Receptor Mediated Antisteroid Action 3110846934, 9783110846935

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Receptor Mediateci Antisteroid Action

Receptor Mediated Antisteroid Action Editor M. K Agarwal

W DE

G Walter de Gruyter • Berlin • New York 1987

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

Library of Congress Cataloging in Publication Data Receptor mediated antisteroid action / editor, M. K. Agarwal. p. cm. Includes bibliographies and indexes. ISBN 0-89925-374-1 (U.S.) 1. Steroid hormones-Receptors-Effect of drugs on. 2. Steroid hormones-Antagonists. I. Agarwal, M. K. [DNLM: 1. Estrones-pharmacodynamics. 2. Receptors, Steroid. 3. Steroids-antagonists & inhibitors. WK150 R2948] RM297.S74R43 1987 615'.36~dcl9 DNLM/DLC 87-27567

CIP-Kurztitelaufnahme der Deutschen Bibliothek Receptor mediated antisteroid action / ed. M. K. Agarwal. Berlin ; New York : de Gruyter, 1987. ISBN 3-11-011355-4 NE: Agarwal, Manjul K. [Hrsg.]

Copyright © 1987 by Walter de Gruyter & 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: Gerike GmbH, Berlin. Binding: Liideritz & Bauer GmbH, Berlin. - Printed in Germany.

PREFACE T h e last few y e a r s h a v e w i t n e s s e d a literal e x p l o s i o n of a d v a n c e s in the field of molecular e n d o c r i n o l o g y . T h e receptor g e n e for all g r o u p s of s t e r o i d hormone r e c e p t o r s has been c l o n e d . Chemical modifications in the s t e r o i d n u c l e u s h a v e p r o v i d e d a whole new a r r a y of materials with specific a g o n i s t / a n t a g o n i s t a c t i v i t y . T h e notion that the s t e r o i d hormone receptor is p r i m a r i l y cytoplasmic has been c h a l l e n g e d . New biochemical a n d immunocytochemical a p p r o a c h e s to p r o b e the receptor a p p r a i s a l of its s t e r e o s p e c i f i c

h a v e permitted f r e s h

configuration.

S t e r o i d hormone a n t a g o n i s t s not o n l y form excellent tools to s t u d y

various

a s p e c t s of the receptor s t r u c t u r e a n d f u n c t i o n b u t t h e y a r e also important in d i a g n o s t i c a n d clinical medicine. A n t a g o n i s t s for all c l a s s e s of s t e r o i d hormones a r e actually u s e d in the treatment of a wide a r r a y of d i s o r d e r s . Medical treatment has now become p o s s i b l e in c a s e s where s u r g e r y to be the o n l y available

used

therapy.

T h e p r e s e n t volume g r o u p s t o g e t h e r latest conceptual d e v e l o p m e n t s a n d o n g o i n g d e s i g n s for a n t a g o n i s t s in all g r o u p s of s t e r o i d h o r m o n e s b y some of the

leading a u t h o r i t i e s actually i n v o l v e d in their r e s p e c t i v e f i e l d s .

the biochemical a n d pharmacological level, v e r y dramatic a d v a n c e s been made in the area of a n t i m i n e r a l o c o r t i c o i d s .

At

have

For the f i r s t time, a

specific p r o b e for the m i n e r a l o - r e c e p t o r has been marketed a n d t h i s book p r e s e n t s the o n l y available review in t h i s a r e a .

D i s c o v e r e d b a r e l y a few y e a r s b a c k , R U 38486 has all b u t

revolutionized

c o n t r a c e p t i o n a n d medical treatment of g l u c o c o r t i c o i d hormone e x c e s s .

All

t h i s information has been r e v i e w e d here c o m p r e h e n s i v e l y . T h e u s e of t h i s antihormone to p r o b e g l u c o c o r t i c o i d

and progesterone receptors,

and

the newly d e v e l o p e d a p p r o a c h of idiotype selection, are also r e v i e w e d in t h i s book for the f i r s t time.

VI

A n t i e s t r o g e n s and antiandrogens have been reviewed both for the fundamentalist, and the clinician

interested in basic mechanisms.

The

influence of antisteroids on modification of behaviour, a n t a g o n i s t s for steroid hormones in insects, and the problem of receptor have also been included to stimulate f u r t h e r

localization

thought.

In this day and age of specialization and literature explosion, it is often a herculean task to keep abreast of developments even in one's own particular field. T h i s book may be used as a source of material that individuals involved constantly in their routine work would not otherwise read. Newcomers may use this volume as a s t a r t i n g point for the history and development of antisteroid research.

backtracking

It is hoped that

c r o s s fertilization of ideas developed in different areas

will

lead to

even greater p r o g r e s s when readily available under a single c o v e r .

Earlier volumes by this editor have covered v a r i o u s biological levels at which the action of hormones can be antagonized. T h i s book concentrates primarily on the antagonism mediated t h r o u g h the specific cellular receptor. The advances summarized here should be of interest to pharmacologists, biochemists, advanced g r a d u a t e s t u d e n t s , and clinicians.

Theoretical criticism of any u n d e r t a k i n g of this sort is within the reach of everyone who wishes to point out that which was not included and could or should have been p r o v i d e d . V a r i o u s degrees of commitment

by

leading authorities do not make it possible to provide a timely c o v e r a g e in every single field. T h a n k s are due to all c o n t r i b u t o r s who mailed their manuscripts in time for a publication rapid e n o u g h to be worthwhile before further research reduces it to a niche in the heap of h i s t o r y . July 1987, Paris

M. K . A g a r w a l ,

Editor

CONTENTS

S t r u c t u r a l D i f f e r e n c e s D i s t i n g u i s h i n g S t e r o i d A n t a g o n i s t s from A g o n i s t s W. L . D u a x a n d J. F . G r i f f i n

1

A n t i h o r m o n e A c t i o n of S t e r o i d s with Modified R i n g L. Starka,

R . Hampl a n d A .

Structure

Kasal

17

A n a l y s i s of the S t r u c t u r e a n d F u n c t i o n of S t e r o i d R e c e p t o r s with the A i d of the A n t i h o r m o n e R U 38486 M. K. Agarwal and G. Lazar Clinical A p p l i c a t i o n s of the Glucocorticoid a n d P r o g e s t i n R U 486

43 Antagonist

L . K . Nieman a n d D . L . L o r i a u x

77

R U 38486 ( M i f e p r i s t o n e ) R e v e r s e s P r o g e s t e r o n e - a n d H y d r o c o r t i s o n e Mediated I n h i b i t i o n of P r o s t a n o i d S y n t h e s i s in C u l t u r e d Myometrial, V a s c u l a r a n d Gut T i s s u e E x p l a n t s J. Y . Jeremy a n d P. D a n d o n a

99

Receptor Mediated A n t i p r o g e s t i n Action of R U 486 M . Kalimi

121

T h e A n t i g l u c o c o r t i c o i d Effects of C o r t e x o l o n e a n d R U 38486 in the Human Leukemic Cell Line C E M - C 7 T . J. Schmidt

139

T h e Mineralocorticoid Receptor a n d the A c t i v i t y of A l d o s t e r o n e Antagonists G . Wambach T h e A n t i m i n e r a l o c o r t i c o i d A c t i o n of T w o Newly D e v e l o p e d Derivatives M . K . A g a r w a l a n d M . Kalimi

169 Spirolactone 197

VIII New Molecular P r o b e s to A s s e s s E s t r o g e n a n d A n t i e s t r o g e n

Actions

E. M. Cormier and V . C. Jordan Antioestrogens and R . I. N i c h o l s o n ,

223

Cancer

K . J . Walker a n d P. D a v i e s

A n t i e s t r o g e n A c t i o n in M C F - 7 T . S . R u h , M. F. R u h ,

275

Cells

R . K . S i n g h a n d W. B . B u t l e r

D e s i g n a n d A c t i v i t y of N o n s t e r o i d

307

Antiandrogens

T . Ojasoo a n d J . P. R a y n a u d

329

Cellular a n d Molecular Effects of A n t i e s t r o g e n s a n d C. V.

Antiandrogens

Ramana M u r t y a n d A . K . R o y

S t e r o i d Hormone A n t a g o n i s t s ,

373

B r a i n Receptor Systems and

Behavior

A. M. Etgen

405

A n t i s t e r o i d A c t i o n in B r a i n a n d C h a n g e s in Animal

Behaviour

C. Fabre-Nys and E. A d k i n s - R e g a n

435

Localization of S t e r o i d Hormone R e c e p t o r s in the Cells b y Immunohistochemistry K . C . R a j e n d r a n a n d I. P a r i k h Antiecdysteroids and

469

Receptors

M. S p i n d l e r - B a r t h and K. D. Spindler

497

AUTHOR

513

SUBJECT

INDEX INDEX

517

STRUCTURAL DIFFERENCES DISTINGUISHING STEROID ANTAGONISTS FROM AGONISTS

W.L. Duax and J.F. Griffin Medical Foundation of Buffalo, Inc., 73 High Street, Buffalo, N.Y.

14203

Introduction Many characteristic hormonal responses of steroids are contingent upon their binding to specific receptors in target tissue (1).

While it is

known that response depends upon interaction of the receptor and nuclear chromatin (2-4), the precise details of this interaction and the role played by the steroid in this process remain 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 compete for the

steroid binding site of the receptor with high affinity demonstrates that the phenomena of binding and activity are at least partially independent. X-Ray crystallographic data provide reliable information on the overall conformation of steroid hormone agonists and antagonists that may be used to gain insight into the conformational flexibility of these molecules, the structural similarities that might account for their binding to a common site and the structural differences that might account for varying degrees of hormonal response. On the basis of conformational analysis of the molecular structures of agonists and antagonists of estrogen, progesterone and corticoid hormones, we have proposed a model for receptor binding and hormonal activity.

The

model suggests that receptor binding is primarily controlled by the interaction of the steroid A ring with the receptor, and the nature and degree of the hormonal response is primarily controlled by the stereochemical features of the.D-ring region of the molecule (5). More recently, examination of the structural features of androgen agonists and antagonists has lead to the conclusion that for this class of

Receptor Mediated Antisteroid Action © 1987 Walter d e Gruyter & Co., Berlin • New York - Printed in Germany

2 hormones the D-ring may be the region critical to receptor interaction and that the stereochemistry of the A ring may control hormonal response (6). This review will summarize the salient features of these two models.

A.

Estrogen Agonists and Antagonists

Structures with high affinity for the estrogen receptor almost without exception contain a phenolic ring that appears to be essential to initiate receptor binding (7,8).

The potent synthetic estrogen diethylstilbestrol

(DES, I) has two phenol rings capable of initiating receptor binding.

In

addition DES is constrained by its central double bond to have an extended conformation with an overall shape and separation of its functional hydroxyl groups similar to that observed in the natural estrogen estradiol (Figure 1).

Fig. 1. A comparison of the observed structures of (a) estradiol and (b) DES. They have in common: overall shape, phenolic ring, and similar separation of hydroxyl groups by a hydrophobic mid region.

Metabolites of DES are of interest due to uncertainty concerning the form responsible for the carginogenic properties of DES and as additional probes of the structure-activity relationships of estrogens.

The low

affinity of indanesterol (Fig. 2b) for the estrogen receptor despite the presence of two phenolic rings indicates that the bent overall shape of the molecule (Fig. 2a) is incompatible with receptor binding (9).

It is

evident that the precise location of steric bulk and functional groups relative to the phenolic ring will govern binding and activity.

The

subtlety of the details of molecular control is exemplified by the case of the racemates of indenesterol A.

Korach finds that only one enantiomer of

indenesterol A (IA) has high affinity for the estrogen receptor (9,10).

3

a)

Fig. 2.

,O 5 H

Bent conformation of indanestrol observed in the solid state.

Model studies using X-ray crystallographically observed structures of IA suggest that it is the a-ring that mimics the estrogen A-ring (9) (Fig. 3) but it is difficult to identify the structural basis for the enantiomeric effect in IA.

It must be related to the topology of the molecule in the

region of the double bond.

In this regard IA has a high degree of

similarity to the crystallographically observed asymmetric form of DES in which the two methyl groups are on the same side of the plane of the double bond.

On the basis of a comparison of the crystallographically

observed structures of estradiol, DES and IA (Fig. 4), we suggested that the more active enantiomer would be the one with C(3)-R chirality (11).

Fig. 3. Comparison of the conformations of IA with estradiol reveal that the best fit is achieved when the a ring of IA is superimposed on the A ring of estradiol with the double bond in the 0 ring overlapping the C(8)C(9) bond in estradiol.

In order to test this hypothesis we undertook the crystal structure analysis of a bromobenzoate derivative of one of the enantiomers.

The

derivative of the inactive enantiomer was used in the X-ray analysis in order to spare the more active enantiomer for further biological testing. The X-ray crystal structure analysis unequivocally demonstrated that as we had postulated, the C(3)-R enantiomer of indenesterol A is more active than the C(3)-S enantiomer (unpublished results).

4 a)

b)

Fig. 4. Stereo views illustrating the best fit of the hydroxyl groups and the hydrophobic bulk of the structure of E (dark solid lines), DES (light solid lines), and IA (dashed lines).

When the phenol rings of a sample of the compounds that compete for binding to the estrogen receptor are superimposed significant differences in the D ring region of the molecules are observed (Fig. 5).

If there is

a close association between estrogens and the receptor it would appear to be limited to the A and B rings.

The receptor is either flexible in the D

ring region or insensitive to it (12,13).

Fig. 5. Superposition of the phenol rings of six compounds that bind to the estrogen receptor, suggests that variability in D-ring orientation is compatible with receptor binding and some degree of activity.

Estrogen antagonists such as trans tamoxifen compete for binding to estrogen receptors and elicit little or none of the characteristic hormonal response.

The clinical utility of the estrogen antagonists is a

result of their competition for the traditional Type I estrogen binding site (14).

5 The 4-hydroxy metabolite of trans tamoxifen (Fig. 6) has been shown to be the potent competitor for the estrogen receptor responsible for the antagonist properties useful in breast cancer therapy (15).

This

observation has led most investigators (16-18) to conclude that, at least in the case of the 4-hydroxy derivative of tamoxifen, it is the a ring that mimics the estradiol A ring in receptor interaction.

If the a' ring

of tamoxifen mimics the A-ring of estradiol when competing for the binding site on the receptor, then the X-ray crystallographic data on the conformation of these molecules can be used to identify the structural differences that may account for the failure of tamoxifen to elicit a significant response.

The atomic skeleton and the van der Vaals envelopes

of estradiol and trans tamoxifen are illustrated if Figs. 7 a and b,

observations

that

was

antiandrogenic

the

repor-

A-nor-5a-androstane

possess

relationships. According

It

estrogenic

found

even

(35)

17a-diethynyl-A-nor-5a-andro-

researchers

study

and

(Anordrin). also

were

and

several

Pincus

of

they

these

form

by

(18)

virtue

with

hormone.

compounds

incubation.

al.

antiestrogens

activity natural

et

One

of

is

appears

of

the

estrogen The high

such

and

to

fast

dis-

receptor

relative but

Mehta

it

compounds

binddecliwas

32 2-ethynyl-5a-A-nor-estrane-2,17B-diol forms

a fast-dissociating

similarly ceptors its

as

(18).

19-nor

17 R - d i o l the

cyproterone

(XXVI)

successfully China In

tation

to

(Dinordrin

have the

steroids

by

been

as

confirmed

by

re-

Anordrin, isomer

of

investigation

Anordrin

a post-coital

binding

has

on

been

contraceptive

between

some

of

to

in

interacted

considerably

sults

in g o o d

were

Moreover, ceptors. ments

some In

for

trogen A-nor

the

them

for

steroids

tested time

formed

receptors, misfit

findings

concerning

estrogen

viously

both

that

had

these

RBA

were

be

a basic

of

of

unexpected

those that

decreasing with

with that

the

rees-

pre-

all increathese

cytosolic

conformational In

fact,

some

these

compounds

as

was

the p h e n o l i c

re-

require-

either

to

fact

suggested

The

i_n v i v o .

androgen

with

components.

interaction

presence

the

similar

complexes

relati-

hydroxyl

structural

The

and

discrimina-

obtained

steroids

values

might

were

these

diffe-

receptors.

receptors.

with

the

hormones.

interacting the

of

to

Surprisingly,

a phenolic data

interacted

temperature

there

receptors the

the

their

enabling

estrogen

with

receptors

and

to

with

androgen

antagonists.

the

of

as

and

applied

and

with

short-lived

so t h a t

between

tested

estrogen

natural

incubation

compounds

were

approximation,

androgen

reported

activities

interact

A-nor

tried

A-nor-5a-androstane-

were

also

other have

Various

agreement

first

hormonal

not p o s s e s s i n g

interaction

and/or

viously

of

the

of

authors

antiimplan-

to

agonists

compounds

The

the

ability

the

conditions

potent

these

56

from

one, a number

(38).

derivatives

affinities

activity

potential their

receptors.

incubation

?)

examined

different

hormonal

tion

the

of

the 2 B - e t h i n y l

Recently,

estrogenic

measuring

A-nor-5a-estrane

sing

receptors, androgen

effectiveness

and

were

(37).

introduced separate

evaluate

Two

II)

baboons

I)

(= a n t i - p r o g e s t i o n a l

analogues

ve

the

(36) .

order

rent

estrogen

which

(2oc,17a-di e t h y n y l - A - n o r - 5 a - e s t r a n e - 2 R ,

(Dinordrin

female

with

does with

anti-fertility

analogue

latter

cycling

The

complex acetate

( X X V , RU 2 6 1 4 3 ) ,

it

hydroxy

with

assumed group

prewas

33 an

essential

the

2-OH

ding

to

prerequisite

group

seemed

estrogen

to

for be

receptors

binding.

an and

one

-hydroxygroup

in t h e

five-membered

play

of

3-hydroxy

the

role

hydrogen

bonding

interaction xy

group

in

that

since

in

equally the of

tors, to

the

3-phenolic

group

classes

those of

2a-

of

also

which

natural

were

stereochemistry

far

less

important.

the

1713-hydroxy

The

group

For of

results

for

the

the

forming

the

From

recep-

extent

receptors,

group the

de-

lies.

androgen

with

ste-

in

androgen

a similar

confirmed

a

surprising

molecule,

with to

the

2-hydro-

substituents

the

2-OH

interaction

by

active.

estrogens

bound

2-

partially

of

quite

and 2B-0H of

bin-

Interestingly,

is

interacted

receptors.

the

could

were

plane

series, for

that

estradiol

site.

effect

average

compounds

however,

of

stereochemistry

this

both

above

suggest

A-ring

group

2R-derivatives

the

the

requirement

might

receptor the

view

ring

from

including

both

of

A-nor

these

the upon

only

point

viate Some

with

depended

reochemical

which

the

In

absolute

seemed

to

be

importance

of

androgen

re-

ceptors. Rousseau stant ding

et

for

al.

rat

protein

vatives with

androgen (ABP)

including

se c o m p o u n d s ABP

with

were

and

the

receptor

nor

to

(estrane

series),

were

of

the

tors

group.

the

other

gesterone

receptor

and

19-A-nor

androstaneboth

the

androgen

to

ABP.

The

were

authors

CH RU X 3 H TA O'HB

£ T3 e a>

£ TJ e « 55 *

55 40

1

Antibody Dilution x 10

J

2

3

Antibody Dilution x 10*

Fig. 16. The Specificity of the Rabbit Idiotype for the Hapten. Antiserum from rabbit immunized with TA-BSA was incubated with various steroids to assess relative binding (see 40).

67 Data in Fig. 17 show that ^H-TA bound to the idiotype could not be displaced by either RU 38486 (the antagonist), RU 28362 (the ideal agonist), cortexolone, or aldosterone. In contrast, nonradioactive TA exhibited dose dependent displacement of 3 H-TA binding to the idiotype antibody. In rat liver cytosol, 3 displacement of bound H-TA was obtained with various cold steroids in the order: TA > RU 38486 > RU 28362 > Aldosterone > Cortexolone

(Fig. 17b). The most striking difference in

these steroid binding proteins is the total lack of binding of 3 H-RU 38486 to the antisteroid antibody as compared to the receptor, and the inability of this synthetic antihormone, 3 and of RU 28362 (the ideal agonist), to displace H-TA bound to the antibody, even at concentration ratios of 1000:1 (40). Antibody

Liver Cytosol

[Steroid] M

[Steroid] M

Fig. 17. The Polyclonal Anti-TA Antibody is Specific for TA, Contrary to the Steroid Binding Site on the Receptor. 3 10 nM H-TA was used to label either the rabbit idiotype or rat liver cytosol. Various nonradioactive steroids were used to displace the bound steroid, as described before (40). These differences were confirmed when

3 H-RU 38486 was used to

label 3GR in rat liver and kidney. Data in Fig. 18 show that 1 nM

H-RU 38486 could be displaced equally well with either

unlabelled TA or RU and less so by Cortexolone = Aldosterone.

68

Since 3h-ru 38486 has no affinity for the mineralocorticoid receptor in the kidney (reviews in 3), and since aldosterone was less effective than TA or RU 38486 in displacing bound 3 H-RU 38486, the renal binding site is quite obviously GR. Kidney Cytosol

Liver Cytosol

• X O •

Fig.

RU38486 TA Aldo Cortexolone

[j_ Comparative Studies on the Glucocorticoid Receptor in Two Target Organs.

3 1 nM H-RU 38486 was used alone, or in presence of various cold steroids, to label GR in hepatic and renal cytosol. For further details see (40). These results were supported by additional studies where

H-TA

was used to label renal GR (not shown). Under these conditions, the displacement of bound

H-TA was in the order: TA =

RU 38486 > Aldosterone > Cortexolone. These data imply strong topological homologies

(perhaps identity) between the steroid

binding sites of the hepatic and renal glucocorticoid receptor. In any event, it is clear from the foregoing that the idiotype appears to contain an active site strictly complementary to TA. The conformation and topology of the steroid binding domain on the receptor appears to be complex, contrary to the notion in vogue where all agonists and antagonists would saturate an identical configuration leading to differences in affinity

(1-5 for various reviews).

69 Further analysis of epitopes near the combining site of the liver glucocorticoid receptor was attempted by raising antiidiotype antibodies to

triamcinolone 6-ketohexanoyl

(TKH)

linked to thyroglobulin, as described previously in detail (39). Data in Table 5 show the influence of three variables on the interaction of the anti-idiotype 8G11-C6 with the Fab fragment of the anti-TA idiotype. Table 5. Interaction of anti-TA Fab with the anti-idiotype

TA. (1 mM) RU 38486 Well Dilution TA-RSA (2 mg/ml) (1 Mm)

Liver Cytosol (5%)

A

2x

0.808

B

4x

1.182

1. 760

1. 884

0.152

c

8x

1.177

1. 597

1. 669

0.149

D

16x

1.188

1. 647

1.899

0.186

E

32x

1. 172

1. 591

1.459

0.271

F

PBS

1. 628

1. 531

1.390

1. 547

G

PBS

1.574

1. 426

1. 737

1. 405

H

Blank

0.005

0. 005

0.007

0.007

-

-

0.119

For details of Eliza method see (39). It is clear from data in Table 5 that liver cytosol competes effectively with anti-TA antisteroid-anti-idiotype confirmed previously

interaction,

(39). Interestingly, both TA and TA-RSA,

used to obtain the antisteroid idiotype, are ineffective. The antihormone RU 38486, too, was totally ineffective in interfering with the anti-TA Fab-anti-idiotype

interaction.

Data in Table 6 show that cytosol from several organs was just as effective as liver cytosol in competing with the anti-TA Fab - 8G11-C6 interaction. It is quite clear that serum, too, competed equally well under these conditions. Thus, the antiidiotype was not very specific for the glucocorticoid receptor.

70

The behaviour of the anti-idiotype was contrary to that of TA which has no affinity for the corticosteroid binding globulin in the serum. In other words, the anti-idiotype was possibly recognizing epitopes adjacent to the active site on both the receptor and transcortin. Table 6. Influence of Various Cytosols and Serum on the Binding of Anti-idiotype to the Idiotype Fab. Well Dilution

Liver

Kidney

Thymus

Spleen

Serum

A

0

0. 076

0. 086

0.052

0..087

0.072

B

2x

0. 055

0. 068

0.047

0,,068

0.066

C

4x

0. 081

0. 056

0.045

0..066

0.075

D

8x

0. 088

0. 087

0.050

0..068

0.076

E

16x

0. 124

0. 121

0.088

0..072

0.074

F

PBS

0. 717

0. 664

0.948

0.. 809

0.755

G

PBS

0. 669

0. 622

0.656

H

Blank

0. Oil

0. Oil

0.014

0.651 0..010

0.010

For details of Eliza see (39). In the anti-idiotype network theory, the ligand for the receptor may provoke idiotype antibodies resembling the receptor and the anti-idiotypes of this subset should mimick the ligand

(39). The results described above indicate that ^both

the transcortin

and the receptor may share a comparable

domain close to the ligand binding sites. A whole family of anti-idiotype antibodies may be helpful in mapping various domains around the active site which, according to the idiotype described earlier, appears to be complex. It would be interesting to see whether a pure agonist antagonist

(RU 28362) and the

(RU 38486) conjugates would elicit a specific

anti-idiotype since neither recognizes the idiotype engendered by Triamcinolone-BSA.

Conclusions and Future Trends The most obvious message that emerges from the present review is the uniqueness of each system subjected to careful analysis No generalizations seem possible from in vitro to in vivo, from one tissue to the other, and from one steroid receptor class to another. To recapitulate just some of these variables, RU 38486 binds to the same population of the glucocorticoid receptor in all tissues studied. Yet, its affinity for rat liver GR is less than that of the agonist triamcinolone exactly in contrast to rat thymus and hepatoma

(12),

(29,30). The

activation of the GR-antagonist complex is complete in rat liver cytosol (27) but only partial in thymocytes

(3, 29, 30)

In the progesterone receptor system, the agonist and the antagonist compete for the same binding site in the calf uterus cytosol but two separate binders were clearly evident in the chick oviduct cytosol, one each specific for RU 38486 and progesterone

(28). The affinity of the antagonist for the

PR was higher than that of the agonist in calf uterus but the reverse was true in the chick oviduct (28). This recalls some of our earliest observations on receptor heterogeneity

(24)

and falls into the category of the estrogen (41) and the mineralocorticoid

(31, 42) receptor series where antagonist

specific binding populations have been reported

(see also

other chapters in this book). This sort of heterogeneity may be due to a combination of separate enhancer elements

(43)

and recent advances in receptor gene cloning may make this sort of reasoning amenable to experimental scrutiny. The most striking differences were noted when receptor stability was considered. At 37° C, GR-TA complexes were far more stable than GR-RU 38486 complexes from rat liver cytosol (27) Paradoxically, the antagonist actually stabilized the progesterone receptor in chick oviduct as compared to PR-progesterone complex at 37° C (28).

72 Like the receptor activation, which proceeds in presence of agonists and antagonists in the GR (27), MR (31), and PR (28) systems, the process of nuclear binding in vivo needs to be reassessed. Although both the GR and the PR receptor complexes are believed to bind to the same hexanucleotide in the promotor (33, 34), this sequence did not compete with the binding of the activated GR-receptor complex to DNA-cellulose which is believed to represent a valid model for in vivo behaviour of different receptors. This concept needs revision not only because receptor activation can not be assessed in presence of phosphate buffers that form the predominant cellular ion in vivo (31) but also because the estro-progestative hormones are now believed to be intranuclear

(see another paper in this

volume). If activation is a means for the transfer of cytosol based corticoid hormone receptor into the nucleus, what is the role of this process in the action of sex steroid hormone receptor that is nuclear based? The idiotype - antiidiotype approach has hitherto been used only in the GR system (39,40). The novelty of this procedure has permitted some cartography of the active site on the rat liver GR. Contrary to the notions in vogue, the steroid binding domain may be far more complex than is generally assumed by competition experiments. Similar studies with nuclear based receptors should prove valuable and this method may permit receptor purification solely by immune selection. Finally, RU 38486 has already proved its usefulness both in diagnostic

(44,45) and clinical

(46,47) medicine, as reviewed

in another chapter. The challenge in future would require dissociation of the antiprogestin from the antiglucocorticoid effects. As a word of caution, it is wrong to assume that all physiological effects of RU 38486 are receptor mediated

(23).

Future screening should be based on physiological action of the derivative in question even if its affinity for the receptor is lower than expected (48, 49).

References 1.

Agarwal, M. K. (ed), Antihormones, Elsevier/North Holland Biomedical Press, Amsterdam, New York, 1979.

2.

Agarwal, M. K. (ed), Hormone Antagonists, Walter de Gruyter, Berlin, New York, 1982.

3.

Agarwal, M. K. (ed), Adrenal Steroid Antagonism, Walter de Gruyter, Berlin, New York, 1984.

4.

Agarwal, M. K. (ed), Principles of Recepterology, Walter de Gruyter, Berlin, New York, 1983.

5.

Agarwal, M. K. (ed), FEBS Letters, 178: 1-5 (1984).

6.

Sakiz, E. in Pharmacology and Clinical Uses of Inhibitors of Hormone Secretion and Action (B. J. A. Furr and A. E. Wakeling, eds), Baillere Tindall, London, 1986.

7.

James, V. H. T. and Few J. D., Clinics in Endocrinol. Met 14: 867-892 (1985).

8.

Nedelec, L., Philibert, D. and Torelli, V. in Proc. 3rd SCI-RSC Medicinal Chemistry Symposium Suppl. 45, (R. W. Lambert ed), Royal Soc., London, 1986.

9.

Chrousos, G. P., Cutler Jr., G. B., Sauer, M., Simons Jr, S. S. and Loriaux, D. L., Pharm. Ther., 20: 263-281 (1983) .

10. Agarwal, M. K., Hainque, B., Moustaid, N. and Lazar, G. FEBS Letters 217: 221-226 (1987). 11. Adaikan,P. G. and Kottegoda, S. R., Drugs of the Future, 9: 755-757 (1984). 12. Lazar, G. and Agarwal, M. K., Biochem. Biophys. Res. Comm., 134: 44-50 (1986). 13. Gjerde, H., Morland, J. and Olsen, H., J. Steroid Biochem 23: 1091-1092 (1985). 14. Mercier, L., Miller, P. A. and Simons Jr, S. S., J. Steroid Biochem., 2J5 : 11-20 ( 1986). 15. Santana, M. A., Bolaz, S. C., Beck; G. and Pogson, C. I., Cancer Lett., 2 3 2 9 - 3 3 7 (1985). 16. Chobert, M. N., Barouki, R., Finidori, J., Ageerbeck, M. Hanoune, J., Philibert, D. and Draedt, R., Biochem. Pharmacol., 32: 3481-3483 (1983). 17. Bakke, 0., Cancer Res., 46: 1275-1279

(1986).

18. Cockayne, D., Sterling, K. M., Shull, S., Mintz, K. P., Illeyne, S. and Cutroneo, K. R. , Biochemistry, 2_5: 32023209 (1986) . 19. Dietrich, J. B., Golaz, S. C., Beck, G. and Bauer, G. J. Steroid Biochem., 24: 417-421 (1986).

74

20. Jeremy, J. Y. and Dandona, P., Endocrinology, 119/ 655-660 (1986) . 21. Grunfeld, J. P., Eloy, L., Moura, A. M., Ganeval, D., Frendo, B. R. and Worcel, M. , Hypertension, ]_: 292-299 (1985) . 22. Lassman, M. N. and Mudlow, P. J., Endocrinology, 94 : 1541-1546 (1974). 23. Lazar, G. and Agarwal, M. K., Biochem. Med., (1986) .

70-74

24. Agarwal, M. K. (ed) Multiple Molecular Forms of Steroid Hormone Receptors, Elsevier/North Holland Biomedical Press, Amsterdam, New York, 1977. 25. Agarwal, M. K. and Philippe, M. , Biochem. Med., 26^: 265276 (1981) . 26. Agarwal, M. K. (ed) Proteases and Hormones, Elsevier/ North Holland Biomedical Press, Amsterdam, New York, 1979. 27. Agarwal, M. K., Lombardo, G., Eliezer, N. and Moudgil, V. K., Biochem. Biophys. Res. Comm., 133 : 745-752 (1985). 28. Moudgil, V. K., Lombardo, G., Hurd, C., Eliezer, N. and Agarwal, M. K., Biochim. Biophys. Acta, 889 : 192-199 (1986). 29. Moguliewsky, M. and Philibert, D., J. Steroid Biochem., 20_: 271-276 (1984) . 30. Gagne, D., Pons M. and Philibert, D., J. Steroid Biochem., 23; 247-251 (1985) . 31. Agarwal, M. K. and Kalimi, M., Biochem. Biophys. Res. Comm., 143: 398-402 (1987). 32. Bourgeois, S., Pfhal, M. and Baulieu, E. E., EMBO J., 3: 751-755 (1984). 33. Dietmar, A., Janich, S., Scheidereit, C., Renkawitz, R., Schütz, G. and Beato, M. , Nature (London), 313 : 706-709 (1985) . 34. Scheidereit, C., Geisse, S., Westphal, H. M. and Beato, M. Nature (London) , 304 : 749-752 (1983) . 35. Miesfeld,R., Rusconi, S., Godowski, P. J., Maler, B. A., Okret, S., Wikström, A. C., Gustafsson, J. A. and Yamamoto K. R., Cell, 46: 389-399 (1986). 36. Hollenberg, S. M., Weinberger, C., Ong, E. S., Cerelli, G. Oro, A., Lebo, R., Thompson, E. B., Rosenfeld, M. G. and Evans, R. M., Nature (London), 318: 635-641 (1985). 37. Agarwal, M. K., FEBS Letters, £2: 25-29

(1976).

38. Giguère, V. , Hollenberg, S. M., Rosenfeld, M. G., and Evans, R. M. , Cell, ±6: 645-652 (1986).

75 39. Cayanis, E., Rajagopalan, R., Cleveland, W. L., Edelman, I. S. and Erlanger, B. F., J. Biol. Chem., 261: 50945103 (1986). 40. Agarwal, M. K. and Cayanis, E., Biochem. Biophys. Res. Comm., 136: 470-475 (1986). 41. Sutherland, R. L., Murphy, L. C., Foo, M. S., Green, M. D. Whybourne, A. M. and Krozowski, Z. S., Nature (Lond) 288: 273-275 (1980). 42. Lazar, G. and Agarwal, M. K., Biochem. Biophys. Res. Comm. 134: 261-265 (1986) . 43. Hammer, R. E., Krumlauf, R., Camper, S. A., Brinster, R. L. and Tilgham, S. M., Science, 235: 53-58 (1987). 44. Bertagna, X. , Bertagna, C., Luton, J.P., Husson, J. M. and Girard, F., J. Clin. Endocr. Metab., 59: 25-28 (1984). 45. Baulieu, E. E. and Segal, S. C. (eds) The Antiprogestin Steroid RU 486 and Human Fertility Control. Plenum Press, New York, London, 1985. 46. Healy, D. L., Chrousos, G. P., Schulte, H. M., Williams, R. F., Gold, P. W. , Baulieu, E. E. and Hodgen, G. D., J. Clin. Endocr. Metab., 863-865 (1983). 47. Nieman, L. L., Morin, Bardin, C. England J.

K., Choate, T. M., Chrousos, G. P., Healy, D. M., Renquist, D., Merriam, G. R., Spitz, I. M. W., Baulieu, E. E. and Loriaux, D. L., New Med., 316: 187-191 (1987).

48. Ottow, E., Beier, S., Elger, W., Henderson, D. A., Neef, G. and Wiechert, R. , Steroids, 4_4: 519-530 (1984). 49. Neef, G., Beier, S., Elger, W., Henderson, D. A. and Wiechert, R. , Steroids, 44: 349-372 (1984). Acknowledgments Some of the studies in this chapter were conducted during the sabbatical leave (MKA) from CNRS, Paris, at Oakland University, Michigan, and Columbia University, New York. G. L. Was the recipient of an International Fellowship from the INSERM. We wish to thank Roussel-Uclaf for the gift of 3 H-RU 38486 and litterature retrieval on this compound. The hexanucleotide sequence in the promotor was kindly supplied by Dr. G. Schiitz. Financial support was made available by the UER Broussais Hotel Dieu. We appreciate the preprints supplied by Drs. D. Philibert and E. Sakiz, Roussel-Uclaf. Dr. C. E. Sekeris, National Hellenic Cancer Institute, Athens, was most helpful with the technique on thymocytes.

CLINICAL APPLICATIONS OF THE GLUCOCORTICOID AND PROGESTIN ANTAGONIST RU 486

L.K. Nieman, D.L. Loriaux Developmental Endocrinology Branch, National Institutes of Child Health and Human Development, National Institutes of Health, Bethesda, MD

20892

Introduction

Clinically useful antagonists of mineralocorticoid, estrogen, and androgen action have been available for many years (1-3).

Despite efforts spanning

almost three decades, useful glucocorticoid and progestin antagonists were not available until 1981, when Roussel UCLAF introduced RU 486, [ 17 R -hydroxy-l

(4-dimethylaminophenyl )-17'i'-l-propynyl-estra-4 ,9-dien-

3-one] a compound with both antiprogestin and antiglucocorticoid activities (Fig 1) (4-7).

RU 486 bound to the rat glucocorticoid receptor with an

affinity greater than that of dexamethasone and to the rat progestin receptor with an affinity greater than that of progesterone (6).

It also

had a weak affinity for the rat androgen receptor (8) but had no affinity for the rat mineralocorticoid or estrogen receptors (9).

ch 3 CH;

\C = C-CH'3

RU 486 Figure 1.

The Structure of RU 486

Receptor Mediated Antisteroid Action © 1987 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

78

Bioassays in rodents showed RU 486 to be a progesterone antagonist without any detectable agonist activity.

RU 486 blocked

progesterone-induced

rabbit uteroglobin raRNA (10), progesterone-induced giant mitochondria in rat endometrium (11), and progesterone-induced sex behavior in the guinea pig (12).

It caused abortion in rats (13), mice (13) and rabbits (14).

RU 486 also opposed glucocorticoid action in in vitro rodent cell systems. It antagonized glucocorticoid-induced cytolysis of lymphocytes

(15),

induction of tyrosine aminotransferase in hepatoma cells (16) and immunosuppression of in vitro antibody responses in mice (17).

It prevented the

suppression of plasma A.CTH levels, thymic weight and potassium excretion caused by glucocorticoid administration (13).

Plasma

adrenocorticotropin

(ACTH) and glucocorticoid levels increased after RU 486

administration,

presumably because of antiglucocorticoid activity at the level of the hypothalamus and pituitary gland (14). Studies in the dog (18), rabbit (14) and rhesus monkey (19-23) have confirmed the antiprogestin and antiglucocorticoid properties of RU 486. Tolerance and toxicity studies in cynomolgus monkeys have given preliminary assurance of the relative safety of the drug.

Signs of adrenal

insufficiency were seen only w i t h large daily doses (100 mg/kg) after one month of treatment (14). On the basis of these preclinical

studies,

investigations of the action of RU 486 in man were begun.

This paper will

review the subsequent studies of the pharmacokinetic properties and a n t i progestin and antiglucocorticoid actions of RU 486 in man.

Pharmacokinetics and Metabolism of RU 486

RU 486 is formulated for human use as a tablet containing micronized RU 486 powder.

The pharmacokinetics and metabolism of RU 486 administered as this

oral preparation have been studied using radioimmunoassay

(24), radio-

receptor assay (25), and high pressure liquid chromatography (26) techniques to measure plasma and urine RU 486 and its metabolites.

Unless a prepara-

tive separation method such as thin layer chromatography is used, the available radioreceptor and radioimmunoassay methods detect both RU 486 and its major metabolites. molecules

separately.

High pressure liquid chromatography measures

these

79 Peak plasma levels of RU 486 are reached within two hours of the administration of a single oral dose of 10 - 25 mg/kg (25,26).

to fasting men and women

The plasma half-life of RU 486 is long, 20 - 24 hours (24-26).

During chronic administration of doses of 15 - 20 mg/kg/day, plasma levels of receptor-reactive material were very stable, ranging between 500 - 1000 ug/dl (25).

RU 486 is metabolized by single or double demethylation of the dimethylamine group and/or hydroxylation of the 17 -propynyl chain (27).

This probably

occurs in the liver, as less than 0.5 percent of a daily dose is detected in the urine by radioreceptor assay.

The demethylated metabolites have been synthesized and tested for biologic activity in the rat.

The N-monodemethylated compound has about

one-third the antiprogestin and antiglucocorticoid activity as the parent compound, and the didemethylated analog is even less potent

(27).

In man, over 95% of circulating RU 486 is bound to plasma proteins. may account, in part, for the long plasma half-life of the compound RU 486 binds to albumin and/or a human

1 acid glycoprotein

(25,28) but not to Cortisol binding globulin (CBG) or binding globulin (TeBG) (29).

This (25,27).

(orosomucoid)

testosterone-estradiol

RU 486 is not bound to plasma proteins in

the rat, rabbit or guinea pig (27).

Antiglucocorticoid Properties of RU 486

The antiglucocorticoid properties of RU 486 have been studied in healthy men.

The morning concentrations of plasma ACTH, S-endorphin and Cortisol

increased after administration of RU 486 at a divided oral dose of 6 mg/kg for 24 hours (30).

This amplification of the normal circadian pattern

persisted for two days.

Administration of RU 486 at different times of day

suggested that the antiglucocorticoid potency of RU 486 was influenced by the circadian rhythm of Cortisol secretion.

Subjects received RU 486

(6mg/kg) as a single dose at 2400h, and one week later, at lOOOh.

Plasma

levels of ACTH, R-endorphin and Cortisol rose only in the morning and were

80 not altered in the evening, regardless of the time of administration of RU 486.

Bertagna and co-workers found a similar increase in late morning

Cortisol levels after administration of the drug at 0200h while afternoon (1400h) administration did not affect evening Cortisol levels (31).

These

results suggest that the disinhibition of the pituitary-adrenal axis by RU 486 occurs only during the early morning hours.

The effect on Cortisol levels of RU 486 given at midnight was dosedependent.

No change was seen at a dose of 2.2 mg/kg, while doses of

4.5 and 6.6 mg/kg increased plasma Cortisol concentrations.

The ability of RU 486 to block dexamethasone action was evaluated to confirm that RU 486 acted via the glucocorticoid receptor.

RU 486 (6 mg/kg)

completely antagonized the inhibitory effect of 1 mg of dexamethasone at midnight (30).

This supports the concept that changes in ACTH and Cortisol

levels are mediated through the glucocorticoid receptor.

Conversely, 2 mg

of dexamethasone blocked the antiglucocorticoid effect of 4 mg/kg RU 486. This suggests that adrenal insufficiency resulting from RU 486 administration could be reversed by glucocorticoid administration.

RU 486 also antagonized the peripheral effects of glucocorticoids.

It

blocked the effects of pharmacologic doses of glucocorticoids on blood leukocytes (32) and antagonized cutaneous steroid-induced (33).

vasoconstriction

This latter phenomenon, produced by three different topical steroids

of differing potency in six normal men, was consistently attenuated or abolished by the oral administration of RU 486 (6mg/kg at 2400 and lOOOh). No peripheral antiglucocorticoid effects were observed after administration of RU 486 alone, presumably because the RU 486-induced increases in ACTH and Cortisol compensate at the target tissue for the antiglucocorticoid action of RU 486.

Generally, the dose of RU 486 required for antiglucocorticoid effect was higher than that required for antiprogestin effect.

For example, the

midluteal phase administration of RU 486 at a dose of ~1 mg/kg for four days induced menses within 48 hours (7).

Despite this antiprogestin

activity, no change in plasma B-LPH or Cortisol levels was observed,

81

suggesting that antiglucocorticoid effects were not significant at this dose.

Thus, in normal individuals, RU 486 blocks glucocorticoid negative feedback at the pituitary gland and induces a compensatory increase in plasma ACTH and Cortisol.

This antiglucocorticoid effect of RU 486 is only apparent

during the morning hours.

It is observed at doses exceeding those required

for an antiprogesterone effect, and can be reversed by administration of a glucocorticoid such as dexamethasone.

The dose-dependent dissociation of

antiglucocorticoid and antiprogestin activity and the homeostatic increase in Cortisol permit use of the drug as an antiprogestin without risking antiglucocorticoid

effects.

The pituitary and adrenal responses to short term administration of RU 486 have also been evaluated in patients with Cushing's syndrome (34). Urinary excretion of Cortisol increased by 50 to 1000%

in four patients

with Cushing's disease after three days of RU 486 administration (400mg/day).

17-Hydroxysteroid excretion and plasma LPH and Cortisol

levels also increased.

The parallel increase in LPH and glucocorticoid

levels suggested that the adrenal response resulted from RU 486 disinhibition of negative feedback at the pituitary gland. The activation of the pituitary-adrenal axis persisted for 3 - 4

days after RU 486 was stopped,

a finding consistent with the long plasma half-life of the compound.

In contrast, RU 486 did not alter the plasma or urinary glucocorticoid concentrations in two patients with nonpituitary-dependent Cushing's syndrome.

The discordant responses of the patients with pituitary and

nonpituitary dependent causes of Cushing's syndrome suggest that RU 486 administration may be useful in the differential diagnosis of Cushing's syndrome.

Further studies will be necessary to define the optimal regimen

for this use.

RU 486 in the Treatment of Cushing's Syndrome

When possible, surgical resection provides optimal therapy for ACTH and cortisol-secreting tumors (35).

Transsphenoidal microadenomectomy

82 remains the treatment of choice for Cushing's disease.

Surgical

treatment

of ectopic ACTH-secreting tumors is not feasible if the tumor is widely metastatic, or if it is occult at the time of presentation.

Patients w i t h

adrenal carcinoma also may have disseminated disease, rendering surgical extirpation unlikely.

For these individuals, an effective medical therapy

for hypercortisolism is desirable.

The medical therapy of Cushing's syndrome has traditionally been directed to the adrenal gland with adrenal enzyme inhibitors such as aminoglutethimide (36), metyrapone (37), ketoconazole (38) and trilostane (39), and with the adrenolytic agent o,p'DDD (40).

These drugs, however,

adequately reduce the hypercortisolism in all patients.

cannot

A further drawback

is the frequent incidence of toxic side-effects, especially at the large doses required for control of hypercortisolism.

RU 486 offers a new approach in the therapy of Cushing's syndrome We have administered this compound for 2 - 1 2

(29).

months to three patients

w i t h Cushing's syndrome caused by the ectopic secretion of ACTH. protocol for treatment was approved under an investigational

The

exemption

for new drugs by the National Center for Drugs and Biologies, DHHS and by the N1CHHD Research Subpanel.

All subjects gave their informed

consent and underwent baseline studies before beginning

treatment.

The assessment of the effectiveness of a glucocorticoid antagonist differs from that of other available medical agents.

With the latter

compounds,

successful inhibition of steroidogenesis can be monitored by measuring plasma Cortisol concentrations or urinary Cortisol or Cortisol metabolite excretion.

In contrast, RU 486 prevents the action of circulating

by occupying its receptor site.

corticoid antagonist can only be assessed indirectly by monitoring and biochemical glucocorticoid-sensitive

Clinical parameters used to follow weight and blood pressure. questionnaire.

Cortisol

Thus, successful treatment w i t h a glucoclinical

measures.

RU 486 treatment included body habitus,

Mood and libido were assessed by a self-report

Metabolic and hormonal measures included fasting and p o s t -

absorptive blood sugar, which are elevated by hypercortisolism, and plasma

83 concentrations of luteinizing hormone (LH), testosterone, testosteroneestradiol binding globulin (TeBG), Cortisol binding globulin (CBG), and TSH, which are suppressed by hypercortisolism. made on blood samples drawn RU 486 dose.

These measurements were

before therapy and prior to each increase in

Plasma ACTH and Cortisol and urinary Cortisol levels were

also measured before and during treatment.

RU 486 was given in divided daily doses beginning at 5 - 10 mg/kg and increasing in 5 mg/kg increments every two weeks to a maximum of 20 mg/kg/day. The first patient to be treated was a 25 year old white man with a widely metastatic carcinoid tumor. ectopic ACTH syndrome.

He presented with features typical of the

These included moon facies, truncal obesity,

muscle weakness, hypertension, diabetes mellitus, hypokalemic alkalosis, hypercortisolism and hyperpigmentation.

He was depressed and complained of

decreased libido.

The patient received RU 486 for nine weeks and showed marked improvement in Cushingoid features.

The physical stigmata of Cushing's syndrome, including

supraclavicular and dorsocervical fat pads and central obesity, regressed. Maximal systolic and diastolic blood pressure decreased steadily during treatment, from 200/120 mm Hg prior to therapy, to 140/90 at its conclusion. The hypokalemic alkalosis resolved as serum potassium levels increased from 1.9 mEq/L to 4.5 mEq/L.

Potassium supplements were discontinued after the

sixth week of therapy.

Both objective and subjective psychological measures improved during therapy with RU 486.

The patient's depression improved, and he reported increased

libido, attention span and sense of well-being.

These subjective improvements

were corroborated by self-rating questionnaire and psychiatric interviews.

Biochemical measures of glucococorticoid action also improved with RU 486 therapy, as shown in Figure 2.

Glucose tolerance improved and plasma

concentrations of testosterone, LH, TeBG, CBG and TSH all returned to the normal range.

No new serum chemistry (creatinine, BUN, SGOT, SGPT) or

chest radiography abnormality or abnormalities in urinalysis, EKG, or physical examination were found during or following therapy.

No signs or

84

A.

B.

Glucose 2 Hr OGTT (mg/dl)

LH (mlU/ml) 30

12 -I

140 -

I

Total Testosterone (ng/dl)

750

250

TeBG (ug/dl)

C B G (Mg/dl)

J

I

J

I

Free Testosterone (ng/dl)

Mean BP (mmHg) ISO

_L Dose RU486 (mg/kg/d) 20 r

10^

•

15

--r

Dose RU 486 (mg/kg/d) F

.

^

— r 45

r

DAY OF ADMISSION Figure 2. The effect of RU 486 treatment on glucocoticoid-sensitive measures. (All ordinate scales are linear.) Panel A: Two hour post-OGTT (normal • ) , h y d r o c o r t i s o n e (•——••), indomethacin (• W) and RU38486 (A A.) on P G I 2 (as 6 - o x o - P G F ^ a ) and PGE2 secretion by gastric explants following an 18 h culture. Untreated tissue release 0.86 ng 6-oxo-PGF]ci and 0.62 ng PGE2/mg wet tissue/l h. Each point represents mean + S.D. (n=6). IC50S in m o l . l - ! for PGI2: betamethasone, 1x10"^; hydrocortisone, l x l 0 ~ 6 ; indomethacin, l x l O - ® ; and RU38486, > l x l 0 - 4 ; for PGE2: betamethasone, l x l O - 7 ; hydrocortisone, l x l O - 6 ; indomethacin, 1 . 2 x l 0 - 6 ; and RU38486, > l x l 0 ~ 4 .

112

HYDROCORTISONE PGE„

PGI„ 0 20CO

co

40 -

I2 >-

60-

UJ X

CO

Q O z < Ico o CC CL

80 -

100-

I

2 3 4 5

|

2

3

4

5

4

5

BETAMETHASONE

o h CO X 2

PGI Q

0

PGE~

2040 60 -

80 •

100-

Figure

I

2

3

4

5

I

2

3

7.

Effect of RU38486 o n h y d r o c o r t i s o n e ( 1 x l 0 ~ 6 m o l . l - 1 ) - and b e t a m e t h a s o n e ( l x l O - ^ m o l . 1 - 1 ) - i n h i b i t e d P G I 2 (as 6-oxo-PGF^a) and PGE2 synthesis by duodenal explants f o l l o w i n g a n 18 h c u l t u r e . Each point represents mean + S.D. (n=6). C o d e : (1) u n t r e a t e d t i s s u e ; (2) g l u c o c o r t i c o i d a l o n e . T h e n same d o s e s of g l u c o c o r t i c o i d + v a r y i n g R U 3 8 4 8 6 , i n mol.l"1: (3) l x l O - ' ; (4) 5 x l 0 - 7 ; (5) 1 x l 0 - 6 . U n t r e a t e d t i s s u e r e l e a s e d 1.4 ng 6 - o x o - P G F ^ a and 0.98 ng PGE2/mg w e t tissue/1 h.

and

negate

Stress gastric

the

ulcerogenic

is a k n o w n

epidemiological

and duodenal

hypersecretion

of

properties

ulcer

adrenal

(47)

of

factor

and

hormones,

NSAIDs

(45,46).

in the g e n e s i s

is a l s o k n o w n including

to

of

elicit

corticosteroids

113

HYDROCORTISONE

PGI,

PGE„

20 •

ço to LU I I-

z >V)

g O z < f— w o

4060

"

80 •

100 .

1 2 3 4 5

1 2

3 4 5

BETAMETHASONE

(E

S

PGE„

PGL

Z o 20 •

40 • 6080 100 L

Figure

1

2 3 4 5

1

2 3 4

5

8.

Effect of RU38486 on hydrocortisone (1xl0 -6 mol.l - 1 )- and betamethasone (3xl0 - ^ mol.I -1 )-inhibited PGI2 (as 6-oxo-PGF^a) and PGE2 synthesis by gastric explants following an 18 h culture. Each point represents mean + S.D. (n=6). Code: (1) untreated tissue; (2) glucocorticoid alone. Then same doses of glucocorticoid + varying RU38486,in mol.l--'-: (3) lxlO - 7 ; (4) 5xl0 - 7 ; (5) 1xlO - 6 . Untreated tissue released 1.32 ng 6-oxo-PGF i a and 0.86 ng PGE 2 /mg wet tissue/l h.

(38).

In this context, the therapeutic administration of

corticosteroids is known sometimes to result in peptic and duodenal ulcers (48).

Since corticosteroids inhibit prosta-

noid synthesis (this paper), it is reasonable to propose that

114 corticosteroid-linked

ulcerogenesis

i n h i b i t i o n of the a n t i u l c e r o g e n s , RU38486 may prove beneficial stressed patients

Concluding

and

in o b v i a t i n g

in p a t i e n t s

potent antagonist

taking

in t h i s p a p e r

i n d u c t i o n of a b o r t i o n , not,

ulcerogenesis

but

earlier,

syndrome,

stressed patients

glucocorticoids)

that RU38486 in

(1,2,3)

in

excess

and patients

is a s s o c i a t e d

with

(as

in

in t h e s e

two major

and gastroduodenal

conditions.

influence many other glucose

diverse Thus

functions will

steroids

therapeutic

the effects

pathological

ulceration.

in the a e t i o l o g y to

the

of this

with Cushing's

novel effects

but

also

thank

Pamela Dale

for

immune

system

RU38486 on also on

these

may prove useful

syndrome.

for p r e p a r i n g

the

of

other

antiglucocorticoid.

Acknowledgement We

of

be

including, of

We

the

shed light not o n l y on the role

its a n t i g l u c o c o r t i c o i d

treating patients

was

Cushing's

Glucocorticoids

functions

in n o r m a l p h y s i o l o g y ,

applications

As

s u p p r e s s i o n of

and protein metabolism,

and brain excitation.

properties

taking

that RU38486 might prove

and

to

the

fully explored.

glucocorticoid

hypertension

and

might p l a y a role

Finally,

a

and

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

that glucocorticoid-induced

beneficial

is

has already been proved

these processes,

adrenal

in

intrauterine

relevant prostanoids

and other

the

corticosteroids.

in v a s c u l a r

as yet, b e e n

discussed

processes:

action

action

as a contraceptive

of RU38486 h a v e

example,

to

Furthermore,

tissue.

as an antiprogestagen,

be successful

confirm

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

tissue and of g l u c o r t i c o i d gastroduodenal

suggested

in p a r t

and PGE2•

Remarks

The data presented

RU38486,

may be due

PGI2

manuscript.

in

115

References 1.

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2.

Healy, D.L. and H.M. Fraser. 1985. The antiprogesterones are coming. Br. Med. J. 290, 580-582.

3.

Crowley, W.F. 1986. Progesterone antagonism. Science and Society. N. Eng. J. Med. 315, 1607-1608.

4.

Hermann, W., R. Wyss, A. Riondel, D. Philibert, G. Teutsch, E. Sakiz and E.-E. Baulieu. 1982. Effet d 1 u n steroide antiprogesterone chez la femme: interruption du cycle menstruel et de la grossesse au debut. C.R. Seances Acad. Sci. (iii) 295, 933-938.

5.

Kelly, R.W., D.L. Healy, M.J. Cameron, I.T. Cameron and D.T. Baird. 1986. The stimulation of prostaglandin production by two antiprogesterone steroids in human endometrial cells. J. Clin. Endo. Metab. 1986, 62, 1116-1123.

6.

Philibert, D. 1984. In: Adrenal Steroid Antagonism (M.K. Aggarwal, ed.) Walter de Gruyter, Berlin • New York, pp 77-101.

7.

Chobert, M.N., R. Barouki, J. Findori, J. Hanoume, D. Philibert and R. Deraedt. 1983. Antiglucorticoid properties of RU38486 in a differential hepatoma cell line. Biochem. Pharmacol. _32, 3481.

8.

Mogiulewsky, M. and D. Philibert. 1984. RU38486: Potent antiglucorticoid activity correlated with strong binding to the cytosolic glucocorticoid receptor followed by an impaired activation. J. Steroid Biochem. 20, 271-276.

9.

Csapo, A.I. 1977. In: The Fetus and Birth: Ciba Symposium 47 (J. Knight and M. O'Connor, eds.) Elsevier, Amsterdam, pp 159-210.

10.

Blackwell, G.J., R. Carnuccio, M. Di Rosa, R.F. Flower, L. Parente and P. Persico. 1980. Macrocortin: a polypeptide causing anti-lipase effects of glucocorticoids. Nature 287, 147-150.

11.

Jeremy, J.Y. and P. Dandona. 1986. Inhibition by hydrocortisone of prostacyclin synthesis by rat aorta and its reversal with RU486. J Endocrinol 119, 661-665.

116

12.

Jeremy, J.Y. and P. Dandona. 1986. RU486 antagonises the inhibitory action of progesterone on prostacyclin and thromboxane A2 synthesis in cultured rat myometrial explants. J Endocrinol 119, 655-660.

13.

Hirata, I.Y., M. Notsue, L. Iwata, M. Parente, M. Di Rosa and R.J. Flower. 1982. Identification of several species of phospholipase inhibitory proteins by radioimmunoassay for lipomodulin. Biochem. Biophys. Res. Comm. 109, 223-234.

14.

Danon, A. and G. Assouline. 1978. Inhibition of prostaglandin synthesis by corticosteroids requires RNA and protein synthesis. Nature 273, 552-554.

15.

Jeremy, J.Y., D.P. Mikhailidis and P. Dandona. 1984. Vascular trauma and prostacyclin release. Microcirc. Endothel. Lymph. 1, 629-642.

16.

Jeremy, J.Y., D.P. Mikhailidis and P. Dandona. 1985. Adrenergic modulation of vascular prostacyclin synthesis. Eur. J. Pharmacol. 115, 33-40.

17.

Jeremy, J.Y., D.P. Mikhailidis and P. Dandona. 1986. Prostanoid synthesis by the rat urinary bladder: evidence for stimulation through muscarine receptor linked calcium channels. Naunyn Schmied. Arch. Pharmacol. 334, 463-467.

18.

Jeremy, J.Y., D.P. Mikhailidis and P. Dandona. 1985. The thromboxane A2 analogue, U46619, stimulates vascular prostacyclin synthesis. Eur. J. Pharmacol. 107, 259-262.

19.

Jeremy, J.Y., D.P. Mikhailidis and P. Dandona. 1986. Muscarinic stimulation of prostacyclin synthesis by the rat penis. Eur. J. Pharmacol. 123, 67-71.

20.

Jeremy, J.Y., D.P. Mikhailidis and P. Dandona. 1985. A comparison of the effects of tiaprofenic acid and indomethacin on in vitro prostaglandin synthesis by rat, rabbit and human stomach tissue. Agents and Actions _12, 205-208.

21.

Ham, E.A., V.J. Cirillo, M.E. Zanetto and F.A. Keuhl, Jr. 1975. Estrogen directed synthesis of specific prostaglandins in uterus. Proc. Natl. Acad. Sci. 72, 1420-1427.

22.

Downie, J., N.L. Poyser and M. Wunderlich. 1974. Level of prostaglandins in human endometrium during the normal menstrual cycle. J. Physiol. 236, 465-472.

117

23.

Maathius, J.B. and R.W. Reilly. 1978. Concentration of prostaglandins F2ct and E2 in the endometrium throughout the human menstrual cycle after administration of clomiphene or an oestrogen-progesterone pill and in early pregnancy. J. Endocrinol. 7_7, 361-371.

24.

Liggins, G.C. and R.N. Howie. 1972. A controlled trial of antepartum glucocorticoid treatment for prevention of the respiratory distress syndrome. Pediatrics 50, 515-525.

25.

Irvine, R.F. 1982. How is the level of free arachidonic acid controlled in mammalian cells Biochem. J. 204, 3-16.

26.

Anggard, E. and B. Samuelsson. 1965. Biosynthesis of prostaglandins from arachidonic acid in guinea pig lung. J. Biol. Chem. 240, 3518-3529.

27.

Bunting, S., R. Gryglewski, S. Moncada and J. Vane. 1976. Arterial walls generate from prostaglandin endoperoxides a substance (prostaglandin X) which relaxes strips of mesenteric and coeliac arteries and inhibits platelet aggregation. Prostaglandins 12, 897-913.

28.

Jeremy, J.Y., D.P. Mikhailidis and P. Dandona. 1986. The effect of nifedipine, nimodipine and nisoldipine on agonist- and trauma-stimulated vascular prostacyclin synthesis. Naunyn Schmied. Arch. Pharmacol. 332, 70-74.

29.

Baenzinger, M.L., F.J. Fogerty, L.F. Mertz and L.F. Chernuta. 1981. Regulation of histamine-mediated prostacyclin synthesis in cultured human vascular endothelial cells. Cell 24, 915-925.

30.

Boeynaems, V.M. and N. Galand. 1983. Stimulation of vascular prostacyclin synthesis by extracellular ADP and ATP. Biochem. Biophys. Res. Comm. 112, 200-212.

31.

Miller, R.L. and K.L. Melmon. 1972. In: Clinical Pharmacology. Basic Principles in Therapeutics. (K.L. Melmon and H.F. Moretti, eds.) MacMillan Co., New York, pp 382-405

32.

Krakoff, L.R., G. Nicholis and B. Amsel. 1975. Pathogenesis of hypertension in Cushing's syndrome. Am. J. Med. 58, 216-218.

33.

Moncada, S. and J.R. Vane. 1979. In: Chemistry and Pharmacological Activity of Prostanoids. (S.M. Roberts and F. Scheinmann, eds. Pergamon Press, Oxford, pp 258-273.

118

34.

Yard, A.C. and P.J. Kadowitz. 1972. Studies on the mechanism of hydrocortisone potentiation of vasoconstrictor responses to epinephrine in the anaesthetised animal. Eur. J. Pharmacol. 20_, 1-9.

35.

Hadhazy, P., L. Nagy, F. Juhasz, B. Malomvogyi and K. Magyar. 1983. Effects of indomethacin and prostaglandin on the tone of human isolated mesenteric arteries. Eur. J. Pharmacol. 91, 477-484.

36.

Kalsner, S. 1969. Mechanisms of hydrocortisone potentiation of responses to epinephrine and norepinephrine in rabbit aorta. Circulation Res. 24, 383-395.

37.

del Favero, A. 1985. In: Side Effects of Drugs Annual (M.N.G. Dukes and L. Beeley, eds.) Elsevier, Amsterdam, p 83.

38.

Liddle, G.W. and K.L. Melmon. 1974. Textbook of Endocrinology (R.H. Williams, ed.) W.B. Saunders Co., Philadelphia, pp 233-283.

39.

Ibels, E.S., J.H. Stewart, J.F. Mahony and A.G.R. Sheil. 1974. Deaths from occlusive arterial disease in renal allograft recipients. Br. Med. J. 552-554.

40.

Harker, L.A., S.M. Scwartz, R. Ross. 1981. In: Clinics in Haematology (C.R.M. Prentice, ed.) W.B. Saunders, London, pp 283-300

41.

Robert, A. 1984. In: Mechanisms of Mucosal Protection in the Upper Gastrointestinal Tract. (A. Allen and G. Felstrom, eds.) Raven Press, New York, p 377.

42.

Chaudury, M.L. and E.D. Jacobson. 1978. Prostaglandin cytroprotection of gastric mucosa. Gastroenterol 74, 59-63.

43.

Levy, M.W. 1974. Aspirin use in patients with major upper gastrointestinal bleeding and peptic ulcer disease. N. Eng. J. Med. 290, 1158-1162.

44.

Chapamn, M.L. 1978. Peptic ulcer: a medical perspective. Med. Clin. North Am. 6_3, 39-51.

45.

Cohen, M.M. and J.M. Pollett. 1976. Prostaglandin E2 prevents aspirin and indomethacin damage to human gastric mucosa. Surg. Forum 27^, 400-401.

46.

Konturek, S.J., T. Radecki and I. Brzozowski. 1981. Aspirin-induced gastric ulcers in cats - protection by prostacyclin. Dig. Dis. Sei. 26^, 1003-1012.

119 47.

48.

Silen, W., A. M e r h a n and J.N.L. Simson. 1981. The p a t h o p h y s i o l o g y of stress u l c e r s . W o r l d of S u r g e r y 165-175. Messer, J., D. R e i t m a n and H.S. S a c k s . 1983. A s s o c i a t i o n of a d r e n o c o r t i c o s t e r o i d t h e r a p y a n d ulcer disease. N. Engl. J. Med. 309, 21-24.

5,

peptic

RECEPTOR-MEDIATED ANTIPROGESTIN ACTION OF RU 486

Mohammed Kalimi Dept. of Physiology, Medical College of Virginia Virginia Commonwealth University, Richmond, VA

23298, USA

Introduction

Progesterone is known to play a prominent role in female reproductive physiology.

Progesterone can promote uterine growth,

inhibit uterine contraction and, depending on concentration, either potentiate or inhibit actions of estrogen.

The role of progesterone in

promotion and formation of a secretory endometrium is crucial for ovum implantation and pregnancy.

Progesterone is also known to stimulate the

secretory activity of the oviduct.

It also promotes viscosity of

cervical mucus and growth and development of the breast.

Many other

biochemical and physiological effects are attributed to progesterone. For example, it is well known that progesterone induces the secretion of avidin by hen oviduct and uteroglobulin in rabbit uterus.

In view of the

significant physiological and biochemical roles of progesterone in female reproductive physiology, it is natural that intense research efforts have been directed toward synthesizing compounds having biochemically and pharmacologically potent antiprogestin effects.

In 1980, researchers at

Roussel-Uclaf synthesized one of the most exciting antiprogestin compounds designated RU 486 (11 - (4-dimethyl amino phenyl)- 17 -hydroxy, 17 - (propyl-ynyl)- estra - 4, 9 - dien - 3-one). This compound showed both antiprogestin and antiglucocorticoid

Receptor Mediated Antisteroid Action © 1987 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

122

properties

i n v i v o as w e l l as i n v i t r o .

devoid of any agonist activity

(1-5).

The R U 486

is r e p o r t e d to b e

The properties of this

pure

antagonist distinguish RU 486 from other antiprogestin compounds R 2 3 2 3 , R M I 1 2 9 3 6 , etc. w h i c h s h o w b o t h p a r t i a l activities 7).

such as

antagonist/agonist

in experimental animal model system a n d in clinical trials

The antiprogestin RU 486 has potential clinical

interest as

(6-

a

t e r m i n a t o r o f p r e g n a n c y a n d p o s s i b l y as a n a n t i c a n c e r a g e n t i n t h e treatment of advanced hormone-dependent breast cancer.

Indeed,

oral

a d m i n i s t r a t i o n o f t h i s c o m p o u n d is k n o w n to i n t e r r u p t the l u t e a l p h a s e the m e n s t r u a l c y c l e a n d e a r l y p r e g n a n c y i n w o m e n ( 8 - 1 0 ) .

The

aspects of R U 486 are r e v i e w e d elsewhere

Beside

clinical importance of this compound,

in this volume.

the r e c e n t a v a i l a b i l i t y

of

clinical obvious

of

t r i t i a t e d R U 4 8 6 m a y r e p r e s e n t a p o w e r f u l n e w t o o l to e x p l o r e t h e

precise

u n d e r s t a n d i n g o f the m e c h a n i s m o f s t e r o i d h o r m o n e a c t i o n a t t h e

molecular

level.

the

Overall,

the e l u c i d a t i o n o f v a r i o u s m e c h a n i s m s b y w h i c h

synthetic drug RU 486 exerts physiological

its p o t e n t a n t i g e s t i o n a l e f f e c t s

and biochemical actions of progesterone

significant scientific

on

is n o t o n l y

i n t e r e s t b u t m a y l e a d to t h e s y n t h e s i s o f

pharmacologically potent compounds with higher antiprogestional

of improved

activity.

T h e b i o l o g i c a l p o t e n c y o f R U 4 6 8 i n v a r i o u s m o d e l s y s t e m is s u m m a r i z e d Table

I.

in

123 TABLE I

Biological potency (in terms of progesterone action) of RU 486 in various model systems.

System

Biological potency

Observed biological effects

Rat uterus (11)

Pure antagonist

Antinidatory and abortive activity

Rabbit uterus(11,19)

Pure antagonist

Inhibition of endometrial proliferation Inhibition of progesterone induced uteroglobulin mRNA

Human endometrium (16)

Partial agonist antagonist

Given alone has some progestomimetic effects Given with progesterone has pure antagonistic effects

Guinea pig nervous (14)

Pure antagonist

Inhibition of sexual behavior system

Rat nervous system (15)

Pure antagonist

Antagonism of progesterone facilitation of estrous behavior

124

Biological potency

System

Observed biological effects

H u m a n b r e a s t tumor cells T47D (17)

Pure antagonist

Complete inhibition of progesterone

induction

of two proteins T47DCO

(22)

Partial agonist antagonist action

Agonist in terms of growth

inhibition

Antagonist in terms of insulin receptor stimulation H u m a n endometrial cancer cells

Pure antagonist

Antimitotic effects

(HEC-1

In this chapter, I will concisely review our current knowledge the receptor-mediated antiprogestin action of RU 486.

regarding

The m e c h a n i s m of

antiglucocorticoid action of RU 486 will be dealt w i t h in a separate chapter in this book.

Receptor binding!: studies In Vitro In order to u n d e r s t a n d the mechanism b y w h i c h RU 486 antagonizes p r o g e s t i n action, it is of fundamental importance to determine w h e t h e r RU 486 exerts its effect b y binding to the same receptor moiety as the agonist without generating any biological responses.

In contrast,

the

125

a n t a g o n i s t m a y b i n d to a n e n t i r e l y d i f f e r e n t p r o t e i n a n d s o m e h o w agonist-receptor

complex interaction or post-receptor

modulate

events.

E x c e p t for one report w h i c h will be m e n t i o n e d later,

current

findings from several laboratories suggest that RU 486 clearly acts b i n d i n g w i t h h i g h a f f i n i t y to the p r o g e s t e r o n e r e c e p t o r o f v a r i o u s tissues.

Philbert

(11) r e p o r t e d a v e r y h i g h r e l a t i v e b i n d i n g

(about 5 times) of R U 486 for rabbit uterine progesterone c o m p a r e d to p r o g e s t e r o n e .

The association constant of

target

affinity

receptor

[ H] R U 4 8 6

for

the r a t u t e r i n e p r o g e s t e r o n e

receptor was about 2 times higher than

of synthetic p r o g e s t i n R5020

(11).

in various target tissues

a f f i n i t y o f t h i s c o m p o u n d f o r the p r o g e s t e r o n e i n the d i s s o c i a t i o n r a t e a t 4 ° C o f progesterone

receptors.

is a l s o

[^H] R U 4 8 6 f r o m r a t

uterine

The half-time of d i s s o c i a t i o n of

higher

apparent

[^H] R U 4 8 6 ,

70 m i n . , a n d 10 m i n . ,

(MBH-PIA) cytosolic progesterone

receptors

b i n d i n g a f f i n i t y w a s c a l c u l a t e d to b e 50 c o m p a r e d to Etgen and Barfield

(15) o b s e r v e d t h a t R U 4 8 6

HPOA cytosols were

(relative

is a c o m p e t i t i v e

inhibitor

(HPOA).

incubated with various concentrations

When

(0.5-20 nM)

of

486 i n the p r e s e n c e or a b s e n c e o f u n l a b e l l e d p r o g e s t e r o n e ,

affinity,

limited capacity binding was detected kd=8.4 nM

injected)

and kd=l.7 nm

(in 8 y g E2 b e n z o a t e

a high

(in o i l

injected 48h before

P r o g e s t i n s w e r e f o u n d to b e the m o s t e f f e c t i v e c o m p e t i t o r binding,

of

progesterone).

[^H] R 5 0 2 0 b i n d i n g i n r a t h y p o t h a l a m u s p r e o p t i c a r e a

[3H] RU

The

B r o w n a n d B l a u s t e i n (14) r e p o r t e d h i g h a f f i n i t y b i n d i n g

R U 4 8 6 to n e u r a l

of

(12,13).

the

higher

receptor

R 5 0 2 0 a n d p r o g e s t e r o n e w e r e f o u n d to b e 16 h o u r s , respectively.

that

I t is n o t e w o r t h y to m e n t i o n t h a t

a s s o c i a t i o n c o n s t a n t o f R 5 0 2 0 w a s r e p o r t e d to b e a b o u t 7 t i m e s than that of progesterone

by

for

death).

[^H] R U 4 8 6

suggesting a n a n t i p r o g e s t i n effect of this c o m p o u n d rather

than

126

an antiglucocorticoid effect in the nervous system.

However, they have

not ruled out the binding of RU 486 to neural glucocorticoid receptor sites beside progesterone receptors.

Gravanis et al. (16) showed high

affinity binding of [ 3 H] RU 486 in cytosols of endometrium obtained from post-menopausal women.

The kd for [ 3 H] RU 486 and [ 3 H] progesterone

binding to the endometrium progesterone receptor was 0.8 nM and 1.2 nM, respectively.

Interestingly, the presence of 30% glycerol in the cytosol

resulted in a significant decrease in the affinity of RU 486 for the progesterone receptor, while the affinity of progesterone for the progesterone receptor remained unchanged. RU 486 (17) is known to suppress the growth of human breast cancer cells in vitro and also inhibits progesterone induction of a number of proteins.

These effects of RU 486 are shown to be mediated by its high

affinity binding to progestin receptors in breast cancer cell lines such as MCF7 and T47D (17).

It was further shown that this growth suppressing

effect was dose-dependent and its magnitude correlated well with the progesterone receptor concentration in these cell lines (17).

The

selective antiprogestronic effect of this compound was demonstrated by the fact that addition of dexamethasone, estradiol or testosterone were unable to reverse the growth inhibitory effect of RU 486, but cells readily responded to low concentration of progestin R5020. Moudgil et al. (18) carried out both competition and stability experiments and conc luded that [ 3 H] RU 486 and [ 3 H] progesterone bind to the

same receptor moiety in the calf uterus cytosol.

Finally, no

immunological differences were observed between [ 3 H] R5020 (agonist) and o [ H] RU 486 (antagonist) receptor complexes of rabbit endometrium cytosol when titrated against five monoclonal antibodies raised against purified

127

R5020-receptor

complex

(19).

T h i s f i n d i n g i n d i c a t e s t h a t t h e r e are

major structural differences between agonist and antagonist c o m p l e x e s to a c c o u n t for the o b s e r v e d d i f f e r e n c e s activity of these two

i n the

receptor

biological

compounds.

I t is a p p a r e n t f r o m the r e v i e w e d l i t e r a t u r e

that R U 486 brings

its a n t i p r o g e s t i n a c t i o n s b y b i n d i n g w i t h h i g h a f f i n i t y to progesterone receptor present in various m a m m a l i a n target Unlike

no

about

the tissues.

the m a m m a l i a n s y s t e m , r e c e n t c h a r a c t e r i z a t i o n o f R U 4 8 6 b i n d i n g

chick oviduct

(avian) cytosol

(20) s u g g e s t s a s e p a r a t e b i n d i n g s i t e

[3H] RU 486 and

[3H] progesterone.

oviduct cytosol

(20), it w a s o b s e r v e d t h a t l i k e

In b i n d i n g studies u s i n g

R U 4 8 6 w h e n a d d e d u p to 50 n M l e v e l w a s u n a b l e

for

chick

[3H] progesterone, to r e a c h the

in

[3H]

saturation.

o In this system,

[ H] R U 4 8 6 w a s n o t d i s p l a c e d b y l a r g e e x c e s s

unlabelled progesterone likewise

ineffective

of

a n d 1000 f o l d e x c e s s o f u n l a b e l l e d R U 4 8 6 o

in displacing

[ H] p r o g e s t e r o n e b i n d i n g .

addition,

d e n a t u r a t i o n s t u d i e s s h o w e d t h a t a l m o s t 40% o f c h i c k

cytosolic

[ H] p r o g e s t e r o n e - r e c e p t o r

incubation at 37°C, whereas

the

Further

[3H] progesterone

and

h e t e r o g e n i t y b e t w e e n t h e s e two c o m p o u n d s

(20).

exchange chromatography on DEAE-sephacel

columns and

physicochemical [ 3 H ] R U 486

labelled cytosol

receptor

binding

For example, b o t h

ion

sucrose-density

g r a d i e n t s a n a l y s i s s u g g e s t the p r e s e n c e o f two l a b e l l e d s p e c i e s [ H] p r o g e s t e r o n e

oviduct

[ 3 H ] R U 4 8 6 c o m p l e x w a s f o u n d to b e

complexes of chick oviduct again revealed macromolecular

case of

In

complex was inactivated within 5 min

s t a b l e u p to 60 m i n o f i n c u b a t i o n a t 37°C. characterization of cytosolic

was

i n the

(two r a d i o a c t i v i t y p e a k s

DEAE-sephacel columns and 4 S and 8 S moieties on sucrose

density

gradients).

(one

O n the o t h e r h a n d , a s i n g l e l a b e l l e d s p e c i e s

on

128

radioactivity peak on DEAE-sephacel column and 4 S

moiety on

sucrose[JH] RU

density gradients) was observed when cytosol was labelled with 486

(20).

R U 486 b i n d i n g

in vivo

Oral doses

( l - 1 0 m g / k g ) o f R U 4 8 6 w h e n a d m i n i s t e r e d to

rats resulted in a dose-dependent decrease of p r o g e s t e r o n e receptors

in rat uterus

overiectomized

in percent free binding

(11).

The total saturation

progesterone receptor b i n d i n g sites in vivo was a t t a i n e d at a dose.

A g o o d c o r r e l a t i o n was n o t e d b e t w e e n the amount of RU

486 induced

responses such as endometrial p r o l i f e r a t i o n and increased volume o f m i t o c h o n d r i a a n d c o m p l e t e o c c u p a n c y o f free p r o g e s t e r o n e (11).

This finding suggests that RU 486

of

lOmg/kg

( l O m g / k g ) r e q u i r e d f o r the c o m p l e t e a n t a g o n i s m o f p r o g e s t e r o n e

binding sites

sites

density

receptor

inhibits

p r o g e s t e r o n e a c t i o n b y b i n d i n g to the p r o g e s t e r o n e r e c e p t o r i n v i v o . R U 486 behaviour

injection

(5mg) w a s a b l e to i n h i b i t the e x p r e s s i o n o f

in o v a r i e c t o m i z e d estrogen-primed guinea pigs treated w i t h

0.lmg progesterone

(14).

This dose

(5mg) r e s u l t e d i n a 44% d e c r e a s e

the n u m b e r o f a v a i l a b l e p r o g e s t i n b i n d i n g s i t e s i n h y p o t h a l a m i c cytosol, w i t h no observable alteration in b i n d i n g affinity Similarly,

sexual

Etegen and Barfield

intracerebral

i n the

h y p o t h a l a m u s o f f e m a l e r a t s w a s a b l e to a n t a g o n i z e facilitated estrous behavior.

or

ventromedial progesterone

T h i s e f f e c t w a s f o u n d to b e m e d i a t e d b y

interference with progesterone receptor binding. present time w h y such a h i g h dose to a n t a g o n i z e the p r o g e s t e r o n e

(MBH-POA)

(14).

(15) d e m o n s t r a t e d t h a t s y s t e m a t i c

i n j e c t i o n o f the 5 m g o f R U 4 8 6

in

(5-10mg/kg)

I t is n o t c l e a r a t

the

o f R U 486 is n e e d e d i n v i v o

induced biological responses when

it

129

displays very potent affinity similar to progesterone and R5020 for the progesterone receptor in vitro (11-15).

For example, R5020 is known to

saturate the free progesterone receptor binding sites in vivo at doses lower than 50 Ug/kg.

One explanation for such a higher in vivo

requirement of RU 486 to obtain complete antiprogestional activity may be due to a higher level of binding of RU 486 to the plasma proteins. However, Mcguilewsky and Phibert (21) have reported that except for humans, other animal species such as rat, rabbit, pig, and monkey, had hardly any detectable specific binding of [^H] RU 486 to plasma proteins. Of course, it is likely that beside other factors, characteristic pharmokinetics and metabolism of RU 486 may account for higher dose requirements of RU 486 in vivo to interact with target tissue cytplasmic progesterone receptors.

Activation and nuclear bindinp studies In mammalian species studied so far, it is apparent that progesterone and RU 486 seem to reversibly compete for the same receptor unit with high affinity (11,14,16,17).

Therefore, it is natural to

presume that antiprogestinic properties of this compound, in spite of its high affinity binding to agonist receptor may reside at the steps beyond initial binding of hormone to the receptors, i.e. impairment at the level of either activation of [^H] RU 486-receptor complex or at the level of nuclear binding of the activated [^H] RU 486-receptor complex when compared with agonist-receptor complex. Moguilewsky and Philbert (21) found no defect in either in vitro o thermal activation or in binding of thermally activated [ H] RU 486receptor complex to the nuclei of rabbit uterus.

They reported that heat

130

treated cytoplasmic [^H] RU 486-progesterone receptor complex of rabbit uterus was bound readily to DNA-cellulose.

Also purified nuclei of

endometrial cells of rabbit uterus when incubated with [^H] RU 486 showed a rapid uptake of specific radioactivity (21).

Since both activation and

nuclear binding were found to be functional with antagonist bound receptor complex, they concluded that the probable reason of antagonistic properties of RU 486 may lie in a step beyond the thermal activation and nuclear binding states, i.e. at the regulatory region of the genes. Rauch et al. (19) studied progesterone induced inhibition of uteroglobin gene expression in the rabbit endometrium of RU 486.

They

have shown that in this system RU 486 was able to totally inhibit the progesterone induction of uteroglobin mRNA without demonstrating any agonistic activity.

High affinity binding of [^H] RU 486 with cytosolic

progesterone receptors has been identified in rabbit uterus previously (11).

Unfortunately, this binding was unable to enhance the

transcription of the uteroglobin gene.

Therefore detailed studies were

conducted by them (19) to characterize whether impairment in the thermal activation or subsequent nuclear binding of thermally activated receptor complex could account for the observed antagonist properties of this compound.

Rauch et al. (19) observed only a slight decrease in the

binding to DNA-cellulose between thermally activated agonist [ 3 H] R5020 receptor complex (47%) and antagonist [^H] RU 486-receptor complex (37%). This slight difference was found to be due to a decrease in thermal activation as well as decrease in affinity of activated complexes to bind to DNA.

This observed difference also persisted when [^H] RU 486 and

[ H] 5020 bound receptor complexes were characterized on phosphocellulose and DEAE cellulose column chromatography.

(These charged resins are

131

k n o w n to d i f f e r e n t i a l l y r e t a i n a n d e l u t e the a c t i v a t e d a n d receptor complexes.)

Decreased affinity of

[%]

nonactivated

RU 486-receptor

to D N A w a s a l s o a p p a r e n t w h e n D N A b o u n d a g o n i s t a n d a n t a g o n i s t were e l u t e d b y h i g h salt.

complexes

F r o m the o b t a i n e d d a t a t h e y c o n c l u d e d

relatively minor differences

in activation and DNA affinity data

n o t a c c o u n t f o r the c o m p l e t e a n t a g o n i s t a c t i o n o f R U 4 8 6 a n d a n t i p r o g e s t o n i c effects of R U 486 lies b e y o n d receptor

(22) r e p o r t e d t h a t b o t h

[3H] R5020 and

that could

therefore,

binding,

a c t i v a t i o n a n d n u c l e a r b i n d i n g o f the a c t i v a t e d r e c e p t o r - h o r m o n e Horowitz

complex

complex.

[ 3 H ] R U 4 8 6 a t 10 n M

c o n c e n t r a t i o n b o u n d e q u a l l y w e l l to two p r o g e s t e r o n e r e c e p t o r

subunits

(mol w t 1 0 8 , 0 0 0 a n d 8 4 , 0 0 0 ) p r e s e n t i n the n u c l e i o f T 4 7 D c o h u m a n b r e a s t c a n c e r ce lis.

The binding of

[3H] R5020

to b o t h p r o g e s t e r o n e

subunits was abolished by unlabeled R5020, progesterone Unlabelled dexamethasone,

receptor

or R U 486.

and hydrocortisone were unable

to

compete.

These results d e m o n s t r a t e d that in intact T47Dco h u m a n cancer cells, 486 binds

s p e c i f i c a l l y to p r o g e s t e r o n e

receptors.

T h e a f f i n i t y o f R U 4 8 6 for the p r o g e s t e r o n e i n t a c t c e l l s w a s f o u n d to b e K d = 2 n M a t 0 - 4 ° C

receptor in vitro and

(22).

In intact cells,

h o u r t r e a t m e n t w i t h 4 n M o f R U 486 w a s a b l e to t r a n s l o c a t e a l m o s t 80% progesterone

r e c e p t o r to h i g h a f f i n i t y n u c l e a r b i n d i n g s i t e s .

c o n c e n t r a t i o n of R U 486 m o b i l i z i n g more than 20,000 fmol r e c e p t o r / m g D N A i n d i c a t e s t h a t the h o r m o n e progesterone

receptor

in

one of

Such a

low

progesterone

is i n t e r a c t i n g d i r e c t l y

with

(22).

The observation of Horowitz activity of R U 486

RU

(22) s u g g e s t s t h a t the

antiprogestin

i n the T 4 7 D c o c e l l l i n e is n o t d u e to the

t r a n s l o c a t i o n o f the a c t i v a t e d R U 4 8 6 - r e c e p t o r c o m p l e x b u t s o m e w h e r e d i s t a l to n u c l e a r b i n d i n g .

However,

impaired

i n t o the

nuclei

the p o s s i b i l i t y

that

132

the binding sites of RU 486-nuclear complexes may differ from those of R5020 or that the fit of RU 486 into the active site of the receptor may differ from that of R5020 is not ruled out by her investigation. It is clear from the studies carried out with rabbit uterus (11,19) and a variant form of human breast cancer cell line (22) that at least in these systems, both agonist and antagonist showed high affinity receptor binding, almost normal activation and subsequent high affinity nuclear binding of the activated complexes.

This suggests that antagonist action

of RU 486 may reside at step distal to nuclear binding, or that there are some conformational changes in antagonist-receptor complex after activation in such a way that these complexes may bind at the nuclear site other than the active site. In contrast to rabbit uterus (19) and human breast cancer cell line (22), certain gross changes were noted between agonist and antagonist bound receptor complexes in some other systems.

For example, Mullick and

Katzenellenbogen (23) using sucrose density gradient technique, characterized the [ 3 H] RU 486 and [ 3 H] R5020 binding with progesterone receptors of MCF7 and T47D human breast cancer cells.

In high salt

o sucrose gradients, salt extracted nuc lear [ J H] 5020 -receptor complexes of both cell lines, sedimented with a single peak as a 4 S species. Interestingly, two distinct 6 S and 4 S species were detected under a similar condition when RU 486-receptor complexes were analyzed.

The [ 3 H]

RU 486 binding was specific to progesterone receptors, since excess of cold R5020, progesterone or RU 486 effectively displaced the [ 3 H] RU 486 binding, while excess cold dexamethasone or hydrocortisone were found to be ineffective.

They ruled out the possibility of association of DNA or

RNA with the 6 S complex, since digestion with either DNAse or RNAse was

133

unable to alter the 6 S form.

However, treatment with either 10 mM

thioglycerol or 3M urea resulted in conversion of 6 S to 4 S form, suggesting that the 6 S may represent an aggregate resulting from association between the two receptor units or between receptor and some other protein.

They speculated the possibility of defective chromatin

binding of this larger aggregate species.

This could explain the

antagonist effect of RU 486 at the molecular level.

Similarly, Hurd and

Moudgil (24) presented evidence showing the impaired transformation (from 8 S to 4 S) of antagonist [ 3 H] RU 486 bound cytosolic progesterone receptor of calf uterus as compared to agonist [ 3 H] R5020.

In 8-30%

linear glycerol gradients containing 0.15 M KC1 and 20 mM sodium molybdate they observed that both [ 3 H] RU 486 and [ 3 H] R5020 receptor complexes sedimented as 8 S species.

Interestingly, on incubation of

samples at 23°C for 10-60 min, in 1-10 mM ATP or with 0.3 M KC1 resulted in conversion of 8 S to 4 S in case of [ 3 H] progesterone bound progesterone-receptor complex whereas similarly treated samples containing [ H] RU 486 bound progesterone-receptor complex displayed only 8 S species and except for a decrease in the 8 S form, there was no conversion of 8 S to 4 S detected.

These results indicate that impaired

transformation of [-"H] RU 486-receptor complex could explain the observed antagonist actions of this compared.

Table II represents the summary

of our current understanding of receptor mediated antiprogestin action of RU 486.

134

TABLE II

Summary of receptor mediated antiprogestin action of RU 486

Model System

I. (i)

Rabbit uteroglobin gene expression (19)

(ii) Estrogen-insensitive T47Dco human breast cancer cells (22) (a)

High affinity receptor binding

(b)

Almost normal activation of the antagonist-receptor complex

(c)

Almost normal nuclear binding of the activated antagonistreceptor complex

(d)

Possible defect may be due to (i) altered conformation of the antagonist-receptor complex resulting in inadequate interaction with the regulatory regions of genes or (ii) steps further to nuclear binding

II. (i)

Calf uterus (24)

(ii) HCF7 cells (23) (a)

Impaired activation of antagonist-receptor complex (impaired conversion of 8 S or 6 S antagonist-receptor complex into 4 S).

It could be summarized from the 1 imited data available at the present time that the antagonistic properties of the RU 486 may reside in its impaired transformation ability as shown in the case of calf uterus

135 or human breast cancer cell lines (23,24) or at the site distal to initial binding, activation and nuclear binding of the activated receptor-steroid complex as shown in the case of rabbit uterus (19). More studies are needed both at the level of activation step and the interaction of the antagonistic receptor complex with the regulatory regions of discrete genes using state of the art techniques such as monoclonal antibodies, recombinant DNA, and immunohistochemical techniques, to precisely understand at the molecular level the antagonistic action of this potent and clinically useful antiprogestinic compound.

REFERENCES 1.

Philbert, D., R. Deraedt, G Teutsch, C. Tournemine, E. Sakiz, 1982. Endocrine society 64th annual meeting, San Francisco, Abstr. 668.

2.

Chobert, M., R. Barouki, J. Finidori, M. Aggerbeck, J. Hanoune, D. Philbert, R. Deraedt, 1983.

3.

Biochem. Pharmacol. 32, 3481-3483.

Healy, D., E. Baulieu, G. Hodgen, 1983.

Fertil. Steril. 40, 253-

257. 4.

Jung-Testas, I., E. Baulieu, 1984.

5.

Moguilewsky, M., D. Philbert, 1984.

J. Steroid Biochem. 20, 301-306. J. Steroid Biochemistry 20.

271-276. 6.

Kendle, K., 1979.

In antihormones (M.K. Agarwal Ed), Elsevier/North

Holland Biomedical Press, pp. 239-305. 7.

Kendle, K., 1982.

In hormone antagonists (M.K. Agarwal Ed), De

Gruyter, Berlin, New York, pp. 233-246.

136 8.

E l g e r , W . , S. B e i e r , K . C h w a l i s z , M . F ä h n r i c h , H e n d e r s o n , G. N e e f , a n d R. R o h d e , 1 9 8 6 . 25,

S. H a s a n ,

J. S t e r o i d

D.

Biochemistry,

835-845.

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R a y n a u d , J . , T. O j a s o o ,

10.

B a u l i e u , E., 1986.

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Philbert, D., 1984. Ed), pp.

1986.

J. S t e r o i d B i o c h e m i s t r y 25,

J. S t e r o i d B i o c h e m i s t r y

25,

811-833.

847-851.

In adrenal steroid antagonism

(M.K.

Agarwal.

77-101.

12.

P h i l b e r t , D., J. R a y n a u d ,

1973.

S t e r o i d s 22,

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P h i l b e r t , D . , T. O j a s o o , J. R a y n a u d ,

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Endocrinology

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B r o w n , T. a n d J . B l a u s t e i n ,

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E t g e n , A. a n d R. B a r f i e l d ,

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G r a v a n i s , A . , G. S c h a i s o n , G. D e B r u x , P. S a t y a s w a r o o p ,

17.

1985.

B r a i n R e s e a r c h 301,

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Endocrinology

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Röbel,

Bordon,

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J. C l i n . E n d o , a n d M e t a b . 60,

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M o u d g i l , V . , N. E l i e z e r , C. L o m b a r d o ,

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Eur. J.

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M c g u i l e w s k y , M. a n d D. P h i l b e r t , RU 486 and fertility control

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Endocrine Society 68th Annual

Endocrine Society 68th Annual

THE ANTIGLUCOCORTICOID EFFECTS OF CORTEXOLONE AND R U 38486 IN THE H U M A N LEUKEMIC CELL LINE CEM-C7

Thomas J. Schmidt Department of Physiology and Biophysics College of Medicine The University of Iowa Iowa City, Iowa 52242

Introduction

Antiglucocorticoids,

by

definition,

are

compounds

which

antagonize or block the biological response to glucocorticoid agonists

in

various

target tissues and cell types.

In the

series

of experiments outlined in this chapter my laboratory

has

compared

the

antiglucocorticoids,

ability

cortexolone

glucocorticoid-mediated line,

CEM-C7.

of and

two

RU

steroidal

38486,

to

block

responses in the h u m a n leukemic cell

W e have utilized CEM-C7 for these comparative

studies because glucocorticoid agonists such as triamcinolone acetonide glutamine (cell

(TA)

elicit

synthetase

lysis)

both

an

activity)

response

anabolic

(1)

(induction

(2) in this cell line, and because of

the availability of a glucocorticoid-resistant subclone, In and

3R7.

addition to investigating the ability of both cortexolone RU

partial two

of

as well as a catabolic

38486 agonist

independent

characterized

to

function activity

agonist-mediated the

antiglucocorticoids CEM-C7.

as

pure antagonists

(elicit no

when given alone) and block these responses,

interaction

of

we

have also

these

two

with cytoplasmic receptors isolated from

W e have analyzed the specificity of this cytoplasmic

binding and have evaluated the potential ability of these two

Receptor Mediated Antisteroid Action © 1987 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

140

steroids to promote in vitro receptor activation (conversion to a DNA-binding form) under a variety of experimental conditions. Subsequently, binding of activated agonist- or antagonist- receptor complexes to DNA has also been studied. The data which will be reviewed indicate that although both cortexolone and RU 38486 function as antiglucocorticoids via their ability to compete with agonists for the steroid binding site within the unactivated cytoplasmic receptor, they do differ significantly in regard to their ability to promote crucial post receptor-occupancy steps, such as activation and DNA binding, which are required if a steroid is to elicit a receptor-mediated biological response. As will be discussed, the experimental results which we have obtained with RU 38486 suggest that once the cytoplasmic receptor has undergone steroid-dependent activation and nuclear translocation, the ligand (agonist or antagonist) may continue to play a major role in terms of modulating the ability of the activated receptors to regulate gene expression.

Background

The cytotoxic effects of glucocorticoid hormones on sensitive lymphoid cells are mediated by intracellular receptors which bind these steroids with high affinity and specificity (for a review see Ref. 3). Once the glucocorticoid molecule has diffused into the target cell and bound to its cytoplasmic receptor, these complexes must undergo a two-step process in order to bind to nuclei and ultimately affect gene expression. The first temperature-dependent step, termed "activation" or "transformation", is thought to involve a conformational_change in the glucocorticoid-receptor complex, resulting in the exposure of positively charged amino acid residues on the surface of the protein (4, 5). These activated complexes exhibit an altered elution profile from

141

anion exchange resins such as DEAE-cellulose (6) and an increased affinity for nuclei and polyanions such as DNAcellulose (4, 7-9). Despite the present uncertainty concerning the underlying biochemical mechanism(s) of activation (for a review see ref. 10), this alteration represents a physiologically relevant step, since it occurs in vivo in a variety of cell and tissue types and is a prerequisite for nuclear binding (11-13). The second temperature-independent step is termed "translocation" and involves the movement of the previously activated complexes into the nucleus where they bind to acceptor sites within the chromatin (14), and, more specifically, to DNA sequences which flank genes whose expression is regulated by glucocorticoids (15-17). The net result of this nuclear interaction is the initiation of messenger RNA synthesis and ultimate translation into specific proteins whose activities constitute the phenotypic response. Glucocorticoid-induced

lymphocytolysis

has been studied for

many years, but the precise mechanism by which these steroids mediate

cell

laboratories

have

lymphocytes amino

death

not

been

elucidated.

Numerous

reported that glucocorticoid treatment of

reduces

acids,

has

and

the transmembrane transport of glucose, nucleosides,

as

well

as

decreases the

synthesis of proteins, lipids, and nucleic acids (for reviews see

refs.

inhibitors was

3,

by

Based on experiments which utilized

of RNA (20-22) and protein (20, 23) synthesis, it

suggested

induced

18, 19). that

specific

glucocorticoids

RNA(s)

and/or protein(s) are

(24, 25).

Although this concept

of glucocorticoid-mediated induction of a "killer" protein(s) received considerable support, other alternative theories for 2+ glucocorticoid-induced cytolysis included enhanced Ca uptake nuclear

(26) and release of free fatty acids which damage the membrane (27).

More recent experiments suggest that

glucocorticoids activate, via a receptor-mediated process, an endonuclease-like

activity

the

lymphocyte

DNA

fragmentation

in

lymphoid cells which cleaves

genome at internucleosomal sites (28). appears

to

precede

This

lymphocytolysis and

142

hence may represent the key triggering event in the steroid regulation of cell death. Consistent with this nuclease theory is the observation that low concentrations of benzamide, a potent inhibitor of poly(ADP-ribose) synthetase (an enzyme involved in DNA repair), enhances the cytotoxicity of glucocorticoids and shortens the interval between hormone addition and the onset of lymphoid cell death (29). A s previously defined, antiglucocorticoids are steroids w h i c h antagonize

or

block

the

biological

responses

to

glucocorticoid agonists (30). The antiglucocorticoid cortexolone has b e e n u s e d extensively for almost twenty years and

has

proven

analysis action.

to

be

an

extremely

useful

tool for the

of the mechanism(s) of glucocorticoid hormone Since early studies w h i c h demonstrated t h a t this

compound could block the inhibitory effects of glucocorticoids on rat thymus cells (31), cortexolone has b e e n reported to function as an antiglucocorticoid in a wide variety

of

target tissues and cell types including:

thymic

lymphocytes (32-36); leukemic lymphocytes and m y o b l a s t s (37); bone (38); chick retina (39); hepatoma tissue culture cells (40);

Hela

cells

the mouse brain block inhibition histamine

(41-43); and the locus ceruleus r e g i o n of (44) . Cortexolone has also b e e n shown to by dexamethasone of the stress response to

(45).

Despite

the

widespread

use

of

this

antiglucocorticoid, the results of experiments in w h i c h the 3 binding of [ H]cortexolone has been studied are somewhat conflicting, especially w i t h regard to nuclear binding. and

Munck

(46) concluded from their studies w i t h intact rat 3 cells that [ H]cortexolone-receptor complexes undergo

thymus

temperature-dependent despite

the

fact

glucocorticoid (36,

Wira

47)

translocation

that

these

and

complexes

response in these cells.

employed

sucrose

bind fail Other

to

nuclei,

to elicit a investigators

density gradient analyses which

indicated that cortexolone-receptor complexes exhibit altered physicochemical complexes.

properties

These

data

when

compared

w i t h TA-receptor

suggested that cortexolone-receptor

complexes either fail to translocate into the nucleus, or, if

143

they do translocate, exhibit a very low affinity for nuclear acceptor sites and hence fail to mediate a biological response. Finally, using autoradiographic procedures, Coutard and Osborne-Pellegrin (48) were unable to detect nuclear accumulation of radioactivity in the mouse brain 3 after injection of [ H]cortexolone. Taken collectively these published data have raised questions concerning the potential ability of cortexolone, once it has presumably bound to unactivated cytoplasmic receptors, to promote receptor activation and DNA binding. In the experiments to be subsequently described, my laboratory has attempted to clarify these discrepancies by analyzing the in vitro binding 3 of [ H]cortexolone to cytoplasmic CEM-C7 receptors (49). More specifically, we have performed a series of experiments which were designed to help us ascertain whether this antiglucocorticoid can promote in vitro activation. Most recently the synthetic steroid RU 38486 (17 3 -hydroxy116 -(4-dimethylaminophenyl)-17a-(l-propynyl) estra-4,9-dien3-one) has been utilized extensively as a pure antiglucocorticoid. Unlike cortexolone, which can be metabolized in the adrenals to a partial agonist (50), RU 38486 is capable of fully antagonizing the acute and chronic effects of glucocorticoids in vitro as well as in vivo without exhibiting partial agonist activity, even when administered at high doses (51, 52). For example, this potent new 19-norsteroid has been reported to: completely antagonize the ability of dexamethasone to inhibit uridine incorporation in isolated thymocytes as well as the ability of this agonist to suppress rat thymus weight when administered chronically (51) ; antagonize the glucocorticoidinduced cytolytic response of murine T-lymphoid cell lines (53); block the dexamethasone induction of hepatic tyrosine aminotransferase (51) and atrial natriuretic factor (54); antagonize dexamethasone-induced suppression of ACTH release by rat pituitary cells in vitro (52); and alleviate many of the symptoms of hypercortisolism in a patient with Cushing's syndrome (55). Several studies have attempted to explain why

144

RU 38486, when given alone, elicits no agonist activity despite the fact that it binds with high affinity to cytoplasmic receptors. As was also the case with cortexolone, the results of these experiments were somewhat conflicting, particularly with regard to nuclear binding of [ 3 H]RU 38486. Mougilewsky and Philibert (56) reported that 3 cytoplasmic rat thymic [ H]RU 38486-receptor complexes can be thermally activated in vitro to a less stable activated form from which the antiglucocorticoid rapidly dissociates. They speculated that this "impaired activation" of cytoplasmic 3 [ H]RU 38486-receptor complexes renders them unable to translocate properly into the nucleus and subsequently initiate a glucocorticoid response. Bourgeois et al. (53) also reported that activated [3H]RU 38486-receptor complexes from rat liver and murine T-lymphoid cells exhibit a lower 3 affinity, as compared to activated [ H]TA-receptor complexes, for both nonspecific DNA-cellulose and for a DNA fragment in the promotor region of MMTV proviral DNA which is known to contain high affinity sites for activated glucocorticoidreceptors. Consistent with these results was the observation that [3H]RU 38486 was less effective than [3H]TA in promoting nuclear translocation of receptors in these murine T-lymphoid cells. In contrast, Coutard and Duval (57) utilized 3 autoradiography to localize [ H]RU 38486 in various glucocorticoid target tissues and detected specific nuclear translocation in brain, liver and kidney. Once again these data have raised questions concerning the potential ability of RU 38486 to promote receptor activation and DNA binding. Again, in an attempt to clarify these discrepancies my laboratory has conducted a series 3 of experiments designed to characterize the interaction of [ H]RU 38486 with cytoplasmic CEM-C7 receptors and to evaluate the potential role of this relatively new antiglucocorticoid in promoting in vitro activation (58). In all of the experiments to be described we have compared the effects of the two antiglucocorticoids, cortexolone and RU 38486, with those of the potent synthetic agonist, triamcinolone acetonide.

145

HjCs, 'N

CHjOH

CH,OH B-

activity

in

16a-methyl-deri. vate

at

all.

and

its

denvates Spirortnont 30

d*rivat*s

100'/.

300

1000

Spironolactone

jog

¡-CM,

J

II

Spirortnont

[—r°

III

IV

MO (CHj^-OH

I

Fig.

5

I

rtlativ*

affinity

to MCf> in

rtlalivr

aniioldoittront

vitro

tfftcl

in

vtvo

A f f i n i t y f o r r a t r e n a l m i n e r a l o c o r t i c o i . d r e c e p t o r s in v i t r o a n d m a x i m a l r e l a t i v e a n t i a l d o s t e r o n e p o t e n c y in r a t s o f s p i r o r e n o n e a n d r e l a t e d c o m p o u n d s in c o m p a r i s o n to s p i r o n o l a c t o n e .

180

Spirorenone

related

30

derivotes

100%

300

1000

rtlative

affinity

to MCR in

rtlalivt

antialdosterone

Spironolactont I—

*-C-CM3

MB (CM,^OH

•Jair

] ig.

6

a

(Fig.

6).

5 , Fig.

new

was

8.6

higher

was

not

reflected

lative

binding

contrast,

showed

in

The

spironolactone

potent

only

50

tone

formation

%

of

mean

that

by

an

affinity ^^> ^2

potent

relative

of

in

in vivo

spirorenone

saturated

derivate

in

vivo

activity

averaged

its

affinity

doubled

than

deri. v a t e

in

being

as

of

vitro

to

its

7.5

was

receptor

spironolactone.

(17a-hydroxy-,

of

however,

binding,

the

re-

73 spirorenone

effects

that

spirorenone

spironolactone

compared

higher

antagonists spirorenone

This,

receptor

well

and

of

spironolactone.

increase of

aldosterone potency

as

17-spiroether

more

group

The

than

the

vivo

nolactone.

The

effect

Affinity for rat r e n a l u i n e r a l o c o r t i c o i d receptors in vitro and m a x i m a l r e l a t i v e a n t i a l d o s t e r o n e p o t e n c y in r a t s o f s p i r o r e n o n e r e l a t e d c o m p o u n d s in comparison to spironolactone.

Spirorenones,

In

vitro

than

times of

that

the

still

of

standard. 1.7

affinity

Modification

spiro-

of

times

being the

17g-hydroxypropyl-derivate

lacof

181

Table

1.

Compound

A f f i n i t y for r e n a l m i n e r a l o c o r t i c o i d r e c e p t o r s ( M C R ) o f r a t s in v i t r o a n d m a x i m a l r e l a t i v e a n t i a l d o s t e r o n e p o t e n c y ( M R P ) in r a t s o f s p i r o n o l a c t o n e d é r i v â t e s in c o m p a r i s o n to s p i r o n o l a c t o n e

Relevant modifications c o m p a r e d to spironolactone

17a-H ydroxypropy1 I

111

IV V VI

7

unsaturated

Uli VIII

IX

X

XI XII

at

184

< 1

No

activity

< 1

No

activity

24

46

( 28-83

)

< 1 54.4

32 34

( 21-45 ( 19-56

) )

152

141

(

98-221)

4. 0

71

(

21-157)

< 1

72

(

21-157)

37 . 1

114

(

51-268)

7. 9 < 1

47 No

(108-387)

compounds

63,73-Methylene (prorenone ) 63,73-Methylene; 17ot-hydroxypropyl, 17 3 - OH A1; 63,73-methylene; 17a-hydroxypropyl, 173-0H 63,73-Methylene17a-propi onate, K ( = K prorenoate)

Methylation

< 1

compounds

A6 (=canrenone) A6-17a-Hydroxyprop y 1, 1 7 3 - 0 H »6,15

6 3 , 7 3 - M e t h y 1ene

M a x i m a l MRP vs spironolactone ( = 100 % ) (95 % c o n f i dence limits) ( % )

compounds

17oi-Hydroxypropyl, 173-0H 17oi-(3-Hydroxy-3, 3-dimethylpropyl), 17 3 - O H 17ot-Hydroxybutyl, 17 3 - OH

II

c6/c

A f f i n i t y for M C R vs spironolactone ( = 100 % ) ( % )

the

D-ring

153-Methyl 16a-Methyl

( 19-153) activity

182

spirorenone) Ihe

in

vivo

resulted activity

spironolactone, very

low.

rolactone did

f)

not

Ihe

relative

tors

and

a

the

an

in

vitro

in

vivo

activity

only

is

no

( r

show

in

negligable

denvate

with

antialdosterone

higher vitro with

nor

affinity

is

in

in

).

vivo

receptor

binding.

receptor

binding

Table the

in

However,

similar

spi-

compound

action

between

< 0.025

activities

of

was

vitro.

and

summarized

that

reversed

mineralocorticoid

correlation p

than

Ihis

in

potency.

however

antialdosterone

= 0.309, vivo

in

included.

vivo

renal

compounds

significant

times

analogue

was in

vitro

relative

tone

vivo

in

rat

do

there

C^

to

compounds with

at

affinity

overall

1.3

antia 1dosterone

capacity

neither

between

spironolactone-like is

still

of

spirorenone

activity

Correlation

loss

binding

configuration

show

a

was

the

finally,

in

to On

and

activity recepof

all

I.

There

vivo

and

several

spironolacthe

other

without

hand,

in

activity.

3

H - dlhydrot*stest»ron* binding In human pros tat* cytoicl

• ».....,

Fig.

7

m3

H-OHT Ih-DHJ

. OUT f J.lSilO'* • OHT IIS x K'


90QQ

»000

7000

tooo 5000

4000

3000

2000

1000

Fig.

I n t e r a c t i o n o f d i f f e r e n t s t e r o i d s IAI11h t e s t o s t e r o n e at h u m a n p r o s t a t e b a n d i n g

8

II

Interaction

a)

Affinities

In a

a

with

for

preliminary

concentration

prostate

unlabelled

DHT

cant

binding

in

dose

a

No

2.5

and

x

alone

androgen

and

M was with

increasing

Gel

filtration

DHT

with

manner.

incubated

was

seen

rone,

hydrocortisone

and

progesterone

tions

(Fig.

renone-derivates (Fig.

for

of

^H-DHT

sites

after

was

only

incubation

sites

were

DHT

found

for

with

and

in

of

significold

with

100-fold

spironolactone

binding

a

by

together

in

(DHT) human

concentrations

revealed

Competition

when

Affinities

sites

with

displacement

displacement

8).

binding

incubated

7).

5a-DHT

binding

^H-dihydrotestosterone

10

labelled

dependent

testosterone DHT.

experiment,

(Fig.

of

prostate

^H-dihydrotestosterone

of

cytosol

human

^H-dihydrosites.

labelled aldoste-

concentrafour

spiro-

compared

9).

Unlabelled binding.

In

DHT

itself

contrast,

was

most

potent

spironolactone

in was

reducing a

weak

the

^H-DHT-

competitor

184 Affinity binding

af xpir»n»!mcHnt M* 5finrn«n»-4»rlrttn situ In humcn prtitat» cfttut

cr0

r r

9

with

^H-dihydrotestosterone

was

fold

reduced

however, binding

by

only

were

more A

centrations

b)

concentrations

sites.

obviously without

the

not

cytosol.

We,

for of

The in

the

four

and

great

is

binding

in

2000

the

most

with

with

binding

fold.

showed

with

Even

bound

The

"^H-DHT was

in

activity

the

binding ligand

the

incubated

for

found

lactone

since

a similar

specific

problem

therefore,

denvates

sites.

spi r o r e n o n e - c o m p o u n d s ,

importance

formation

the

four spirorenone-denin h u m a n p r o s t a t e

competing

reduction

600

»o.r«,i,-o«

spironolactone,

^H-methyltrienolone

avoiding

spirorenone

%

of

for

%.

active

50

lactone

Affinities

receptors

11

between

was

Methyltrienolone

the

n

Affinity o ^ spironolactone and v a t e s for H-DHT-bi.nding sites cytoso1 .

Fig.

000

'H-OHT-

.¿¿r^ xtr* oOT^

0

10

far

SHBG

the

in

con-

structure

compound (low)

affinity.

sites for

androgen

contamination

spironolactone

^H-methyltrienolone

and

and

of

four

cytosol

185

3

H

-mothyItrionolono-binding

sitos

in

human

p r o i t o t o

cytosol

methyllrienolon* cpm/1 roc lion methwltrienolone 2 5 1 IO'OM Spironolactone

•

• methyl

fraction»

Fig.

10

of 500

tnenoione

pi

A f f i n i t y of s p i r o n o l a c t o n e and spirorenone-denuates for H - m e t h y l t r i e n o l o n e b i n d i n g s a t e s in h u m a n p r o s t a t e c y t o s o l w h e n i n c u b a t e d an 1 0 - f o l d e x c e s s .

A f f i n i t y of 3

methyltr

spironolcctono

H-mothyttrionolono-binding

end

spiroronono s i f t

in

-dorivatos human

for

p r o s t c f o

cytosol

lenolort*

¡5

mothjrltritnolone * 10-4 ft

•

-

Spironolactone

•

methyltrienolone

• sptrotenones

fraction»

F ig.

11

0f 500

l-IV

jit

A f f i n i t y of s p i r o n o l a c t o n e and sp] r o r e n o n e - d e n v a t e s for H - m e t h y l t r i e n o l o n e b i n d i n g s i t e s in h u m a n p r o s t a t e c y t o s o l w h e n i n c u b a t e d in 1 0 0 - f o l d e x c e s s .

186

prepared from 10,

2.5 100

When

from

human

x

^ - 2.5

10

and

1000

prostate

spironolactone

bated

in

10

fold

seen

and

only

binding

by

Finally,

with

of

used,

and

in

(Fig.

the

ligand

was

tested

in

displaced

"^H-methyltrienoione

are

3

obviously

H-mfthyllritnolonr

methyl

trienolone

cpm /

fraction

less

- binding

10).

When

50

incu-

in

100

of

bound

fold %

was

methyltnenolone,

spironolactone 2 lowered the

however

active

were

reduction

^H-methyltnenolone

spirorenone-derivates

of

a

11). of

binding

fold

(Fig.

no

displacement

excess

ren ones

1000

representing

compounds

unlabelled

reduction

ranging

steroids.

spirorenone

spirorenones a

selected

cold

observed

were

a minimal

the

trienolone The

the

spironolactone

however

^ M were

Concentrations

concentrations,

was

concentrations

with

10

excess

higher

"^H-methyltrienolone higher

x

fold

tissue.

than

t i l t s

in

by

same

by

and

(Fig.

12).

concentrations,

only

40

%.

The

spironolactone

human

me thy 1 -

proitat*

spiro-

in

cytosot

A

iOOO-

•—• —o

3500-

3000-

'H - methyl trienolone 15 M 10-' M •

spironolactone

•

methyltrienolone

• spirorenones

2500-

2000-

15 00-

1000-

500-

fraction»

Fig.

12

of

250

pt

A f f i n i t y of s p i r o n o l a c t o n e and s p i r o r e n o n e - d e r i v a t e s for H - m e t h y l t r i e n o l o n e b i n d i n g s i t e s in h u m a n p r o s t a t e c y t o s o l w h e n i n c u b a t e d in 1 0 0 0 - f o l d e x c e s s .

I-IV

187

competing human

with

the

prostate

ligand

androgen

methyltrienolone

for

binding

at

the

receptors.

Discussion

The

observations,

tissues mone

is

receptors

competitive

and

a unique

ship

compounds

of

tion at

in

in

3)

less

since ties of

ty

data

these

were

and

independent under

was

with

in

with

ring

compared

the

toad

method

from

with

further and

allowing

(27,

a

and

receptor

data

binding

antagonistic

a

of

as

properties

of

The

unclear activi-

of in

seven vivo

the

of

activi-

shortbetween

the

system

the

compounds

a discrepancy

activity

well

relationship

differentiation Moreover,

to

compounds.

the

of

reduc-

the ^--lac-

biological

means

24

aldosterone

greater

The

of

comparison

activity

revealed

antagonistic

relation-

remained

28).

by

activity.

pharmacokinetic The

the

by

unsaturation

parent

studies

binding

bladder

in

show

their

target hor-

affinity

1)

unsaturation

affinities

antagonistic

affinity

higher

2)

or

the

cytoplasmic

in

In

inhibited

demonstrated

compound

investigated

investigation.

binding

renal

already

vitro

the

spironolactone

agonistic

to

not

is as

relation

antagonistic

circuit- current agonistic

in

at

cytoplasmic

structure-affinity

thej^-lactone

compounds.

studied

rat

They of

step

investigated

r i n g B, of

these

compounds

was

to

at

such

19-nor-spironolactones

of

agonistic,

these

in

affinities

the of

study

a potential

(26).

aldosterone

initial

a 1 . first

opening

significance

to

affinity

position

and

this

of

binding

antagonists

analogues

spironolactone. as

with

vitro

receptor

C^/Cy

tone

that

et

action

specific

chance

F"under

spironolactone receptors

the

by

aldosterone

offered

activity.

that

initiated

whereas

is

between compounds

manifested

significant

tremendous

dissociation

agonistic

activity. Recent the

studies

relative

confirmed

affinity

for

the

mineralocorticoid

receptor

between in

vitro

188

and of

the

in

vivo

pharmacological

antimineralocorticoid

more

potent

renal

vity med

by

the and

with

same

which

cannot

the

to

an

be

either

studies

low

again

structure

of

in

depending

on

observations et

al.

of

chain

a

the

ronolactone

series

while

appeared

its

to

antagonism,

It

is

have

in

takes

place

chain

at

that by

C-17

derivate

vivo

the

rat

aldosterone

and in

the

importance

for

the

complete

by

an

to

spirolactone

of

structure

at

et

of

binding

loss

f under

in

vivo

receptors.

the

for the

the

in

a 1.

17-spirolactone

in

one the

in

the

leads binding

C-17.

Simi-

(26)

and

ring,

the

a carboxyl

17a-hydroxypropyl

was

for

the

a

striking

higher

the

cytoplasma

is

implies active

conversion hydroxyl

group

and

of

to

its spire-

a blockade essential

that

this

of

for

compound

metabolites

mi n e r a l o c o r t i coi d

the

that

mineralocorticoid that

more

side

result:

than

Assuming

result or

vivo of

a

showed

vivo

this

to

oxidation to

in

a

prorenone,

group,

sites

vivo

affinity

likely

in

ring

binding

acid,

both

ring

and

^--lactone

or

negligable.

competitive

then

with

prorenone

of

affinity

be

binding

converted

for

containing

activity

aldosterone

is

prore-

to

aldosterone

remaining

acti-

that

vivo

Opening

made

without

17(B-hydroxy

pharmacological

ceptor

the

for

vivo

c a n re n o n e

almost

also

in

(30) .

compounds

prototype and

were

an

affinity an

be

hypothesis,

demonstrate

receptor.

to

vivo

perfor-

active

affinity

in

were

inactive was

spironolactone,

capacity,

the

was

clearly

or

Peterfalvi

for

found

and

studies

in

17-spirolactone

a partial

Among

the

groups

was

without despite

compounds

however,

to

lar

both

vitro

Further

affinity

lactonized

extremely

e.g.

almost

converted

certain

17-0-methyl-5,6-dihydrocanrenoic

mineralocorticoid

ring,

in

receptors

support

are

K+-canrenoate

despite Our

group

respectively,

(30).

vitro.

was

for

Prorenone

both

spironolactone.

a considerable

sites

intact

to

canrenoate

c a n re n o n e and

however

mi n e r a l o c o r t i c o i d

superior

noate

compounds.

spironolactone

K+-prorenoate

(29). rat

than

activity

which

receptors. active

group

further

of

substances the

side

a closure

to

189

the

spirolactone

Peterfalvi the

et

lactonic

They

found

a 1.

that

as

in

for

ring

already

C-17

cannot

the

closure

additional

at

be

is

other tion

side

compound of

the

affinity

but

also

this

to

their

the

the

side

vitro

and

-

chain the

to

the

is

one

of

in

the

case

did

a clear

The

double

bond

action The

in

vitro

second

group

76-methylene prorenone, in

vitro

the

groups but

by

as

as

only

in

of

to

C-16

vivo

for

The

the

affinity

et

the

al.

As

in

a minor

the

affinity

extent

the

of

Canre-

the

in

in vivo.

spironolac-

sites

vivo.

the in

of

for

receptor

in

moiety,

17a-hydroxypropyl

abolished

the

for

basis

17a-hydroxy-

slightly

compared

activity

(29).

of

the

to

compounds

6(3,7gmethylene

both

low

receptors

increased

when

only

B-ring

activity

effects

7a-dithioacetylated

pharmacological

practically

reduced

well

of

and

the

the

metabolites

affinity

not very

extent.

the

spironolactone

show

C-15

Claire

introduction

active

of

between

series.

the

the

oxida-

derivates

variable

antialdosterone

exthe

Nevertheless, on

at

its

for

an

by

is

17a-hy-

investigation.

features

a

antialdosterone

increases

and

extensively

not

to

vivo

two

of

Thus,

answered

affinity

- or

receptor

of

by the

formation

abolished. be

-

chain

closure.

affinity

sites

(31).

derivate

ring

is

only

the

compound

caused

a

structural

This

study

compound

pharmacological

reasonable

but

one

ring

vivo

and

a corresponding

As

and

in

tone

propyl

impede

group

receptor a

of

a

in

assumption

chain

17a-hydroxybutyl

of

activity. acid,

This

vitro.

present

influence

presented

importance

inactive

17a

however,

of

was

the

biochemical

aldosterone

in

mineralocorticoid

can,

Modifications

none

the

in

activity

spironolactone

to

antialdosterone

A branched

- seems

mechanism

additional

chain

hydroxyl

the

assay

supported

compounds.

17a

pointed the

lactonized,

receptor

droxy-3,3-dimethylpropyl tended

for

17-0-methyl-5,6-di.hydrocanrenoic

which

as

(30)

ring

derivative well

ring.

as

canrenone.

is

the

shown

receptor

vivo,

the

as

previous

6(3, by sites

shown group,

and

176-hydroxyl

for

the

receptor

antialdosterone

acti-

190

vity. of

In

the

case

prorenone,

was

lost

but

comparison The

to

the

vates

methylated

in

receptor

their

affinity

spirorenone

lites

markedly

than

the

to

v ivo

form

the was

receptor kept

(twice

that

of

derivate for

The the

the

in

spirorenone

transformed

probably

the

C^/C2

for

this

of

be

the

might

accorderi-

strong

be,

that

saturated result

finding

similar

activity of

(0.73

into

of

times

the that

a more

acitve

antimineralocorticoid one

of

these

compound.

active

Another

be

a slower

than

of

the

C^/C^

the

spironolac-

indicates,

would

spirorenone

a

mineralocorticoid

binding

must

It

in

showed

affinity

This

achieve

was

spironolactone

spironolactone).

to

elimination

different.

derivate

vivo.

is

open

D-nng.

but

higher

planation

the

for

vivo

was

in

at

activity

saturated

tone)

vity

vivo

C^/C^

of

in

receptors

its

war

capacity

power

and

saturated

metabolite

binding

mi.neralocorticoid

in

C^/C2

affinity

the

the

prorenone.

with

potency

of

prorenoate,

anti.aldosterone

for

dance

Spirorenone

potassium

a part the

affinity

of

acti-

metabo-

possible

ex-

iriactivati.on saturated

and

com-

pound. It

has

been

essential

postulated

for

the

activity

(30,32).

was

less

active

and

in

Thus,

vitro, from

spirolactone aldosterone The

and

a

ronolactone

in

its

vitro

this

activity

(markedly

was

compound

has

to

aldosterone

but

still

potent

than

concluded,

spiroether

containing group, in

lower

be

the

be

derivate

ring

at

C^

is

antagonistic of

spirorenone

active

in

vivo

spironolactone. that

ring

not

lead

only to

the

active

compounds.

capacity

practically

more

can

also

derivate

binding

of

congener,

it

173-hydroxy

pharmacological

while

the

moreover,

but

spirolactone

17-spiroether

antagonistic

spirorenone

chain

The

result

form

the

development

than

and

that

that

showed

vivo in for

a

was

17a-hydroxypropyl a

striking

higher

comparison the

result:

its

that

spi-

in

of

spirorenone)

mineralocorticoid

negligable. converted

to

than

side

This

result

vivo

to

an

receptor

implies active

that

meta-

191

b o 1 i t e . It active group ther has

is

likely,

substance of

the

been

form) (33,

34).

vates or

of

The

loss

of

ceptor

the

droxyl

of

C ^

the

at

the

the

as

results

were

found In

of

aldosterone

teers

been

at

use

the

gestagen

In

of

these

taken

the

steroids

into

investigation and

in

in

vivo

with

active

by

deri.-

shortening complete

of

the due

of

reto

the

a hy-

(unpublished).

activity

vitro.

in

Similar

spirolactone is

which

limited

et

configuration

a potent

series

studies

also

is

aldosterone

their

of

aldo-

compounds

possesses

a

higher

spironolactone.

(35), in

this

new

healthy

However, their

type

volun-

the

clini-

affinity

there

consideration.

In

is

the

confined

vitro

for

to

binding

affinity

antagonistic

relative

receptor,

^H-aldosterone

However,

in

spironolactone.

compounds

mineralocorticoid be

is

for

for

receptors.

comparing

various

than

chain

classical

preliminary

doses

a new

a

probably

spirorenone

the

(open form

C^

oxydation

MCR

properties

antagonists

lower

cal

in

and

vivo is

pharmacological

represents

than

at

in

the

the

step

Peterfalvi

in

diastereomers

conclusion, and

by

spirolactone

for

other

potency

shown

the

latter

branching,

17a-si.de

the

ring

results

This

after

the of

blocking

aldosterone

pharmacological has

of

affinity for

antagonist

As

closure

end

the

(32).

activity vitro.

fur-

17a-hydroxypropyl

the

chain

and

lactonic

pointed with

The

an

hydroxyl

canrenoate

its

lactonic

also

senes,

completely

well

with

in

the

to

group

ring.

with

experience

reversion

abolished

sterone

the

of

a carboxyl

potassium

17a-side

ring

as

compounds

own

activity

the

group

Finally, at

of was

antialdosterone

binding

hindrance

vivo

of

u i. v o c o n v e r s i o n

oxydation to

for

spironolactone

lengthening

by

C ^

equilibrates

activity our

in

17-sp1ro 1 actone

importance

From the

at

demonstrated

rapidly

antialdosterone a 1 . (30).

the

this

place

chain

to

well

which

takes

side

a closure

that

for

affinities

are

the

a number

receptor

to the

the

of

of renal

aspects

to

system,

between

cytoplasmic receptor

for of

assay

competition

effect

a

receptor a given

the

steroid sites. com-

192

pound 37).

could In

rences

the in

ween

which

tance. with

high

tone

^H-DHT

Fig.

9).

The

TEBG

compared

Competition contrast,

tone

in

state

by

does

the

these

in

of

bind

bet-

prostate

critical

of

prostate

transport

androgen to

are

For

principal

the

less

in

imporbind binding

spironolaccytosol

protein

might

(see

obviously

more

to

methyltrienolone,

in

affinity

cannot

therefore

present

studying

the

affinity,

compounds

is

discussed

studies for

with

the

antagonistic

understanding

of

the

metabolism

action

compounds.

of

search

well

receptor

vivo

is

the

modula-

compound.

one

cannot

a given the

receptors

more

and

for

the

and

compound.

usefulness

for

as

expect

sites

mineralocorticoid

steroids, as

pro-

into

receptor

mineralocorticoid

in

androgen

in

the

for

vivo

human

necessary.

demonstrate

expected

activity

of

above,

affinity in

to

in

site

spironolac-

transferee!

activity

vivo

activity

the

be

receptor

than

binding

binding

between

antimineralocorticoid

androgen active

protein

reasons

However,

mineralocorticoid

lower

Anti.androgenic

agreement

mineralocorticoid

a

results

and

effect

new

human

tested

binding

suggesting

the

of

in

this

synthetic

derivates

condition.

of

is

interaction

competition

^H-methyltnenolone

Testing

vitro,

and

-antago-

testosteron-estradiol

the

at

the

receptors

chosen

serum

binding

or

5a-dihydrotestosterone

spiroreriones

metabolism

the

ligand

reflect

However,

a complete

considering

and

diffe-

metabolism

with.spironolactone.

with

reducing

vivo

are

(36,

vivo.

androgen

binding

four

cytosol

sites.

ted

for

Spirorenone

(38).

in

Therefore,

reflect

there

action

aldosterone-agonisti.c

the

the

hand,

distribution,

and

to

antagonistic

other

when

testosterone

(IEBG).

with

the

emerge

affinity

partially

the

substance

addition,

Both,

globulin

in

a

spironolactones In

on

or

absorption,

effect

of

problems

tissue.

agonistic

bioassay,

action

Similar

an

intestinal

excretion nistic

induce

or

of in anti-

specific a

better

mechanism

of

193

Summary

We

tested

to

spironolactone

at

rat

renal

sterone son

the

in

group

of

in

and

the a

steroids

compete

with

vivo.

showed

17-spirolactone

a

to

a

group

reduced

activity

the

7a-thi.oacetyl

the

in

by

41

and

at

the

52

%

D-ring vivo

were

receptor

vivo

3-8

in

in

as

more

was

receptor

activity

affinity

for

androgen The

of

potent

in

as

for

however,

than

binding

of

and

in

of

vitro

not

activity

in

(prore-

activity group

activity

of

its

Taken

replace

of

comvivo.

a methyl

two

new

deriTheir together,

testing

compounds. receptors

provides

into

insights

re-

action

mineralocorti.coid

sites

in

did

binding

and

four

human

the

the Comand meta-

spirorenone-derivates

prostate

compete

spirorenones the

suggesting c y t o s o l ic

a

cytosol

significantly

reflecting

spironolactone

for

loss

spironolactone.

Using

more

lower

androgen

was

with

competition

showed

compounds

in

increased.

does

a

B-positi.on

and

lone

four

compari-

antimineralocorticoids.

spirorenones

ligand,

in

vitro in

globulin.

four

in

unsaturated

the

testosterone-estradiol-binding

the

antialdo-

17a-hydroxypro-

reduction

slightly

spironolactone

dihydrotestosterone

as

vivo

a

C , /C 6 /

group

well

affinity

affinity

potential

The

tested.

binding

the

antialdosterone

Spirorenone

only

by

Introduction

antimineralocorticoid

of

in

a similar

vitro.

times

between

biological bolism

resulted and

the

vivo

respectively.

affinity

measuring in

in

similar

for

and

resulted

both

of

parison

rats

spironolactone,

increased

vates

structures

vitro

ring

reduction

none)

in

in

adrenalectomized

without

Compared

Substitution

both

with

^H-aldosterone

receptors

176-hydroxyl

affinity

pounds

to

17

spironolactone.

Replacement

ceptor

of

cytoplasmic

activity

with

pyl

ability

for

also ^H-

the

methyltneno-

displacement

affinity

of

receptor.

than

these

194 Re f e r e n c e s 1.

Kagawa,

C.M.:

Endocrinology

2. C o r v o l , P . , C l a i r e , B . : K i d n e y Int. 2 0 ,

67,

125-132

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3. F u n d e r , J . W . , F e l d m a n n , D . , H i g h l a n d , E . , Biochem. Pharmacol. 23: 1493-1501 (1974)

K.,

Rossier,

Edelman,

1.5.:

4. M a r v e r , D . , S t e w a r t , J . , F u n d e r , J . W . , F e l d m a n n , D . , E d e l man, I.S.: Proc. Nat. Acad. Sei. (USA) 71, 1 4 3 1 - 1 4 3 5 (1974) 5. C l a i r e , M . , R a f e s t i n - O b 1 i n , M . E . , M i c h a u d , A . , R o t h - M e y e r , C., C o r v o l , P.: E n d o c r i n o l o g y 104, 1 1 9 4 - 1 2 0 0 (1979) 6. E r b l e r , 366-372

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A b s h a g e n , U., S p o r e , S . , S c h ö n e s h o f e r , R e n n e k a m p , M., O e l k e r s , W. : Clin. Sei. 310s (1976)

11. L o r i a u x , R.: Ann. 12.

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D.L., Menrad, Int. M e d . 8 5 , H.P., Potts, (1978)

13. M e n a r d , R . A . , 94, 1628-1636

R., T a y l o r , A., 630-636 (1976)

G.O.:

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B.,

A.A., Mitchell, A (1976)

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B.W.:

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19.

Pita, J.C., Lippmann, M.E., E n d o c r i n o l o g y 97, 1521-1527

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R o u s s e a u , G., B a x t e r , J.D., F u n d e r , T o m k i n s , G . M . : J. S t e r o i d . B i o c h e m .

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W a m b a c h , G . , H i g g i n s , J . R . : In A n t i h o r m o n e s ( E d . M . Agariwal) p. 1 6 7 , E l s e v i e r / N o r t h H o l l a n d B i o c h e m i c a l Amsterdam (1979)

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29.

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Z a g a r e l l a , J., H r i b a r , J., Ther. 19, 1 5 8 - 1 6 4 (1976) A n t i - A l d o s t e r o n e s , in: R.J. Dorfman, Academic

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in C l i n i c a l Excerpta Medica T.,

196 Acknowledgements W e w o u l d l i k e to e x p r e s s o u r g r a t i t u d e to P r o f e s s o r R. W i e c h e r t , D r . W . E d e r a n d D r . U. K e r b , S c h e r i n g A k t i e n g e s e l l s c h a f t B e r l i n a n d B e r g k a m e n , W - G e r m a n y , for the g e n e r o u s s u p p l y of the s t e r o i d s u s e d in t h i s w o r k . W e t h a n k M r . M. B u s e a n d M r s . G. S u c k a u for e x c e l l e n t a s s i s t a n c e . P a r t of the d a t a a r e p u b l i s h e d in a c o n t r i b u t i o n to B i o c h e m i c a l P h a r m a c o l o g y , 32, 1 4 7 9 - 1 4 8 5 ( 1 9 8 3 ) . T h e s e s t u d i e s w e r e s u p p o r t e d by g r a n t s f r o m M i n i s t e r i u m für W i s s e n s c h a f t u n d F o r s c h u n g d e s Landes Nordrhein-Westfalen.

THE A N T I M I N E R A L O C O R T I C O I D

ACTION OF TWO NEWLY

SP I R O L A C T O N E

DEVELOPED

DERIVATIVES

M . K. A g a rwa1 Centre

U n i v e r s i t a i r e d e s C o r d e l i e r s , 15 rue E c o l e de 7 5 2 7 0 P a r i s C e d e x 06, F r a n c e

Médecine,

M. Kai imi Department

of P h y s i o l o g y ,

M i n e r a l o c o r t icoi d h o r m o n e s mammals w h o s e

dysfunction

Medical

regulate

the

syndromes

(l- »). A l t h o u g h

aldosterone

corticoid

hormone, minute

amounts

in p a t h o l o g y to the o n e

accepted

and

appropriate

the

target

chemical

analysis

revealed

that

molecular various only

organ of the

becomes

these

the mi n e r a l o c o r t icoid

agonists may

s u c h that

for

subsequent

transfer

(9).

by p r o g e s t i n s reviewed

receptor receptor

occupancy activation

(MR)

Physicoexhibits action

saturation

of

of

to c o m p e t e

(5-6) and/or to

to

impede

impaired

at the

level

of the c e l l u l a r

in an e a r l i e r

volume

receptor in t h i s

The a n t i m i n e r a l o c o r t icoid a c t i o n of s p i r o l a c t o n e to

an

with with

nuclear

i n h i b i t i o n of the mi n e r a l o c o r t i co id a c t i o n

by o n e of us

in r e l a t i o n

bind of

reviews).

shown

leading

It

however

physiological

was

noxious

steroids

has

receptor

an a n t a g o n i s t

also

reviews).

(5-6).

binder

C 7 — 8 for

are

cytoplasm

proceed via preferential

model

The

kidney

the

a s u b s p e c i e s of the MR s y s t e m

the a g o n i s t

hormonal

intracellular

mineralo-

particularly

C+ for

in

related

steroids

in the cell

s u c h as the

balance

and

is the m a i n

syndrome

receptor

Virginia

hydrosodic

of o t h e r

that

t h a t all

same

heterogeneity

In the c l a s s i c a l the

property

s u c h as the C o n n ' s

was generally

of

leads to h y p e r t e n s i o n

1

endowed with similar

College

receptor

conformations

will

be

Receptor Mediated Antisteroid Action © 1987 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

has

been

series

(3).

derivatives

reviewed

here.

198 Experimental

Procedures

Male,

Wistar

rats

mized

5-7 d a y s

and water

ad

(200-250

prior

to o r g a n

libitum

were

sacrificed

n a t ion,

the o r g a n w a s

l ipid f r e e

cytosol

105,000

g.

60 m i n ,

followed

under

Blood was

allowed

by

60 m i n

g to o b t a i n

a clear

serum.

For

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

binding

M Tris-HCl C) w i t h

pH

in p r e s e n c e Free C)

7.4),

of

steroids

an e x c e s s were

in p r e s e n c e

0.25%

dextran

(3000

g,

10 ml

of

vector the

quantitated

and

kinetics,

using

as d e s c r i b e d

the

left

before

studies,

also

cytosol

in t h e T r i s 35°

C for

and

processed

protein

various

as b e f o r e . T h e

(12).

for

of

or

choice. min,

charcoal

+

fluid

in p r e s e n c e

of

spectrometer.

(10) and

dependent

the

steroid-

concentration under

was

similar

of condi-

(11).

was

buffer.

at

radioactivity

studied

steroid

as a b o v e ,

method

a single

min

centrifugation

in a P a c k a r d

in d e t a i l

radioactive

(2.5%

0.01

alone

(10

supernatant

Time

(60

steroid

by

10,000

(in

material

incubation

Bradford

saturation

then

CPM/mg

the

was

incubated

and counted

protein.

10 n M of

treated

vials

at

cytosol

charcoal

as C P M / m g

hormone,

for

sedimented

resulting

and

room temperature

radioinert

(Amersham)

by

buffer,

at

0.2 ml

further

expressed

sodium molybdate were

the

The

exsanguiat

tritiated

activated

cocktail

For d e n a t u r a t i o n with

the

by

to s c i n t i l l a t i o n

radioactive

tions,

0.2 ml 4° C ) .

the ACS

Protein was results

of

of

removed

initial

food

u1tracentrifugation

serum, was

of

T-70) which was

10 m i n ,

transferred

amount

by

on

house.

C, a n d c e n t r i f u g e d

studies,

or w h o l e

the d e s i r e d

the

by

to c l o t

at

animal

anesthesia

with

prepared

adrenalecto-

and m a i n t a i n e d

controlled

ether

perfused was

bilaterally

ablation

in a c l i m a t e

Animals a

g) were

incubated with

Samples periods

(2 h,

or w i t h o u t

are

mM

in tripl i c a t e of

time,

determination

results

C) 10

of

expressed

charcoal protein as

199 To a s s e s s

receptor

following

the

of

at

choice

molybdate.

activation,

2 h equlibration 4° C,

( 1 . 4 mg

incubated

for

of

prior

45 m i n

to c o u n t i n g gradient

sucrose

gradients

30 m M

KC1

buffer

Clow

was

steroid.

finally were

40,000

rpm.

Charcoal 5 ml

estimate

was

in 10 m M

gradients).

incubated

(2 h at

treated

of

10 m M

the

Fractions

of

sedimentation

Double

labelled

ion e x c h a n g e

cytosol, tritiated powder/ml with

prepared

incubated

0.2

M sodium and

this

20°

at

a Gilford

gradients.

assessed (4.6

by

was

Samples

rotor with

C)

cytosol

0.4 ml

S)

in

and

references

on

procedures

for

4° C ) w i t h

of c h o i c e

as

coefficients

by

(MKA)

in 0 . 0 0 2

(2 h at

steroid

us

employed

chromatography

performed of

thereafter

same

tritiated

4° C.

the

serum albumin

Bovine S) w e r e

(14).

was

t o p of

plus

at

the

and

collected

the

fluid.

and Ames

by o n e

from

7.5

in the

SW

was

(7.1

pH

at

radioactivity

globulin

DE-52) was

precooled

the

5-20%

10 n M

in a S p i n c o

The

fluid

(45 m i n

as a b o v e ,

8 drops were

starting

buffer

sodium molybdate,

4° C

and

(13).

prepared

4° C ) w i t h

gradients

18 h at

and

linear,

incubation

and c e n t r i f u g e d

for

buffer

on

Tris

Cytosol

an a d d i t i o n a l

Martin

(Whatman

performed

DNA-

agitation.

in d e t a i l

resistant

established

ug

cellulose)

occasional Tris

100

sodium

before

prepared

receptor

DNA/ml

C,

steroid

10 m M

as d e s c r i b e d

apparatus,

gamma

of

scintillation

scintillation

human

tritiated

1 ml

layered onto

Densiflow

25°

in t h e

salt

centrifuged

at

overnight

or a b s e n c e

charcoal

left

mixed with

thymus

4° C w i t h

analysis

first

the

or a b s e n c e

ul w e r e

calf

was

thrice with

Following

in p r e s e n c e was

at

suspended

Density

200

native

samples were washed was

with

in p r e s e n c e

Aliquots

cellulose

pellet

cytosol

to

the m e t h o d

of

DEAE-ce11u1ose

originally

purpose

phosphate

the d e s i r e d then charcoal

(15-18).

buffer amount

pH of

treated

The 7.5, the

(50

mg

c y t o s o l ) . Rat b l o o d s e r u m w a s s i m i l a r l y incubated 14 C - c o r t i sol a n d t h e n c h a r c o a l t r e a t e d as a b o v e , MCi

separately.

The

serum

and

the c y t o s o l

were

then mixed

and

200 loaded onto initial

a DE-52

0.002

this

low s a l t

ient

(begun

e a c h of flow

rate

buffer, the

0.002

of

processed

M phosphate

at

the

column

buffer.

arrow

in t h e

M and 0.2

determination A1iquots

ACS

radioactivity

fluid

for

described

sodium

(1

x 130 c m ) ,

separation.

3

30 m l / h

H-RU

filtration,

phosphate

activity

Uclaf,

with

of

a

of

2 ml

pH

7.5,

were

absorbance

measurements spectral

of

at

30 ml

4°C,

10 ml

taking

a

for

of

into

as

at

and

280 m u a n d

overlap,

of

grad-

collected

at

the

20 ml

linear

figures) consisting

were mixed with

the

account

previously

equilibrated

mixture

at

C and

2 ml

Roma invi11e.

3

as a b o v e

processed

H-ZK

0.01

charged

for

ion

collected

for

with

Sweden) M

with

the

exchange

at a f l o w

absorbance

rate

and

radio-

the

corres-

(15-18).

reference

steroid

and eluted 7.5, w e r e

were

as a b o v e

Ci/mM,

radioinert

pH

prepared of

determination

(50

U 1 t r o g e 1-ACA-'+'t ( P h a r m a c i a ,

+ 0.1 M N a C l ,

Fractions

26752

ponding

of

1 ml

and

passage

eluted

in

(15-18).

cytosol-serum of

of

background,

For m o l e c u l a r columns

After

equilibrated

M phosphate,

Fractions

conductivity. quenching,

x 25 c m )

protein was

30 m l / h .

for

CO.5

(lot 9 1587

X 3025 A),

and

7) w e r e

obtained

(lot

2 0 9 6 - 2 ; 87 C i / m M )

ES

from

Rousseland

the c o r r e s p o n d i n g c o l d m a t e r i a l w e r e p r o c u r e d f r o m S c h e r i n g , 3 14 1 , 2 , 3 , H - a 1 d o s t e r o n e C+5 C i / m M ; b a t c h 3 4 ) a n d 4 C-

Germany. cortisol The

(55 m C i / m M ;

radiochemical

layer c h r o m a t o g r a p h y . Reagent We w i s h Losert

grade to

thank

Broussais

provided

All

for

D.

other and

Dieu,

Virginia by

of

aided and

Affiliate.

Schering,

in all

from

Amersham.

cases

were

in

high

thin

purity

Sigma.

gift

Paris,

purchased

97%

chemicals

Philibert

the

This work was

Hotel

Association, also

Dr.

57) w e r e

exceeded

from Merck

(Schering)

respectively.

batch

purity

(Rousse 1-Uc 1 af) and RU

26752

by g r a n t s by

ZK

from

the A m e r i c a n

Some

Germany.

and

financial

Dr.

W.

91587, the

UER

Heart support

was

201 Results Data

presented

kidney,

ZK

in F i g .

91 587

- MR

formation

reached

remained

unaltered

renal

MR could

steroid

began

labelled then

a more

since

in b o t h

( m i n e r a l o c o r t icoid

for

at

least

be m a x i m a l l y to d i s s o c i a t e

progressive

specific

that,

a maximum within

the M R w i t h i n

the o r g a n this

1 show

the

receptor)

first

only

soon after. followed with

RU

time,

both

antagonists

were

they

show

no a f f i n i t y

for o t h e r

the

26752

labelling

MR

classes

RU

2 h and heart,

first

the

and

With

after

and

complex

60 m i n

In t h e by

decl ine. A l t h o u g h

differences

heart

8 h thereafter.

labelled

30 m i n

the

RU

26752, the 26752

a rapid

and

reasonCs)

for

are

not

clear

at

specifically of

receptors

C19-20).

Fig.

1. T e m p o r a l S a t u r a t i o n of By T w o A n t a g o n i s t s .

the Mi n e r a l o c o r t ico i d

Receptor

Renal c y t o s o l w a s i n c u b a t e d w i t h 100 nM of e i t h e r 3 H RU 2 6 7 5 2 ( x - - - x ) or 3 H ZK 9 1587 (x x). C n r d i a c c y t o s o l w a s similarly i n c u b a t e d w i t h e i t h e r 3 H RU 2 6 7 5 2 ( 0 ) o r 3 H ZK 9 1 5 8 7 C • ). A l i q u o t s of 200 u1 w e r e c h a r c o a l t r e a t e d at t h e i n d i c a t e d t i m e p e r i o d s f o r d e t e r m i n a t i o n of r a d i o a c t i v i t y . All p o i n t s a r e a v e r a g e of t h r e e s e p a r a t e d e t e r m i n a t i o n s . S i m i l a r r e s u l t s h a v e b e e n d e s c r i b e d by o n e of us in a n e a r l i e r p u b l i c a t i o n us i ng o n 1y RU 2 6 7 5 2 ( 1 1) .

202 Data

shown

in F i g .

antagonist

binding.

ZK

91587-MR

by

the

Similar

used with site

kidney

specififc

the a g o n i s t

since the

did

a more

any

attest

(Fig.

two

and

ZK

(11)

a 1dosterone-MR

10 nM

further

aldosterone

abundant

range

site

to t h e

target 91587

at a l l , w h e r e a s

RU

at

did

not

26752

for

heart bind was

of

fact

the

and

of

concenwas of

affinity

the

kidney.

ZK

91587

the

liver

antagonists Furthermore,

serum

only

a

for

plateau

that

lung o r

to b l o o d

bound

^H-

26752

a

MR-

followed

the e x i s t e n c e reached

The

specificity

organs

of

100 nM

^H-RU

little

complex all

slope

until

when

with

(2,3,11).

affinity

initial

suggest

antagonists

specificity

10 n M s t e r o i d w a s

and

the

exhibit

cortin

an

until

cytosol

not

in the

the

obtained

cytosol for M R

organs,

formation

of

confirm

results were

for

well

before

In b o t h

complex

saturation

tration.

2a f u r t h e r

trans-

minimally

2b).

0)

o Q-2 O)

E

X 50

nM STEROID Fig.

Q_ O 0

2. ZK 9 1 5 8 7 B i n d s t h e M i n e r a l o c o r t i c o i d Target Organs only.

_

•

Receptor

_ in

the

200 u1 c y t o s o l , in t r i p l i c a t e , f r o m v a r i o u s o r g a n s w a s i n c u b a t e d f o r 60 m i n at 4° C w i t h the i n d i c a t e d c o n c e n t r a t i o n of the r a d i o a c t i v e s t e r o i d a l o n e or in p r e s e n c e of o n e t h o u s a n d f o l d e x c e s s of the c o l d m a t e r i a l w h i c h w a s s u b t r a c t e d f r o m the f o r m e r to a c c o u n t for the n o n - s p e c i f i c b i n d i n g . F o r F i g . 2b, 200 y1 s e r u m w a s u s e d in p l a c e of c y t o s o l . F o r f u r t h e r d e t a i l s s e e the s e c t i o n o n m e t h o d s . l t m u s t be n o t e d t h a t in all o u r s t u d i e s u n d i l u t e d s e r u m w a s u s e d , c o n t r a r y to s o m e l a b o r a t o r i e s w h e r e a s e r u m d i l u t i o n of 1:20 w a s u s e d l e a d i n g to e r r o n e o u s c o n c l u s i o n s ( 2 1 ) .

203

V

1

c 2 e O )

E

-9

u

-8

-7

M

Fig.

3•

RU

26752

Does

Not

-6

-5

STEROID

Displace

Aldosterone

in Rat

Kidney.

Renal c y t o s o l w a s i n c u b a t e d a l o n e w i t h ^ H - R U 2 6 7 5 2 ( O ) , and in p r e s e n c e of e i t h e r c o l d RU 2 6 7 5 2 ( £ D ) or c o l d a l d o s t e r o n e ( # ) , at the i n d i c a t e d c o n c e n t r a t i o n s of t h e c o m p e t i t o r . All s a m p l e s w e r e run in tripl icate and c a l c u l a t e d as C P M / m g prote i n (11).

The

specificity

several could

other

not

easily

be

of

displaced

competed

Similar

out

results

26752.

Independent specififc

26752

available for

the

suitable

for

however,

that

established be

kept

various

the

in m i n d means

binder, Thus,

in the

all

with

of

in t h e

receptor. has

physicochemica1 in the

of

makes

steroid

arsenal

was

in p l a c e

bind

of

ZK

91587

It

is

already

of

RU

an

anta-

>

is RU

clear, been

(7),

analysis

of

the

was

particularly

hormones

of

but used.

agonists,

them

by

^ H - R U 26752

aldosterone

existence

not

specificity

this

of

the

organs

confirmed that

material

91587

does

heterogeneity

classes

currently

for

that the

ZK

was

3 show

homologous

target

analysis

to MR

in F i g .

non-radioactive the

obtained

receptor

for

binding

Data

confirmation

MR

(22). MR

by

when

were

gonist also

antagonist

experiments.

MR

well and by

biochemist.

must

204 Denaturation upto

60 m i n

MR, more

studies at

so

C

if

were

absence

100%

had been stable

of m o l y b d a t e

35°

C and

was

90%

in the heat first more

studies 30 m i n .

of M R .

tion

than

of M R - ^ H - R U

With

these

the

and

differences

further

the

confirmed

again

that

species,

label

exhibits

in p l a c e protected only

was

reported (not

the

even at all.

popula10-100

both

the

range. (22)

and

shown).

a

MIN 35° C

10 nM

against

denatura-

for

1-10 nM s t e r o i d cytosol

of

effect

to h e a t

affinity

almost

within

during

labelled with

been

even 26752

this was

two d i s t i n c t

sensitive

cardiac

H-RU

complete

protective

suggest

in the

3

of

denaturation

no

previously

with

(12)

complex

heat

is m o r e

antagonists have

used

molybdate

26752

91587,

abundant

population

Similar

was

exhibited

studies

antagonist,

agonist were

^H-ZK

The more

n M of e i t h e r

26752

In a d d i t i o n ,

and m o l y b d a t e

Collectively, tions

H-RU

here.

denaturation rapid

3

30 m i n

to

incubation.

100 n M a l d o s t e r o n e

at

60 m i n . nM

with

complexes

used

during

The d i s s o c i a t i o n

from MR within 100

was

included

(12).

stable

40%

complete

10 m i n w h e n

4 reveal

Nearly

dissociated by

in F i g .

10 n M a l d o s t e r o n e

if m o l y b d a t e

The c o m p l e x e s in the

35°

shown

205

b

c

MIN 35° C

Fig,

h. D e n a t u r a t ion K i n e t i c s of t h e Renal M i n e r a 1 o c o r t i c o i d R e c e p t o r in P r e s e n c e of A g o n i s t s a n d A n t a g o n i s t s .

For e x p e r i m e n t a l d e t a i l s s e e M e t h o d s s e c t i o n a n d ( 1 2 ) . All v a l u e s a r e a v e r a g e of t h r e e s e p a r a t e e x p e r i m e n t s d o n e o n separate cytosols. N o molybdate C • ); w i t h 10 m M m o l y b d a t e ( 0 ).

206 Data

in

Fig.5a

underwent

show

heat

did

not

obtained

cal

just

confirming permit when

derivatives

MR

as

as

earlier

cardiac

action

at

the

2 67 5 2 - r e c e p t o r

at

did the

assessed

by

complex

binding

to

a 1 dosterone-receptor

studies

(12).

all.

cytosol

spirolactone of

3h - R U

act¡vat i on,

well

activation

rat

of

mechanism

renal

dependent

DNA-ce11u1ose, complex,

that

was

not level

However,

Similar

used (5b).

appear of

the

^H-ZK

results

to

Thus,

the

possess

cellular

91587

were two

identireceptor.

a ALDOSTERONE

Fig.

5.

RU 2675!

ZK 91587

T h e r m a l A c t i v a t i o n of the M i n e r a l o c o r t icoid in p r e s e n c e o f A g o n i s t a n d A n t a g o n i s t s .

For d e t a i l s 4° C ( Q );

see 25°

Methods and (12). Incubations C w i t h (£77] ) or w i t h o u t ( ^ 1 )

Receptor

as f o l l o w s : 10 m M m o l y b d a t e .

207 Data

in

Fig.

6 present

activated

and

Molybdate

stabilized

the

7 S

vation,

the

region

^H-RU

26752,

predominant the

4 S

in

of

in

7 S to

7 S

region

with

peaks

of

thermal

equal

Under

these

^H-ZK

91587-MR

magnitude

activation

Similar

results

used

place

of

were

a

of

MR

the

the

seen

antagonists receptor

(see

With as

a in

26752-MR

with

the

nonactivated, buffer 4 S

this

cardiac

showed

regions

profile.

cytosol

was

cytosol.

steroid earlier

mechanism

m i nera1ocorticoids.

rat

as

the

alter

the

shoulder

^H-RU

salt

7 S and

further when

low

in

acti-

of

region.

smaller the

S form,

in

shift

sedimented

conditions,

in t h e

not

obtained

renal

confirm

did

4

MR

gradients.

sedimented

h S

the

the

Thermal

in a

kidney

in t h e

two

the

the

both

density

buffer.

largely

and

terone

from

salt

resulted

revealed

stabilized

and

low

of

complex

activation,

aldosterone.

dissociate

sucrose

thermal

agonist

activation

of

stabilized

the

molybdate

data

on

analysis

Upon

was

These

MR

molybdate,

complex

peak

ever

a 1dosterone-MR

presence

complex

in

the

molybdate

region.

first

nonactivated

in a b s e n c e

a 1dosterone-MR

the

of

Similar have

also

another

dependent on

DNA-ce11u1ose

action

of

the

differences been

two

in

and

this

with

6

E a o

FRACTION

8 NOS

the

volume).

ALDOSTERONE

12

thermal

further

the

CM

k

in

synthetic

between

observed

chapter

i

.15 E

differences

a n t i-

agonists proges-

208

_

E

1-

eg

6

E a

o

A

FRACTION

BSA

I

12

8

NOS

IK 9158?

1 Globulin

I

CM

O

E a. o

U

FRACTION

Fig.

6.

8

12

NOS

Sucrose Density Gradient Analysis Minera1ocortIcoid Receptor.

of

the

Renal

F o r e x p e r i m e n t a l d e t a i l s s e e thn s e c t i o n o n M e t h o d s . I n c u b a t i o n s w i t h o u t C • ) o r w i t h ( 0 ) 10 m M m o l y b d a t e .

Receptor depends system

heterogeneity upon

(7

matography 3

H-RU

vity

range

0.006

M

100 nM

H-ZK

again

995-1675

H-ZK

nM

in t h e 9 1587,

H-ZK

studies two

the

either

one

Transcortin

both

0.002

of

3

that

(Fig.

of

the

two

or

the

(MR^)

With

slope

that

the

the

10 nM

of

first

is q u i t e

of

distinct.

E a o

20

FRACTION NOS

30

U0

MR^

of

(Fig.

in F i g .

mechanism

With

radioacti

and

populations

the

and

(15-18). bound

labelled

and

elution

7c),

MR.

It

1

(10-

slope

antagonist) corresponds

antiminera 1ocorticoids

10

7a)

prewash

whereas

of

(conducti-

( T ) and

7b). be

chro-

peak

(Fig.

position

second

as M R ^

confirm

single

MR^

and

fractionation

phosphate

26752,

MR^

MR ^ c o u l d

existence

M

fact

exchange

respectively H-RU

in the

prewash

only

agonist

further

the

ion

- 0.02!+ M

in the

91587) eluted

nM of

confirmed

in p l a c e

ionic

and

labelled

Siemens) called

both

to c o n c l u d e

(1-10

the

low

established

steroid,

(MR,,) r e g i o n s ,

however,

These of

from

obtained

confirming

3

micro

91587

be

the

Double

in 0 . 020

aldosterone

is t e m p t i n g 100

nM)

phosphate 3

a well

DEAE-ce11ulose

C100

of

could

region 3

tissue,

reviews).

distinct

position

vity

on

267 52

clearly

the

for

is n o w

to

MR^

action

b

210

c

Fig.

7.

Ion E x c h a n g e C h r o m a t o g r a p h y C o r t ico id R e c e p t o r .

of

the

Renal

Mineralo-

2 ml renal c y t o s o l w a s i n c u b a t e d w i t h the a n t a g o n i s t in e a c h c a s e a n d 1 ml b l o o d s e r u m e q u i l i b r a t e d w i t h 0.1 mC i o f l i + C C o r t i s o l w a s u s e d as a p o s i t i o n m a r k e r . 100 n M s t e r o i d w a s u s e d for F i g u r e s a a n d b but o n l y 10 nM ^ h - Z K 9 1 5 8 7 w a s u s e d for F i g . 7c. . . 3 H C • ); C ( 0 ); A280.

211

Data

in

this

Figure exchange

for

the

ion

The

use

of

of

us

nearly

two

entity

the

since

almost

as

decades

peak

or

well

even

Tissue

with

corticoid (25).

with

be

10

is

any

time

but

was

H-ZK

that

in

the have

relevant

been

to

ask

antagonists

would

renal

replaced

another

cytosol

few

The

organs

Data

in

3

that

Fig.

population With

H-ZK

in

the

the

relative to

MR^

be

kidney dance

the

contrasted

a

(Fig.

receptor

to

the

maintained

is

of

Further

required

The

to

as

in nM

>

MR^

the 3

as

in

< MR^

to

constitute

studies,

to

a

a given could

target the

the

7a). the

(Fig.

in

mechanism stimulus

to

receptor

8b)

abun-

the drug.

of is

possibly

unequivocally.

MR ^

the

relative

equally

MR^

However,

explain

not

the

of

pharmacological

the

respond

one

heart

the

well

particularly

these

if

both

8b).

the

words,

homeostasis

whether

(Fig.

26752,

situation

could

when

gluco-

earlier

labelled

(Fig.

18).

(26).

kidney

H-RU

MR^

response

were

answer

various

similarly

26752

observed

organs

organs

of

(4,

mineralo-specific

H-RU

In o t h e r

also

action

described

100

MR^

the

7b).

3

subpopu1 ation

between

if all

vicissitude. tion,

of

were

with

could

organism.

nM

just

place

physiological

differences

discrimination ted

heart, in

(15-18)

question

behave

been

confirm

minera 1ocorticoids

100

proportions

cytosol of

to

that

populations

differential Such

8 show

by

previously

responds

9 1587

and

is

was

heart h a s

These

described

the

either

agonists

MR

elution

with

labelled

91587.

antagonize

of

when

receptor

at

3

one

assessment

26752,

nM

by

addition,

a distinct

mineralocorticoid

organ.

In

mineralocorticoid

populations

therefore

permits

ideal

species.

perfected

columns.

H-RU

just

with

24),

are

receptor

marker,

observed 3

nM

conditions

various

(23,

differences

receptor

It w a s

of

prewash

not

these

position

back

progestins

dependent

that

different

100

observations as

a

in t h e

exclusively

earliest

as

between

it c o u l d

transcortin,

show

separation

transcortin

r e p r o d u c i b i 1 ity observed,

also

any

presenbe one

purifica-

212 a

FRACTION

Fig.

8.

NOS

Ion E x c h a n g e C h r o m a t o g r a p h y R e c e p t o r from Rat H e a r t .

of

the

Mineralocorticoid

2 ml c a r d i a c c y t o s o l w a s i n c u b a t e d w i t h the a n t a g o n i s t in e a c h c a s e a n d 1 ml b l o o d s e r u m w a s e q u i l i b r a t e d w i t h 0.1 uCi of - C o r t i s o l w a s u s e d as a p o s i t i o n m a r k e r . 100 n M 3 H - s t e r o i d w a s u s e d in all c a s e s . 3

H

C •

);

C 0 );

A00n.

213 Molecular

filtration

antagonists dalton daltons quite

labelled

region CFig.

from

In p r e v i o u s

studies, and

Despite

91587 on

peak

these has

been

regions,

in m o l e c u l a r whereas

50,000 region.

to e l u t e

these

in

conditions RU

density

aldosterone

in c h a r g e

were .

dalton

weights,

charge

both

110,000

about

shown

under

identical differ

that

populations

similarity

populations

of

65,000

carry

near

in t h e

entity

in t h e

aldosterone dalton

revealed

component

b o t h of

on D E A E - c e 1 1 u 1 o s e )

bound MR

columns

lighter

transcortin

65,000

bound components single

by a

9). A g a i n ,

the

C15-18).

a heavier

followed

distinct

110,000

on Ultrogel

as well

26752 Cone

and

ZK

(two

peaks

DEAE-ce11ulose).

FRACTION NOS Fig.

9. M o l e c u l a r F i l t r a t i o n R e c e p t o r on Ultrogel

of the Renal Columns.

Mineralocort i coid

100 n M t r i t i a t e d s t e r o i d w a s u s e d to s a t u r a t e k ml c y t o s o l ; 2 ml s e r u m w i t h 0.2 uCi ^ ^ C - C o r t i s o 1 w a s u s e d as a p o s i t i o n m a r k e r . For f u r t h e r d e t a i l s see M e t h o d s and ( 1 5 - 1 8 ) . ltf ' « ( • ) ; C C 0 ); Aosn.

214 Data MR

in F i g .

labelled

could more

be

with

abundant was

26752

kidney, CFigs.

9b vs vs

the m o l e c u l a r less

abundant

lighter,

component when

10b).

the

organs

when

tissue

specific

gave

^H-RU

the

elution relative

component

26752

was

differences

10a),

91587 was to the

similar

However,

1 ighter

Whereas heavier,

CFig.

^H-ZK

10b). As c o m p a r e d 91587

filtration

two a n t a g o n i s t s .

as a

reversed

CFig. ^H-ZK

heavier

the

resolved

abundance RU

10 s h o w

was

noted

rat

H-RU

26752

followed

by

the

relative in p l a c e

situation profile

in of

in t h e these

the two

confirm

above.

16

x

E

Q. O

•15

50

25

FRACTION

NOS

E a o

FRACTION Fig.

10. M o l e c u l a r Receptor.

Filtration

of

NOS Cardiac

of

heart

24i

to

a

the

in t h e

abundance

Again,

cardiac

used

reversed

employed.

of 3

Minera 1 ocorticoid

215

Discussion

The

results

ces

in t h e

described mechanisms

derivatives finally

of

known

side

effects

side

properties,

The

and

is

that

molecule

was

reviews

The

^H-RU

by

higher

dependent

the

effects

3

more

hormones, 3

26752

or

these

new

receptor. inferred

H-ZK

abundant,

The by

abundant

a

cold

presence

authors

estrogen

has

the

by

derivatives

site

that

1 ittle than

are of

using

studies

receptor is

of

are

two

in

RU

binding

to

a

rather for

target

classical

ZK

all

for

the MR

estrogen

(11).

range

was

10-100

nM

confirmed

9 1587,

which

serum

carriers,

for

the of

suggest

time

mineralo^H-RU

that

both

mi n e r a l o c o r t i c o i d has

also

spiro1actones aldosterone

for

agonist,

binding

in

sites

similarly

specific

(for

26752

displacement

tritiated

the

nM

largely

than

26752,

receptor

kidney

between

n o n - r a d i o a c t ive

has

RU

the

possibly

- MR

1-10

of

material

rat

numbered

possible

with

was

26752

were

MR

of

another

position

latter

the

steroids,

specific

(see

molecule,

denominated

site

not

lack

system

affinity

the

for

absence that

and

91587

competition

contain

The

7a

which

H-RU

in

the noxious

antigestationa1

in w o m e n

the

the

observations

affinity

organs

in

sites

supremacy

to

a n t imi n o r a l o c o r t icoi d .

and

of

of

These

minera1ocorticoid

of

receptor

devoid

action,

substance,

for

kinetics

overall

from

corticoid

that

residue

in a

of

These

kinetics.

cytosol

The

The

are

important

spirolactone

ideal

differen-

synthesized are

molecule.

like

spirolactone

binding

(11).

an

propyl

classes

that

parent

native

being

side

newly

analogues

methoxy-carbony 1 derivative,

showed

cross

in

the

another,

26752

the

a

two

important

i rregular i t i es The

resulted

2-4).

two

aldosterone

followed

to

for

see

suggested

the

specificity

lacking

responsible

from

of

of

Such

antiandrogen

volume).

reveal

compounds

of

menstrual

far

increased

which

action

those

include

introduction

parent

by

at

effects

in t h i s

therefore,

of

paper

spiro1actone.

arriving

many

chapter

in t h i s

been the and

receptor

shown

been in

(22). to

antagonist, that (27).

is

more

216 The

importance

spirolactone Most

of c h e m i c a l

molecule

dramatic

t i o n of mitted

the

hormone

process

whether

this

these

is o b v i o u s l y

processes

receptor

is n o t

possible on the

l a c k of

activation explanation

latter was

this

glucocorticoid

just

as well

acetonide

or

the

contrary

of

of

compartment

sort

are

receptor

antig1ucocorticoid permit

The

influence

in t h i s

for

to

the

in v i v o very

so c a l l e d lose or

same

antagonist

Furthermore,

longer

suggestions

be

the

considered

action

where

inhibiting

the

of

the

transfer

is g e n e r a l l y

Thus,

either RU with

on

only

the

been

p r e c e d e d by

It c a n

(28)

subject

safely

situations,

or

between

nuclei,

is

by

be

time

binding

in n o w a y

activated

activity

(29

for

and of

in vivo,

reviews).

the

glucocorti-

separate here

either

that

from target

to e i t h e r or

DNA

no

in organs

In a d d i t i o n ,

target

on

triamcinolone

various

system.

work

cortexolone,

be s a i d

this

judged

but

limited

at all

at

hormone-receptor

recent

GR c a n

progestin

the

by

the a g o n i s t

38486

possible

activation,

isolated

s p e c i f i c.

has

volume.

seem

f r o m an

supported

activation

38486

systems

extrapolations vitro

thermal

also (GR).

of

not

RU

it

(9).

in p r e s e n c e

of

In o t h e r w o r d s ,

can no

that

proby

activation.

by

due

interfere

adopted

an a g o n i s t

to p r e v i o u s

91587)

in v i v o

the a c t i v a t i o n

agonist.

to be a c t i v e

CZK

determined

that

antiminera1ocorticoid

the a n t a g o n i s t

receptor

ions

per-

natural

to be

be o p e r a t i v e

event,

receptor

did

chapters

the

the

another

coid

It r e m a i n s

activa-

26752)

the

to the c o n f o r m a t i o n

receptor

for

believed

Conclusions the

of

activation

(RU

the

studies.

thermal

as

phosphate

In a n y

unique

of

to t h e n u c l e a r

receptor

as well

also

of

to d i s t i n g u i s h

a valid

7 of

subsequent

in t h e

antihormone

just

would

(12).

not

basis

spirolactone,

one

completely.

in p r e s e n c e

merely

in all

observed

predominance

cess

in p o s i t i o n

the m e t h o x y - c a r b o n y 1 d e r i v a t i v e

differences

to t h e

with

of M R

Whereas

process

these

primarily

confirmed were

to p r o c e e d

aldosterone,

inhibited

the

was

differences

receptor.

this

substitution

the

cellu-

hormone

217

In

the

first

receptor buffer as

but

with

this

ever

complex

disaggregated

other

was

not

permit

forms. the

did

above

on

total

further

either

aldosterone

ZK

91587

-

Rather

stabilized the

protection as

an

ting

the

whatever

liver

60

min

for

against receptor

specific

known. alter

C or

35°

C

so

in

the

for

data

lacking

values.

had

no

these

was with

Similarly, in

presence

influence

on

differences

were

antiprogestin

RU

actually

receptor

system

(30).

totally

Such

not

This

(GR)

comparisons

in

cytosol

the is

Such

organism

The

were

and

reason(s)

with

differences

where

a

continuously.

at

any

38486, protecthe

stable

underscore

stability endowed

RU in

whereas

complexes

cytosol

afford

same

characterization

stability.

almost

oviduct

did

ineffective

receptor - GR

38486

in c h i c k

progesterone,

was

(28).

whole

protection

populations.

acetonide

tissue

prevails

inactive

cortexolone

denaturation

but

extrapolations.

receptor

and

did

receptor

differences

Perhaps

activa-

confirm

altogether

Again,

receptor

generalized

importance 37°

at

individual

largely

intermediate

glucocorticoid

need

almost

these

thermal

active

the

heterogeneous

in t h i s

upto

divided

4 S units

with

just

26752,

under

upon

receptor

26752

hormone,

triamcinolone

that

the

C,

against

RU

progesetrone

native

and

and

complex.

agonist

not

or

antig1ucocorticoid, rat

35°

gave

suggesting

paradoxically,

whereas

at

protected

receptor

dependent

(31)

salt

Whereas

RU

was

into

reported

low

cellulose.

26752

of

(1-4).

complex

further

the

activation,

antagonist

h o r m o n a l 1y

were

aldosterone

H-RU

molybdate

dose

between

studies

with 3

91587;

thermal

dissociation

aldosterone-

in

4 S populations,

change

receptor DNA

region

receptors the

receptor the

conclusions

denaturation

H-ZK

-

gradients,

7 S

4 S after

with

mere

distinction

the

hormone

not

words,

glucocorticoid

almost 3

which

Similar

obtained

In

91587 7 S and

sucrose

in

to

of

the

In o t h e r

on

confirmed

^H-ZK

into

conditions, tion.

classes

largely

unactivated equally

analysis sedimented

the

caution for

these

35°

C

are

factors

may

temperature

assume of

218 Ion e x c h a n g e

chromatography

on D E A E - c e 1 1 u 1 o s e

the mi n e r a l o c o r t icoi d r e c e p t o r forms whose Both the

genesis

aldosterone low

^H-RU

ionic

26752

consisted H-RU

bound MR.

of M R £

phosphate.

organ

1 i ver

C 18) .

these

kidney

evidence

for

and

the

with

is u n c l e a r

molecular

all

classes (32-34). ting

a mixture

of

two

In o t h e r

from

two

M and with

the

nontarget

ionic

forms

Here,

the

populations words,

these

the

MR^

molecular

ionic c o m p o n e n t s

receptor

the

resin

largely

columns.

of

and

transcortin.

in the

these

gave

in

progesterone

M phosphate

a r e of

of

the

homogeneous

populations

litterature of

steroid

How

then

in the c e l l .

To

actually

hormones.

hormone

receptors

to u n d e r s t a n d coded

in an e a r l i e r

the

these

of

extend

some

abounds The

of gene

has now

two

observed

the

the

(35).

earl¡est

receptor

new

various

been

receptor

back

receptor

for

relationships,

publ i c a t i o n

our

decades

in e v e r

appearannce

by a s i n g l e

modifications

understand

and

heterogeneity

steroid

species

post-trans1 ationa1

confirm

heterogeneity

of m o l e c u l a r

classes

receptor

advanced

bound

91587,

coeluted

both

two

the

ZK

serum

both

within

of

91587

receptor

24-). The

of

consists

on

in 0 . 0 2 0 - 0 . 0 2 4

lacking

alike

the

ZK

and

on Ultrogel

whether

chromatography

of

demonstrations for

or

data with

23,

of 0 . 0 6 totally

tissue.

columns.

demonstration (7,

conditions, aldosterone,

the

that ionic

transcortin

blood

to M R ^

heterogeneity

26752

the

sizes

on Ultrogel

These

RU

or

from

of

region

with

into

component

retained

very

ant¡hormones

forms.

aldosterone

lacking

distinct

and

the M R ^

1 0 0 - 1 2 0, 00 0 d a l t o n s .

component It

steroid

aldosterone

filtration

two

5 0 - 6 5 , 00 0 a n d

the

populations

but w e r e

from molecular

hormone of

was

confirmed

be s e p a r a t e d

labelled

exclusively

in the M R ^

target

Further

quite

antagonists

transcortin

came

The

bound

Under

two

upon

91587

and MR^, with

both

267 5 2 w a s

R-5020,

ZK

prewash which

respective1y, 3

depends

and

could

cloned

of

isoaccep-

gene? are

Perhaps

operative

a model

was

219

Persepectives

and

Conclusion

Mineralocorticoid substances and

including

other

most

means

promising

already

hormone

in

attempted

in o r d e r

increase in t h i s the in

the

the

native

It

duction

of

a

a molecule receptor species

the

called

(9). was

nM

hormone.

Type

bind

1-10

in

The of

existence

confirmed

and

of

an

specificity

for

both

These

antagonist (22)

in

mineralocorticoid

to

resolve

possible

receptor

protein.

It for

is

however

MR

vectors

since in

that

neither

non-target

specificity,

both

is

receptor

intro-

produced specific

abundant affinity

for

increased

of

to

such of

with

was

system makes

derivatives

action

with

(27). it

and in

are

are

1 iver. quite

a

Gene

even

transcortin,

the

so

affinity

heterogeneity

serum

the

minera1oreceptor

keeping

modifications

as

in

supposed

variable

in

receptor

these

bind

theirmechanisms

the

sites

that

specific

and

appearnce

organs

increase

derivative.

variance

with

p o s t - t r a n s l a t ional

clear they

estrogen

the

at

receptor

vector

to

are

antagonists

of

point

an

for

methoxy-carbony 1 derivative

similar

difficult

the

to

more

little

cloning

the

another,

and

chapter

aldosterone

had

independently

been

that

which

the

subs-

effects

c o n f i g u a r t ion the

the

affinity

lead

demonstrate 7a

(18)

far

another the

spirolactone

and

range

91587).

agonists

in

the

range,

nM

have

side

(see

saturated

I mineralocorticoid

all

The

(ZK

that

molecule

action

chapter

By

Chemical

increase

the

various

Spiro1actones,

(2-4).

necessarily

of

residue

introduction

7 position

not

are

by

progestins

(2-4).

undesirable

that

action

10-100

(36,37), before

spirolactone

does

antagonized

context

ant i hormone

26752)

the the

natural

the

to

in in

be

decades

attenuate

in t h i s

propyl

(RU

in t h i s since

is c l e a r

physiological

described

the

to

receptor

Results

with

use

the specific book).

cellular

repeatedly

materials

titutions

can

glucocorticoids

reviewed

in c l i n i c a l

action

more may

the

specific nor

Despite

to this

distinct.

220 ^H-RU

26752

receptor

- MR c o m p l e x

complex

activation. activation RU

26752

similar vated

35°

C but

underwent

^H-ZK

91587

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

ZK

ZK

was

when

studies

action

differ

of

RU

reveal

guration. any one

in the

Thus,

is

addition, elution

of

the

new

activation

desired

receptor

the MR

by

quite

nonacti7 S and

heat.

S

In

DEAE-ce11ulose but

only

one

in the

could

imposed

unique

and

be e x p l o i t e d

generating may

be

in t h e

upon

the

dictates in the

receptor

resins

7a

that confi

receptor

by

subsequent screening

of

variants

function

fruitfully

from affinity

mechanism

derivatives

substitution

physiological

derivatives

on

differences

of

This may of

even

in t h e

altered

observed

C.

in a m a n n e r

however,

Spirolactone

nature

capable

the

such

sucrose

employed.

inherently

behaviour.

new derivatives endowed with

35°

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

steroid

receptor

at on

further

important

two,

thermal

receptor

to s a t u r a t e

26752 was

these

solely

not

hormone thermal

permit

was centrifuged

forms were

used

comparable

the

- MR complex;

this was

ionic

91587

be e l u t e d These

and

two

the

7 S to 4 S s h i f t

thermal

- MR complex

91 587

compartments

of

of

than

did not

destabilized underwent

as a result

addition, when

greatly

- MR c o m p l e x

gradients

unstable

Contrarily, and

at

was more

in v i v o .

utilized for

In

in

the

eventual

pur i f i cat i on. Note:

The

trivial

follows:

RU

names

are

as

21,

1 7-carbo 1 actone;

of

the

two a n t a g o n i s t s

employed

here

267 5 2 ; 7 a p r o p y 1 - 3 - o x o - 1 7 a - p r e g n - ' t - e n e ZK

16 8 - m e t h y 1 e n e - 3 - o x o - 1 7

91 5 8 7 ;

7 a - m e t h o x y - c a r b o n y 1 - 1 5 £3,

a-pregn-^-ene

21,

17-carbo 1actone.

221

References

1.

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3.

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Endocr.

N E W M O L E C U L A R PROBES TO ASSESS ESTROGEN A N D A N T I E S T R O G E N ACTIONS

E t h e l M. Cormier and V. Craig

Jordan

D e p a r t m e n t of H u m a n O n c o l o g y , University of W i s c o n s i n , M a d i s o n , W i s c o n s i n 53792

Introduction M a n y b r e a s t c a n c e r s grow in direct response to e s t r o g e n stimulation

(1, 2).

As early as 1896, B e a t s o n

demonstrated

that o o p h o r e c t o m y of premenopausal w o m e n w i t h breast r e s u l t e d in tumor regression in some p a t i e n t s

(3).

cancer The

for this o b s e r v a t i o n was unknown until more than fifty later.

The synthesis and use of t r i t i u m - l a b e l e d

reason years

estrogens,

w i t h h i g h s p e c i f i c activity, led to the discovery of a receptor p r o t e i n w i t h both a high binding affinity and specificity

(4-7).

ligand

The m e a s u r e m e n t of e s t r o g e n receptors

b i o p s i e s of breast cancer patients

in

(8-12) indicated a m e c h a n -

ism of h o r m o n e - s t i m u l a t e d growth and p r o m p t e d d e v e l o p m e n t

of

e s t r o g e n a n t a g o n i s t s for breast cancer therapy

The

(13, 14).

c o r r e l a t i o n b e t w e e n response to endocrine therapy and

the

p r e s e n c e of e s t r o g e n receptors in human breast cancer

(15-17)

has f o c u s e d m u c h a t t e n t i o n on the molecular m e c h a n i s m s of estrogen action.

The ultimate aim is to u n d e r s t a n d the

role

of e s t r o g e n in the development of breast cancer, so that i n f o r m a t i o n can be used for the d e s i g n of e f f e c t i v e a n d u l t i m a t e l y the p r e v e n t i o n of the d i s e a s e .

this

treatments

It is c l e a r ,

h o w e v e r , that a complete understanding of the mode of

estrogen

a c t i o n t h r o u g h its receptor must be p u r s u e d since each s t e p m a y lend itself to m a n i p u l a t i o n or d i s r u p t i o n by natural or s y n t h e t i c agents.

This review will focus on the c h a n g i n g

Receptor Mediated Antisteroid Action © 1987 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

224 c o n c e p t s concerning the d e v e l o p m e n t of breast cancer w i t h a n o v e r v i e w on new probes to assess estrogen a c t i o n a n d new t h e r a p i e s for

A.

treatment.

M o d e l s for steroid receptor

1) C l a s s i c a l m o d e l . cell m e m b r a n e s

action

Steroids appear to diffuse freely

(18, 19).

through

R e t e n t i o n of the s t e r o i d is a c c o m -

p l i s h e d by high affinity binding to receptor sites.

In a

f u n c t i o n a l sense, a receptor must display a dual role, that of r e c o g n i t i o n and a c t i v a t i o n .

The receptor p r o t e i n m i g h t

p l i s h b o t h roles by either possessing two f u n c t i o n a l or two c o n f o r m a t i o n a l states

accom-

domains

(see Figure 1).

The c l a s s i c a l m o d e l of e s t r o g e n receptor a c t i v a t i o n has

been

a c c e p t e d as the p a r a d i g m to explain e s t r o g e n a c t i o n a n d the a c t i o n of m o s t steroid hormones

(20).

In the a b s e n c e of

h o r m o n e , the e s t r o g e n receptor is located in the c y t o p l a s m of the cell

(see Figure IB).

W h e n hormone is bound, an

estrogen-

receptor complex is formed w h i c h then becomes t r a n s f o r m e d an a c t i v e state.

Receptor

transformation has b e e n

ized by the change in the c o n f o r m a t i o n of the complex.

This c o n f o r m a t i o n a l change causes a shift in the (20).

cell g r o w t h are

trans-

including

stimulated.

The e x i s t e n c e of d i f f e r e n t binding and a c t i v a t i o n tions has been well d o c u m e n t e d for the e s t r o g e n

conforma-

receptor

A c t i v a t i o n of the e s t r o g e n receptor m o d i f i e s

r e c e p t o r ' s intrinsic activity as well as the value.

gradient

The resulting a c t i v a t e d complex then

l o c a t e s to the nucleus where biological responses

(21-23).

character-

ligand-receptor

s e d i m e n t a t i o n c o e f f i c i e n t from 4S to 5S in sucrose analysis

into

sedimentation

Receptor a c t i v a t i o n is d e p e n d e n t upon both

b i n d i n g a n d temperature

(21).

the

estrogen

A u t o r a d i o g r a p h i c and cell

225

F i g u r e 1. S c h e m a t i c representation of ligand-receptor r e c o g nition and activation. A) The m e m b r a n e - b o u n d e p i d e r m a l g r o w t h factor receptor p o s s e s s e s two structural domains. An epid e r m a l g r o w t h factor (EGF) binding d o m a i n a n d a c a t a l y t i c k i n a s e d o m a i n w h i c h is a c t i v a t e d by E G F binding and w h i c h c a u s e s the p h o s p h o r y l a t i o n and a c t i v a t i o n of a number of enzymes. B) In the classical e s t r o g e n receptor m o d e l , e s t r o g e n (E 2 ) freely diffuses through the cell m e m b r a n e and b i n d s to a c y t o p l a s m i c receptor. A n e s t r o g e n - e s t r o g e n receptor complex with a s e d i m e n t a t i o n value of 4S is f o r m e d . A c t i v a t i o n of this complex is a c c o m p a n i e d by a change in the s e d i m e n t a t i o n value to 5S. The 5S complex interacts w i t h nuclear acceptor sites to switch on DNA synthesis and m R N A production. f r a c t i o n a t i o n studies show that at 4°C, uterine

tissue

i n c u b a t e d w i t h t r i t i u m - l a b e l e d estradiol retains most of label in the c y t o p l a s m i c fraction

(21).

At a higher

the

tempera-

ture of 37°C, m o s t of the estradiol is a s s o c i a t e d w i t h the nuclear

fraction.

Estrogen treatment both _in vitro a n d

226 in v i v o leads to receptor decrease in the cytosol, by an increase in nuclear receptor levels

paralleled

(22, 23).

The

s e d i m e n t a t i o n coefficient of the e s t r a d i o l - e s t r o g e n complex in salt-containing

receptor

sucrose g r a d i e n t s c h a n g e s w i t h

nuclear uptake from 4S to 5S (24, 25).

Activated

estrogen

receptor increases mRNA pools and p r o t e i n synthesis as a result of DNA acceptor site interactions 2) Nuclear e s t r o g e n receptors.

(26-31).

The current e s t r o g e n

receptor

m o d e l c o n t e n d s that the u n o c c u p i e d e s t r o g e n receptor is a l m o s t e x c l u s i v e l y nuclear

(see Figure 2).

As early as 1977, a small

number of reports had q u e s t i o n e d the dogma that the receptor was c y t o p l a s m i c

( 32-36).

unoccupied

Studies by Linkie et al.

s h o w e d that the 4S s e d i m e n t a t i o n form of the e s t r o g e n

receptor

c o u l d be t r a n s f o r m e d to the 5S form w i t h i n the nucleus 33).

These d a t a q u e s t i o n e d if the classical m o d e l of

a c t i v a t i o n p r e c e d i n g nuclear translocation was

(32, receptor

correct.

S h e r i d a n a n d coworkers found receptor l o c a l i z a t i o n to be highly d e p e n d e n t upon tissue p r e p a r a t i o n

(34-36).

These

a u t h o r s s u g g e s t e d that receptors were in e q u i l i b r i u m

between

the n u c l e u s and cytoplasm and that p r e p a r a t i o n of cells

in

h y p o t o n i c buffers caused a receptor

that

redistribution such

nuclear receptors relocated into the fraction c o n t a i n i n g higher buffer

the

volume.

E n u c l e a t i o n studies in GH3 rat p i t u i t a r y tumor and M C F - 7 breast cancer cell lines, using differential

centrifugation

and c y t o c h a l a s i n - B to extrude the nucleus, showed that b i n d i n g of tritiated estradiol o c c u r r e d mostly in those tions containing the n u c l e o p l a s t s

the frac-

(an intact nucleus w i t h a

small perimeter of t r y p a n - b l u e - e x c l u d i n g c y t o p l a s m i c m e m b r a n e ) (37, 38).

Similar results were also o b t a i n e d in GH3 c e l l s

the a b s e n c e of c y t o c h a l a s i n - B e x p e r i m e n t s , a cytoplast membrane)

(38).

In the GH3 cell

(cytoplasm in a

in

line

trypan-blue-excluding

layer virtually free of contaminating nuclei

was

227 IGFR

k:

TGFIi

39KD

protein inT

•»«. •TGF-O

CELL PROLIFERATION Figure 2. Schematic representation of unoccupied nuclear estrogen receptors and a nuclear estrogen-estrogen receptor transformation. Receptor activation by estrogen may cause a decrease in the activation and secretion of transforming growth factor beta (TGF-6) and the 39 KD protein concurrently with the activation and increased secretion of transforming growth factor alpha (TGF-a), epidermal growth factor (EGF), and insulin-like growth factor I (IGF-I). Each of these hormones may then interact with their receptors (EGFR and IGFR, respectively) in an autocrine or paracrine fashion to enhance cell proliferation. obtained; nucleoplast fractions contained both nucleoplast and whole cells.

Nucleoplasts which are between 80 to 95% pure

were obtained from MCF-7 breast cancer cells (39) in preliminary studies done in this laboratory

(see Table 1).

Again,

the majority of the unoccupied receptor remained nuclear Table 2).

(see

Enucleation studies on cells from other estrogen

228

Estrogen receptor (ER) fmol ER/ pmol ER/ Protein/DNA mg protein mg DNA W e cells

17.6

124

2.2

12.5 3.1

159 706

2.0 2.2

Fractions: Mixed Nucleoplasts

T a b l e 1. MCF-7 cells were e n u c l e a t e d by a p r o c e d u r e that u s e d a c o n t i n u o u s Percoll gradient (37) w i t h p a r t i a l s e p a r a t i o n of fractions. The m i x e d fraction was h e t e r o g e n e o u s a n d c o n t a i n e d n u c l e o p l a s t s , cytoplasts, and partially e n u c l e a t e d cells, w h i l e the n u c l e o p l a s t fraction was a p p r o x i m a t e l y 90% p u r e . E s t r o g e n receptor, p r o t e i n and DNA were m e a s u r e d in e a c h f r a c tion. The results show that the c o n c e n t r a t i o n of e s t r o g e n receptor per mg DNA remains constant b e t w e e n w h o l e cells a n d n u c l e o p l a s t s , but the c o n c e n t r a t i o n of e s t r o g e n receptor per mg p r o t e i n increases dramatically in n u c l e o p l a s t s c o m p a r e d to w h o l e cells. P r o t e i n per DNA ratio d e c r e a s e s b e t w e e n w h o l e cells and nucleoplasts. target tissue have also concluded that u n o c c u p i e d receptor resides in the nucleus

estrogen

(40).

A l t h o u g h e n u c l e a t i o n studies cannot c o n c l u s i v e l y e x c l u d e p o s s i b i l i t y that the estrogen receptor remains in the v o l u m e of c y t o p l a s m surrounding the nucleus,

the

small

immunocyto-

c h e m i c a l studies using highly specific m o n o c l o n a l

antibodies

to the e s t r o g e n receptor have also indicated that

unoccupied

e s t r o g e n receptors are p r e d o m i n a n t l y localized in the

nucleus

of a number of target tissues including h u m a n breast

tumor

s a m p l e s , MCF-7 breast cancer cells, h u m a n and rabbit

uterine

229

Number of estrogen receptors per cell or nucleoplast Experiment I

II

Whole cells

24,200

26,600

Nucleoplasts

20,300

18,700

T a b l e 2. In two e n u c l e a t i o n experiments of MCF-7 cells, up to 84% of the u n o c c u p i e d estrogen receptor is r e c o v e r e d in the n u c l e o p l a s t fraction. t i s s u e as well as rabbit oviduct, corpus luteum, m a m m a r y g l a n d , p i t u i t a r y , and liver

(41).

The u n i f o r m

staining o b s e r v e d suggests that receptors are

nuclear distributed

t h r o u g h o u t the nucleus and are not c l u s t e r e d a r o u n d the nuclear membrane.

Furthermore, treatment of cells or a n i m a l s

w i t h e s t r o g e n does not increase the nuclear intensity.

Together the enucleation and

staining

immuno-staining

t e c h n i q u e s p r o v i d e strong evidence for a nuclear

receptor.

In further support of a nuclear receptor, the binding

kinetics

for the e s t r o g e n receptor in vivo appear similar to that of receptor i m m o b i l i z e d on hydroxylapatite

(42-44).

Since

230

r e c e p t o r s behave as if they are immobilized a n d since

estrogen

a c t i o n is targetted for the nucleus then these k i n e t i c

studies

suggest that e s t r o g e n receptors may be nuclear a n d a l s o a s s o c i a t e d w i t h nuclear

acceptors.

S t u d i e s in immature rats injected w i t h

low-affinity,

n o n s t e r o i d a l estrogens also imply the p o s s i b i l i t y of a n u c l e a r e s t r o g e n receptor

(45).

Experiments in this laboratory

an e x c h a n g e assay o b s e r v e d that c y t o s o l i c e s t r o g e n

using

receptors

d i d not d e c r e a s e w h e n immature rats were t r e a t e d w i t h

low-

a f f i n i t y , n o n s t e r o i d a l estrogens a l t h o u g h u t e r i n e w e i g h t progesterone

receptor levels increased

(45).

We

hypothesized

that tissue h o m o g e n i z a t i o n caused u n o c c u p i e d nuclear to b e c o m e c y t o p l a s m i c .

and

receptor

T h e r e f o r e , treatment of target

tissues

w i t h l o w - a f f i n i t y compounds, which readily d i s s o c i a t e f r o m the r e c e p t o r , w o u l d follow the d i s t r i b u t i o n p a t t e r n of receptor.

The n u c l e a r - a s s o c i a t e d receptor is

unoccupied

therefore

b e l i e v e d to bind estrogen and undergo t r a n s f o r m a t i o n into a n active complex.

Once activated, the r e c e p t o r - l i g a n d

complex

m a y then interact with DNA acceptor sites w h i c h lead to biological 3.

response.

B i o l o g i c a l l y active u n o c c u p i e d receptor.

A third p o s s i b l e

m o d e l is that u n o c c u p i e d receptors are not only located in the n u c l e u s but are also active.

Significant a m o u n t s of

u n o c c u p i e d nuclear estrogen receptor can be found in h u m a n b r e a s t tumor b i o p s i e s and cultured breast cancer cell (46-49).

A nuclear location of receptor in MCF-7

cancer cells has been p r o p o s e d

(48).

lines

breast

H o w e v e r , this c l a i m w a s

later r e t r a c t e d since major c y t o p l a s m i c c o n t a m i n a t i o n found in the MCF-7 cell nuclear p r e p a r a t i o n s

(50).

was

But,

light of the recent finding that phenol red, a pH dye

in

indi-

cator in high c o n c e n t r a t i o n s in tissue culture m e d i a , is a weak e s t r o g e n

(51, 52), the concept of active

unoccupied

e s t r o g e n receptor should be reconsidered for several

reasons.

231 E x p e r i m e n t s done by K a t z e n e l l e n b o g e n et a^.

(personal

communi-

cation) a n d in this laboratory demonstrate that the c o n t r o l g r o w t h rate of MCF-7 cells in the absence of phenol red is initially slower than in the presence of p h e n o l red, but a d a p t s w i t h time to that of the original phenol red g r o w t h response

(53).

This a d a p t i o n to a

nonestrogen-containing

e n v i r o n m e n t has a strong correlation w i t h an increase e s t r o g e n receptors, a l t h o u g h progesterone receptor remain very low.

It can therefore be a r g u e d whether or not

the increase in g r o w t h is stimulated by the i n c r e a s e d of u n o c c u p i e d

in

levels number

receptor.

Interestingly,

it is well d o c u m e n t e d that p o s t m e n o p a u s a l

have higher e s t r o g e n receptors in their breast tumors premenopausal women

(54).

women

than

This o b s e r v a t i o n m a y e x p l a i n the

m e c h a n i s m of tumor growth in postmenopausal w o m e n in the a b s e n c e of m e a s u r a b l e estrogens.

Another recent

report

involving g l u c o c o r t i c o i d receptors has indicated that

the

i n t e r a c t i o n of u n o c c u p i e d g l u c o c o r t i c o i d receptors w i t h nuclear c o m p o n e n t s may be able to turn on steroid genes

(55).

responsive

Thus, MCF-7 growth in p h e n o l - r e d - f r e e

media

a f f o r d s a unique system to look at the induction of g r o w t h in the a b s e n c e of

B.

estrogens.

E s t r o g e n s and phenol

red

It is a p p a r e n t , in hindsight, that m u c h of the d i f f i c u l t y

in

d e m o n s t r a t i n g an e s t r o g e n - s t i m u l a t e d g r o w t h in MCF-7

breast

cancer cells was due to the presence of phenol red.

We

c u r r e n t l y find that there is a very potent

growth-promoting

response e v o k e d in MCF-7 cells at very low c o n c e n t r a t i o n s estradiol

(see Figure 3A).

of

Half-maximal s t i m u l a t i o n is m e a -

s u r e d at 1 0 - 1 2 M to 1 0 - 1 3 M in a 5-day g r o w t h assay.

At

these

232

B 30

20

25 I5

a>

^ 20 15

ED=n « 5XI0"I3M

10

o» 10

5

Contri

•13 -12 -II -10 Log Cone (M)

>> Ll_ Ll. _ -I O O + T * UJ UJ

F i g u r e 3. A) G r o w t h curve of MCF-7 cells g r o w n in 24-well tissue culture plates and treated w i t h v a r i o u s c o n c e n t r a t i o n s of e s t r a d i o l - 1 7 6 . G r o w t h is m e a s u r e d as yg of DNA. B) G r o w t h of MCF-7 cells m e a s u r e d as ug of DNA after 7 days of t r e a t m e n t 8 w i t h L Y 1 5 6 7 5 8 (LY, 10 ) , epidermal g r o w t h factor (EGF, 10 M) or a c o m b i n a t i o n of both and c o m p a r e d w i t h no t r e a t m e n t (C). e s t r a d i o l c o n c e n t r a t i o n s , cell p r o l i f e r a t i o n is more to e s t r o g e n s t i m u l a t i o n than p r o g e s t e r o n e receptor G r o w t h s t i m u l a t i o n of MCF-7 cells at e s t r o g e n

sensitive

synthesis.

concentrations

u n d e t e c t a b l e by radioimmunoassay d e m o n s t r a t e the p o s s i b l e s e n s i t i v i t y of breast cancer to e s t r o g e n s t i m u l a t i o n .

This

s y s t e m p r o v i d e s a model to evaluate agents useful in the p r e v e n t i o n of breast cancer and also the o p p o r t u n i t y r e e v a l u a t e the current concepts concerning e s t r o g e n

to action.

233 C.

E s t r o g e n s and growth

factors

G r o w t h factor induction by estrogen has been p o s t u l a t e d to be a p o s s i b l e m e c h a n i s m by w h i c h the steroid p r o m o t e s g r o w t h h u m a n breast cancer

(56-59).

in

Breast cancer cell lines have

b e e n s h o w n to synthesize insulin-like g r o w t h factor I and II a n d the e s t r o g e n - i n d u c i b l e transforming g r o w t h factor (56, 58, 60).

alpha

F u r t h e r m o r e , injecting a t h y m i c mice w i t h a c i d -

e x t r a c t e d m e d i a , c o n d i t i o n e d by e s t r o g e n - s t i m u l a t e d

MCF-7

c e l l s a n d charcoal stripped to remove free e s t r o g e n ,

causes

the g r o w t h of implanted MCF-7 tumors (56).

condi-

Whereas,

t i o n e d m e d i a from u n t r e a t e d cells only weakly growth.

stimulated

T h e r e f o r e , if a n t i e s t r o g e n binding to the

receptor

i n h i b i t s the a c t i v a t i o n of positive g r o w t h factors, then the a d d i t i o n of a g r o w t h factor back into the m e d i a should the inhibitory effect.

strate that epidermal growth factor

(EGF) can o v e r c o m e

g r o w t h i n h i b i t i o n of MCF-7 cells caused by the LY156758

reverse

Studies done in this laboratory

(see Figure 3b) (61).

demonthe

antiestrogen,

However, the reversal of

g r o w t h i n h i b i t i o n by E G F was no greater than the sum of g r o w t h f o u n d in the E G F - t r e a t e d and a n t i e s t r o g e n - t r e a t e d

groups.

T h i s a d d i t i v e effect suggests that E G F - i n d u c e d cell

prolifera-

tion is i n d e p e n d e n t of the estrogen receptor system. e x p e r i m e n t s strongly support the concept that other can replace e s t r o g e n in supporting tumor

These factors

growth.

S e v e r a l lines of evidence point toward an important

relation-

s h i p b e t w e e n e s t r o g e n and epidermal g r o w t h factor receptors the p r o g r e s s i o n of breast cancer

(62-66).

Binding

in

studies

p e r f o r m e d on cell cultures and breast tumor b i o p s i e s

have

d e m o n s t r a t e d that a negative c o r r e l a t i o n exists b e t w e e n

the

levels of e s t r o g e n receptor and the levels of e p i d e r m a l

growth

factor

(EGF) receptor

(60-64).

In a d d i t i o n , greater

numbers

of E G F receptors are found in m e t a s t a t i c tumors than in the p r i m a r y tumor

(62).

These o b s e r v a t i o n s imply a p o s s i b l e

234 m e c h a n i s m for e n d o c r i n e therapy failure as the cancer more hormonally

becomes

independent.

E n c o m p a s s i n g the o l d and new concepts of e s t r o g e n

receptor

m e c h a n i s m s and its role in the d e v e l o p m e n t and c o n t r o l of breast cancer, a series of a p p r o a c h e s to the t r e a t m e n t of h o r m o n e - d e p e n d e n t breast cancer can be formulated.

These

s h o u l d focus on the p r e v e n t i o n of 1) e s t r o g e n b i o s y n t h e s i s e s t r o g e n receptor binding, 2) a c t i v a t i o n and/or

of the receptor, 3) acceptor site binding, 4) m R N A a n d / o r t r a n s l a t i o n , a n d 5) growth-factor receptor b i n d i n g .

and

transformation synthesis

induction a n d

A l t e r n a t e strategies to regulate g r o w t h of

b r e a s t cancer will be d i s c u s s e d later in this a r t i c l e .

Antiestrogens E n d o c r i n e m a n i p u l a t i o n , especially in the form of

antiestrogen

therapy, has become an important a l t e r n a t i v e to the use of c h e m o t h e r a p e u t i c agents in the treatment of b r e a s t cancer

(67-71).

hormone-dependent

The reason for this is the

target

t i s s u e - s p e c i f i c activity and lack of side effects of antiestrogens

(13, 14).

To date, four major classes of

e s t r o g e n s have been reported (see Figure 4). substituted triphenylethylenes, hydroxylated

T h e s e are

the

triphenylethyl-

enes, b i c y c l i c a n t i e s t r o g e n s and s u b s t i t u t e d s t e r o i d s 73).

anti-

(13, 72,

Since the literature pertaining to these c o m p o u n d s

been extensively

reviewed

has

(13, 14), this chapter will focus o n

those a n t i e s t r o g e n s w h i c h have current interest for c l i n i c a l use or as new probes for experimental

future

research.

W i t h few e x c e p t i o n s , none of the present n o n s t e r o i d a l p o u n d s are true e s t r o g e n a n t a g o n i s t s since none appear block receptor a c t i v a t i o n completely

(74, 75).

For

a n t i e s t r o g e n s may have either agonist or a n t a g o n i s t

comto

example, action

235

0(CH,),N, 501-509.

3.

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4.

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5.

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6.

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7.

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9.

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Donnell, M.S., Glenn, B.A., Meyer, A. and Donegan, W.L. (1979). Estrogen receptor protein in intracranial meningiomas. J. Neurosurg. 50, 499-502.

146.

Blankenstein, M . A . , Blaauw, G., Lamberts, S.W.J, and Mulder, E. (1983). Presence of progesterone receptors and absence of oestrogen receptors in human cranial meningioma cytosols. Eur. J. Cancer Clin, oncol. J 9 , 365-370.

147.

Blaauw, G.,, Blankenstein, M.A. and Lamberts, S.W.J. (1986). Sex steroid receptors in human meningiomas. Acta Neurochir. _79> 42-47.

148.

Blankenstein, M.A. , Berns, Thijssen, J.H.H. (1986).

and Martinez, R. (1985). J. Neurosurg. 62, 162-164. Hormone

Receptors

in

receptors in

Meningiomas.

J.

P.M.J.J., Blaauw, G., Mulder,, E. and Search to estrogen receptors in human

305 meningioma tissue sections with a monoclonal antibody against human estrogen receptor. Cancer Res. 4268-4270. 149.

Blankenstein, M . A . , Blaau, G., van't Verlaat, J.W., van der Meulen-Dijk, C. and Thijssen, J.H.H. (1987). Steroid receptors in cerebral tumours, possible consequences for endocrine treatment? In: Hormonal Manipulation of Cancer: Peptides, Growth Factors, and New (anti) Steroidal Agents (Ed. J.G.M. K l i j n et al.) Raven Press, New York pp61-70.

ANTIESTROGEN ACTION IN MCF-7 CELLS

T.S. Rah, M.F. Ruh, R.K. Singh Department of Physiology, St. Louis University School of Medicine, St. Louis, MO 63104 W.B. Butler Michigan Cancer Foundation and Department of Biochemistry, Wayne State University, Detroit, MI 48201

INTRODUCTION Non-steroidal antiestrogens inhibit estrogen stimulated cellular functions by competing with estrogens for the estrogen receptor (1,2).

However, the antiestrogen-receptor

complex is inefficient and does not promote maximal cell stimulation.

In fact, the estrogen receptor when bound by

antiestrogen may itself produce specific inhibitory actions (3,4).

In order to better understand the mechanisms whereby

antiestrogens inhibit estrogenic responses recent studies have utilized high affinity radiolabeled antiestrogens (5,7) which have allowed a much more accurate assessment of the physicochemical characteristics of antiestrogen-receptor complexes as well as their interaction with nuclear acceptor sites.

However, the cellular mechanisms whereby these

non-steroidal antiestrogens provoke both agonist and antagonist responses is still not clear. Multiple chromatin acceptor sites in mammalian chromatin have been demonstrated which indicate different degrees of estrogen receptor binding capacity and affinity for estrogenversus antiestrogen-receptor complexes (8-10).

Antiestrogen

interaction with the estrogen receptor appears to alter the receptor such that its physicochemical characteristics differ

Receptor Mediated Antisteroid Action © 1987 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

308 f r o m t h a t of the e s t r a d i o l - r e c e p t o r the b i o l o g i c a l

complex

Thus,

r e s p o n s e s of a n t i e s t r o g e n s m a y be the

result of s u c h a l t e r e d r e c e p t o r p r o p e r t i e s binding.

(11-13).

end

induced by

ligand

T h e r e f o r e , the d i s t i n c t i o n b e t w e e n e s t r o g e n -

antiestrogen-receptor chromatin acceptor differences

b i n d i n g to m u l t i p l e sets of

s i t e s m a y be a result of m o r e

in initial l i g a n d - r e c e p t o r

and

different fundamental

interaction,

i.e.,

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

Since

the

antiestrogens

compete w i t h e s t r o g e n s for the e s t r o g e n r e c e p t o r , t h e r e

would

not be s u f f i c i e n t e s t r o g e n - r e c e p t o r

with

the s p e c i f i c a c c e p t o r specific cellular

c o m p l e x to i n t e r a c t

s i t e s to e l i c i t m a x i m a l

responses.

In a d d i t i o n ,

estrogen-

antiestrogen-

receptor c o m p l e x e s m a y e x e r t an e f f e c t of t h e i r o w n b y interacting with different acceptor

sites

(9).

This

review

will s u m m a r i z e the r e s u l t s f r o m our l a b o r a t o r y w h i c h the h y p o t h e s i s t h a t a n t i e s t r o g e n - r e c e p t o r altered

complexes

interaction with chromatin acceptor

have

s i t e s in M C F - 7

cells, a well established human breast cancer cell Our l a b o r a t o r y uses the t r i p h e n y l e t h y l e n e H1285

support

line.

antiestrogen,

[4-(N,N-diethylaminoethoxy)4'-methoxy-a-(p-

hydroxyphenyl)a'-ethylstilbene] affinity

(14), w h i c h h a s a h i g h

(Kd 0.23 nM) for the e s t r o g e n

receptor

in M C F - 7

cells

(Kd for e s t r a d i o l = 0.25 nM) and h a s b e e n d e m o n s t r a t e d to have very potent antiestrogen activity

in MCF-7

H1285 w a s d e t e r m i n e d to be a m o r e p o t e n t

cells.

i n h i b i t o r of

p r o l i f e r a t i o n t h a n the w i d e l y s t u d i e d a n t i e s t r o g e n

tamoxifen,

but H 1 2 8 5 w a s e q u i p o t e n t w i t h t r a n s - h y d r o x y t a m o x i f e n H1285 evoked minimal receptor

increases

in c e l l u l a r

l e v e l s and no i n c r e a s e

suppressed plasminogen activator

(15).

progesterone

in p l a s m i n o g e n

a c t i v i t y over a b r o a d range of c o n c e n t r a t i o n s , estrogens.

cell

activator and

it

activity stimulated

by

In a d d i t i o n , H 1 2 8 5 h a s a v e r y low a f f i n i t y

estrogen-noncompetible

antiestrogen binding

sites.

for

309 CHROMATIN ACCEPTOR

SITES

It is g e n e r a l l y a c c e p t e d t h a t an i n t e r a c t i o n steroid hormone required

receptors and nuclear "acceptor

between sites"

in o r d e r for s t e r o i d h o r m o n e s to r e g u l a t e

expression.

H o w e v e r , there

is some c o n t r o v e r s y c o n c e r n i n g

nature of s p e c i f i c n u c l e a r b i n d i n g s i t e s steroid hormone

receptors.

(acceptor

sites)

T h e r e have b e e n n u m e r o u s

interaction with target cell

receptor

components,

s u c h as b i n d i n g to n u c l e a r m e m b r a n e s , R N A ,

m a t r i x , D N A s e q u e n c e s and a c c e p t o r p r o t e i n - D N A

nuclear

another.

chromosomal

a c c e p t o r p r o t e i n s as c a n d i d a t e c o m p o n e n t s of a c c e p t o r receptors.

The term "acceptor

which confer specific high affinity binding receptor c o m p l e x e s .

sites

s i t e " as used

this chapter will signify nonhistone protein-DNA w i t h the s t e r o i d

nuclear

complexes.

N o n e of t h e s e m o d e l s are m u t u a l l y e x c l u s i v e of one Our l a b o r a t o r y h a s c h o s e n to s t u d y n o n h i s t o n e

the for

models

for s t e r o i d

for s t e r o i d

is

gene

in

complexes

in the

interaction

In our a p p r o a c h to the

study of the m e c h a n i s m s of a c t i o n of s t e r o i d h o r m o n e s , we have used r e c e p t o r b i n d i n g to a c c e p t o r p r o t e i n - D N A nucleoacidic protein

(NAP).

NAP

is d e f i n e d

sites

termed

(16,17) as e i t h e r

that partially deproteinized chromatin fraction with acceptor activity

(native NAP) or c e r t a i n

nonhistone proteins

chromosomal

r e c o n s t i t u t e d to DNA f o r m i n g an

protein-DNA complex with enhanced acceptor (reconstituted

receptor-acceptor

is to s e l e c t i v e l y s t r i p n o n h i s t o n e p r o t e i n s

chromatin with chaotropic agents i n t e r a c t i o n of s t e r o i d - r e c e p t o r fractions

acceptor

activity

NAP).

One a p p r o a c h we use to study s t e r o i d sites

enhanced

(native N A P ) .

from

(18) and to s t u d y the c o m p l e x e s w i t h the

residual

This chromatin preparation serves

functions;

r e m o v a l of s p e c i f i c p r o t e i n s w h i c h m a s k

sites a n d ,

if n e e d e d , the e x t r a c t i o n of the a c c e p t o r

c o m p o n e n t s of t h o s e same s i t e s . "acceptor a c t i v i t y "

Thus,

"acceptor

two

acceptor protein

sites" or

(high a f f i n i t y , s p e c i f i c b i n d i n g

of

310 r e c e p t o r s to c h r o m a t i n )

c a n be u n m a s k e d and m e a s u r e d

A n o t h e r a p p r o a c h to the s t u d y of a c c e p t o r

sites

(Fig.

involves

d e t e c t i o n of s p e c i f i c a c c e p t o r p r o t e i n s w h i c h d i s p l a y a c t i v i t y w h e n r e c o n s t i t u t e d to D N A . proteins

hydrochloride and receptor (Fig.

chromatin

( G d n » H C l ) , r e c o n s t i t u t e d to h o m o l o g o u s

1).

the

acceptor

guanidine

interaction with these nucleoacidic

(reconstituted NAP) assay

The nonhistone

(CP) are r e m o v e d b y s t e p - e l u t i o n w i t h

1).

DNA,

proteins

is m e a s u r e d using a s t r e p t o m y c i n

filter

T h i s a s s a y c o u p l e d w i t h v a r i o u s CP

p u r i f i c a t i o n t e c h n i q u e s w i l l a l l o w us to c h a r a c t e r i z e p r o t e i n c o m p o n e n t of a c c e p t o r

s i t e s , and u l t i m a t e l y

the

to

c h a r a c t e r i z e the DNA s e q u e n c e s w h i c h are c o n s t i t u e n t s of

the

sites.

STUDIES UTILIZING NATIVE NAP Experiments

utilizing partially deproteinized

chromatin

f r a c t i o n s f r o m a v a r i e t y of s y s t e m s h a v e d e m o n s t r a t e d

the

s p e c i f i c i n t e r a c t i o n of s t e r o i d r e c e p t o r s w i t h a c c e p t o r (8,16-25).

S t u d i e s of the b i n d i n g of v a r i o u s s t e r o i d

receptors to c h r o m a t i n a c c e p t o r

sites revealed that

sites

hormone

certain

nonhistone nuclear proteins may mask specific steroid

receptor

b i n d i n g s i t e s on c h r o m a t i n and r e m o v a l of t h e s e p r o t e i n s the c h a o t r o p i c salt g u a n i d i n e h y d r o c h l o r i d e f a c i l i t a t e the in vitro b i n d i n g of s t e r o i d to s p e c i f i c a c c e p t o r

e x t r a c t e d by 2 M Gdn-HC1 molarity.

1%-2%

After

receptor

s i t e s on the c h r o m a t i n .

of the h i s t o n e s and 80%-85% approximately

(Gdn-HC1)

all

are

extracts

s e l e c t i v e e x t r a c t i o n of t h e s e

p u r i f i e d s t e r o i d receptor

b i n d i n g of

nonhistone fractions

S e v e r a l p a r a m e t e r s of

s p e c i f i c i n t e r a c t i o n of receptor w i t h a c c e p t o r

in

partially

to the r e s i d u a l c h r o m a t i n

can m e a s u r e a c c e p t o r a c t i v i t y . determined.

Essentially

n o n h i s t o n e p r o t e i n s w i t h e a c h increase

p r o t e i n s from c h r o m a t i n - c e l l u l o s e ,

be

complexes

of the n o n h i s t o n e p r o t e i n s

and 3-8 M Gdn-HG1

with

may

the

s i t e s can

then

311

NUCLEI

CYTOSOL

PURIFICATION

Fig. 1. O u t l i n e for the a s s a y of n u c l e a r a c c e p t o r sites. T h i s figure p r e s e n t s the a p p r o a c h used in our l a b o r a t o r y for the c e l l - f r e e b i n d i n g a s s a y s for nuclear acceptor sites. The p r e p a r a t i o n s of p a r t i a l l y deproteinized chromatin-cellulose, reconstituted n u c l e o a c i d i c p r o t e i n s (NAP) as w e l l as p a r t i a l l y p u r i f i e d e s t r o g e n receptor are o u t l i n e d .

312 We have acceptor

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

s i t e s for e s t r o g e n - a n d

complexes

MCF-7 cells

(9).

Triphenylethylene

and

antiestrogen-resistant

T w o s u b l i n e s of M C F - 7 c e l l s , E - 3 a n d antiestrogens

culture.

T h u s , E - 3 c e l l s are t e r m e d

are e s t r o g e n - s e n s i t i v e . antiestrogen-sensitive

H1285(10~7

antiestrogen-sensitive Both

Chromatin was prepared

by 1-8 M G d n - H C 1

and antiestrogen-resistant

to unmask acceptor

sites.

[ 3 H ] e s t r a d i o l - and

(30-fold)

sublines

from MCF-7

l i n k e d c o v a l e n t l y to c e l l u l o s e , a n d d e p r o t e i n i z e d

used for c h r o m a t i n b i n d i n g The following

assays

Partially

[3H]H1285-receptor

activity for

(acceptor

(residual chromatin

results were obtained:

from antiestrogen-sensitive sites)

cells

(1)

revealed maximal

unmasked by

receptor complexes acceptor

activity

and 4 M G d n - H C 1

(E-3 or R R ) . from either

(Fig. 2 A ) .

complexes from either increased binding

By c o n t r a s t ,

[ H]H1285-

cell type demonstrated

(2) For the c h r o m a t i n cells,

1 M

prepared

[3H]estradiol-receptor

for c h r o m a t i n p r e p a r e d f r o m

cells. However,

unmasked fraction

[3h]H1285-receptor

an

in b i n d i n g to the 4 M G d n * H C 1

(Fig. 2 B ) .

required

antiestrogen-

complexes

(3) T h e s e a c c e p t o r

undenatured estrogen

sites

receptor and

competitively

inhibited by radioinert estrogen

demonstrating

the s p e c i f i c i t y a n d s a t u r a b i l i t y of

sites.

maximal

s e n s i t i v e or r e s i s t a n t c e l l s s h o w e d

showed a drastic decrease

acceptor

either

3

activity unmasked at 1 M and 6 M G d n - H C 1 ,

as w a s d e m o n s t r a t e d

chromatin

Chromatin binding

in t h e c h r o m a t i n f r a c t i o n s u n m a s k e d b y

from antiestrogen-resistant

sensitive

were acceptor

1 M and 6 M Gdn-HCl

[3H]estradiol-receptor complexes prepared from

type of M C F - 7 c e l l s

cells,

sequentially

complexes prepared by step-elution from DEAE-cellulose sites).

growth

i n h i b i t i o n of E - 3 c e l l g r o w t h at 9 - 1 2 d a y s of

w h e r e a s R R c e l l s are a n t i e s t r o g e n - r e s i s t a n t .

purified

RR,

inhibit the

of E - 3 b u t h a v e no e f f e c t on the g r o w t h of R R . M) c a u s e d 80%

chromatin

antiestrogen-receptor

in a n t i e s t r o g e n - s e n s i t i v e

were used.

of

B i n d i n g of

receptor complexes

[ 3 H ] e s t r a d i o l - or

on

were

receptor these

[3H]H1 285-

in the p r e s e n c e of u n l a b e l e d

ligand-

313

receptor complexes was determined for those subtractions of deproteinized chromatin demonstrating the greatest acceptor activity (1 M, 4 M and 6 M Gdn-HC1).

A 2-fold excess of

radioinert estradiol- or H1285-receptor complexes was able to compete with and displace [^Hjestradiol-receptor

complexes

from the 1 M acceptor site with a respective binding decrease of 75% and 70% which compares well to a theoretical maximum of

GdnHCI(M)

Gdn-HCI(M)

Fig. 2. Comparison of binding of [3HJestradiol- and [3h]H1285-receptor complexes to chromatin subfractions from antiestrogen-sensitive (A) and antiestrogen-resistant (B) MCF-7 cells. MCF-7 cytosol from antiestrogensensitive (E-3) (Panel A) and antiestrogen-resistant (RR) (Panel B) cells was charged with 10 nM [^h]estradiol or 30 nM [ 3 H]H1285 for 2 hr at 4C. after charcoal-treatment the cytosol was loaded onto a pre-equilibrated DEAEcellulose column (9 ml). After washing with 50 ml of buffer containing 0.1 M KC1, the receptor complexes were eluted with 0.3 M KC1. Chromatin-cellulose (40u g DNA) from E-3 cells (A) or RR cells (B) was extracted with 0-8 M Gdn•HC1, washed to remove the Gdn»HCl and incubated with an aliquot (0.12 pmol) of [ 3 HJestradiol- (• •) or [3H]H1285- (O o) receptor complexes for 60 min at 4C. Radioactivity was extracted with absolute ethanol and counted. Each value was corrected for intact chromatin binding. (Reprinted from ref. 9).

314

(B)

(A) 1M Gdn-HCI

(C)

4M Gdn-HCI

100 £ (0 a>

6 M GkkrHCI X

80

x ® a

E

o

60

a a> o

40

o

a> DC •o c 3

o

I

20

CD

+ dR

+

+

+

E2R

HR

dR

[3H]E 2 R

• • E2R

HR

[3H]H1285R

4. dR

>

•

E2R

HR

[3H]E2R

Fig. 3. Specificity and saturability of binding of [^Hlestradiol- and [^h]H128 5-receptor complexes to chromatin acceptor sites. Chromatin-cellulose prepared from E-3 cells (40 u g DNA) was extracted with Gdn-HC1 and then washed to remove the Gdn-HCI. Partially purified radiolabeled receptor complexes (0.14 p m o l / a s s a y ) from E-3 cells were used to determine chromatin binding activity in the presence of 2-fold excess unlabeled estradiol-receptor complexes (E2R) and unlabeled H1285-receptor complexes (HR). The 100% value (control) was obtained with 2-fold excess heat-denatured receptors (dR) to keep the protein content constant (700 v g / a s s a y ) . (Reprinted from ref. 9).

315

67% for a two-fold excess radioinert receptor

(Fig. 3A) .

However, with the 6 M site (Fig. 3C), the binding of [^H]estradiol-receptor complexes was reduced more (76%) by 2-fold excess estradiol-receptor but reduced only 46% by the H1285-receptor, again corresponding to the direct binding data shown in Fig. 2.

In addition, binding to the 4 M site by

[3H]H1285- receptor complexes

(Fig. 3B) was reduced more

(68%) by Hl285-receptor and reduced less (30%) by estradiolreceptor, which is similar to the binding affinity in Fig. 2.

indicated

These results indicate not only that the receptor

binding to E-3 chromatin acceptor sites -is competable but also that these sites can be quite specific for a particular ligand-receptor complex, i.e., estradiol- or Hl285-receptor complexes. Thus, the different binding characteristics of antiestrogen-receptor complexes (compared with estrogenreceptor complexes) to chromatin acceptor sites from antiestrogen-sensitive cells may be responsible for the inhibitory effects of antiestrogens on cell growth.

In

addition, the acquisition of antiestrogen resistance to certain sublines of MCF-7 cells appears to result from alterations in the state of chromatin rather than the receptor itself since estrogen receptors from E-3 or RR sublines exhibited very similar chromatin binding

profiles.

STUDIES UTILIZING RECONSTITUTED NAP The chromatin-cellulose assay described in the previous section suggests that certain salt concentrations

remove

masking proteins, which allows for increased acceptor activity, and the next subsequent higher salt concentrations remove these exposed acceptor site proteins.

Support for

these results requires characterization of the removed acceptor proteins.

To do this, the chromosomal proteins

(CP)

316 conferring

a c c e p t o r a c t i v i t y m u s t be e x t r a c t e d using the

next

h i g h e r c o n c e n t r a t i o n s of a c h a o t r o p i c a g e n t , the p r o t e i n s be f r a c t i o n a t e d , and t h e s e p r o t e i n s m u s t be a s s a y e d acceptor activity by reconstituting

onto D N A .

The latter

a l l o w s t h e s e h i g h l y h y d r o p h o b i c p r o t e i n s to be s o l u b l e salt b u f f e r s a n d , t h e r e f o r e a l l o w s for r e c e p t o r assays.

the i n t e r p r e t a t i o n of the c h r o m a t i n - c e l l u l o s e

binding

studies

proteins from antiestrogen-sensitive

cells.

We

d e t e c t i o n of s p e c i f i c a c c e p t o r p r o t e i n s w h i c h ,

chromatin refined

differences

by

A f t e r o b t a i n i n g MCF-7

c h r o m a t i n w h i c h was t h e n b o u n d to h y d r o x y l a p a t i t e , first r e m o v e d w i t h 3 M NaCl

chromatin proteins

(CP) w e r e

The

reconstitution

to DNA w e r e p e r f o r m e d using the m e t h o d s d e s c r i b e d (16-18).

the

complexes.

p r e p a r a t i o n of the n o n h i s t o n e p r o t e i n s a n d the

histones were

is

when

r e c o n s t i t u t e d to D N A , d i s p l a y a c c e p t o r a c t i v i t y b e t w e e n e s t r o g e n - and a n t i e s t r o g e n - r e c e p t o r

laboratory

that

studies.

W e i n v e s t i g a t e d the c h a r a c t e r i s t i c s of MCF-7

Spelsberg's

step

in low

R e s u l t s f r o m our l a b o r a t o r y h a v e d e m o n s t r a t e d

supported by reconstitution

must

for

and the

(E-3)

the nonhistone

r e m o v e d w i t h a s e r i e s of

s t e p - e l u t i o n s b y 1-8 M Gdn»HC1

a n d t e r m e d CP1 to CP8 on the

b a s i s of m o l a r i t i e s of the salt.

S a m p l e s of

nonhistone

p r o t e i n f r a c t i o n s w e r e t h e n r e c o n s t i t u t e d to h u m a n DNA to nucleoacidic protein

(NAP)

fractions.

The

reconstituted

form NAP

f r a c t i o n s w e r e a s s a y e d for t h e i r a b i l i t y to b i n d [^Hjestradiol- and

[3H]Hl285-receptor

using the s t r e p t o m y c i n a s s a y [^Hjestradiol-receptor

(16).

complexes

Results

b i n d i n g to p r o t e i n - D N A

r e c o n s t i t u t e d w i t h CP2 and C P 7 .

However,

receptor c o m p l e x e s b o u n d not o n l y to the

(Fig. 4)

revealed

enhanced

complexes

3

[ H ] H 1285reconstituted

site b u t in a d d i t i o n b o u n d to a new CP5 s i t e , w h e r e a s to the CP7 site w a s n e a r l y a b s e n t as c o m p a r e d to estradiol-receptors.

These

r e s u l t s s u p p o r t our

f i n d i n g s of u n m a s k e d a c c e p t o r subfractions

(Fig. 2).

were s a t u r a b l e

The

sites

in r e s i d u a l respective

binding

[^H] previous chromatin

r e c o n s t i t u t e d CP5 and CP7

(Fig. 5) for t h e i r

CP2

receptor

sites

317

1


\E 0 . 4 o E o. 0.3 OC

o

I-

CL LU O

111

cr

Z> O m

J

I

I

I

I

I

I

I—

I

2

3

4

5

6

7

8

G d n H C I (M)

Fig. 4. Binding of [ 3 HJestradiol- and [ 3 H]H1 285receptor complexes to chromatin acceptor sites in MCF-7 antiestrogen-sensitive cells. For these reconstitution studies, histones were removed by 3 M NaC1 and MCF-7 chromatin nonhistone proteins were fractionated from chromatin-hydroxylapatite by step-elution using Gdn-HC1 (1-8 M). Samples of the extracted proteins were reconstituted to human DNA by reverse gradient dialysis. The receptor complexes were then incubated for 60 min with each reconstituted nucleoacidic protein (NAP) (70u g DNA) or DNA. Acceptor activity for [ 3 H ] estradiol- or [3h]H128 5-receptor complexes was then assayed using the streptomycin filter assay. Binding to DNA was subtracted from these values.

318 c o m p l e x e s w i t h K d s of 0.17 and 0.35 nM, r e s p e c t i v e l y The c o n c e n t r a t i o n of a c c e p t o r sites/cell.

(Fig.

sites was approximately

In a d d i t i o n a 2 - f o l d e x c e s s of

(Fig. 7). total

with

for the CP7 site w h e r e a s a 2 - f o l d

e x c e s s u n l a b e l e d H 1 2 8 5 - r e c e p t o r s , b u t not competed with

4000

unlabeled

e s t r a d i o l - r e c e p t o r s , b u t not H 1 2 8 5 - r e c e p t o r s , c o m p e t e d [^Hlestradiol-receptors

6).

[^H]H1285-receptor

Specific binding

estradiol-receptors,

c o m p l e x e s for the CP5

site

is e s t i m a t e d to be a b o u t 50%

of

binding.

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

responses

induced b y

estrogens

Fig. 5. S a t u r a t i o n k i n e t i c s for b i n d i n g of [^H] e s t r a d i o l - and [ ^ h ] H 1 2 8 5 - r e c e p t o r c o m p l e x e s to N A P f r o m c h r o m a t i n p r o t e i n f r a c t i o n CP7 and C P 5 , r e s p e c t i v e l y . MCF-7 n o n h i s t o n e p r o t e i n s f r a c t i o n a t e d f r o m c h r o m a t i n b e t w e e n 4M and 5M Gdn-HC1 and b e t w e e n 6M a n d 7M Gdn-HC1 were r e c o n s t i t u t e d to D N A by reverse g r a d i e n t dialysis. I n c r e a s i n g c o n c e n t r a t i o n s of [ ^ H j e s t r a d i o l or ['h]H 1 2 8 5 - r e c e p t o r c o m p l e x e s were i n c u b a t e d w i t h r e c o n s t i t u t e d N A P from CP7 ( [ ^ H ] e s t r a d i o l - r e c e p t o r s ) or CP5 ( [ 3 h ] H 1 2 8 5 - r e c e p t o r s ) and b i n d i n g a s s a y e d .

319

.BOUND RECEPTOR ( p m o l / m g DNA) Fig. 6. The d a t a in F i g . 5 w e r e a n a l y z e d using p l o t s (see t e x t ) .

versus a n t i e s t r o g e n s , e s p e c i a l l y since the CP5 p r o t e i n s s e e m to be a b s e n t

(or m o d i f i e d )

in

acceptor

antiestrogen-

resistant MCF-7 c e l l s since b i n d i n g by r e c e p t o r s measurable.

sites

responses.

is not

In a d d i t i o n , our d a t a using v a r i a n t s of MCF-7

cells s u p p o r t our h y p o t h e s i s t h a t m u l t i p l e acceptor

Scatchard

regulate v a r i o u s d i f f e r e n t

Thus,

sets of

chromatin

biological

in a r e s i s t a n t line o n l y one a c c e p t o r

site

m a y be a f f e c t e d a l t e r i n g o n l y one g r o u p of r e s p o n s e s .

MCF-7

antiestrogen-resistant

growth

RR cells fail to r e s p o n d to the

inhibiting e f f e c t s of a n t i e s t r o g e n s b u t a n t i e s t r o g e n s

can

320 still i n h i b i t the e s t r o g e n (9).

In the a n t i e s t r o g e n

i n d u c t i o n of p l a s m i n o g e n r e s i s t a n t MCF-7 v a r i a n t

activator

R27,

a n t i e s t r o g e n s , w h i l e not i n h i b i t i n g c e l l g r o w t h , not o n l y induce p r o g e s t e r o n e

r e c e p t o r w h i c h t h e y do in n o r m a l

c e l l s , b u t also g a i n the a b i l i t y to induce the sensitive

52K p r o t e i n

CO llJ X UJ

o o

oc

o

H Q. UJ

Ul CC O

Z

(26,27).

NAP-5

120

MCF-7

estrogen

NAP-7

1.

100 80 60 40 20

o

CD

+ dR

+ ER [3h]hr

+ HR

+ dR

+ ER

+ HR

[3h]er

Fig. 7. C o m p e t i t i v e b i n d i n g of e s t r o g e n - r e c e p t o r c o m p l e x e s to r e c o n s t i t u t e d N A P f r o m CP5 and C P 7 . P a r t i a l l y p u r i f i e d [ 3 H ] e s t r a d i o l - or [ 3 H ] H 1 2 8 5 receptor c o m p l e x e s w e r e used to d e t e r m i n e b i n d i n g to NAP from CP7 and C P 5 , r e s p e c t i v e l y , in the a b s e n c e of (100%) or in the p r e s e n c e of 2 - f o l d e x c e s s u n l a b e l l e d e s t r a d i o l - r e c e p t o r c o m p l e x e s (F,R) or H l 2 8 5 - r e c e p t o r c o m p l e x e s (HR). P r o t e i n c o n t e n t w a s k e p t c o n s t a n t w i t h h e a t - d e n a t u r e d r e c e p t o r p r e p a r a t i o n s (dR). Receptor b i n d i n g w a s a s s a y e d using the s t r e p t o m y c i n filter a s s a y .

321 DISCUSSION We have demonstrated that estrogen- and receptor c o m p l e x e s d i f f e r characteristics.

antiestrogen-

in their c h r o m a t i n

binding

T h e s e r e s u l t s w e r e o b t a i n e d using

different approaches;

i.e., b i n d i n g of r e c e p t o r to

d e p r o t e i n i z e d c h r o m a t i n f r a c t i o n s and to nucleoacidic protein fractions.

two partially

reconstituted

W e as w e l l as o t h e r s

have

d e m o n s t r a t e d m a n y p r o p e r t i e s of the s p e c i f i c i n t e r a c t i o n of steroid 28-30).

receptors with chromatin acceptor Steroid receptors

sites

(8,9,17,22,

i n t e r a c t in a s a t u r a b l e m a n n e r

intact or p a r t i a l l y d e p r o t e i n i z e d c h r o m a t i n

with

subfractions.

S p e c i f i c c h r o m o s o m a l p r o t e i n s a p p e a r to be a s s o c i a t e d

with

a c c e p t o r site a c t i v i t y , since r e m o v a l of t h e s e p r o t e i n s b y salt or p r o n a s e d e c r e a s e s

receptor b i n d i n g

(8,16,31), and

s p e c i f i c p r o t e i n f r a c t i o n s w h e n r e c o n s t i t u t e d to D N A acceptor activity that there

(16,30-32).

We have recently

is b o t h t a r g e t t i s s u e , s p e c i e s , a n d

s p e c i f i c i t y for c h r o m a t i n a c c e p t o r chromatin acceptor conformational

sites

sites c a n d i s t i n g u i s h

changes

determined receptor

(8,24,25,30).

That

structural/

i n d u c e d in the e s t r o g e n receptor

e s t r o g e n s v e r s u s a n t i e s t r o g e n s u p p o r t s our p r e v i o u s (33) w h i c h s u g g e s t e d t h a t e s t r o g e n

only

enhance

by

findings

receptor b o u n d b y

estrogen

u n d e r g o e s a c t i v a t i o n to a d i f f e r e n t d e g r e e w h e n c o m p a r e d

with

estrogen receptor bound by antiestrogen.

that

activation/transformation

It is p o s s i b l e

is a m u l t i s t e p p r o c e s s .

l a b o r a t o r y as w e l l as o t h e r s

Our

(11-15,34) have s u g g e s t e d

a n t i e s t r o g e n b i n d i n g m a y a f f e c t the m o n o m e r - d i m e r

that

equilibrium.

W e h a v e s u g g e s t e d t h a t the e s t r o g e n r e c e p t o r e x i s t s as a d i m e r w h i c h can r e a d i l y d i s s o c i a t e

into m o n o m e r s .

Estradiol

to the r e c e p t o r w o u l d not impede d i m e r d i s s o c i a t i o n . a n t i e s t r o g e n b i n d i n g to the receptor m a y inhibit

binding However,

full

d i s s o c i a t i o n and the c o m p l e x w o u l d remain as a m o d i f i e d (12,13,32).

Therefore, antiestrogen binding would

receptor c o n f o r m a t i o n t h a t is d i f f e r e n t t h a n that

dimer

induce induced

a by

322 estrogen binding (32,35).

Recently, a monoclonal antibody was

developed which discriminates between receptor bound by estrogen and receptor bound by antiestrogen complexes (36,37), a further indication that antiestrogens induce a structural change in the receptor.

These receptor alterations could at

least explain the observed differences in salt-resistant nuclear binding between estrogens and antiestrogens and the inability of antiestrogen-receptor complexes to bind the total complement of estrogen receptor-acceptor sites.

Ultimately,

these conformational differences are manifested in the antagonist actions of antiestrogen. Estrogen receptors bound by estradiol interact with several classes of nuclear binding sites to provoke maximal cellular responses.

Although antiestrogen-receptor

complexes

bind well to some sites, these complexes appear to bind poorly to other nuclear sites which may explain the antiestrogen effects of these compounds.

In addition, our results lend

support to the idea that antiestrogens have effects of their own on cells in addition to acting as estrogen antagonists and, in some cases, agonists.

It had previously been

suggested by one of us (3) that the estrogen receptor bound by antiestrogen could specifically act in the nucleus to alter gene expression such that cell division or tumor growth is inhibited.

Our current chromatin binding data demonstrate

that antiestrogen-receptor complexes bind MCF-7 E-3 chromatin in a way that is similar to estrogen-receptor complexes, such as the 1 M site (CP 2), but also in ways that are different, such as less binding to a 6 M site (CP 7) and new binding to a 4 M site (CP 5).

Clone RR is resistant to growth inhibition

by antiestrogens but is still sensitive to estrogens, which induce plasminogen activator and the 52K protein.

The RR

appears to lack that portion of the chromatin binding (4 M site or CP 5) which is unique for antiestrogen-receptor complexes. Lippman's group has recently demonstrated that MCF-7 cells

323 produce both growth stimulating factors as well as a growth inhibiting factor.

Whereas estrogens stimulate growth

stimulating factors, antiestrogens increase the production of a tumor growth inhibiting factor, TGF-g(4).

Could the 4 M

site, i.e., CP 5 in association with specific DNA sequences, be somehow responsible for the production of TGF-(5 by antiestrogen?

Antiestrogen-resistance

(lacks of inhibition of

cell growth) may be correlated with lack of binding by estrogen receptor to the antiestrogen-specific acceptor sites as well as lack of TGF- ¡3 production.

The MCF-7 clone RR,

however, retains the ability for antiestrogens to inhibit estrogen induced plasminogen activator.

Could the 6 M site

(CP 7) which binds estrogen receptor bound by estrogen be responsible for stimulation of plasminogen activator?

Since

antiestrogens compete with estrogens for the estrogen receptor, there would not be sufficient estrogen receptor to interact with the specific acceptor sites to elicit the response, e.g. plasminogen activator production. Although we used a chaotropic agent, Gdn-HC1, as an in vitro tool to expose certain chromatin acceptor sites, it is quite possible that _in vivo some acceptor sites are made available (unmasked) at different times during the several hours of a response to estrogens by subtle but specific alterations in nonhistone masking proteins.

These alterations

may be enzymatic, may include changes in the degree of phosphorylation, acetylation, peptide cleavage, or subunit loss or may be the result of other conformational changes in nonhistone proteins induced by estrogen receptor binding to a few initially open sites.

Thus, both the masking and

unmasking of acceptor sites in vivo would be envisioned as physiological events.

Such continual exposure of new classes

of acceptor sites _iri vivo is consistent with the "sequential" (38) or "ratchet" (39) model of steroid hormone action. laboratory has speculated that _in_ vivo nuclear acceptor sites would be sequentially unmasked and masked with time

Our

324 UNMASKING a MASKING OF NUCLEAR A C C E P T O R S I T E S ESTROGEN

EARLY TERM ER

J

ER

J Mort«d Acceptor Sit»

VS A N T I E S T R O G E N

INTERMEDIATE TERM ER

J

ER .

ER

X

I

CIXpcS

1

UNMASK Cap

EARLY MASK RESPONSES

|

I Matkad Acceptor Slut

•

t

UNMASK cap MASK B

EARLY RESPONSES

L A T E TERM ER

ER

ER

I

ER

ER

I I I

G D ^ d b o D C D C D | I

I|UNMASK E 8F

B

AER AER

ACTION

|

|

STIMUf LATION OF GROWTH

AER

T I

|

AER

|I

AER .

|

MASK MASK AA

INHIBITION OF GROWTH

STIMULATION OF GROWTH

AER

AER •

|

I LACK OF GROWTH

Fig. 8. Model suggesting the effects of unmasking and masking of nuclear acceptor sites _in vivo. Acceptor proteins A-F would be sequentially unmasked and masked with time of exposure of target tissue to estrogen or antiestrogen. Receptor complexes initially bind to available unmasked sites which initiate early responses. With time some of these sites may become masked while others become unmasked to initiate other sets of responses. Antiestrogen-receptor complexes would be unable to bind certain acceptor sites (i.e., D) thus preventing completion of agonist responses, resulting in "antiestrogencity". It is also possible that antiestrogens initiate inhibitory responses of their own by interacting with different sites (i.e., C) .

325 after exposure to hormone bound receptor.

The ability of

antiestrogens to elicit some agonist responses, inhibit some estrogen-induced responses and possibly to have unique actions can be explained by this model (Fig. 8).

Both estrogen- and

antiestrogen-receptor complexes could elicit early responses by binding unmasked acceptor sites in intact chromatin. However, only estrogens would allow complete stimulation upon continued exposure to estrogen by binding of receptor to newly unmasked acceptor sites.

In addition, antiestrogen could

initiate inhibitory responses of their own by interacting with different sites. The receptor/acceptor interactions described in our studies fulfill many of the criteria of a physiologically significant binding system.

What is unknown is the exact

nature of the protein and DNA components of the acceptor site. Does the estrogen receptor bind to a nonhistone protein-DNA complex, to a nonhistone protein alone or to a DNA sequence alone?

Nonetheless, from previous and current evidence, it is

clear that certain nonhistone proteins are implicated in the specificity of acceptor sites for steroid receptors. Ultimately the acceptor site components will have to be isolated and highly purified to determine the role these sites may have in steroid hormone action. In summary, what is beginning to emerge is a picture of steroid receptor action in which the effect of a ligand on a cell depends on at least three things; the ligand (agonist or antagonist), the nature of the receptor (ligand specificity, chromatin affinity and other properties when a ligand is bound), and the state of the available chromatin acceptor sites (unmasked or masked, present or absent).

Changes in any

one of these can affect the range of responses of a cell to a steroid hormone or related ligand.

326

Acknowledgement This work was supported by grants HD-13425, RR-05388 and CA-43868 awarded by the National Institutes of Health.

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DESIGN AND ACTIVITY OF NONSTEROID ANTIANDROGENS T. Ojasoo, J.P. Raynaud Roussel-Uclaf, 75007 Paris, France Introduction Anti-androgens block the production, transport and/or action of active endogenous androgens and, as such, may be (a) inhibitors of testosterone synthesis or secretion by the testes, (b) competitive inhibitors of enzymes involved in the formation and metabolism of testosterone, (c) inhibitors of androgen uptake by target organs and of androgen binding to receptor proteins. Category "a" comprises inhibitors of gonadotropin release by the hypothalamopituitary unit and is represented by estrogens and LHRH-superagonists and antagonists ; category "b" Includes Inhibitors of the formation of adrenal androgens and of the cytochrome-Pi 5o dependent 5a-reductase and aromatase systems, that influence the in situ formation of dihydrotestosterone (DHT) and of estrogen in target tissues ; compounds of category "c" block the direct effect of androgen and therefore can be considered antineoplastic agents acting to directly inhibit androgen-promoted cell growth. Some might even exert such activity by Interference with protein targets other than the hormone binding site of the androgen receptor. A single compound may belong to more than one category. Table 1 broadly classifies the major antiandrogens of disclosed chemical structure that are presently in clinical development or use for the treatment of androgen-sensitive benign and malignant diseases. The first pituitary hormone inhibitors used as antiprostatic cancer agents were estrogens, in particular diethylstilbestrol and its diphosphate, dienestrol, ethynyl estradiol and conjugated estrogens. These, however, have fallen into disfavour because of the associated cardiovascular complications [1, 2]. High doses of

Receptor Mediated Antisteroid Action © 1987 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

330 estrogen are cytotoxic [3] but are also carcinogenic [4], Weaker estrogens such as bifluranol [5] or lower dose schedules are now promulgated but they may Induce androgen receptor (AR) [6] and consequently increase the androgen-sensitlvlty of the prostate [7, 8]. A recent report, however, suggests that estrogens downregulate AR in cultured human mammary cancer cells [9]. Some estrogens also inhibit 5o-reductase activity [10]. Continuous administration of LHRH-analogs reputedly castrates as effectively as estrogens [11] but the down-regulation of pituitary receptors only occurs after an initial early increase in LH and testosterone. The design and activity of these compounds have been the subject of excellent reviews [12-15]. The longer-acting forms available as biodegradable implants [16-18] may well supercede less convenient administration forms (subcutaneous injections or nasal sprays). The pituitary inhibitory activity of ant1androgenic progestins will be discussed below. Ehzyme inhibitors used to obtain an antiandrogenlc action should be specific. In prostate cancer patients who have relapsed after a first-line endocrine therapy, compounds like aminoglutethimlde, ketoconazole and other imidazole derivatives [19] have led to regression or stabilization of disease in about 30% of cases [20-24]. These compounds also act to inhibit Cortisol as well as adrenal androgens and the aromatase complex of enzymes. Patients therefore require corticosteroid replacement therapy and this may well affect the efficacy of the treatment. The design of new compounds has been directed towards specific inhibitors of the NADPH-dependent enzyme, 5a-reductase, which converts testosterone to DHT and of aromatase, also a microsomal P4 5 o enzyme, that converts androgens into estrogens. Older men have higher plasma estrogen levels than younger men [25] and elevated 5a-reductase activities have been reported in BPH stroma [26, 27] and in the skin of hirsute women [28, 29], Several progestins, in particular the much studied 6-methylene progesterone, are inhibitors of 5a-reductase [30, 31] and there now exists a new generation of inhibitors belonging to the 4-azasteroid family. Excellent

331

TABLE 1 .

Antiandrogens In Clinical Development and Use. COMPOUND

ACTIVITY

PROPOSED INDICATIONS

Estrogens QHS

Potent estrogen

Prostate cancer

Weak estrogen

Benign prostatic hypertrophy

Estrogen Antineoplastic

Prostate cancer

LHRH-receptor desensitizor

Prostate cancer

Initially anticonvulsant Adrenal inhibitor

Second-line prostate cancer therapy

Antifungal Adrenal inhibitor

Second-line prostate cancer therapy

H5C2 Diethylstilbestrol and its diphosphate

H0

CH CH

~ ~\ V"0H

\ / /

HsC

CiHs

p

Bifluranol (Biorex, UK)

(CICH 2 CH 2 ) 2 N C O O

JK

J

M

Estramustine (Leo, S) LHRH - analoqs ;

D-Trpß D-Leu&, Pro-NH Et?

Decapeptyl (Ipsen F )

: Leuprolide

6

( Abbott USA )

D - S e r ( t Bu) ,Pro-NH Et9 : Buserelin

(Hoechst FRG )

D-Ser (t BU)6, AZ Gly

( ICI UK )

: Zoladex

Inhibitors of adrenal steroid biosynthesis and of aromatase C2HS

0

Aminoglutethimide (Ciba, CH)

»^Ä

C H j C - N^

N—y\

\ ) - O C H 2 - 4F,

= /

O r5"0-cl I

0

ci

>

—'

Ketoconazole (Janssen, NL)

332 TABLE 1 . Contd. COMPOUND

ACTIVITY

PROPOSED INDICATIONS

Competitive inhibitors of 5d -reductase H3c

CH,

k A

Keratolytic

Acne

Antiandrogen

Benign prostatic hypertrophy

Progestin Antiandrogen

Acne Hypersexuality Prostate cancer

Antiandrogen

Benign prostatic hypertrophy

Antiandrogen

Benign prostatic hypertrophy

Progestin Antiandrogen

Prostate cancer

COOH

C H j

Retinoic acid (Hoffmann-La Roche, CH)

oXX; CH3

4-aza 3-oxo steroids

: MK906

(Merck Sharp and Dohme, USA)

Pituitary inhibitors that are also competitive inhibitors of androqen receptor bindina and of adrenal steroid biosvnthesis

^

Jl

Cyproterone acetate (Schering, FRG)

Pituitary inhibitors that are also competitive inhibitors of androqen receptor bindina and of 5a -reductase

QI^+^SS^

Oxendolone (Takeda, Japan) TSAA - 291

Lipido-steroid complex extracted from the plant Serenoa repens Permixon (Pierre Fabre. F)

Pituitary inhibitors that are also competitive inhibitors of androqen receptor bindina

„c

H— 285. 66. Keeping, H.S., Lyttle, C.R. 1982. Endocrinology 111, 2046. 67. Charaness, G.C., Bannayan, G.A., Laudry, Jr. L.A., Sheridan, P.J., McGuire, W.L. 1979. Biol. Reprod. 21_, 1087. 68. Nguyen, G.L., Giambiagi, N., Mayrand, C., Lecerf, F., Pasqualini, J.R. 1986. Endocrinology V\9_, 978. 69. Hsue, A.J.W., Ericksson, G.F., Yeu, S.S.C. 1978. Nature 273, 37. 70. Bichon, M., Bayard, F. 1979. J. Steroid Biochem. 10, 105. 71. Kniefei, M.A., Katzenellenbogen, B.S. 1981. Endocrinology 108, 545. 72. Huppert, L.C. 1979. Fertil. Steril. 31, 1. 73. Sakai, F., Chiex, F., Clavel, M., Colon, J., Mayer, M., Pommatau, E., Saey, S. 1978. J. Endocrinol. 76, 219. 74. Amara, J.F., Dannies, P.A. 1983. Endocrinology 112, 1141. 75. Amara, J.F., Dannies, P.A. 1986. Mol. Cell Endocrinol. 47, 183. 76. Martinez, A.C., Anara, J.F., Dannies, P.S. 1986. Mol. CeTl Endocrinol 48, 127. 77. Blue, M.L., Williams, D.L. 1981. Biochem. Biophys Res. Commun.98, 785. 78. Canopy, F., Williams, D.L. 1980. Biochemistry 19, 221979. Sutherland, R.L., Jordan, V.C. 1981. In: Non-Steroid anti-estrogens: Molecular Pharmacology and antitumor Actions. Academic Press, Sydney. 80. Riegel, A.T., Jordan, V.C., Bain, R.R., Schoenberg, D.R. 1986. J. Steroid. Biochem. 24, 1141. 81. Tata, J.R. 1976. CeTl 9, 1. 82. Aitken, S.C., Lippman, M.E., Kasid, A., Schoenberg, D.R. 1985. Cancer Res. 45, 2608. 83. Bronzert, D.A., Monaco, M.E., Pinkus, L., Aitken, S.C., Lipman, M.E. 1981. Cancer Res. 41_, 604. 84. Kasid, A., Davidson, N.E., Gelman, E.P., Lipman, M.E. 1986. J. Biol. Chem. 261_, 5562. 85. McGuire, W.L., Carbone, P.P., Vollmar, E.P. 1975. In: Estrogen Receptors in human breast, cancer. Raven Press, New York. 86. Leung, B.S., Sasaki, G.H. 1975. Endocrinology 97, 564. 87. Leung, B.S., Mosley, S., Davenport, C.E., Krippaehne, W.W., Fletcher, W.S. 1975. In: Estrogen Receptor in prediction of clinical responses to endocrine ablation. McGuire, W.L., Carbone, P.P. and Vollmer, E.P. (eds.) Raven Press, New York. 88. Leung, B.S. 1978. In: Hormones, Receptors and Breast Cancer. (McGuire, W.L. ed.) Raven Press, New York, pp. 219. 89. Edwards, D.P., Murthy, S.R., McGuire, W.L. 1980. Cancer Res. 40, 1722. 90. DeSombre, E.R., Argoblast, L.V. 1974. Cancer Res. 34, 1971. 91. Horwitz, K.B., McGuire, W.L. 1978. In: Breast Cancer: Advances in Research and Treatment Vol. 2, (McGuire ed.) Plenum Publishing Corp., New York, pp. 155. 92. Patterson, J.S., Battersby, L.A. 1980. Cancer Treat. Rep. 64, 775.

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100. McGuire, W.L., Edwards, D.P., Adams, D.J., Savage, N., 1981. In: Non-Steroidal Anti estrogens: Molecular Pharmacology and Antitamor Activity (Sutherland, R.L. and Jordan, V.C., eds.) Academic Press, New York, pp. 339. 101. Katzenellenbogen, B.S., Norman, M.J., Eckert, R.L., Peltz, S.W., Mangel, W.F. 1984. Cancer Res. 44, 112. 102. Westley, B., Rochefort, H. 1980. Cell 20, 353. 103. Sheen, Y.Y., Katzenellenbogen, B.S. 19$7. Endocrinology 120, 1140. 104. Westley, B., May, F.E., Brown, A.M., Knust, A., Chambón, P., Lippman, M.E., Rochefort, H. 1984. J. Biol. Chem. 259, 10030. 105. Vignon, F., Lippman, M., Newala, H., DeRocq, D., Rochefort, H. 1984. Cancer Res. 44, 2084. 106. Sheen, Y.Y., Katzenellenbogen, B.S. 1987. Endocrinology 120, 1140. 107. Wang, J.L., Hsu, Y.M. 1986. Trends Biochem. Sei. JJ_> 23* 108. Geller, J., McCoy, K. 1974. Acta Endocrinol. (Copenhagen) 75, 385. 109. Liao, S. 1974. Biochem., Ser. One 8, 154. 110. Tezon, G.G., Vazquez, M.H., Blaquier, J.A. 1982. Endocrinology 111, 2039. 111. Bullock, L.P., Barthe, P.L., Mowszowicz, I., Orth, D.N., Bardin, C.W. 1975. Endocrinology 97, 189. 112. Mowszowicz, I., Bieber, D.E., Chung, K.W., Bullock, L.P., Bardin, C.W. 1974. Endocrinology 95, 1589. 113. Sundaram, K., Cao, Y.Q., Wang, N.G., Bardin, C.W., River, J., Vale, W. 1981. Life Sei 28, 83114. Lecompte, P., Wang, N.G., Sundaram, K., River, J., Vale, W., Bardin, C.W. 1982. Endocrinology 111, 1. 115. Imperto-McGinley, J., Guerrero, L., Guatier, T., Peterson, R.E. 1974. Science _186, 1213. 116. Brooks, J.R., Bapista, E.M., Berman, C., Ham, E.A., Hichens, M., Johnston, D.B.R., Primka, R.L., Rasmusson, G.H., Reynolds, G.F., Schmitt, S.M., Arth, G.E. 1981. Endocrinology 109, 830. 117. Brooks, J.R., Berman, C., Glitzer, M.S., Gordon, L.R., Primka, R.L., Reynolds, G.F., Rasmusson, G.H. 1982. Prostate, 35. 118. Wenderoth, U.K., George, F.W., Wilson, J.D. 1983. Endocrinology 113, 569. 119. Liang, T., Brady, E.J., Cheung, E.H., Saperstein, R. 1984. Endocrinology 115, 2311. 120. Liang, T., Cascieri, M.A., Cheung, A.H., Reynolds, G.F., Rasmusson, G.H. 1985. Endocrinology 117, 571.

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121. Hudson, R.W., Mofitt, P.M., Owens, W.A. 1983. Can. J. Biochem. Cell Biol. _61_, 750. 122. Rasmusson, G.H., Liang, T., Brooks, J.R. 1983. In: Gene Regulation by Steroid Hormones II (Roy, A.K. & Clark, J.H. eds.) SpringerVerlag, New York, pp. 311. 123. Pajunen, A.E.I., Isomaa, V.V., Janne, O.A., Bardin, C.W. 1982. J. Biol. Chem. 257, 8190. 124. Swank, R.T., Paigen, K., Granschow, R.E. 1973. J. Mol. Biol. 81, 225. 125. Toole, J.J., Hastie, N.D., Held, W.A. 1979. Cell J7, 441. 126. Catterall, J.F., Watson, C.S., Kontula, K.K., Janne, O.A., Bardin, C.W. 1986. Adv. Exp. Med. Biol. 196, 213. 127. Catterall, J.F., Kontula, K.K., Watson, C.S., Seppanen, P.J., Funkenstein, B., Melanitou, E., Hickock, N.J., Bardin, C.W., Janne, O.A. 1986. Ree. Prog. Horm. Res. 42, 71. 128. Kontula, K.K., Seppanen, P.J., Van Duyne, P., Bardin, C.W., Janne, O.A. 1985. Endocrinology J_1_6, 226. 129. Newmann, F., Wallrabe, R.B., Elger, W., Steinbeck, H., Han, J.D., Kramer, M. 1970. Ree. Prog. Horm. Res. 26, 337. 130. Mowszowicz, I., Bieber, D.E., Chung, K.W., Bullock, L.P., Bardin, C.W. 1974. Endocrinology 95, 1589. 131. Gupta, C., Bullock, L.P., Bardin, C.W. 1978. Endocrinology 102, 736. 132. Bullock, L.P., Barthe, P.L., Mowszowicz, I., Orth, D.N., Bardin, C.W. 1975. Endocrinology 97, 189. 133. Bardin, C.W., Bullock, L.P., Shenins, R., Mowszowicz, I., Blackburn, W.R. 1973. Ree. Prog. Horm. Res. 29, 65. 134. Coffey, D.S. 1974. In: Male Accessory Sex Organs, Structure and Function in Marwnals (Brandes, D. ed.) Academic Press, New York, pp. 307. 135. Böhmer, T., Hansson, V. 1975. Mol. Cell. Endocrinol. 103. 136. Lasnitzki, I., Röbel, P. 1976. Adv. Bio Sei. 3, 175. 137. Kitamura, Y., Uehida, N., Odaguchi, K., Yamaguchi, K., Okamoto, S., Matsumoto, K. 1980. J. Steroid Biochem. .13, 333. 138. Kitamura, Y., Okamoto, S., Hayata, I., Uehida, N., Yamaguchi, K.. Matsumoto, K. 1979. Cancer Res. 39, 4713. 139. Cambray, G.J., King, R.J. 1976. J. Endocrinol. 71., 43. 140. Schroeder, F.H., Richards, B. 1985. In: Progress in Clinical and Biological Research - Therapeutic Principles in Metastatic Prostatie Cancer (Riss, A.R. ed.) New York, 185A. 141. Newmann, F., Steinbeck, H. 1974. In: Handbook of Experimental Pharmacology (Eichler, 0., Fareh, A., Herken, H. & Welch, A.D., eds.) Springer, Berlin, 35, pp. 235. 142. Newmann, F., Humpel, M., Senge, Th, Schenek, B., TUnn, U. 1982. In: Prostate Cancer (Jacobi, G.H. and Hohenfeilner, R.F. eds.) Williams & Wilkins, Baltimore, pp. 269. 143. Luderschmidt, Chr., Plewig, G. 1977. Archs. Derm. Res. 258, 185. 144. Steinbeck, H., Newmann, F. 1971. J. Reprod. Fert. 26, 59.

STEROID HORMONE ANTAGONISTS, BRAIN RECEPTOR SYSTEMS AND BEHAVIOR

A.M. Etgen Departments of Psychiatry and Neuroscience, Albert Einstein College of Medicine, Bronx, N.Y. 10461

Introduction The antiestrogenic properties of the first nonsteroidal estrogen antagonist MER-25 were described by Lerner et al. (1) almost thirty years ago. In the ensuing three decades dozens of additional agents which antagonize the physiological effects of steroid hormones have been developed and characterized. The reasons for this intensive research effort are at least twofold. First, steroid hormone antagonists have potential therapeutic utility, e.g., in the treatment of acne, hirsutism, steroid-dependent breast and prostate cancers, and as fertility and antifertility agents. The clinical uses of antisteroids are discussed in several of the other chapters in this volume. Second, such compounds are potentially powerful tools for elucidating the cellular and molecular mechanisms of steroid hormone action. This chapter will focus on the use of such antagonists to identify the sites and mechanisms of steroid hormone regulation of animal behavior. The generally accepted model for the molecular mechanism of steroid action originated from early studies of estrogen action in the uterus (2,3). Briefly, steroids enter both target and nontarget cells by passive diffusion. Target cells contain intracellular macromolecules ("receptors") which bind the steroid with high affinity, limited capacity, and relative stereospecificity. Binding of steroid to the receptor

Receptor Mediated Antisteroid Action © 1987 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

406

initiates conformational alterations in the steroid-receptor complex, converting it to a form with enhanced affinity for intranuclear chromatin "acceptor" sites. Interaction of steroid-receptor complexes with chromatin initiates changes in gene transcription (i.e., RNA synthesis), and these alterations in gene expression are believed to mediate the physiological responses of target tissues to steroid hormones (for reviews see 4-7). There is still some disagreement regarding the subcellular compartmentation of steroid receptors (i.e., cytosolic vs. nuclear) in the absence of hormone (8-11). For purposes of the present discussion, the term "cytosol receptor" is used to designate the state of the receptor with lower affinity for nuclear components, and "nuclear receptor" refers to cytosol receptors that have translocated to the cell nucleus and/or converted to a state with increased affinity for cell nuclear components. As reviewed below, the availability of steroid antagonists has allowed researchers to ask whether receptor-mediated modulation of gene expression in brain cells can account for the behavioral effects of steroids. To date most investigations have used antisteroids to study the neural sites and mechanisms of steroid regulation of reproductive (sexual) behavior. Thus most of this chapter will review experiments in which the interactions of antiandrogens, antiprogestins, or antiestrogens with brain steroid receptor systems have been correlated with changes in sexual behavior. The handful of studies that have examined other behaviors will also be described.

Antiandrogens The growth and function of male secondary sex structures (especially the seminal vesicles and ventral prostate) and, in most species, the display of male sexual behavior are

407

androgen-dependent processes. By the mid-197 0s a variety of steroidal (e.g., cyproterone acetate) and nonsteroidal (e.g., flutamide) antagonists of androgen-receptor binding in peripheral tissues had been reported to inhibit androgen action in the prostate and seminal vesicles (see 12-14). Until recently, however, attempts to modify androgen-dependent male reproductive behavior with such antiandrogens have proven relatively unsuccessful. Administration of peripherally potent antiandrogens, even in doses that induced sterility or decreased seminal vesicle and prostate weights, failed to alter sexual behavior in adult male rats or guinea pigs (1520). Similarly, flutamide had no effect on intermale aggression in male mice even though prostate weights were decreased (21). There is one early report that cyproterone acetate reversibly reduced sexual activity in male rats (22) and Sodersten et al. (20) found that flutamide partially inhibited testosterone-induced ejaculatory behavior in castrated male rats. The latter authors suggested that the observed ejaculatory failure probably resulted from flutamideinduced alterations in penile morphology rather than actions of the compound in the brain since the animals showed normal mounting and intromission behavior. Similar interpretations of flutamide's effects of male sexual behavior were made by Gray (23). Moreover, one laboratory (24) reported that both systemic injections of cyproterone acetate and intrahypothalamic implants of the free alcohol cyproterone actually enhanced sexual behavior in adult male rats. A likely explanation for these negative results is provided by studies of the cellular actions of antiandrogens in the brain. Whalen et al. (25) demonstrated that cyproterone acetate administration did not alter the uptake of radiolabeled testosterone by the pituitary, hypothalamus, preoptic area or cortex even in doses which significantly reduced hormone uptake by the seminal vesicles and penis. In vitro competition studies also found that flutamide and BOMT,

408 another potent antiandrogen in the periphery, were poor competitors for 3 H-androgen binding sites in brain cytosols from male mice (21,26) and rats (27,28).

These results

suggest that most antiandrogens fail to modify androgendependent behaviors because they fail to interact with brain androgen receptor systems.

The data also support the proposal

of Sheridan (29) that the brain may have as many as three distinct receptors for androgens which differ in some ways from peripheral androgen receptors. One recent study successfully correlated the behavioral effects and brain androgen receptor binding properties of the potent antiandrogen Sch 16423, a hydroxylated metabolite of flutamide (28) .

At a dose of 15 mg/day, Sch 16423 reversibly

depressed the restoration of male sexual behavior in castrated rats receiving testosterone replacement via Silastic capsules. In vivo injections of the compound reduced cell nuclear binding of radiolabeled androgen in the brain

(hypothalamus-

preoptic area-amygdala-septum) and pituitary; at concentrations of 10 uM or greater Sch 16423 also competed for binding of the synthetic androgen pituitary cytosols.

3

H-R1881 in brain and

Thus Sch 16423 may be the first useful

antagonist for studying the role of brain androgen receptor systems as mediators of the behavioral effects of androgens.

Antiprogestins In most female mammals ovarian estrogens and progestins synergize to control the onset, quality and duration of female reproductive behavior (for reviews see 30,31).

The most

effective treatment for inducing sexual behavior in ovariectomized rodents is sequential treatment with estrogen followed 36-48 hours later by progestin

(32,33).

A number of

laboratories have shown that estrogen treatments which facilitate female reproductive behavior also induce the

409

synthesis of progestin receptors in the hypothalamus and preoptic area, leading to the hypothesis that progestin receptors may mediate the behavioral effects of progesterone in the brain (for reviews see 31,34-36). For many years the search for agents which specifically antagonize the physiological effects of progestins was relatively unfruitful (see 37,38). However, a recently developed synthetic steroid, RU 38486, has been reported to antagonize a number of progestational responses in peripheral reproductive tissues, presumably because it acts as a competitive inhibitor of intracellular progesterone-receptor binding (39-44). Although RU 38486 has some progestin agonist activity (45,46) and is also a potent glucocorticoid receptor antagonist (47-49), the compound has already proven to be an effective tool for evaluating the neuroanatomical sites and molecular mechanisms of progestin regulation of female reproductive behavior. The first behavioral study with RU 38486 (50) showed that the drug inhibits progesterone activation of sexual behavior in ovariectomized, estrogen-primed female guinea pigs. This inhibitory action can be prevented by giving very high doses of progesterone but not Cortisol (36,51), suggesting that the behavioral effects of RU 38486 involve antagonism of progestin rather than of glucocorticoid action. The behavioral antagonism was correlated with the capacity of in vivo drug injections to reduce the concentration of progestin receptors in cytosols of hypothalamus-preoptic area from estrogen-primed guinea pigs. In addition, RU 38486 competed in vitro for progestin but not estrogen binding sites in brain cytosols (50). Etgen and Barfield (52) subsequently demonstrated that the compound competes for progestin binding sites in female rat brain cytosols and that injections of RU 38486 inhibit female reproductive behavior in estrogen-primed rats a$ well. They also showed that 3H-RU 38486 binding in rat brain

410

cytosols is increased by estrogen priming and is displaced by progestins (and to a lesser extent by glucocorticoids). These experiments all support the hypothesis that intracellular progestin receptors in the hypothalamus and/or preoptic area mediate progesterone facilitation of female sexual behavior in rodents. Interestingly, Richmond and Clemens (53) recently reported that RU 38486 does not antagonize the activation of sexual receptivity by cholinergic drugs in estrogen-primed female rats, suggesting that brain progestin receptors do not mediate the cholinergic facilitation of female reproductive behavior. In addition to the evidence cited above that RU 38486 competes for brain cytosol progestin receptors, Brown and Blaustein (51) have shown that the compound induces accumulation of progestin-receptor complexes in guinea pig hypothalamic cell nuclei. However, nuclear accumulation of brain progestin receptors following RU 38486 administration is detectable only when exchange assays are conducted at elevated temperatures. Thus RU 38486 may dissociate more slowly from brain progestin receptors than does progesterone itself. These slow dissociation kinetics may explain the observation that RU 38486 given alone makes animals unable to respond behaviorally to progesterone given 24 hours later (50,53; Vathy, Etgen and Barfield, unpublished observations). Blaustein and colleagues have also proposed that the termination of female sexual behavior is attributable to the loss of progestin receptor complexes from hypothalamic cell nuclei (50,54,55). This hypothesis was tested using RU 38486 in estrogen-primed guinea pigs (51). Injection of the drug 4 hours after progesterone had no effect on the latency of lordosis responses, but the display of sexual behavior in treated animals terminated several hours earlier than in animals given vehicle injections. This shortened period of behavioral receptivity in RU 38486-treated guinea pigs was

411

correlated with reductions in the concentration of progestin receptors in hypothalamic cell nuclei 2 and 6 hours after drug administration. Thus studies using RU 38486 are also consistent with the hypothesis that the loss of progestin receptors from hypothalamic cell nuclei may be a mechanism whereby the period of sexual behavior is terminated, at least in female guinea pigs. RU 38486 has also been utilized to examine the neural sites of progesterone stimulation of female sexual behavior (lordosis). Etgen and Barfield (52) applied the drug in bilateral intracerebral implants to the preoptic area, ventromedial hypothalamus, habenula and interpeduncular region of estrogenprimed rats 1 hour prior to systemic progesterone injections. Implants of RU 38486 into the ventromedial hypothalamus but not the preoptic area or interpeduncular region of the midbrain suppressed lordosis in a significant percentage of animals. These data confirm the results of recent progestin implant studies which point to the ventromedial nucleus of the hypothalamus as the primary neural locus of progesterone facilitation of female sexual behavior (56,57). Moreover, some RU 38486 implants in the habenula also inhibited progesterone-dependent lordosis, suggesting that progestinreceptor interactions in the habenula may also play a role in behavioral regulation. In summary, the steroidal antiprogestin RU 38486 antagonizes progesterone facilitation of female sexual behavior in estrogen-primed rodents when administered either systemically or intracerebrally. It is also clear that this progestin antagonist binds directly to brain progestin receptors, and the evidence presented to date is consistent with the interpretation that RU 38486 exerts its effects on reproductive behavior by interfering with progesteronereceptor interactions in neural tissues. Thus further studies with this compound should yield new insights into the cellular

412

and molecular mechanisms underlying progestin modulation of sexual and other behaviors.

Antiestrogens By far the most extensively characaterized steroid antagonists in both neural and peripheral tissues are the antiestrogens. Historically, analyses of antiestrogen action in the uterus provided some of the first direct evidence that modulation of gene expression by intracellular steroid receptors is a critical molecular event underlying target tissue responses to hormones (e.g., 5,58-61). More recent work has suggested that relatively antiestrogen-specific binding sites exist ubiquitously in vertebrate tissues (see 62-64) and that there may be an endogenous ligand for these binding sites (65). However, the physiological function of the antiestrogen binding sites is still undetermined; this issue and a variety of the clinical and basic research uses of estrogen antagonists in peripheral tissues are reviewed in other chapters of this volume. While studies of the effects of estrogen antagonists on neural estrogen binding and behavior are neither as numerous nor as systematic as those on the uterus and on breast cancer, antiestrogen modulation of both female sexual behavior and eating behavior has been reported (for earlier reviews see 66,67). In addition, a substantial amount of information regarding antiestrogen interaction with brain estrogen receptors is also available. The remainder of this chapter will review these data in some detail since they provide the best example of the use of steroid antagonists as probes of the sites and molecular mechanisms of hormonal regulation of behavior.

413

Effects of antiestrogens on female sexual behavior Arai and Gorski (68) were the first to report that the nonsteroidal antiestrogen CI-628 inhibited female rat sexual behavior when administered concurrently with estrogen. Delaying CI-628 treatment until 24 hours after the estrogen injection or until the time of progesterone injection (48 hours post-estrogen) did not attenuate sexual receptivity. Additional experiments in a variety of laboratories subsequently showed that a large number of estrogen antagonists, including MER-25, CI-628, clomiphene, nafoxidine, and tamoxifen, are effective inhibitors of estrogen-stimulated female sexual behavior in rats, hamsters and guinea pigs (for reviews, see 69 and chapter in this volume by Fabre-Nys). These behavioral studies also established some important general characteristics of antiestrogen action. First, the degree of behavioral inhibition is dependent on the dose of both estrogen and antiestrogen. Second, the temporal sequence of estrogen and antiestrogen administration is a critical variable; with the possible exception of guinea pigs (70,71) antiestrogens are effective behavioral antagonists only when given at or near the time of estrogen injection. Thus the behavioral inhibition exerted by estrogen antagonists is likely to be due to antagonism of estrogen rather than of progesterone action and is unlikely to reflect nonspecific or toxic effects of the compounds. Komisaruk and Beyer (72) demonstrated further that MER-25 does not block lordosis responses induced by cervical probing plus flank-perineum stimulation. This observation suggests further that antiestrogens act directly on the brain rather than by altering peripheral sensory input. The latter conclusion has been confirmed by experiments showing that implants of antiestrogens directly into the brain can block the activation of female rat sexual behavior by systemic estrogen priming (73-75). In fact the latter two studies have provided

414

c o n v i n c i n g e v i d e n c e t h a t e s t r o g e n a c t i o n in t h e

ventromedial

hypothalamus

(see 76)

is n e c e s s a r y a s w e l l a s s u f f i c i e n t

p r i m e f e m a l e s e x u a l b e h a v i o r in o v a r i e c t o m i z e d r a t s . in c o n t r a s t w i t h t h e m i x e d e s t r o g e n activity of antiestrogens

to

Finally,

agonist/antagonist

in m o s t t a r g e t t i s s u e s ,

these

compounds appear to be almost pure antagonists w i t h regard to e s t r o g e n f a c i l i t a t i o n of f e m a l e r e p r o d u c t i v e b e h a v i o r . enclomiphene has b e e n reported to have very w e a k

Only

estrogen

a g o n i s t e f f e c t s o n f e m a l e s e x u a l b e h a v i o r in g u i n e a p i g s 79) a n d i n r a t s

(80).

Hence in-depth examination of

(77-

estrogen-

antiestrogen interactions with brain estrogen receptors may a n ideal s y s t e m i n w h i c h t o e l u c i d a t e t h e m o l e c u l a r of s t e r o i d r e g u l a t i o n of b e h a v i o r .

However,

it m u s t b e

recognized that the major estrogen receptor-containing of t h e b r a i n

be

mechanisms

(hypothalamus, preoptic area, amygdala)

s i t e s o f e s t r o g e n r e g u l a t i o n of g o n a d o t r o p i n

regions

are also

release,

aggression, maternal behavior and eating behavior. o n e m u s t b e c a u t i o u s in i n t e r p r e t i n g r e p o r t s of

Therefore,

antiestrogen

a c t i o n in t h e h y p o t h a l a m u s a s b e i n g u n i q u e l y r e l a t e d t o t h e c o n t r o l of f e m a l e s e x u a l

behavior.

Effects of antiestrogens on brain uptake and retention

of

estrogen There are positive and negative reports of r e d u c t i o n o f h y p o t h a l a m i c u p t a k e of

3

antiestrogen

H-estradiol.

Several

i n v e s t i g a t o r s d e t e c t e d n o s i g n i f i c a n t i n h i b i t i o n of in v i v o 3

H - e s t r a d i o l u p t a k e i n t o w h o l e h o m o g e n a t e s of r a t

by nafoxidine

(81), M E R - 2 5

(82) o r C I - 6 2 8

hypothalamus

(83), e v e n w h e n t h e

l a t t e r a n t a g o n i s t w a s a d m i n i s t e r e d in a p a r a d i g m in w h i c h female sexual behavior was inhibited (85)

(84).

Walker and

found similarly that CI-628 did not reduce

u p t a k e of

3

H - e s t r a d i o l benzoate by guinea pig

Feder

cellular

hypothalamus;

however, CI-628 does not block female reproductive behavior this species Powers

(77).

Since Meyerson and Lindstrom

(87) r e p o r t e d t h a t C I - 6 2 8 a n t a g o n i z e d

(86)

estradiol

and

in

415

benzoate- but not estradiol-induced lordosis behavior, the reported failure of antiestrogens to reduce hypothalamic estradiol uptake might not be unexpected. Landau (88), however, also found that CI-628 did not effect in vivo uptake and retention of 3H-estradiol benzoate by hypothalamic whole homogenates. In spite of the observations cited above, many laboratories have demonstrated that antiestrogens can inhibit cellular uptake and retention of radiolabeled estradiol by the hypothalamus in vivo. Four studies reported that clomiphene depressed hypothalamic uptake of 3H-estradiol (81,89,90) or 3 H-estradiol benzoate (85) in rats and guinea pigs, respectively. The first three studies must be interpreted cautiously because racemic clomiphene was administered, and the trans isomer is an estrogen agonist (see 91,92). Both CI628 (82,93) and nafoxidine (82,94) are also capable of decreasing 3H-estradiol uptake and retention by whole homogenates of female rat hypothalamus. There are no positive reports of MER-25 inhibition of brain estradiol uptake or retention. There is no obvious explanation for the differences in the reported effects of estrogen antagonists on neural uptake of estradiol. The discrepancies cannot be attributed to simple differences in doses, temporal sequence, or route of administration of estrogen and antiestrogen. One can only conclude that some antiestrogens can, under some conditions, interfere with cellular uptake and retention of estradiol by the hypothalamus; therefore, neural uptake and retention are probably prerequisites for estrogen activation of female sexual behavior.

416

Antiestrogen interactions with cytosol receptors in vitro The hypothalamus, preoptic area and amygdala, like other estrogen target tissues, contain specific, high affinity, saturable cytosol estrogen receptors (e.g., 95-97). If behavioral responses to estrogen are, like uterine growth, mediated by a receptor system, one would predict that antiestrogens can compete with estradiol for receptor binding in brain cytosols. Indeed several laboratories have reported that nonsteroidal antiestrogens compete for specific estradiol binding in hypothalamus-preoptic area cytosols (97-102). Moreover, the relative affinities of estrogen antagonists for hypothalamic cytosol receptors correlates well with their potency as inhibitors of estrogen-stimulated female sexual behavior. These data are consistent with the hypothesis that brain estrogen receptors mediate estrogenic activation of female reproductive behavior. The precise manner in which antiestrogens interact with estrogen receptors is still unclear. It is widely accepted that antiestrogens operate by competing directly for the estradiol binding site on the receptor, i.e., by competitive inhibition. This notion is supported both by kinetic studies of estrogen-antiestrogen competition for uterine cytosol binding sites (e.g., 61,103) and by the observation that 3 Hantiestrogens can bind directly to estrogen receptors in a variety of tissues (104-108), including brain (109; Etgen, unpublished observations). However, the results of other kinetic studies suggested that many nonsteroidal antiestrogens, including tamoxifen, were allosteric rather than simple competitive inhibitors of estradiol binding (110). Our studies of antiestrogen interactions with neural estradiol receptors support the view that one cannot explain all of the influences of antiestrogens, at least on brain receptor systems, by competitive inhibition. We examined the competition by unlabeled estradiol, tamoxifen and nafoxidine

417

for 3H-estradiol binding sites in female rat hypothalamuspreoptic area cytosols when the concentrations of both the 3 Hestradiol and the competitors were varied over a large range (100). When these data were analyzed by the method of Scatchard (111), we found evidence of mixed competitivenoncompetitive inhibition of 3H-estradiol binding by both antiestrogens, i.e., either or both the apparent K