although occupation of either class of receptors by VIP or secretin causes activation of adenylate cyclase and increased cyclic AMP (1), it is only the increase in cyclic AMP mediated by the VIP-preferring receptors that causes stimulation of pancreatic enzyme secretion. What function, if any, is altered by the increase in cyclic AMP caused by the secretin-preferring receptors is not known.

533 CCK RECEPTOR ANTAGONISTS In general, the structural requirements for occupation of a peptide hormone receptor are quite stringent in that only other peptides will bind to the hormone receptor and the peptides that do bind are usually fragments of the hormone or analogues with a similar chemical structure.

One striking exception to

this general pattern occurs with the opiate receptors (4,5). These receptors can interact not only with peptides (enkephalins and other opioid peptides) but also with morphine and structurally related alkaloid compounds.

Two other exceptions

to the general principle that only peptides will bind to peptide hormone receptors are illustrated by the CCK-receptor antagonists (Fig. 2). Serendipitously we found that butyryl derivatives of cyclic GMP will competitively antagonize the actions of CCK on pancreatic acinar cells by inhibiting the interaction of CCK with its cell surface receptors (6,7).

As illustrated in Figure 3-left, in-

creasing concentrations of dibutyryl cyclic GMP (Bt2CGMP) cause a parallel, rightward shift in the dose-response curve for CCKstimulated amylase secretion from pancreatic acini.

With a

fixed concentration of CCK (1 nM), increasing concentrations of Bt2cGMP cause a progressive decrease in CCK-stimulated enzyme secretion as well as CCK-stimulated calcium outflux and these changes can be accounted for completely by the ability of the cyclic nucleotide analogue to inhibit binding of CCK to its membrane receptors on pancreatic acinar cells (Fig. 4-left). In addition to Bt2CGMP, monobutyryl derivatives of cyclic GMP also antagonize the interaction of CCK with its membrane receptors (6,7).

Because native cyclic GMP does not inhibit the

action of CCK, the presence of at least one butyryl moiety appears to be essential for cyclic GMP to inhibit interaction of CCK with its receptors (6,7). In inhibiting the action of CCK, 2 2' N -monobutyryl cyclic GMP is less potent than the 0 -monobutyryl derivative which, in turn, is less potent than the

534

CCK-8 (log M)

Figure 3 Effect of Bt^cGMP (left panel) and proglumide (right panel) on the stimulation of enzyme secretion caused by the C-terminal octapeptides of CCK. (CCK-8 is ten times more potent potent than CCK and has the same efficacy). Results are from reference 8.

Figure 4 Abilities of Bt 2 cGMP, proglumide and benzotrijat to inhibit binding of 1 2 5 I-CCK, CCK-stimulated outflux of Ca and CCK-stimulated amylase secretion. Results are expressed as the percentages of the values obtained with no added antagonist. Results are from reference 8.

535 dibutyryl derivative (6,7). Another class of non-peptide antagonists of the interaction of CCK with its membrane receptors on pancreatic acini are proglumide, a derivative of glutaramic acid, and benzotript, a derivative of tryptophan (8) (Fig. 2). As illustrated in Figure 3right, increasing concentrations of proglumide, like Bt2cGMP, cause a parallel, rightward shift in the dose-response curve for CCK-stimulated amylase secretion from pancreatic acini. With a fixed concentration of CCK (1 nM), increasing concentrations of proglumide or benzotript cause a progressive decrease in CCK-stimulated enzyme secretion as well as CCK-stimulated calcium outflux and these changes can be accounted for completely by the abilities of the antagonists to inhibit binding of CCK to its membrane receptors on pancreatic acinar cells (Fig. 4-center and -left). The abilities of Bt2CGMP, proglumide and benzotript to antagonize the action of CCK is specific for those peptides that interact with CCK receptors (i.e., CCK, gastrin and caerulein). These antagonists do not inhibit the actions of secretagogues that have a mode of action similar to that of CCK but which act through different receptors (e.g., muscarinic cholinergic agonists, bombesin and structurally related peptides, physalaemin and structurally related peptides) and do not alter the actions of secretagogues whose effects are mediated by cyclic AMP (i.e., cholera toxin, VIP, secretin). When pancreatic acinar cells are first incubated with CCK, washed to remove the free peptide and then reincubated in fresh incubation solution, there is significant residual stimulation of enzyme secretion (Fig. 5), and this residual stimulation reflects persistent occupation of CCK receptors by the peptide (9,10).

The magnitude of residual stimulation reflects the

amount of persistently bound CCK which, in turn, is influenced directly by the amount of CCK that is bound during the first

536

2nd INCUBATION (min)

Figure 5 Ability of the C-terminal octapeptide of CCK to cause residual stimulation of enzyme secretion from pancreatic acinar cells and the ability of Bt^cGMP to reverse this residual stimulation. Acini were first incubated for 10 minutes at 4°C with or without CCK-8, washed and reincubated for 60 minutes at 37°C. Amylase secretion is expressed as the percentage of the amylase activity in the cells at the beginning of the second incubation that was released into the extracellular medium during the second incubation. Results are from references 9, 10 and 11.

incubation and inversely by the rate at which the bound peptide dissociates when the acini are washed and reincubated

(9,10).

After induction of residual stimulation by first incubating acinar cells with CCK, adding a CCK-receptor antagonist immediately abolishes this stimulation (Figs. 5 and 6).

Thus, CCK-

receptor antagonists such as Bt2CGMP, proglumide or benzotript not only antagonize CCK-induced stimulation of pancreatic enzyme secretion but also reverse the residual stimulation of enzyme secretion caused by first incubating acinar cells with CCK.

Moreover, the relative potencies with which the various

antagonists reverse CCK-induced residual stimulation are the same as those with which these antagonists inhibit binding of

537

Figure 6 Abilities of Bt 2 cGMP, proglumide and benzotript to reverse the residual stimulation of enzyme secretion caused by the C-terminal octapeptide of CCK. Values for amylase release are expressed as the percentage of the residual stimulation caused by 10 nM CCK-8 alone. Results are from reference 8.

CCK to its receptors and the accompanying change in acinar cell function (compare Figs. 2 and 4).

Finally, the abilities of

CCK antagonists to reverse CCK-induced residual stimulation, like their abilities to antagonize the action of CCK, are fully reversible, and the antagonist-induced reversal of residual stimulation is accompanied by restoration of full responsiveness to CCK (8,9,10,11). The configuration of the dose-response curve for CCK-stimulated enzyme secretion (Fig. 2), and the ability of CCK to induce residual stimulation of enzyme secretion (Fig. 5) coupled with the abilities of CCK-receptor antagonists to prevent the actions of CCK (Figs. 3 and 4) as well as their abilities to reverse CCK-induced residual stimulation of enzyme secretion (Figs. 5 and 6) led us to develop an hypothesis to characterize

538 CONDITIONS

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594 TRH

antagonliti

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Several hundreds of TRH analogues have been synthesized.

Structure-

activity relationships have been extensively studied and it has been well established that agonist activity is highly dependent upon preserving the essential features of the side chain of the three constituent aminoacids (73,74,75,76).

Whereas several analogues with agonist activity have been

synthesized, it has been difficult to obtain antagonists (2,77,78).

Only

very recently, two potent antagonists, capable to block the action of TRH In vivo, have been synthesized (79).

In these TRH derivatives, the C-

terminal amide has been replaced by a diazomethyl or a chloromethyl group. Opioid-peptide

antagonliti

:

The various effects of the opioid peptides can be blocked by morphine antagonists such as naloxone, naltrexone or diprenorphine (3). hundreds of enkephalin analogues have been synthesized (3,80). a small number has been fully tested for antagonist potency. 2

gonists have not been found.

Several So far only Full anta-

N-allyl derivatives of [D-Ala ]-Met-enke-

phalin or Leu-enkephalin are compounds with mixed agonist-antagonist properties (81,82). Recently, p-endorphin fragments antagonizing the analgesic action of pendorphin or morphine have been synthesized.

p-endorphin-[6-31] and p-

endorphin-[20-31] were found to inhibit both morphine and p-endorphin analgesia.

p-endorphin-[l-5] - [16-31] only antagonized morphine analgesia

(83). Vaioptiziiln

antagonliti

:

A series of vasopressin antagonists have become available. structure and potency is summarized in Table 2.

Their peptide

Most of these compounds

not only antagonize vasopressor activity but have also an anti-oxytocic activity.

An important achievement is that some of the most potent anta-

gonists have virtually lost antidiuretic activity. 0-the.i peptide.

antagonliti

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Up to our knowledge, no compounds have been prepared yet which block the action of somatostatin, VIP, neurotensin and substance P.

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598 active plasma concentrations of the DA antagonist perphenazine In vivo correspond to the active concentrations of this drug In vWio

(99).

(95) How-

ever, other data suggest that blockade of anterior pituitary DA receptors may not be the only mechanism underlying the release by DA antagonists.

In vivo

stimulation of PRL

It has been shown that the high plasma PRL

levels in rats obtained by median eminence lesions or treatment with amethyl-p-tyrosine (an inhibitor of DA synthesis) are depressed but cannot be returned to normal by infusing DA at plasma concentrations several times higher than those present on the average in portal blood (20,26).

Yet

treatment with DA antagonists increases plasma PRL levels up to 300 ng/ml or higher (100,101,115,127), which are values equaling those obtained after median eminence lesions or treatment with a-methyl-p-tyrosine (20j26,127). Furthermore, we have recently shown that DA or DA agonists can stimulate PRL secretion in cultured pituitary cells above the level of spontaneous release even in the presence of DA antagonists (123).

Finally, it should

be reminded that various DA antagonists, besides their anti-dopaminergic activity, also interfere with serotoninergic, adrenergic, cholinergic or histaminergic mechanisms (124) and the latter mechanisms are also involved in the regulation of PRL secretion (125).

Thus, in addition to blockade

of anterior pituitary DA receptors, other mechanisms may be responsible for the In vivo

rise of PRL release after treatment with DA antagonists.

The benzamides sulpiride and metoclopramide, for example, enhance PRL secretion In vivo

much more than would be expected from their potency in 3 blocking anterior pituitary DA receptors labeled with H -spiperone (97). With respect to the mechanism of action of DA antagonists in rising PRL secretion, recent observations show that DA is internalized by a DA receptor-mediated mechanism and that DA antagonists decrease internalization (126,127).

The authors speculate that internalization of DA may be re-

quired to elicit its inhibitory effect on PRL release.

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Several groups have demonstrated the presence of specific binding sites using either tritiated agonists (DA, dihydroergocriptine, RU-24213) or tritiated antagonists (haloperidol, spiperone, domperidone) in bovine

599 (31,32,33,34), rat (30,35,38), sheep (37), steer (37) and human pituitaries (39), including PRL cell adenomas (36,39). DA antagonists compete with a 3 3 similar high affinity with both H -DA and H -spiperone binding sites. In contrast, DA and DA agonists of the catechol series, such as apomorphine, 3 ADTN or RU-24213, display a much higher competition affinity for H -DA 3 sites than for H -spiperone sites. DA agonist of the ergot-alkaloid series, 3 such as bromocriptine and dihydroergocriptine behave in competing with H 3 spiperone or the H -catechol-like agonist RU-24213 binding like DA antagonists do.

Based on biphasic displacement curves by agonists, it has 3 been proposed that catechol-like DA agonists displace H -antagonists from two different binding sites (possibly two different receptors), one for which agonists have high affinity, another for which they have low affinity (128).

Antagonists have equal high affinity for both sites. Others,

on the other hand, propose a unitary model (129).

The DA receptor complex

would consist of 3 subunits, one on which agonists bind with high affinity and two on which antagonists bind.

The model postulates cooperative

phenomena induced by antagonist binding which would dramatically decrease the binding affinity for catechol-like agonists. Influence

o{,

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:

It is well known that DA antagonists increase PRL release to a higher extent in women than in men (95,112,120,130).

This sex difference seems to

be based on a direct effect of estrogens on pituitary PRL release. Estrogen treatment causes a profound desensitization of dopaminergic inhibition of PRL release in rat pituitary cell monolayer cultures (131,132) whereas vivo

In

administration of estrogens to ovariectomized rats causes a 4-fold

higher increase of plasma PRL levels

upon

treatment

gonist thioproperazine than in controls (132).

with the DA anta-

The underlying mechanism

does not appear to be a change in the number or affinity of DA receptors in the pituitary but at a step subsequent to binding of DA to its receptor (35).

Receptor-mediated internalization of DA by PRL cells is sig-

nificantly inhibited by estrogen (133,134).

600

Paradoxical

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o^ PA antagonists

:

Several groups of investigators observed that DA antagonists at relatively high concentrations inhibit PRL release from rat pituitary cell cultures (32,99,103,135).

Pimozide, clopimozide and penfluridol were among the

most potent in this respect (32,99,103).

Similar inhibitory actions have

been found as far as 6H (137) and LHRH-stimulated LH secretion are concerned (103,136).

Also insulin secretion from isolated islets is inhibited

by these compounds (138).

Although several investigators have claimed the

effect was due to a partial agonist nature of DA antagonists (32,135), others have shown that the effect is caused by an interaction with Ca + + dependent mechanisms linked to the stimulus-secretion coupling (103) and not related to DA receptor activation (137).

In fact, high concentrations

++

of these drugs also antagonize Ca -dependent smooth muscle contractions of the caudal artery of the rat (103).

Moreover, it has been suggested

that pimozide and trifluoperazine inhibit in

vitno

glucose-induced

insulin release by interfering with the action of calmodulin (138).

Acknowledgements : The authors wish to thank Miss M. Bareau for excellent secretarial assistance.

Personal work mentioned in this chapter was sustained by grants

from I.W.O.N.L., F.G.W.O. and the Onderzoeksfonds, K.U.Leuven.

RIA-kits

were obtained through the U.S. National Pituitary Agency, NIAMDD, Bethesda, Maryland.

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LUTEINIZING HORMONE RELEASING HORMONE ANALOGS AS ANTIHORMONES

Frederick Bex and Alan Corbin Endocrinology Section, Wyeth Laboratories, Inc. Box 8299, Philadelphia, PA 19101 U.S.A.

Introduction Although the ultimate pattern of pituitary gonadotropin secretion ife the result of a complex interaction of a variety of factors, primary regulatory control is provided by the hypothalamic decapeptide, luteinizing hormone releasing hormone (LHRH).

In view of the crucial role performed by

this peptide in normal reproductive function, its availability, made possible by historic isolation, characterization and synthetic efforts (1,2, 3,4,5), was thought to provide a potentially significant means for the diagnosis and treatment of hypothalamically related hypogonadal conditions. While useful as an adjunct to existing methods for differentiating hypothalamic from pituitary dysfunction, LHRH has yet to achieve the therapeutic success that was anticipated.

Since, initially, these discouraging

clinical results were presumed to be a function of the extremely short in vivo half-life of LHRH, attention was directed towards synthesizing derivatives which would mimic the parent compound's LH-releasing property (agonists) but possess prolonged functional activity and/or increased potency. An integral part of these synthetic efforts originally had involved the identification of structures which lacked LHRH activity (LH release) but retained the ability to compete with LHRH for binding to its site(s) of

pyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 1 2 3 4 5 6 7 8 9 10 Amino acid sequence of LHRH

© 1982 Walter de Gruyter & Co., Berlin • New York Hormone Antagonists, Editor M. K. Agarwal

610 action.

Theoretically, these compounds (antagonists) would inhibit endog-

enous regulation of pituitary gonadotropic function and thereby offer a potentially effective new method for the control of fertility through ovulatory blockade. Exceptionally productive peptide programs have resulted in reports on nearly 1000 analogs of LHRH, including very potent and long-acting agonists ("superagonists") as well as a rapidly expanding list of antagonists. However, the extensive biological testing accompanying these developments has revealed the pharmacological nature of the LHRH class of peptides and the processes that they control to be far more complex than originally predicted.

This has, in turn, led to dramatic changes in concepts, con-

cerning the application of these compounds.

In this regard, it has been

demonstrated in a variety of laboratory species of both sexes, and recently in the clinic as well, that LHRH and, to a greater extent, its agonistic analogs, possess very potent antifertility properties (for recent review see 6,7).

While this activity appeared to contradict the integral

role that the parent molecule performs in normal reproductive function, and was for this reason termed "paradoxical", recent evidence has allowed partial resolution of the conflicting pro/anti-fertility classification of LHRH and LHRH agonists.

Inappropriate patterns and levels of pituitary LH

secretion induced by these compounds have been found to inhibit, in terms of functional activity (desensitization) and actual numbers (downregulation), gonadal LH receptors as well as receptors for other pituitary hormones, FSH and prolactin, thereby disrupting pituitary dependent-gonadal steroidogenesis.

Moreover, specific binding of LHRH and its analogs to

the gonads has been identified and this, also is associated with steroidogenic inhibition.

Thus, there are definite but as yet not completely de-

fined "physiological" limits for LHRH or LHRH-like analogs which if exceeded, result in inhibition of the reproductive process.

The identifica-

tion of this property has provided the basis for an LHRH agonist approach to contraception now undergoing extensive clinical investigation and has also served to redirect strategies for the therapeutic use of these compounds towards achieving more physiologically appropriate levels and patterns of gonadotropins.

611 While the following review is primarily concerned with the subgroup of the LHRH class of peptides which fulfills the classical definition of an antagonist, where appropriate, comparisons will be made with the current status of the agonists which, in broader perspective and in terms of practical utility, can be considered as antagonists of the normal physiological process controlled by endogenous LHRH.

Identification and Development of LHRH Antagonists Crucial to LHRH antagonist development, which historically represented the first goal of investigations into the structure-activity relationship of the parent compound, was the demonstration that the binding and gonadotropin-releasing portions of the peptide could be selectively dissociated. Deletion of histidyl in the second position was the first modification identified as producing a compound retaining the ability to reversibly compete with LHRH but possessing reduced intrinsic LH-releasing activity (8-,9).

This was followed by a series of analogs involving the substitu-

tion of various amino acids in position 2, the most promising of which appeared to be D-Phe^-LHRH(IO).

Further improvement in antagonist activi-

ty occurred as the result of combining second position modifications with substitutions of certain D-amino acids (e.g. Ala, Leu, Trp, Phe) in the 6 position.

The elimination of the 10 position glycine yielding a C-termi-

nal Pro' ethylamide (Fujino modification)(11,12,13,14), for the purpose of increasing receptor binding and resistance of the peptide to enzymatic degradation actually led to reduced activity.

This is in contrast to the

increased potency produced by both the 6 and 10 position modifications in the superagonist class of LHRH analogs.

Representatives of the doubly substituted (positions 2 and 6) antagonists were the first to be evaluated for their ability to inhibit spontaneous reproductive events (i.e. ovulatory LH surge, ovulation, cyclicity, gestation) .

Both iji vitro and jji vivo evaluations had relied exclusively on,

and still overly emphasize, models which measure the ability of potential antagonists to prevent LH release in response to exogenously administered LHRH.

Such test systems have included cultured pituitary cells(9), iso-

612 lated whole rat pituitaries(15), ovariectomized, steroid-blocked rats(12, 13,16,17,18) immature male rats(9,13), adult, castrated, estrogen-treated male rats(9), and intact adult male and female rats(16).

However, inhibi-

tion by the antagonists of a single precisely timed artificial LHRH stimulus, as identified in these models, does not necessarily reflect potency in countering the spontaneous, dynamic release of LHRH in the normal cycling animal.

In fact, it has since been established that not all com-

pounds exhibiting high jLn vitro antagonist potency have significant antireproductive activity(19,20). 9

In this regard, D-Phe2-D-Ala6[DesGly10]-

2

Pro -NHEt-LHRH and D-Phe -D-Ser6-LHRH, both potent inhibitors of LHRH-induced LH release iji vitro, were unable to prevent ovulation at high dosages(6).

The difficulty in achieving ovulatory inhibition, and the dis-

crepancy with iji vitro evidence, was most likely due to the fact that only a fraction of the proestrous gonadotropin surge is required for ovulation, thereby necessitating quite extensive and prolonged inhibition of its endogenous LHRH stimulus(21).

The importance of this limitation was further

illustrated by the report that only minimal effects on the ovulatory rate 9 f\ in hamsters were produced by the compound D-Phe -D-Leu -LHRH, at dosages that almost completely suppressed the pre-ovulatory LH and FSH surge(22). However, a similar LHRH derivative in terms of structure and in vitro activity, D-Phe^-D-Ala^-LHRH, represented the first antagonist for which antireproductive activity was directly established.

D-Phe2-D-Ala^-LHRH

was found to significantly depress the pre-ovulatory, proestrous gonadotropin surge and completely inhibit ovulation in normally cycling unanesthetized rats and, upon pre-coital administration, prevented pregnancy in subsequently inseminated recipients(14,23,24).

Administration of this

peptide on the immediately preceding metestrus or diestrus also suppressed the proestrous LH surge and ovulation(25).

These extended effects appear-

ed not to be the result of the prolonged active half-life of D-Phe2-DAla^-LHRH but possibly to a neural component subserving its anti-ovulatory activity resembling that observed for the barbiturates.

This concept was

supported both by the fact that a mating stimulus could override the metestrus - or diestrus (not proestrus) administered ovulatory block and by the ability of D-Phe^-D-Ala^-LHRH to inhibit the release of endogenous LHRH at the hypothalamic level(26) .

613 Although the demonstration of the functional activity of D-Phe2-D-Ala6LHRH was a significant and encouraging step in antagonist development, the dosage and delivery requirements (12-15 mg/kg given over a 2.5 hour perio6-LHRH sub-

might be amenable to modification(19).

stituted in position 1 with either Acetyl(Ac)-dehydro-Pro, Ac-D-Ala, AcD-Phe or D-pyroGlu resulted in the most potent and long acting antagonists that had yet been reported(34,35,36,37) .

D-pyroGlul-D-Phe2-D-Trp3»6-LHRH

was capable of completely inhibiting ovulation when administered as a single subcutaneous injection just prior to the proestrous ovulatory gonadotropin surge (200-250 yg/rat) or given either earlier that morning (500 yg/rat) or on the afternoon of the preceding day (1500 yg/rat)(37).

More-

over this was the first antagonist for which post-coital administration was shown to inhibit pregnancy in rats(38). The identification of antagonists of the potency of D-pyroGlu^/Ac-Prol-DPhe2-D-Trp3>6-LHRH required nearly eight years of remarkably comprehensive and creative synthetic effort and resulted in literally hundreds of analogs including such novel structures as those containing more than ten residues (N-terminal extensions)(39), those incorporating indomethacin, aspirin, or additional N-terminal peptide sequences (branched chain analogs) (40,41) , cyclic structures(42,43), as well as those in which the amide bond was replaced with CH2S at various sites(44).

The information

concerning the conformational and functional aspects of LHRH structure revealed by these studies has greatly facilitated continuing efforts to improve antagonist design.

This has been evidenced recently by the identi-

fication of certain additional modifications which have resulted in antagonists possessing sufficient potency to enable the extensive pre-clinical and clinical testing necessary to realistically evaluate their prospective utility as fertility regulating agents.

Notably, it has been demonstrated

on the basis of an earlier observation(24) that halogenation of the second position Phe in the tetra-substituted (positions 1,2,3, & 6) antagonists dramatically improved their activity.

Approximately 60 yg of Ac-D-Phe^-

3

D-pCl-Phe^-D-Trp »6-LHRH completely inhibited ovulation in rats when administered immediately prior to the gonadotropin surge(41).

Even greater

anti-ovulatory potency (in the range of 10-25 yg/rat) has been reported for a variety of similar structures including Ac-Gly-*--D-pCl-Phe2-D-Trp3» LHRH(45), Ac-D-Phe1-D-pCl-Phe2-D-Trp3'6-D-Ala10-LHRH, Ac-D-pCl-Phe 1 » 2 ^Trp3-D-Phe6-D-Ala10-LHRH(46), Ac-dehydroPro1-[pF or pCL]-D-Phe2-D-Trp3»6LHRH, and Ac-dehydro-Pro1-pCl-D-Phe2-D-Trp3»6-Na-MeLeu7-LHRH(47).

The

615 ability of Ac-dehydro-Prol-pCl-Phe2-D-Trp3,6-N6-LHRH, was used(74).

However, this

compound was ineffective in blocking ovulation when the treatment overlapped the initiation of the preovulatory surge.

The requirements for

large amounts of compound, prolonged treatment regimens and precise timing, as evident in these studies, present impractical limitations on development of the antagonist approach to contraception.

Hopefully, the

considerably more potent antagonists presently available will provide more encouraging prospects. Clinical studies In men, a massive single intramuscular injection of 90 mg of D-Phe2-DTrp^-D-Phe^-LHRH effectively attenuated the gonadotropin response to exogenous LHRH(32).

Similar dosages of D-Phe2-D-Trp3-D-Phe6-LHRH administered

to women on day 12-14 of the cycle reduced the gonadotropin response to LHRH and also blocked ovulation and disrupted cyclicity(46). 1

A prelimi-

2

nary study with the more potent analog, Ac-D-Phe -D-pCl-Phe -D-Trp3,6-LHRH revealed that doses of 10 mg given on day 12 of the cycle blocked ovulation in two of the four women tested(46).

Concluding Remarks In contrast to the limited information concerning the effects of the antagonists in humans, the agonist approach to contraception has undergone extensive clinical investigation and is in a relatively advanced stage of development(6,7).

The agonists presently available, generally involving

modifications in the 6 and 10 positions of the parent compound (e.g. D-Ser (TBu)6-Des-Gly10-Pro9-NHEt-LHRH, D-Trp6-Des-Gly10-Pro9-NHEt-LHRHX ha« been found to produce rapid luteolysis, progesterone decline and premature onset of menses following short term administration during the early and mid-segments of the luteal phase.

This potential post-ovulatory luteal

phase approach to contraception has been complemented with drug administration over an entire menstrual cycle, leading to inhibition of ovulation, shortened luteal phase and subsequent menstruation.

The doses re-

quired to produce these effects are quite reasonable (ug quantities) and the efficacy of the nasal spray mode of drug delivery has provided a sim-

619 pie and practical means of administration.

Extensive toxicologic, patho-

logic and ancillary pharmacologic studies, as well as a long clinical record, indicate that the agonists possess an excellent therapeutic margin. It is ironic, not only that substances originally thought of as promoting fertility are now being developed as fertility regulators, but that progress towards this latter end has far surpassed that of the LHRH antagonists, which represented the initial LHRH subclass to receive attention foy this purpose.

While it is hoped that the recent identification of an

extremely potent generation of antagonists will allow appropriate clinical evaluation, extensive reproductive, ancillary pharmacologic and safety testing must be performed before this class of LHRH derivatives can realistically be considered as the basis for a new method of birth control.

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Endocrinology 108, 1998-2001 Horm. Metab.

Biol. R e p r o d .

ANTIGONADOTROPIC, ANTISTEROIDOGENIC AND ANTISTEROIDAL ACTIVITIES OF AGONIST ANALOGS OF LUTEINIZING

HORMONE-

RELEASING HORMONE AS REVEALED BY THEIR ANTIREPRODUCTIVE ACTIVITIES.

Brian H. Vickery Department of Physiology, Institute of Biological Sciences, Syntex Research, 3401 Hillview Avenue, Palo Alto, California 94304, USA.

Since 1975 a growing volume of literature has dealt with actions of luteinizing hormone-releasing hormone

(LHRH)

agonists which are at variance with the known physiological function of the hormone.

LHRH was known to stimulate

gonadotropin synthesis and release at the level of the pituitary and, therefore, indirectly to mediate puberty in both sexes, folliculogenesis and ovulation in females and spermatogenesis hormone

in males.

The gonadotropins,

(LH) and follicle stimulating hormone

luteinizing (FSH), are

also responsible for gonadal steroidogenesis with the steroids produced in response to the gonadotropins both in maturation and function of the reproductive

acting tracts

and in development of secondary sexual characteristics sexual

and

behavior.

Systemic administration of amounts of LHRH agonists which are substantially greater than are needed to stimulate gonadotroph in release have been shown to delay onset of puberty in both sexes, inhibit ovulation, luteal and pregnancy in females and spermatogenesis

function

in males, and

to have a suppressive effect on gonadal steroid production

© 1982 Walter de Gruyter & Co., Berlin • New York Hormone Antagonists, Editor M. K. Agarwal

624

and a c t i o n

in b o t h s e x e s .

unexpected,

These

findings were

b e e n t e r m e d the p a r a d o x i c a l e f f e c t s of L H R H Investigation agonists

initially

r e m a i n to a l a r g e e x t e n t u n e x p l a i n e d

and

agonists.

into the a n t i r e p r o d u c t i v e e f f e c t s of

is r e v e a l i n g c o m p l e x

a g e n t s and g o n a d o t r o p i n

inter-reactions

and s t e r o i d

LHRH

between

r e c e p t o r s , and

inter-reactions,

together

w i t h the d e t e c t i o n of

these

even

e f f e c t s e x e r t e d upon s t e r o i d o g e n i c e n z y m e s y s t e m s . m a t e r i a l s at p o i n t s d i s t a n t

have

These

LHRH-like

from the h y p o t h a l a m u s , s u c h

the g o n a d s and the p l a c e n t a ,

as

are r e q u i r i n g a r e d e f i n i t i o n

of

the f u n c t i o n of L H R H .

E f f e c t s in F e m a l e s .

T h e e a r l i e s t hint of the a b i l i t y of L H R H to i n t e r f e r e reproductive processes B a n i k and G i v n e r

in f e m a l e s c o m e s from the d a t a of

(1) w h o d e m o n s t r a t e d

t h r e e d a y s in rats p r e v e n t e d m a t i n g

that i n j e c t i o n

and c o n c e p t i o n .

w a s e x p l a i n e d as b e i n g due to d i s r u p t i o n of c y c l e s i n d u c t i o n of o v u l a t i o n at i n a p p r o p r i a t e estrous cycle. explain. noted

Subsequent

s t a g e s of

E v i d e n c e of c h a n g e s

in c i r c u l a t i n g

documented post-coital contraceptive J o h n s o n et al

This by

the

steroids

(2)

and u t e r o t r o p i c

a n a l o g , D - L e u ^ L H R H e t h y l a m i d e , to d e l a y v a g i n a l and i n h i b i t n o r m a l o v a r i a n g r o w t h

was effects

potency opening

in the i m m a t u r e

i n h i b i t n o r m a l e s t r o u s c y c l i c i t y and c a u s e

rat a n d

to

reversible

a t r o p h y of the o v a r i e s and u t e r u s of the a d u l t

rat.

I n h i b i t o r y e f f e c t s of L H R H a g o n i s t s on the o v a r i a n

response

in rats w e r e t h e n d o c u m e n t e d

for the first time an e x t r a p i t u i t a r y

to

which

(3) t h e n s h o w e d the h i g h

to e x o g e n o u s g o n a d o t r o p i n

every

findings were more difficult

in the r e p o r t of C o r b i n and B e a t t i e

of L H R H .

with

site of a c t i o n of

and these

625 agents was revealed by duplication of the suppressive effects of the LHRH agonists in hypophysectomized (4).

animals

A similar lack of an obligatory role of the pituitary

in the postcoital contraceptive actions of these agents has now been demonstrated

(5, 6).

Ovulation inhibition with LHRH agonists has been established in rats (7-11), dogs (12), cattle Table 1) and women

(13), monkeys

(14-18,

(19-26).

Table 1. Effect of Daily Intramuscular Injection of DNal(2)^LHRH on Occurrence of Ovulation in Rhesus and Cynomolgus Monkeys. Rhesus

Dose pg/day

No. of Animals

Cynomolgus

No. of Ovulations Per Treated Cycles

No. of Animals

No. of Ovulations Per Treated Cycles

5

15/15

3

8/8

0.31

3

6/9

3

0/8

1.25

3

1/9

5.00

3

0/9

20.00

2

0/6

80.00

3

0/9

The ovulation inhibition and associated continuous diestrus in rats at low dose levels, (7, B. Vickery, G. McRae, unpublished), appears to be due to a luteotropic effect as it is associated with increased ovarian weight and enhanced progesterone secretion.

It was suggested

(7), although not

substantiated, that there had been a shift from predominant estrogen to progesterone synthesis.

At higher dose levels

(8) however, or following twice daily administration

(9),

ovarian and uterine weights are markedly decreased, plasma

626 e s t r a d i o l l e v e l s can be r e d u c e d rats and p l a s m a p r o g e s t e r o n e dose-related

fashion.

to those in

ovariectomized

l e v e l s are r e d u c e d in a

T h e s e e f f e c t s are m a n i f e s t

in s p i t e

of i n c r e a s e d l e v e l s of g o n a d o t r o p i n s w h i c h m a y be

elevated

to the range n o r m a l

for o v a r i e c t o m i z e d

animals

(8, 9).

In

f a c t , the e l e v a t e d l e v e l s of g o n a d o t r o p i n h a v e led to the oft-quoted

t h e o r y that the g o n a d a l and s t e r o i d e f f e c t s

due p r i m a r i l y receptors

to d o w n - r e g u l a t i o n of g o n a d a l

(27-31)

as has b e e n S h o w n for other

h o r m o n e s and their

r eceptors

(32)•

e v i d e n c e , h o w e v e r , have p o i n t e d

protein

S e v e r a l l i n e s of

to the c o n c l u s i o n

is not the o n l y m e c h a n i s m of a c t i o n i n v o l v e d . l e v e l s of D A I a L H R H e t h y l a m i d e w h i l e c a u s i n g circulating

l e v e l s of LH i n d i s t i n g u i s h a b l e

p r o v o k e d by higher shown

Low

(9, T a b l e

In a d d i t i o n , c o n t i n u o u s

from

l e v e l s but d i d c a u s e the same a n t i g o n a d a l

circulating

as o n c e or

animal

and

twice d a i l y

injection.

in v i t r o of

(34, 35),

The direct

i n c l u d i n g e s t r a d i o l and p r o g e s t e r o n e (34, 35, F i g .

by a n t a g o n i s t i c a n a l o g s

1) w h i c h c a n be

(38) has not yet b e e n

The direct gonadal

first

through specific receptors some paracrine

to F S H

synthesis,

shown mediated

for an L H R H - l i k e m a t e r i a l

function.

of

inhibition

antagonized

a c t i o n s are p r o b a b l y

w h i c h m a y e v e n be p r o d u c e d l o c a l l y subserve

f o l l o w e d by

rat g r a n u l o s a cell s t e r o i d o g e n i c r e s p o n s e

by L H R H a g o n i s t s in v i v o .

agonists

(28, 31), and then the d e m o n s t r a t i o n ,

their d i r e c t g o n a d a l a c t i o n stimulation,

(4) w a s

from

gonadotropin

T h e d i s c o v e r y of a n t i g o n a d o t r o p i c e f f e c t s of L H R H in the h y p o p h y s e c t o m i z e d

been

2).

(33) was not a s s o c i a t e d w i t h e l e v a t e d

the s u g g e s t i o n

dose

those

a d m i n i s t r a t i o n of a g o n i s t

antisteroidogenic effects

this

elevated

to c a u s e c y s t i c o v a r i e s , and no e f f e c t on

pellets

that

d o s e l e v e l s of the a g o n i s t , h a v e

l e v e l s of p r o g e s t e r o n e

are

gonadotropin

(42, 43) and

(36-41)

perhaps

627 Table 2. Effect of Twice Daily Administration of D A l a 6 LHRH ethylamide to Female Rats Upon Circulating Levels of LH and Progesterone. 3 Mean LH per mg

Mean Serum Level + S.E. Dose No. of yg/day Animals

-

0.08

10 10

Progesterone ng/mL 18.1 + 1.6

34.5 +

16.0 + 1.6

10

4.9 + 0 . 4 b

2.00

10

6.4 + 0.6

b

10.00

10

7.2 + 1 . 4 b

0.40

LH ng/mL

Pituitary yg 16.3 + 1.8

5.3

172.6 + 2 3 . 6

b

7.9 + 7.9°

232.0 + 20. 3

b

7.4 + 0 . 5 b

210.6 + 12.3

b

7.5 + 0. 4 b

249 .6 + 22.8 b

6.2 + 0. 3 b

Endocrinology a = McRae, G . , Vickery, B.H.: b = p < 0.001 compared to control c = p < 0.01 compared to control Ovulation

104, 182A

(1979)

inhibition by these agents in primates is also

associated with lowered estradiol levels ranging from the equivalent of early follicular phase levels in rhesus Fig. 2), to a 22% decrease in amenorrheic menstruating women

(44,

versus

(23) , to a "severe suppression"

in 3 of 5

women receiving 14 daily injections of 10 yg of DSer(BUT) 6 LHRH ethylamide

(24).

Although a fall in

circulating gonadotropin levels is routinely observed primates

(16, 44), including women

in

(25) treated with LHRH

agonists, other levels of antagonism appear to be involved in the suppression of ovulation as neither

estrogen

challenge in stumptailed macaques

exogenous

gonadotropin challenge in rhesus

(17) nor

(44) provoke their

normal

responses. On the basis that LHRH agonists interfered with ovulation and steroidogenesis, it was proposed

that these agents might

628

Fig. 1. Effect of Various Concentrations of LHRH or D N a l ( 2 ) 6 L H R H on FSH-stimulated Estrogen Production by Rat Granulosa Cells in vitro (A.J.W. Hsueh, B.H. Vickery, unpublished). Methods used were as described by Hsueh et al (35) .

A D N a l i 2 ' 6 LHRH

A

3000

LHRH

2000

1000

FSH

10"

10"10

10"9

10"8

PEPTIDE CONCENTRATION

10"

M

be useful in treatment of steroid-dependent neoplasms of the female reproductive system.

The potent analog DLeu^LHRH

ethylamide was used to test this proposal in female rats bearing either a large spontaneous mammary tumor or tumors induced with dimethyl-benzanthracene

(45).

Significant

tumor regression was obtained in either case.

The work was

confirmed using D S e r ( T B U ) 6 , A z a G l y 1 0 L H R H which was shown to be as effective as either ovariectomy or the antiestrogen tamoxifen in decreasing the number of newly formed

tumors

and in regressing pre-existing tumors, when the tumors were estrogen-receptor

positive

(47, 48).

Serum estradiol

levels

in this case were shown to be lowered by the treatment, and in keeping with this, only equivocal data were obtained on tumors which were not estrogen-receptor positive.

A similar

629 Fig. 2. Effect of Chronic Daily Injection of V a r i o u s Dose Levels of D N a l ( 2 ) 6 L H R H to Female Rhesus Monkeys on Their Peripheral Serum Levels of Estradiol (B.H. V i c k e r y , G.I. McRae, unpublished). 320 300

Control #443

280 260 240 E

220

Q.

200

"> 5> ENDOCRINE HORMONE: HYPOTHALAMUS PLACENTA

PARACRINE HORMONE: GONADS MALE ACCESSORY ORGANS UTERUS PLACENTA ADRENAL TUMORS

GONADOTROPIN SYNTHESIS/RELEASE

GONADOTROPIN MODULATION STEROIDOGENESIS RECEPTOR MODULATION IMMUNE SUPPRESSION

Acknowledgements

I am grateful to Ms. Georgia McRae and to Dr. Jessie C. Goodpasture for their patient attempts to improve the readability of this manuscript.

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67A

(1979)

SECTION 3: MISCELLANEOUS HORMONES AND ANTAGONISTS

HORMONAL INHIBITION OF ADENYLATE CYCLASE: A POSSIBLE FOR PHYSIOLOGICAL

L e e E. L i m b i r d , V a n d e r b i l t U n i v e r s i t y S c h o o l of D e p a r t m e n t of P h a r m a c o l o g y , N a s h v i l l e ,

Medicine,

Tennessee

The delicate balance which characterizes physiological

MECHANISM

ANTAGONISM

regulatory mechanisms results

the control

from the

appro-

priate interplay between hormones mediating activating inhibitory responses.

of

and

It is c l e a r t h a t a v a s t n u m b e r of

hor-

mones and drugs mediate their effects via stimulation of

mem-

brane-bound adenylate cyclase and elevation of CAMP concentrations.

More recently

intracellular

it h a s b e e n

appreciated

t h a t a l a r g e n u m b e r of h o r m o n e s a n d d r u g s , a c t i n g v i a tors discrete

from those which stimulate adenylate

a r e c a p a b l e of a t t e n u a t i n g b a s a l a n d p a r t i c u l a r l y stimulated adenylate cyclase activity. m o n e s h a v e b e e n t a b u l a t e d by J a k o b s receptors

recep-

cyclase, hormone-

These inhibitory

(1) a n d i n c l u d e

in t h e h e a r t , o p i a t e r e c e p t o r s

hor-

muscarinic

in b r a i n a n d

cultured

c e l l s a n d c t 2 - a d r e n e r g i c r e c e p t o r s in a d i p o s e a n d p l a t e l e t s . should be c l a r i f i e d that inhibition of adenylate however,

is n o t a l w a y s c o u p l e d to i n h i b i t i o n o f

function.

cyclase, physiological

F o r e x a m p l e , a 2 - a d r e n e r g i c a t t e n u a t i o n of

cyclin-stimulated adenylate cyclase

prosta-

in h u m a n p l a t e l e t s

c o m i t a n t w i t h a c t i v a t i o n of p l a t e l e t a g g r e g a t i o n . avoid confusion,

It

inhibition and attenuation will

Thus,

to

refer

t h r o u g h o u t t h i s c h a p t e r to d i r e c t e f f e c t s o n a d e n y l a t e c a t a l y t i c a c t i v i t y s t u d i e d in b r o k e n c e l l s u n l e s s

is c o n -

cyclase

otherwise

indicated. S e q u e n c e of E v e n t s in H o r m o n a l A c t i v a t i o n o f A d e n y l a t e

Cyclase

To u n d e r s t a n d the rationale of c u r r e n t experimental p r o a c h e s to e l u c i d a t i o n of h o r m o n a l cyclase,

i n h i b i t i o n of

it is h e l p f u l to r e c a l l t h e c u r r e n t

conceptualization

of t h e s e q u e n c e o f e v e n t s in h o r m o n a l a c t i v a t i o n o f

© 1982 Walter de Gruyter & Co., Berlin • New York Hormone Antagonists, Editor M. K. Agarwal

ap-

adenylate adenylate

662 cyclase (2).

Hormone mediated

transmembrane signaling in-

volves at least three separate membrane components:

the spe-

cific receptor for hormones or drugs (R), the catalytic moiety responsible for synthesis of ATP to cyclic AMP (C), and a GTP binding regulatory protein (s) (G) responsible for mediating the effects of GTP on receptor-agonist interactions and expression of catalytic activity (3).

The molecular interac-

tions among these components have been explored in most detail in catecholamine-sensitive adenylate cyclase systems, since the availability of both B-adrenergic agonists and antagonists has allowed investigators to focus on those molecular events uniquely promoted by agonist occupancy of the B-adrenergic receptor and not mimicked by antagonist occupancy of the receptor.

Agonist occupancy of the B-adrenergic receptor

results in the promotion or stabilization of an R-G complex (4,5), and probably provides the molecular interaction responsible for agonist-induced release of GDP from G (6).

Occupan-

cy of the now unoccupied G by GTP results in dissociation of the R-G complex (5) and the stabilization of G-C interactions (7), resulting in the synthesis of cAMP (8).

It should be

pointed out that the synthesis of cAMP from the physiological substrate, ATP*Mg ++ , requires the concerted effort of the G-C complex (8).

Synthesis of cAMP persists until the G-C complex

is destabilized (7) by hydrolysis of GTP to GDP (9).

Modula-

tion of receptor-agonist interactions by GTP and appearance of so-called receptor "affinity states" can be understood in light of the above scheme (10).

Thus, the receptor in asso-

ciation with G, i.e. the R-G complex, possesses a higher affinity for agonists than the GTP-dissociated receptor, R. Receptor-antagonist interactions are independent of the state of physical association of R with G.

Hence, in target mem-

branes washed reasonably free of endogenous guanine nucleotides, agonist competition curves for radiolabeled antagonist binding are shallow (pseudo Hill coefficient < 1), indicating a heterogeneity of "affinity states" of the receptor (i.e. RG H T , R T N ) for agonists.

In the presence of GTP, these

663 agonist

competition curves are shifted to the right and to a

"normal steepness", indicating that the agonist is now interacting with a homogeneous population of receptor affinity states (i.e. R dissociated from G) possessing a lower affinity for agonist (10) . The scheme described above is certainly an over-simplification of what will ultimately be understood about hormonal activation of adenylate cyclase, but provides a skeleton onto which extant data in the literature can be attached.

The

recent purification of G from rabbit liver indicates that this protein contains three non-identical subunits of 52,000, 45,000 and 35,000 Mr, and that changes in the stoichiometry of these subunits may correlate with or be responsible for changes in expression of adenylate cyclase activity (11). Hormonal Inhibition of Adenylate Cyclase Activity In much the same way that the primary role of hormones in stimulating adenylate cyclase activity is felt to be to facilitate the access of GTP to G and hence to increase the rate of G G T p - C encounters, a reasonable number of investigators feel that hormonal attenuation of adenylate cyclase is also a reflection of the ability of the inhibitory hormones to facilitate the interaction of GTP at the GTP binding regulatory protein(s) mediating decreases in catalytic activity

(12,13).

Thus it is helpful to summarize the inhibitory effects of GTP on adenylate cyclase activity. The investigation of the bimodal effects on adenylate cyclase activity has been explored most rigorously in the rat adipose system (12).

At low concentrations, GTP enhances

basal and hormone-stimulated adenylate cyclase activity, whereas at high concentrations, activity is inhibited.

The

point at which GTP affects on adenylate cyclase activity change from stimulatory to inhibitory varies with the target membrane, but typically is in the range of 0.1 to 1.0 pM. interesting feature of the GTP inhibitory phase is that it

An

664 cannot be mimicked by hydrolysis-resistant analogs of GTP, e.g. Gpp(NH)p.

Thus, in marked contrast to the persistent activa-

tion of adenylate cyclase by these analogs (12), the lability of the terminal phosphate appears to be critical for inhibition of adenylate cyclase, suggesting a phosphotransferase reaction might be involved in the inhibitory system. Inhibition of adenylate cyclase by hormones requires GTP, and the EC^Q for GTP in mediating inhibition is typically tenfold higher than that for mediating hormonal activation of cyclase but identical to that observed for GTP inhibition of "basal" catalytic activity (12).

Hormonal inhibition of ade-

nylate cyclase is also not observed in the presence of hydrolys-is-resistant analogs of GTP. The similar requirement for GTP in mediating hormonal activation and attenuation of adenylate cyclase has posed the question of whether or not the same G component, or same population of G components, is capable of signaling both excitatory and inhibitory signals to the catalytic subunit.

Several

indirect lines of evidence argue that two discrete populations of GTP-binding proteins are involved in these opposing regulatory effects.

First, the differing EC^Q for GTP in eliciting

activation versus inhibition of adenylate cyclase is consistent with two independent GTP binding sites possessing two different affinities for GTP.

Second, the ability of Gpp(NH)p to persis-

tently activate adenylate cyclase is in distinct contrast to the inability of hydrolysis-resistant analogs of GTP to inhibit the enzyme.

Finally, protease digestion (14,15), M n + +

(12,16), and sulfhydryl directed reagents (14) all selectively abolish either the activating or the inhibiting component of adenylate cyclase in various target membranes.

A limitation,

however, to all such indirect studies is that the same G component (or complex) might mediate both activation and attenuation of adenylate cyclase, but might possess two (or more) GTP binding sites and hence two functional domains which are differentially affected by the above perturbants.

More direct

data have been obtained by isolating membrane effector compo-

665 nents by virtue of their association with the receptor coupled to attenuation of adenylate cyclase, as described below. Characteristics of Hormone Interactions with Receptors Coupled to Attenuation of Adenylate Cyclase

The availability of radiolabeled agonists rine) and antagonists

([^H]yohimbine,

3 ([ H]epineph-

[^H]dihydroergocryptine

(DHE) or [^H]phentolamine) for identifying a-adrenergic receptors has directed a lot of biochemically-oriented research on hormonal inhibition of adenylate cyclase to 012-adrenergic receptors,

and in particular to the ra2-adrenergic receptors of

the platelet.

In much the same way that guanine nucleotides

mediate (^-adrenergic attenuation of basal or PGI2 (PGE-^) stimulated platelet adenylate cyclase, guanine nucleotides (Gpp(NH)p >_ GTP _> GDP >>> GMP; no effect of App (NH) p) also modulate 012 receptor affinity for agonists without altering receptor-antagonist interactions petition curves for

(19-21).

Thus, agonist com-

[ Hjantagonist binding are shallow in the

absence of exogenous guanine nucleotides and are shifted to the right and to normal steepness in the presence of GTP. Computer modeling of these competition profiles has suggested that, in a manner analogous to the 3-adrenergic receptor system coupled to activation of adenylate cyclase, agonist occupancy of platelet 012-adrenergic receptors results in formation or stabilization of a high-affinity receptor-agonist interaction which is reversed or prevented by the addition of guanine nucleotides

(19).

Biochemical studies in our laboratory

have indicated that digitonin solubilization of the unoccupied human platelet a2 receptor results in a loss of ability of the Heterogeneity of ct-adrenergic receptors: a-adrenergic receptors are subdivided into ui or a 2 subtypes based on the relative pharmacological potency of adrenergic agents (17). Fortuitously, the pharmacological subtypes correspond to the effector system to which the receptor is coupled. Thus, a 2 receptors appear to be coupled to attenuation of adenylate cyclase, whereas a 1 receptors are implicated in phosphatidylinositol turnover and increases in cytosolic C a + + levels (18).

666 receptor to form the so-called

"high affinity" state or to be

modulated by guanine nucleotides

(20,21).

However,

["^H]epi-

nephrine agonist occupancy of the platelet az receptor to solubilization high-affinity

stabilizes the guanine-nucleotide

form of the receptor.

sensitive

This solubilized

stabilized complex sediments more rapidly in sucrose gradients than either unoccupied

prior agonist-

density

or antagonist-occupied

tors, suggesting that agonist-occupancy

recep-

of the 012 receptor may

promote a physical association of the a receptor with the GTP binding protein which modulates receptor affinity However, this ["*H]epinephrine-ct2 32 contain the

P-ADP-ribosylated

component responsible This observation

for agonists.

receptor complex does not 42,000 Mr subunit of the G

for activation of adenylate cyclase

is in distinct contrast to the

(23).

8-adrenergic

receptor system where agonist occupancy of the receptor stabi32 lizes its interaction with the P'ADP-ribosylated 42,000 Mr subunit of G

(5).

Thus, the data obtained with the

ergic receptor system of human platelets provide

ot2-adren-

definitive

evidence that the GTP-binding protein which associates

with

the agonist-occupied

affinity

a-receptor and modulates receptor

for agonists is independent of the 42,000 Mr subunit of the G component coupled to activation of human platelet adenylate clase.

cy-

Further studies will be needed to document the b i o c h e m -

ical nature of the CX2-receptor-associated and its functional efficacy

GTP-binding

protein

in mediating inhibition of

adeny-

late cyclase. The Probable Role of Sodium in Hormonal Attenuation Adenylate

of

Cyclase

N a + ion has also been demonstrated

to permit or

hormonal attenuation of adenylate cyclase suggested that the ability of N a

+

(1).

enhance

It has been

to permit or enhance

detec-

tion of hormonal attenuation of adenylate cyclase may be due to the observed ability of N a + to abolish the inhibitory of the bimodal effect of GTP on adenylate cyclase, and

phase

thus

667 increase the sensitivity for detection of hormonal inhibition of adenylate cyclase (13).

This might explain why different

target membranes demonstrate different dependencies on Na + for expression of hormonal inhibition of adenylate cyclase, since higher concentrations of GTP inhibit adenylate cyclase to differing extents in different target membranes (12,13). In addition to its effects on hormonal attenuation of adenylate cyclase, Na + ion also modulates receptor-affinity for agonists in a manner which appears synergistic with GTP (20). In contrast to GTP, however, Na + does not eliminate the heterogeneity of receptor-agonist interactions, manifested by the appearance of shallow agonist competition binding curves in the absence and presence of Na + ion (24) .

Another aspect of the

effect of Na + not shared by guanine nucleotides is the ability of Na + to perturb receptor-antagonist interactions in both membrane and solubilized preparations (25).

What remains to be

documented is whether or not these effects of Na + have a fundamental role in regulating the physiological function mediated via hormone receptors coupled to attenuation of adenylate cyclase . Despite the superficial phenomenological similarity between hormonal systems coupled to activation and attenuation of adenylate cyclase in terms of their requirement for GTP to elicit effects on adenylate cyclase activity and in the ability of GTP to modulate receptor-agonist interactions, certain fundamental differences between the two systems exist.

Thus,

biochemical studies suggest that a GTP-binding component distinct from that which activates adenylate cyclase modulates receptor affinity for agonists at attenuating receptors. Whether or not this receptor-associated GTP-binding component also conveys inhibitory signals to the catalytic subunit remains to be elucidated.

Furthermore, Na + ion appears to modu-

late hormonal systems coupled to adenylate cyclase attenuation without reported effects on stimulatory adenylate cyclase systems.

Finally, one critical difference between hormonal sys-

tems coupled to stimulation of intracellular cAMP levels and

668 those coupled to attenuation of cAMP elevation is the documentation for stimulatory systems that changes in cAMP levels indeed mediate, at least in part, the physiological effects characteristic of the stimulatory hormone.

However, for inhib-

itory systems there are as yet no rigorous studies which unequivocally demonstrate that decreases in cAMP levels are responsible for mediating the physiological effects of hormones coupled to inhibition of adenylate cyclase.

Thus, hormonal

attenuation of adenylate cyclase remains simply a postulated mechanism for hormonal antagonism of cAMP-mediated physiological effects.

References 1. 2.

Jakobs, K.H.: Mol. Cell. Endocrino. 16, 147-156 (1979). Limbird, L.E.: Biochem. J. 195, 1-13 (1981).

3.

Ross, E.M., Gilman, A.G.: Ann. Rev. Biochem. 49, 533-564 (1980). Limbird, L.E., Lefkowitz, R.J.: Proc. Natl. Acad. Sei. USA 74, 228-232 (1978).

4. 5.

Limbird, L.E., Gill, D.M., Lefkowitz, R.J.: Acad. Sei. USA 77, 775-779 (1980).

6.

Cassel, D., Seiinger, Z.: Proc. Natl. Acad. Sei. USA 75, 4155-4159 (1978). Pfeuffer, T.: FEBS Lett. 101, 85-89 (1979). Ross, E.M., Howlett, A.C., Ferguson, K.M., Gilman, A.G.: J. Biol. Chem. 253, 6401-6412 (1978).

7. 8. 9. 10. 11. 12. 13.

Qassel, D., Levkovitz, H., Seiinger, Z.: Res. 3, 393-406 (1977).

Proc. Natl.

J. Cyclic Nucl.

Kent, R.S., DeLean, A., Lefkowitz, R.J.: Mol. Pharmacol. 17, 14-23 (1980). Northup, J.K., Sternweis, P.C., Smigel, M.D., Schleifer, L.S., Ross, E.M., Gilman, A.G.: Proc. Natl. Acad. Sei. USA 77, Cooper, D.M.F., Schlegel, W., Lin, M.C., Rodbell, M.: J. Biol. Chem. 254, 8937-8941 (1979). Cooper, D.M.F., Londos, C.: IN PRESS (1981).

Horiz. Biochem. Biophys.

669 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

Yamamura, H., Lad, P., Rodbell, M.: J. Biol. Chem. 252, 7964-7966 (1977). Pinkett, M.O., Jaworski, C.J., Evaine, D., Anderson, W.B.: J. Biol. Chem. 255, 7716-7721 (1980). Michel, T., Hoffman, B.B., Lefkowitz, R.J.: Clin. Res. 29, 566A (1981). Hoffman, B.B., Lefkowitz, R.J.: New Eng. J. Med. 302, 1390-1396 (1980). Fain, J.N., Garcia-Sainz: Life Sci. 26, 1183-1194 (1980). Hoffman, B.B., Michel, T., Kilpatrick, D.M., Lefkowitz, R.J., Tolbert, M.E.M., Gilman, H., Fain, J.: Proc. Natl. Acad. Sci. USA 77, 4569-4578 (1980). Limbird, L.E., MacMillan, S.T., Smith, S.K.: Adv. Cyclic Nucl. Res. 14, IN PRESS (1981). Smith, S.K., Limbird, L.E.: Proc. Natl. Acad. Sci. USA 78, July (1981). Gill, D.M., Meren, R. : Proc. Natl. Acad. Sci. USA 75, 3050-3054. Smith, S.K., Limbird, L.E.: Submitted. Michel, T., Hoffman, B.B. and Lefkowitz, R.J.: Nature 288,709-711 (1980). Limbird, L.E., Speck, J.L.: Manuscript in Preparation.

NALOXONE AND NALTREXONE:

Endorphin Antihormones

Mark S. Gold, A. Carter Pottash Fair Oaks Hospital, Summit, New Jersey Psychiatric Diagnostic Laboratories of America, Summit, N.J. Yale University School of Medicine, New Haven, Connecticut

Introduction Much of our current knowledge concerning the structure and function of the opiate antagonists derives from a well-defined body of knowledge regarding structure, pharmacology, physiology and psychological effects of the opiate agonists, particularly morphine and its structural derivatives.

Although

knowledge about the psychological effects of opium date from approximately the Third Century, B.C., it was not until the mid-nineteenth century with the production of relatively pure alkaloid derivations of opium, the invention of the hypodermic needle and the subsequent increasing use of parenteral morphine for analgesia that the problems of addiction prompted the search for analgesics with lower addictive potential.

This

research led to the eventual discovery of the relatively pure opiate antagonists naloxone and naltrexone whose chemical structures bear strong resemblance to the parent compound morphine and its dimethylated derivative thebaine. Pharmacology The opiate antagonists naloxone and naltrexone are similar with regard to metabolic fate and excretion, both being metabolized in the liver, primarily by conjugation with glucuronic acid.

They differ greatly, however, in relative activity after

© 1982 Walter de Gruyter & Co., Berlin • New York Hormone Antagonlsts, Editor M. K. Agarwal

672

oral administration, naloxone being so rapidly metabolized in its first passage through the liver that it retains only about one fiftieth of its potency when orally administered as compared to its activity when given parenterally.

Following in-

travenous administration, naloxone has a peak effect within 20 minutes, has a half-life in plasma of about one hour and a duration of action of one to four hours.

In contrast, nal^

trexone retains most of its efficacy when given orally owing primarily to protection from metabolic degradation in the liver provided by the cyclopropylmethyl nitrogen.

substitution on the

After oral doses of over 100 mg, peak concentrations

of naltrexone in plasma are attained in 1-2 hours and then slowly decline with a half-life of 10 hours.

Opiate antagonist

activity is sustained for 48-72 hours, owing partly to the presence of the breakdown product 6-naltrexol which is a weak antagonist but has a longer half-life.

In view of lack of mor-

phine-like subjective effects, lack of tolerance, absence of physical dependence and persistence of ability on chronic administration to precipitate abstinence syndrome in morphinedependent subjects the abuse potential of naloxone and naltrexone is virtually zero. Opiate Receptors Perhaps the most exciting discovery and advance in the field of neuroscience during the past decade was the discovery of peptides in the human brain with pharmacological activity similar to morphine and other opiates. This discovery did not occur accidentally but was the outcome of a systematic and intensive search for endogenous substances with opiate-like activity based largely on pharmacological observations of opiate antagonist activity under certain experimental conditions. The discovery of the existence of endogenous opiate receptors and endogenous opiate-like substances has stimulated a search for the physiological role of these substances.

Based on the

673

physiological and pharmacological effects morphine and related opiate compounds, a number of possible functions have been proposed to be influenced or mediated by endogenous opiate systems including pain modulation, respiratory, appetite and temperature regulation, motor activity, sleep and affective modulation learning and memory, "anti-psychotic" activity, mediation of reward and pleasure states and neuroendocrine regulation. The opiate antagonists, particularly naloxone, have provided a major investigative tool for evaluating possible contribution of endogenous opiates in regulation of the above functions. Endorphin Functions Analgesia produced by focal electrical brain stimulation in rats(l,2,3) and in man(4,5) is partially antagonized by naloxone, although not invariably (6,7).

These observations

support the hypothesis that analgesia produced by focal brain stimulation is mediated by the release of enkephalin in midbrain presumably activating inhibitory pathways to the spinal cord's dorsal horn. Naloxone has been shown to produce dose related suppression of appetitive behavior in rats deprived of food for 4 8 hours (8) a finding confirmed in a number of other studies(9,10). Further evidence for endogenous opiate modulation of feeding drives comes from the finding that genetically obese rats and mice have elevated levels of pituitary B-endorphin(11,12). The finding that naloxone potentiates acute and chronic heatinduced behavioral changes in rats has been used to support the hypothesis that endorphins may specifically function in adaptation to heat stress (13). Excessive grooming in animals induced by morphine and B-endorphin are reversed by Naloxone(14,15,16,17).

The level of

general activity has been reported to increase after enkephalin ICV or into the nucleus accumbens in animals (18,19), although

674 a majority of studies describe decreased motor activity and even catatonic-like immobilization, reversible by naloxone (20,21,22). The finding that naloxone treated rats were shown to attend to novel stimuli for longer periods of time has been used as argument for a role of the endogenous opiate system in focused attentional behavior (23).

Discovery of New Anti-withdrawal Treatments Investigators using single neuronal recording techniques and microiontophoresis reported that endogenous opiates and exogenous opiates decreased nucleus locus coeruleus (LC) firing rates and that this effect was specifically reversed by the opiate antagonist naloxone (24).

These data suggested that

the specific opiate receptors on the LC which might normally utilize endorphins as a natural neurotransmitter inhibit LC firing rate and modulate ascending NE activity and release (25).

These data suggested to us that a critical endorphin-LC

connection might exist and be related to some opiate effects and possibly play a critical role in opiate withdrawal(24,25) and naturally occuring panic states.

The NE hyperactivity or

LC hypothesis predicted that drugs which specifically inhibit the LC by an action at specific presynaptic LC receptors, would be found to have marked antiwithdrawal efficacy in man. Clonidine, lofexidine, nonaddicting opiate receptor agonists, vasopression, or GABA agonists like baclofen may be found to have potent antiwithdrawal efficacy (26,28). We have previously suggested that opiate withdrawal may result from a sudden lack of exogenous opiates and an inadequate endorphin reserve (25,29). This endorphin-noradrenergic locus coeruleus hypothesis (24,29) also suggests that prolonged abstinence, post-detoxification depression, and other affec-

675 tive symptoms as well as relapse may result from a prolonged endorphin deficiency. Endorphin Reserve Test The endorphin or opiate antagonists naltrexone and naloxone have been reported to cause a marked increase in plasma ACTH and Cortisol levels in normal man(29).

Naloxone blocks

opiate/endorphin receptor sites and through a feedback mechanism causes the release of available pro-ACTH/endorphin procursor stores to overcome the blockade.

This results in large

increases in plasma ACTH and Cortisol levels(30).

We have

used intravenous naloxone(20 mg) as a provocative test of the brain's endorphin system by measuring ACTH and Cortisol response of methadone addicts and normal controls and have clearly demonstrated impaired response in chronic methadone addicts.

These impaired response data for recently detoxified

addicts suggest that the Endorphin precursor-Endorphin-ACTH system or reserve is not functioning normally.

The fact that

response was decreased even in addicts who had mild symptoms and a stress response to the large dose of naloxone (which would be expected to increase ACTH and Cortisol), suggest that chronic methadone administration decreases the synthesis, storage, and quantity or functional integrity of the endorphin endogenous opiate reserve.

The preliminary data reported here

support the endorphin-LC hypothesis (24-27).

These data also

suggest that prolonged abstinence, post-detoxification depression and other affective systems which contribute to relapse may result from a prolonged endorphin deficiency.

These

naloxone test data are also consistent with animal data which have shown reduced brain B-endorphin immunoactivity in rats addicted to morphine for 3 months or longer and decreased B-endorphin immunoactivity and Naltrexone test response in chronic male opiate addicts (29).

676

Naltrexone's Clinical Ability In addition to naloxone and naltrexone's utility in neuroscience and as a provocative stimulus to test endorphin reserve, Naltrexone is very useful in the ongoing medical treatment of recovering opiate abusers and addicts.

Naltrexone

can be given three times a week as a prophylactic maintenance treatment of opiate-free addicts to prevent relapses. Naltrexone Advantages 1.

Oral administration

2.

Long half-life

3.

No abuse potential

4.

Reasonable cost

5.

Therapeutic efficacy

6.

Antagonizes or blocks the euphoric effects of opiates

7.

Antagonizes or blocks the development of physical dependence

8.

Few opiate-like effects

9. 10.

Non-addicting Tolerance does not develop to its opiate antagonism

11. 12.

Absence of serious side effects Absence of toxicity in chronic use

13.

Reduction of opiate craving

14.

May promote deconditioning to external environmental cues which provoke conditioned abstinence syndrome and craving by not allowing opiate use in response to subtle environmental events

15.

Extinction of opiate-seeking behavior by blockade of euphoria and other reinforcing properties of opiate use and opiate tolerance

16.

Provides the needed opiate-free time to allow for alteration in life course and psychological change in a structured psychotherapeutic milieu

17.

May replenish/normalize opiate-induced depletion/ derangement of endorphin system

677 18.

Allows for the early re-entry of recovering addict into the workplace so that the psychological treatment program which must include work, can be continued outside of the hospital in a residential setting

19.

Can be given chronically to the thinking and working addict who has significant opiate access to eliminate or markedly reduce relapse .

References 1. 2.

Akil, H., Mayer, D.J., Liebeskind, J.C.:Science 191, 961-2 (1976). Mayer, D.J., Hayes, R.L. :Science 188 419,941-3 (1975).

3.

Mayer, D.J., Liebeskind, J.C.: Brain Res 6 8,73-93(1974).

4.

Richardson, D. E. , Akil, H.: J Neurosurg Part 1 4J7, 178-83, and Part 2 47, 184-94 (1977).

5.

Adams, J.E.: Pain Vol. 2 161-66 (1976).

6.

Pert, A., Walters, M. :Life Science .19 1023-32

7.

Yaksh, T.L., Young, J.C., Rudy, T.A.: Life Science 3J5 1193-1198 (1976).

8.

Holtzman, S.G.: Life Science

9.

Gellert,V.F.,Sparber, S.B.: J Pharm Exp Ther 201, 44-54 (1977).

(1976).

1465-70 (1975).

10.

Goldberg, S.R.,Mossi, W.H., Goldberg,D.M.: J Pharm Exp Ther 196 625-36 (1976).

11.

Margules, D.L., Moisett, B., Lewis, M.J.,Shibuya,H., Pert, C.B.: Science 202 988-91 (1978).

12.

Garthwaite, T.L., Kalkhoff, R.K., Guansing, A.R., Hagen, T.C., Menahan, L.A.: Endocrinology 105 (5) 1178-82(1979).

13.

Holaday, J.W., Law, P.Y., Loh, H.H., Li C.H.: J Pharmacol Exp Ther 20J3 176-83 (1979) .

14.

Catlin, D.H., Hui, K.K., Loh, H.H., Li, C.H.: Adv Biochem Psychopharmacol .L8 341-50 (1978) .

15.

Gispen, W.H., Buitelaar, J., Wiegant, V.M., et al: Eur J Pharmacol ¿9 393-7 (1976).

16.

Veith, J.L., Sandman, C.A., Walker, J.M., Coy, D.H., Kastin, A.J.: Physiol Behav 20^ (5) 539-42,(19 78).

17.

Wrigant, V.M., Colbern, D., van Wimersma Greidanus, T.J., Gispen, W.H.: Brain Res Bull 3 (2) 167-70 (1978).

18.

Katz, R.J., Carroll, B.J., Baldright, G.: Pharmacol Biochem Behav 8J4) 493-6 (1978).

19.

Pert, A., Sivit, C.: Nature 265

20.

Chang, J.K., Fong, B.T., Pert, A., et al: Life Sei lj3 (12) 1473-81 (1976).

21.

Izumi, K., Motomatsu, T., Chretien, M.: Life Sei 20(7) 1149-55 (1977) .

22.

Jacquet, Y.F., Maries, N.: Science 194 (4265) 632-5 (1976).

23.

Segal, D.S., Brown, R.G., Bloom,F., Ling, N., Guillemin, R. : Science 198 (4315) 411-14(1977).

24.

Gold, M.S., Kleber, H.D.: Drug & Alcohol Dependence (4) 419-424 (1979).

25.

Gold, M.S., Byck, R. , Sweeney, D.R., Kleber, H.D.: Biomedicine 30 1-4 (1979) .

26.

Gold, M.S., Pottash, A.C., Extein, I., Kleber, H.D.: Lancet II 1078-79 (1980).

27.

Gold, M.S., Pottash, A.C., Sweeney, D.R., Kleber, H.D.: JAMA 243 343-46 (1980).

28.

Gold, M.S., Pottash, A.L.C., Sweeney, D.R., Kleber, H.D: Amer J Psychia 137 3 (1980) .

29.

Gold, M.S., Pottash, A.L., Extein, I., Kleber, H.D.: Lancet II 973 (1980) .

30.

Gold, M.S., Pottash, A.L.C.: Advances in Substance Abuse, Vol. 3 in press.

(5595) 645-7 (1977).

ANTIHISTAMINES J. DRY * 4*

i

et A. PRADALIER

4*

Professeur de Clinique Médicale - Service de Médecine Interne, et Centre d'Allergie de l'Hôpital Rothschild 33 boulevard de Picpus, 75571 PARIS CEDEX 12. Maître de Conférence Agrégé : Service de Médecine Interne et Centre d'Allergie de l'Hôpital Rothschild (Service du Pr. J. DRY).

SUMMARY Compounds with antihistamine activity are competitive antagonists of histamine for its specific cellular receptor. The may be distinguished into two groups, H^ and U^ depending upon the nature of the receptor for which they exhibit preferential affinity. In recent years, cimetidine has been shown to react specifically with H^ receptors and this has been exploited in gastroenterology for treatment of gastric ulcers. Anti-H^ type of compounds are mostly used in the treatment of allergy, insomnia and traveller's sickness, and current research is directed to eliminating their undesirable side effects. INTRODUCTION Histamine was synthesized by Windhaus and Vogt as early as 1907 but it was only in 1937 that Ungar and coll. (45) demonstrated the antihistamine (AH) activity of various compounds. In the following years, various substances (antergan 1942, mepyramine 1944 promethazine and diphenhydramine 1946 etc.) with greater antihistamine like activity, and lesser toxicity, were tested on smooth muscle preparations, histamine shock in the guinea pig, and greater reactivity against the anaphylactic shock.

© 1982 Walter de Gruyter & Co., Berlin - New York Hormone Antagonlsts, Editor M. K. Agarwal

680 Rapidly, a number of molecules endowed with antihistamine like activity were proposed but it was only in 1972 that Black and al. (4) discovered anti-H2 active substances that were effective against those aspects of histamine that were resistant to the classically known histamine antagonists viz : histamine induced gastric secretion of acid, and action on rat uterine muscle. These differences are based upon the demonstration of two Distinc type of histamine receptors viz : H^ and H^. The present article is intended to discuss the pharmacology and the therapeutic uses of the various anti-H^ anti-I^ compounds. It will be shown that the competitive antagonism for the receptor predicts better preventive action than the curative effect. ANTI H^ HISTAMINE ANTAGONISTS Anti-Hj^ antagonists are competitive inhibitors of histamine for its attachment to the H^ site in various tissues. They reversibly block the receptor sites for histamine in such tissues. They do not interfere with histaminase activity in vitro. In concentrations higher than that commonly used in the clinic they do exert an stimulatory effect on histamine liberation in vivo. A number of other effects of antiH.^ antagonists include : sedation, anticholergic influence. On the other hand, they do not oppose the action of other substances (slow reacting substance, eosinophilic chemotactic factor, platelet activating factor, serotonin, kinins, prostaglandins) liberated at the same time as histamine during the anaphylactic reaction. Structure-activity relationships of these substances is rather complex but the majority of the active substances molecule and this relates them chemically to the histamine molecule. A. PHARMACOLOGICAL PROPERTIES_: The single most important pharmacological property of these materials is to antagonise

681

histamine at various levels of action and in various tissues. 1°- Antagonism in the smooth muscle : a) isolated ileum of the guinea pig : all anti-H^ antagonists oppose histamine mediated longitudinal contraction of this tissue. This effect is reversible and depends upon the chemical nature of the antagonist. The inhibitory action of various anti-H^ antagonists in the guinea pig ileum treated with histamine, acetylcholine, 5-hydroxytryptamine, nicotine, K + , Ba + + has permitted an establishment of either a total or a partial specificity of the antagonist action ; this is supported by the work of Schild with the concept of pA£ mediation. Anticholinergic activity also contributes to the activity of antihistamine phenothiazine derivatives whereas antiserotonine activity plays a role in the action of cyproheptadine. b) bronchial smooth muscle : Anti-H^ antagonists inhibit histamine action on the tracheal, bronchial or bronchiolar fragments of the human or the guinea pig tissue, on the isolated guinea pig lung, and in the guinea pig in vivo. Histamine induced bronchiolar spasms are reversed by a preventive injection of an Anti-H^ agent. c) other smooth muscles : Anti-H^ antagonists inhibit the histamine induced contraction of the uterus from the guinea pig, rabbit and the cat ; on the other hand, they are inactive on the bronchiolar contraction in the beast, tracheal contraction in the cat, and the uteral relaxation in the rat, that follows histamine release in these tissues (44). These last three preparations, on the other hand, are reactive to the anti-I^ antagonists. 2°- Cardiovascular antagonism : vascular effects of anti-H.^. Histamine provokes a diminution in the arterial tension of the cat and the dog, in a dose dependent manner. Anti-H

682

partially reverses this effect of histamine and it is totally abolished by a combined anti-I^ and anti-H2 therapy. Similarly, anti-H1 diminish, but do not eliminate, the elevation of the arterial tension that follows histamine administration in the beast. On the other hand, they inhibit the contraction of pulmonary vessels following histamine application, and pulmonary hypertension in the dog and the rabbit, as a result of histamine administration in vivo. Thus, blood vessels are endowed with both anti-H^ and anti-f^ receptors. Cardiac effects of anti-H^^. Anti-H1 stimulate cardiac contractibility (dromotrope + effect) ; anti-B^ antagonise histamine mediated chronotrope and ionotrope positive effects. Selected anti-H^ (antazoline) possess antiarythmic properties. 3°- Antagonism of histamine induced local vascular effects and vascular permeability. Anti-Hl inhibit the influence of intradermally injected histamine. Doses required to reduce 50% o£ the observed effect are : chlorpheniramine 8 mg ; promethazine 18 mg, chlorcyclizine 38 mg, mepyramine 105 mg. Antiprurigenous effect is also clear under these conditions. Discreet local anaesthesia may also be produced by promethazine but this does not parallel the antihistamine effect of this phenothiazine derivative, and does not appear to be involved in the antiprurigenous action of these drugs. AntiHj^ also exercise an antagonistic effect on the peripheral vascular effects of histamine as well as the capillary permeability modifications observed under these conditions. Anti-H^ have a minor antagonistic effect in the edema produced by the intraperitoneal or intravenous injection of formaldehyde or hyaluronidase. Anti-H^ are effective in controlling the modifications induced by histamine liberators (40-80 in the dog and dextran in the rat), probably because histamine alone is responsible for the observed effects.

683 4°- Action on exocrine secretion. Histamine accelerates gastric,salivary and tear gland secretion and this is antagonised by anti-I^ antagonists, except for the hydrochloric acid liberation in the animal as well as the man. 5°- Effect on the central nervous system. Application of histamine in different portions of the cortex or the under cortex, as well as intracerebral injection of histamine, has revelated that histaminergic fibres may conceivably be related to the centers of food intake, vomitting and anorexia. It is interesting to correlate the antiemetic action and thirst inhibition of selected anti-histamines ; in addition, certain of these strongly inhibit the vomitting induced by apomorphine. H^ antagonists also diminish the excitability of vestibular neurones and this is used as an argument to explain the beneficial role of these materials in combatting the traveller's sickness in man. Promethazine, alimemazine, doxylamine, ethymemazine, diphenydramine, and dexchlorpheniramine all possess a strong hypnotic and sedative action. Although this was said to be related to their anticholinergic activity, the finding of histamine receptors in the central nervous system may be of potential interest in interpreting these older observations. H^ antagonists traverse the blood brain barrier and potentiate the action of alcohol, barbiturate type of hypnotics, tranquilisers and neuroleptics. In addition theypossess epileptic effect in higher doses and also stimulatory effect on respiration. On the other hand, certain anti-H^ compounds, such as phenindamine, are endowed with stimulatory effects in the central nervous system. This paradoxical phenomenon may be observed in some subjects, especially children with other anti-H

antagonists.

684 6 o - Anti-anaphylactic and anti-allergic actions. Anti-H^ action varies according to the animal species ; rabbit and beasts are better protected than the rat or the man. The effect on cutaneous anaphylaxis, or passive intraperitoneal anaphylaxis, is rather poor. Bronchiolar spasms produced by house dust or pollen in man are poorly protected by anti-H^, although in some reports thiazinamium may be effective under some of these conditions (Simonsson and BoojeNoord in 16). 7°- Local anesthetic action. All of the antihistamines exert some local anesthetic action. It is some structural resemblance between antihistamines and local anesthetics but there is no correlation between local anesthetic and antihistamine properties. Changes in cell membrane permeability might be involved in the mechanism of this effect (39). B. THE VARIOUS ANT I-H ^PRODUCTS . There is a great variety of anti-H^ antagonists, usually classified according to their chemical nature, whose therapeutic indications are quite comparable, and whose side effects are numerous. CHEMISTRY : Antihistamines can be classified somewhat arbitrarily into groups, on the basis of their structure or of their relationship to the aminoethyl side chacri of histamine. - Ethylenediamine series : derivatives of the general basic structure : ^ N-C-C-N^ including : i * mepyramine Néo Antergan , tripelennamine Pyribenzamine , antazoline Antistine , methapyriléne, thenyldiamine, chloropyramine, histapyrrodine, clemizol, methapheniline...

685 - Aminoethyl ethers group. Derivatives of the general basic structure -0-C-C-N