Pharmacology of an antiandrogen, anandron, used as an adjuvant therapy in the treatment of prostate cancer

J. steroid Biochem. Vol. 24. No. 1. pp. 139-146, Printedin Great Britain. All rights reserved

1986 Copyright

f

0022-4731/86 $3.00 + 0.00 1986 Pergamon Press Ltd

PHARMACOLOGY OF AN ANTIANDROGEN, ANANDRON, USED AS AN ADJUVANT THERAPY IN THE TREATMENT OF PROSTATE CANCER MARTINE MOGUILEWSKY*,

and *RousselLUclaf.

JEAN FIET~, COLETTE TOURNEMINE*

JEAN-PIERRE RAYNAUD*

35 Boulevard des Invalides,

Paris 75007 and tH6pital

St-Louis,

Paris 75010,

France

Summary-To improve the inhibition of prostate cancer growth obtained by surgical or chemical castration (estrogens or LHRH analogs), blockade of the action of residual androgens of adrenal origin has been proposed. Among antiandrogens acting through the androgen receptor (AR), the nonsteroid anandron (RU 23908) has several advantages over available compounds: megestrol acetate and cyproterone acetate, both steroids, bind substantially to other hormone receptors (progestin, gluco- and mineralocorticoid): and anandron binds only to AR. The nonsteroid flutamide is a prodrug converted to the active metabolite, hydroxyflutamide; anandron is well absorbed on oral administration of an active dose and intact compound disappears slowly from plasma. This may explain why, although in vitro anandron interacts very transiently with AR, in vivo a high level of untransformed anandron is present at the receptor site to induce its antiandrogenic activity. Animal experiments confirm that anandron can counteract the effect of adrenal androgens and inhibit the LHRH analog-induced initial increase in androgen (“flare-up”). Thus, in rats castrated either surgically or by buserelin or DES and supplemented with adrenal androgens (since endogenous adrenal secretion is very low in this species compared to man), anandron decreased prostate weight to control levels. The administration of buserelin to intact rats over 15 days resulted in a significant increase in prostate weight between Days 1 and 5. The addition of anandron to the buserelin inhibited this increase and, furthermore, led to a far greater decrease in prostate weight than that due to buserelin alone at 15 days, indicating a synergy of action.

INTRODUCTION

Ever since Huggins and Hodges proposed castration as a therapy for prostate cancer over 40 years ago [ 11, this has been considered the standard form of therapy by both urologists and oncologists. The most recent development in the field has been the introduction of reversible medical castration as an alternative to surgical castration following the demonstration of the effect of LHRH analogs so-called “paradoxical” [2-51 which, at pharmacological doses, reduce, via desensitization of pituitary LHRH receptors, plasma testosterone concentration to castration levels. However, the exquisite hormone dependence of prostate cancer cells should offer other, maybe more effective, means of combatting the disease. Attempts have thus been made to develop compounds that suppress the biosynthesis of androgens (enzyme inhibitors) or that prevent the binding of endogenous androgens to their receptor (antiandrogens). Each treatment so far has obvious drawbacks: -orchiectomy often does not elicit the full unconditional consent of the patient and necessitates hospitalization; although a psychologically-favored -estrogens, means of achieving castration, give rise to cardiovascular complications in many patients that are costly and that can result in discontinuation of treatment [6, 71; -LHRH analogs bring about a castration level of testosterone only after an initial rise that is often

reflected in a “flare-up” of the disease [8-lo]; -enzyme inhibitors act unspecifically on enzymes involved in the biosynthetic pathways of both androgens and glucocorticoids and can only be used in conjunction with some form of replacement therapy that often cancels the benefits [ll-131; -antiandrogens specifically block the action of androgens, whether of testicular or adrenal origin, at all target sites, but blockade of pituitary receptors interferes with the feedback mechanism which controls testosterone concentration [14]; consequently, although administration of anti-androgens may well block androgen action, it increases testicular androgen production which in turn counteracts the antiandrogen effect in peripheral organs (i.e. the prostate) [15-l 71. In the human, the adrenals are an important source of weak androgens. Some of them can be converted into testosterone and dihydrotestosterone (DHT) in the prostate cell and thus sooner or later reactivate tumor growth [18-221. Castration does not suppress the secretion of these androgens. A logical step would therefore seem to be the combination of castration with an antiandrogen to achieve total androgen blockade[23-261. In cases. where castration is achieved by LHRH analogs, the antiandrogen would have the additional effect of preventing the adverse effect of the testosterone surge. We have therefore undertaken to verify these hypotheses in an animal model designed to artificially 139

MARTINE M~GUILEWSKY et

140

al.

Flutamide

Cyproterone

reproduce animal

conditions

prevalent

RU 23908 IANANDRON

acetate

Fig. 1. Structures of antiandrogens

in man.

on the market or in clinical

Disregarding

tumor

models too remote from human prostate cancer cells, we studied instead the rationale of our combined therapy in rats supplemented with exogenous adrenal androgens to levels comparable to man. The fact that the rat normally secretes only a marginal amount of adrenal androgens is often neglected in the interpretation of experiments in this species [27]. Amongst the large number of available steroid and nonsteroid anti-androgens, only few are either on the market or in clinical trials (Fig. I). Megestrol acetate is prescribed in association with estrogens on the west coast of North America [28]; cyproterone acetate has been selected for many oncology trials in Europe [29]; flutamide has been under investigation in the U.S.A. for at least a decade and is now marketed in some countries [30]; anandron, a more recent compound, is being tested in a double blind-clinical trial in Europe following the promising results obtained by F. Labrie’s group [25,31-331. We decided to select anandron (5,5-dimethyl-3-[4-nitro-3-(trifluoromethyl)phenyl]-2,4_imidazolidinedione) for our studies for the reasons outlined below. INTERACTION OF ANTIANDROGENS WITH CYTOSOL STEROID HORMONE RECEPTORS

As shown in Table 1, the relative binding affinity (RBA) profiles of steroid and nonsteroid antiandrogens for the cytosol receptors of the five steroid hormone classes are very different. Whereas nonwith antiandrogens competed only steroid [-‘HItestosterone binding for the androgen receptor Table

I.

RBAs of antiandrogens

trials for prostate

1

cancer

therapy.

(AR), steroid antiandrogens, with structures closer to that of the natural hormone, also inhibited the binding of [3H]promegestone to the progestin receptor (PR), of [3H]dexamethasone to the glucocorticoid receptor (GR) and of [ 3H]aldosterone to the mineralocorticoid receptor (MR). Moreover, although the RBAs of all compounds for AR decreased with incubation time, indicating that the androgenreceptor complexes formed are more short-lived than the testosterone-receptor complex, steroid antiandrogens exhibited a higher affinity for AR than nonsteroids. This could explain the weak agonist activity of megestrol acetate or cyproterone acetate under certain conditions [35. 361. Amongst nonsteroid antiandrogens, flutamide had a very weak RBA for AR while hydroxyflutamide interacted with the same affinity as anandron. This could explain why flutamide is practically devoid of activity in uitro and needs to be converted into hydroxyflutamide in ciao to exert its antiandrogen effects [37, 381. Anandron was therefore selected for further study since, unlike steroid antiandrogens, it does not interact with steroid hormone receptors other than AR and is thus devoid of hormonal side effects and since, unlike flutamide, it does not need to undergo conversion to an active metabolite [33, 391. KINETICS OF INTERACTION OF ANANDRON WITH AR In vitro

interaction

As shown in Table I and Fig. 2a, the RBA of anandron for the prostate cytosol AR decreased

for cytosol steroid

hormone

receptors*

AR 30 min Cyproterone acetate Megestrol acetate Flutamide Hydroxytlutamide Anandron

20 39 0.3 4.5 4.5

24 h

PR 2h OC

GR 4hO C

MR I h0 C

ER 2hOC

8 5 < 0.1 0.5 0.5

60 I20 < 0.1 < 0.1 < 0.1

5 50
I 3 to.1 < 0.1
< < < < <

*AR. PR, CR. MR and ER: androgen (rat prostate), progestin (rabbit uterus). mineralocorticoid (rat kidney) and estrogen (mouse uterus) receptors, respectively. described previously [34]. The RBAs of testosterone, progesterone, dexamethasone, AR. PR. GR. MR and ER were taken to be equal to 100.

0.1 0.1 0.1 0.1 0.1

glucocorticoid (rat thymus), The RBAs were measured as aldosterone and estradiol for

Pharmacology of anandron

IN VITRO

012

4

incubation time (hours)

(lOma/kd

0.01 M Tris-HCl (pH 7.4) 0.25 M sucrose, 0.002 M dithiothreitol] of ventral prostates from castrated (CX) adult male rats. Aliquots were incubated for different time intervals with 5 nM [3H]methyltrienolone at 0°C in the presence or absence of increasing concentrations of either testosterone or anandron (S-25,000 nM). The RBA of anandron was determined by a DCC adsorption technique, as previously described [16]. The RBA of testosterone was taken to be equal to 100. (b) In vivo interactionwithcytosol AR. Groups of 5 CX adult male rats received oral administration of lOmg/kg of anandron or solvent (controls) and were killed at different time intervals after treatment. Cytosols from pooled prostates were prepared and incubated for I h at WC with 10 nM [3H]methyltrienolone in the presence or absence of 1 PM radioinert methyltrienolone and in the presence of 1 PM of triamcinolone acetonide to determine free specific androgen binding sites [17]. Bound [‘Hlmethyltrienolone was determined by a DCC adsorption technique. The results are the means of 2-4 determinations (W----O). Plasma kinetics. Groups of adult male rats received an oral administration of 10 mg/kg of [ “C]anandron. Blood was drawn at different time intervals after treatment. The radioactivity associated with anandron was determined in plasma after extraction and separation by TLC (0 -- - - 0) [33,39].

time at 0°C indicating that anandron forms a fleeting complex with AR [33]. No metabolic degradation occurs under these conditions. with

the rat (Fig. 2b), absorption was rapid (halflife = 0.34 h) and followed by a high plateau of unchanged compound (5-6 pg/ml, accounting for 95-70% of the radioactivity) which persisted for 6 h and then disappeared slowly (terminal half-life of 7 h) [33,39]. ANTIANDROGEN ACTIVITY OF ANANDRON ON THE “CASTRATED” RAT PROSTATE

Fig. 2. Kinetics of interaction of anandron with cytosol AR of rat prostate. (a) In vitro. Cytosol was prepared by centrifugation for 1h at 105,OOOg of a homogenate [in

markedly

141

incubation

In vivo interaction Following oral administration of lOmg/kg of anandron to castrated rats, only 2@-40% of prostate androgen binding sites were unoccupied over the first 16 h (Fig. 2b). The high and prolonged occupation level of cytosol AR in vivo, which contrasts with the rapid dissociation observed in vitro, is explained by the long plasma half-life of anandron [33]. After an oral administration of 10 mg/kg of [ 14C]anandron to

The sustained in viva interaction of anandron with AR led to only very weak nuclear retention of the receptor in the prostate of castrated rats and thus to no agonist activity, i.e. no increase in prostate weight even after an 8-day treatment with 10 mg/kg (Fig. 3). Testosterone propionate treatment (0.25 mg/kg), on the other hand, gave rise to a high nuclear AR level and a marked increase in prostate weight. Because anandron sequesters AR in the cytosol compartment, it prevents testosterone binding and thus impedes androgen action. The antiandrogen activity of anandron on the prostate is fully manifest in castrated animals but weakened in intact animals by the compensatory increase in testicular secretion resulting from interference with the hypothalamo-pituitary-gonadal axis [16, 171. As demonstrated below, it can be used against adrenal androgens left intact by any form of castration and against the known testosterone surge occurring after LHRH analog administration. Antiandrogen activity of anandron against adrenal androgens

As illustrated in Fig. 4, the plasma concentrations of androgens of adrenal origin are totally different in humans and rats. Human adrenals secrete very

control*

H Fig. 3. Androgenic and antiandrogenic activity of anandron in castrated adult male rats. Groups of 5 adult male rats (200 g) castrated for 24 h received daily for 8 days an oral dose of anandron (10 mg/kg/day), an S.C. injection of testosterone propionate (0.25 mg/kg/day) or both treatments. Control rats were injected with solvent only. The rats were killed 2 h after the last treatment, ventral prostates were removed, weighed, pooled and homogenized in 5 vol of buffer. A crude nuclear pellet was obtained by three IO-min centrifugations at 800 g and suspended in an equal volume of buffer. Androgen binding sites were measured after 24 h incubation at 0°C with increasing concentrations of [3H]methyltrienolone with or without a IOO-fold excess of unlabeled methyltrienolone and in the presence of a lOO-fold excess of triamcinolone acetonide. Nuclei were then reprepared and washed three times with buffer and the radioactivity incorporated was counted.

MARTINE MOGUILEWSKYet ul.

142

DHA

1 lphydroxy

10.4-4.3)

~ost~~ione

DHAs

F

(60-2310)

1001

Bndrostenedione

(0.28-2.23)

I

50(1

I

Fig. 4. Plasma concentrations of androgens in the human and in the rat. Androstenedione. 1I/?-hydroxyandrostenedione and DHA were measured simultaneously by radioimmunoassay after celite chromatography [40] in the plasma of orchiectomized prostate cancer patients (aged between 46 and 70. n = 15; 0) and of orchiectomized adult male rats (n = 5); !ZI).DHAs was determined in the same plasmas by radioimmunoassay using a DHA-3CMO-BSA antiserum (Kit ER-660) (EIR-5303 Wiirenlingen. Switzerland). Means & SD and ranges are represented.

high levels of androgens, especially dehydroepiandrosterone sulfate (DHAs), whereas, in rat plasma, adrenal androgens are very low or even undetectable. To measure the antiandrogen effect of anandron against adrenal androgens in an experimental model closer to the human, rats were implanted with osmotic minipumps containing the four adrenal androgens (Table 2). Controlled doses were released

Table 2. Inhibition

by anandron

of the e&t

over a period of 1.5days to obtain circulating Levels (0.7&-0.1, t.3i:0.2, 3.7kO.6, 684+55ng/ml, respectively for androstenedione, 1Ifi-hydroxyandrostenedione, DHA and DHAs) in the same range as those recorded in orchiectomized humans (Fig. 4). As shown in Table 7 (Experiment l), when intact rats receiving adrenal androgens had both their sources (endogenous and exogenousj of androgens suppressed (orchiectomy or DES treatment and

of adrenal

androgens

m castrated Prostate

Circulating Testicular E.rperiment I

+ _ _ _ _

Orchiectomy

_ _

DES

androgens -.. ----Adrenal

+ + + + + f t -I-

:

-

Experiment 2 Busereiin

Orchiectomy

+ t-

-

+

-

+ +

i- _

+ -

Anandron (ma/rat/day) 0 0 0

0.4

1; 0 0 0.4 2 IO 0 0 0.2

1 5

0

MeUl rt SEM 275.2 23.8 105.2 59.3 40.0 24.4

male rats”

weight (mg)

i: 23.2 _t I .7 * 20.3 t 6.3 & 7.7 + 3.0

33.0 2 6.0 173.6 * 22.3 123.3 * 9.4 73.5 t_ x.1 30.X + 2.6 774.0 * 5x.5 534.6 i 23.1 5419 +: 37.4 41 I .o+4X.6 216.3 _+ 14.8 41.9 * 3.0

% Control 9 38 21 15 9 I’ 63 4s 27 II 69 71 53 28 5

‘Groups of 5 adult male rats (around 25Og in Experiment I and4008 in Experiment 2) were implanted with osmotic minipumps (Alzet) releasing controlled doses of adrenal androgens (androstenedione 96 pg/24 h, I Ifi-hydroxyandrostenedione 6Ofig,Q4 h. DHA 144 pg/24 h and DHAs 2400 pg/24 h) in order to obtain circulating levels equivalent to those recorded in humans (see the text). Testicular secretion was suppressed either by orchieclomy, DES treatment (IO fig/rat/day) or buserelin (250 n&at/day). Some rats received oral administration ofanandron from the first day of castration, All rats were killed I5 days after the beginning of treatment. Prostates were removed and weighed.

Pharmacology of anandron

of adrenal androgen supplementation), their prostates atrophied down to 9 and 12% of control, respectively. When only testosterone was suppressed either by orchiectomy or by DES treatment, prostate weight levelled off at 38% (for orchiectomized rats) and 63% (for DES-treated rats) of control due to the trophic effect of adrenal androgens transformed into active androgens. Although DES treatment led to a decrease in prostate weight comparable to orchieetomy in the absence of adrenal androgen supplementation, indicating that testicufar secretion had been totally suppressed by estrogens. the trophic effect of adrenal androgens was more important in estrogen-treated rats. This could be due to the induction of AR by estrogen treatment in the prostate [41], hence enhancing the androgen sensitivity of the organ. In this experiment, the AR concentration in DES-treated rat prostate was 4.4 pmolig tissue while AR concentration in orchiectomized rat prostate was only f .8 pmol/g tissue. When anandron was administered to orchiectomized or DES-treated rats. the effect of adrenal androgens was totally counteracted by a dose of 10 mg/rat/day. In the rat, testosterone secretion is less effectively suppressed by daily S.C. injections of the LHRH analog buserelin (250 ng) than by other types of castration since these injections act principally on the testes (desensitization of LH receptors) rather than on the pituitary as in the human [42]. Pituitary receptor down-regulation can only be maintained in the rat by repeated administration at short time intervals or by continuous administration by infusion or implants [5,42]. This may expfain why in Experiment 2 (Table 2) prostate weight was only decreased to 69% of control in adrenal-androgen-supplemented rats treated with buserelin. Combining anandron with buserelin, however, fed to a marked further decrease (to 28% of control at S mg/rat/day). PROSTATE

01 0

143

5

10

15

DAYS

Fig. 5. Inhibition of the effect of the initial rise in testosterone induced by buserelin treatment in the rat. Groups of 5 adult male rats received daily for IS days a S.C.injection of buserehn (2.50ng~rat~dayj. an oral administration of anandron (5 mg/rat/day) or both treatments. Control intact rats and a group of castrated rats received solvent only. The rats were killed after 2, 4 or IS days of treatment or of castration. Prostates were removed and weighed. Prostate weights are represented as a percent of that of intact control rats and are the means of 3 experiments.

Anti~ndroge~ activity of a~a~dro~ against the rise in testosterone induced by LHRH anaIogs

When intact rats (unsupplemented with adrenal androgens) received daily S.C. injections of buserelin (250 ng/rat/day) (Fig. 6), their prostate weights were increased during the first few days of treatment. The castrating effect of the peptide started only after 6 or 7 days in contrast to the immediate atrophic effect of orchiectomy. When anandron (5 mg/rat~day) was administered together with buserelin, the “flare-up” effect of buserelin was totally inhibited and the onset of the castrating effect of the combination was very similar to that achieved by orchiectomy.

SEMINAL VESICLES

TESTES

BUSERELIN fng/rat/day)

Fig. 6. Potentiation by anandron of the “castrating” effect of buserelin in the rat. Groups of 5 adult male rats received daily for 15 days a S.C. injection of buserelin, an oral administration of anandron (5 mg/rat/day) or both treatments. Control intact rats or castrated rats received solvent only. Rats were killed 24 h after the last treatment. The prostates, seminal vesicles and testes were removed and weighed. Organs weights are represented as a percentage of that of control intact rats [organs from orchidectomized (cx) rats =O%].

MARTINE MOGUILEWSKK et (11.

144

POTENTIATION OF THE CASTRATING EFFECT OF LHRH ANALOGS BY ANANDRON IN THE RAT

When buserelin (50-1250 ng/rat/day) was administered S.C. daily for 15 days to male rats, prostate and seminal vesicles were decreased in a dose-dependent manner but the inhibition was never as important as that produced by orchiectomy (inhibition considered as 100%) since testosterone was never totally suppressed by this mode of treatment in the rat (Fig. 6). The combination of anandron (5 mg/rat/day) with low doses of buserelin led to a total decrease in prostate and seminal vesicle weights due to an additivity of effects. Anandron impedes the action of any residual testosterone on the prostate and seminal vesicles and buserelin prevents the rebound testosterone increase induced by anandron. However, whereas anandron alone has a weak trophic effect on the testes and buserelin a weak atrophic effect (related to testicular desensitization), the combination of both compounds led to a marked decrease in testicular weight more characteristic of a synergistic than an additive effect. DISCUSSION

Amongst antiandrogens at present under clinical study or on the market, nonsteroids (flutamide and anandron) offer the advantage over steroids (megestrol acetate and cyproterone acetate) of interacting only with AR and not with other steroid hormone receptors. They are thus devoid of other hormonal or anti-hormonal activities. Moreover, the structural similarity of steroid antiandrogens and natural hormones implies better recognition by AR than for nonsteroids. Their higher RBAs can, in certain experimental conditions, be related to a weak androgenie effect [35,36]. In contrast, nonsteroid antiandrogens can be considered to be “pure” antiandrogens. These antiandrogens are active on all target cells containing AR and thus inhibit the negative feedback of androgens at the pituitary level, giving rise in the case of nonsteroids to an increase in testicular secretion [15, 161. Since megestrol acetate and cyproterone acetate are progestational, their antigonadotropic activity compensates for a time their antiandrogen activity on the pituitary, but their hypothalamic anti-androgen action leads to an increase in LHRH which enhances LH and testosterone secretion during treatment [43]. Therefore, anti-androgens are more effective on prostate cells when the compensatory increase in testosterone secretion is suppressed by surgical or chemical castration. Unlike flutamide which needs to be converted into its hydroxy metabolite to interact with AR [38], anandron is not a prodrug but is an antiandrogen per se [33.39]. It is thus the anti-androgen of choice to be added to classical treatments suppressing testicular androgens. Anandron interacts very transiently with AR in

vitro while, in z&o, the interaction with androgen binding sites is very prolonged. This discordance can be explained by the kinetics and metabolism of the compound: the slow disappearance rate from plasma and the low rate of metabolism maintain a permanently high level of unchanged compound in the vicinity of the target cell, which can reassociate with the receptor as and when dissociation occurs. However, in spite of this sustained interaction with the receptor in vitro, little receptor is retained in the nucleus, much less than with agonist hormones, so that no androgenic effect of anandron is observed [33]. Since anandron sequesters AR in the cytosol compartment, it can impede the action of any androgen. It can therefore be used as an adjuvant therapy to chemical or surgical castration to counteract the effect of adrenal androgens in prostate cancer patients. Since the rat secretes very low levels of adrenal androgens, the advantage offered by an antiandrogen over castration alone cannot be demonstrated unless adrenal androgens are added in circulating amounts equivalent to those recorded in humans. In this way, we have shown that the activity of adrenal androgens is far from negligible since they have at least 3&40% of the trophic activity of total androgens on the prostate and that anandron can totally block their activity. Anandron can thus offer a real improvement upon classical treatments. Anandron would also be effective in patients castrated by estrogens but, according to our animal experiments, higher doses might be required here to counteract the greater activity of adrenal androgens resulting from higher AR concentrations in the prostate. The estrogen inducibility of androgen binding sites [41, 331 has also been demonstrated in the Dunning rat tumor model [44], the androgen-dependent Shionogi carcinoma [45] and in DES-treated prostate cancer patients [46]. This method of castration should be progressively replaced by other types of castration not only because of its known cardiovascular side effects but also because it increases the sensitivity of prostatic cells to androgens. When combined with castration by an LHRH analog, anandron inhibits not only the activity of adrenal androgens but also any temporary increase in androgen secretion due to the LHRH analog treatment. In rats whose prostate weights were temporarily increased by buserehn treatment, anandron was able to counteract this increase. Finally, we have shown that, in the rat, anandron potentiates the castrating effect of an LHRH analog. Daily S.C. injections of the LHRH agonist for 15 days do not totally suppress testicular secretion, unlike continuous administration which can lower testosterone to castration levels [5,27]. Lower S.C. doses of buserelin were necessary to decrease prostate weight to castrate levels in the presence of anandron because anandron counteracts the effect of this residual testosterone on the prostate. Since the combination of

Pharmacology

both compounds greatly decreases testes weight, potentiation by anandron of the effect of the LHRH analog might occur not only in the prostate but also either directly in the testes where, as mentioned earlier, the LHRH analog also exerts a castrating effect in the rat, or in the pituitary where anandron has been shown to increase the sensitivity of pituitary cells to LHRH administration [16]. LHRH receptors have also been identified in rat Leydig cells [47] and are thought to mediate inhibition by LHRH agonists of Leydig cell function, resulting in a decrease in testosterone secretion. Anandron might increase the sensitivity of LHRH receptors to LHRH agonists in the testes as well as in the pituitary. This could lead either to a faster castrating effect of buserehn or to an action of lower doses of LHRH analog in the presence of anandron. Whether such an effect also exists in the human is under investigation and may be highly relevant to the clinical use of LHRH analogs.

of anandron

11.

12.

13.

14.

15.

16.

17. REFERENCES 1. Huggins C. and Hodges C. V.: Studies of prostatic cancer. I. Effect of castration, estrogen, and androgen injections on serum phosphatases in metastatic carcinoma of the prostate. Cancer Res. 1 (1941) 293-297. 2. Auclair C., Kelly P. A., Coy D. H., Schally A. V. and Labrie F.: Potent inhibitory activity of (D-Leu6, DesGlv-NH!“) LHRH ethvlamide on LHihCG and PRL tesiicular receptor leveis in the rat. Endocrinology 101 (1977) 189&1893. 3. Sandow J., Von Rechenberg W., Jerzabeck G. and Stall W.: Pituitary gonadotropin inhibition by a highly active analog of luteinizing hormone-releasing hormone. Ferf. Steril. 30 (1978) 2055209. A.: LHRH 4. Schally A. V.. Coy D. H. and Arimura agonists and antagonists. Int. J. Gynaec. Obstet. 18 (1980) 318324. 5. Sandow J. and Beier B.: LHRH agonists-mechanism of action and effect on target tissues. In EORTC Genitourinary Group Monograph 2, Part A : Therapeutic Principles in Metastatic Prostatic Cancer (Edited by F. M. Schrijder and B. Richards). Liss, New York (1985) pp. 121-142. 6. Byar D. P.: The Veterans Administration Cooperative Urogical Research Group’s studies of cancer of the prostate. Cancer 32 (1973) 11261130. Hedlung P. 0.. Gustafsson H. and Sjogren S.: Cardiovascular complications to treatment of prostate cancer with estramustine phosphate (Estracyt’K ) or conventional estrogen. A follow-up of 212 randomized patients. Stand. J. Ural. Nephrol. (Suppl.) 55 (1980) 103~105. Kerle D., Williams G., Ware H. and Bloom S. R.: Failure of long-term luteinising hormone releasing hormone treatment for prostatic cancer to suppress serum luteinising hormone and testosterone. Br. med. J. 289 (1984) 468-469. Debruyne F. M. J., Karthaus H. F. M., Schrijder F. H., de Noogt H. J, de Jong F. H. and Klijn J. G. M.: Results of a Dutch phase II trial with the LHRH agonist Buserelin in patients with metastatic prostatic cancer. In EORTC Genitourinary Group Monograph 2, Part A: Therapeutic Principles in Metastatic Prostatic Cancer (Edited by F. H. Schrtider and B. Richards). Liss, New York (1985) pp. 251-270. 10. Donnelly K. J.: Contmuous subcutaneous admmtstration of “Zoladex” (ICI 118, 63&an LHRH ana-

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