LHRH and Analogues as Potential Therapy for Benign

Prostatic Hyperplasia and Hormone-Dependent Cancers

C. AUCLAIR,*M. STERNAND M. L. GIVNER

In adult male rats, daily S.C. injections of 0.008-125 pg luteinizing hormone releasing hormone
(LHRH) or its analogue (D-Ala6,des-Gly-NH2I0)LHRHethylamide, led to significant differences
in inhibitory effects on testicular function and accessory sex organ weights. The analogue was
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at least 35 times more potent than LHRH in reducing testicular LH/hCG receptors and 350
times more effective in decreasing plasma testosterone concentrations. In a second study, adult
male rats treated with 25-2500 pg LHRH daily for a 3-week period showed 85%-90% decrease
in plasma testosterone concentrations. Treatment with 25 or 250 pg LHRH effected a maximal
25% and 40% decrease of the ventral prostate and seminal vesicles, respectively, without
affecting the testicular weight, the latter being reduced by 15% with the higher dose of 2500
pg. The LHRH analogue is proposed for the treatment of prostatic and breast cancers while
native LHRH could be an effective therapy for benign prostatic hyperplasia.

Key Words: LHRH, (D-Alas,des-Gly-NH,’o)LHRH ethylamide;Testicular LH/hCG receptors; Testis; Prostate;

Testosterone.
For personal use only.

INTRODUCTION
Benign prostatic hyperplasia is a common disorder that produces clinical symptoms
in 30%-40% of men over 60 years old [9] and is a frequent cause of hospitalization and
surgery for urinary obstruction. To avoid prostatectomy, various hormonal manipu-
lations have been attempted, including castration [ 121, estrogens [ 181, androgens [ 6 ] ,
and androgen-estrogen combinations [141. Although these therapies often resulted in
some clinical improvement, the side effects were sometimes undesirable and unac-
ceptable.
Administration of luteinizing hormone releasing hormone (LHRH) agonistic ana-
logues were shown to cause a “chemical-like’’ castration associated with reduced sex
steroid production and decreased accessory sex organ weights [ l , 2, 51. The goal of
this study is to verify the concept that LHRH and its potent analogues, e.g., (D-
Ala6,des-Gly-NH2I0)LHRHethylamide, are potentially useful for treating benign pros-
tatic hyperplasia (BPH) and hormone-dependent prostatic and breast cancers.

MATERIALS AND METHODS

Adult male S-D rats weighing 275-325 g were housed five per cage in an air-conditioned and
light-controlled room and given food and water ad libitum. In the first protocol, the animals (eight

Received January 2, 1981; revised April 3, 1981.

From the Ayerst Research Laboratories, P.O. Box 6115, Montreal, Quebec, Canada, H4R 156.
Address reprint requests to: M. L. Givner, Ayerst Research Laboratories, P.O. Box 6115, Montreal,
Quebec, Canada H4R 156.
*Present address: Roussel Canada Inc., 4045 Cote Vertu, Montreal, Quebec, Canada H4R 2E8.
237
8 Elsevier North Holland, Inc., 1981, ARCHIVES OF ANDROLOGY 7937-244 (1981)
52 Vanderbilt Ave., New York, New York 10017
014&5016/8 I /070237-08$02.50
 238 C. Auclair, M. Stern and M. L. Givner

per group) were injected S.C. once daily for 7 days with 0.008-125 pg of LHRH or (D-Alab,des-
Gly-NH2")LHRH ethylamide or the vehicle alone (saline-gelatin, 1%). All animals were killed
by decapitation on the morning of day 8 , 2 4 hr after the last injection. In the second experiment,
rats (10 per group) were injected S.C. daily for 1-3 consecutive weeks with either 25, 250, 2500
pg of LHRH or the vehicle (saline-gelatin, 1%) alone. Rats were killed 24 hr after the last
injection. The testes, seminal vesicles, ventral prostate, and plasma were weighed or collected
arid then li.oLen ( - 20RC) for storage.
Ovine LH (NIH-LH-S21; 2.5 x NIH-LH-S1) was generously supplied by the NIAMDD Pi-
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tuitary Hormone Distributing Program, NIH, Bethesda, MD. Highly purified human chorionic
gonadotropin (hCG) (1 I ,OOO IU/mg), LHRH and (D-Ala6,des-Gly-NH2")LHRHethylamide were
supplied by Drs. K. Singh, K. Sestanj, and H. Immer, respectively, from Ayerst Research
Laboratories, Montreal. Radioiodination of hCG and measurement of testicular LH/hCG receptor
levels were performed as previously described [2]. Plasma testosterone concentrations were
measured using a standard radioimmunoassay technique with a specific single antibody (devel-
For personal use only.

8o t
\ 8
@--Q

D
Testis Weight
Seminal Vesicle Weight
Ventrol Prostrate Weight
- - U Bound hCG
Y Plasma Testosterone

04, ' 1
0 0.008 0.04
1 1
1
I
125
[D-Ala6,des-Gly-NH1"] LHRH-EA (pg)
FIGURE 1. Effect of a 7-day treatment with (D-Ala6,des-Gly-NH,'">LHRHethylamide on testic-
ular LHlhCG receptor levels, plasma testosterone concentrations and testicular and accessory sex
organ weights.
 LHRH and Analogues in BPH and Cancer 239

oped against 19-carboxymethylether-BSA, Radioassay Systems, Inc., Carson, CA) and 2,4,6,7-
3H-testosterone. The limits of the assay were 20-500 pg testosterone per tube.

RESULTS
Subcutaneous injections for 7 consecutive days of LHRH or (D-Ala6,des-Gly-
NH210)LHRHethylamide caused a dose-dependent inhibition of the testicular function
as evidenced by decreased testicular LHhCG binding sites and plasma testosterone
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concentrations (Figs. 1, 2). The LHRH analogue induced a significant (p < 0.01) 43%
loss of LH receptors in animals treated with only 0.008 pg daily, the maximal inhibition
being seen at doses ranging from 0.04 to 1 pg (Fig. 1). Treatment with increasing doses
of the LHRH analogue also caused a graded 80% decrease of plasma testosterone

120-

110-

100-
For personal use only.

90 -

-
-2
c
C
8
80

70-
,,
, I,,
Y

c
0 60-
'la,,
i--4- -/
C
0,
3 50-
L
0,
- -- - -
40 - \

\
\

30 - Testis Weight '4..

O--- 0 Seminal Vesicle Weight *',
2o - Ventral Prostrate Weight %
',

- -0
.+-**'a=-
0- - - 0 Bound hCG
- p----d Plasma Testosterone

I I f I I I
O-;b 0.04 0.2 1 5 25 125
 240 C. Auclair, M. Stern and M. L. Givner

concentrations from 5.2 ? 0.3 to 1.0 2 0.2 ng/ml. The near maximal inhibition was
reached with daily doses of 0.04 pg or more. The decrease observed in circulating
levels of testosterone is of the same order of magnitude and is parallel with the reduction
of testicular LH receptor concentrations. The weights of the testes, seminal vesicles
and the ventral prostate were also significantly ( p < 0.01) reduced after a7-day treatment
with 1 pg or more of the LHRH analogue (Fig. 1).
In contrast to its analogue, a 7-day treatment with LHRH at doses up to 125 pug had
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no effect on the weight of the testes and the ventral prostate even though the natural
peptide induced a marked reduction of testicular LWhCG receptor levels and plasma
testosterone concentrations (Fig. 2). At the highest concentration used (125 pg), LHRH
caused a 55% maximal reduction of testosterone levels (Fig. 2), while the analog
produced a 80% decrease (Fig. 1). The calculated EDSosfor testicular LH/hCG receptor
levels are 0.35 pg for LHRH and 0.01 pg for the analogue.
Treatment with 25 or 250 pg LHRH for 3 consecutive weeks caused a maximal 25%
and 40% decrease of the ventral prostate and seminal vesicle weight, respectively,
while it did not affect the weight of the testes (Table I). At the two lower doses, the
inhibitory effect of LHRH on accessory sex organ weights was significant after 2 weeks
of treatment, while at 2500 pg daily, it already reduced the weight of accessory sex
For personal use only.

TABLE 1 Effect of a 1 to 3 Weeks' Treatments with LHRH on the Weights of Seminal Vesicles, Ventral
Prostate and Testes (Mean & SEM)

WEEKS OF TREATMENT
TREATMENT"
0 1 2 3

Testes (g)

Control 2.99 t 0.09 2.81 t 0.14 3.16 t 0.10 3.14 f 0.14

LHRH 25 pg 2.79 f 0.12 3.06 f 0.05 3.11 f 0.14
250 Pg 3.01 f 0.08 2.88 t 0.14 3.17 t 0.10
2500 pg 2.75 t 0.12 2.65 f 0.09' 2.74 t O.lOb

Seminal Vesicles (mg)

Control 295 t 10 305 f 10 354 f 10 353 f 16

LHRH 25 pg 281 t 14 247 f 9 260 t 13'
250 j ~ g 298 t 10 249 f 18' 210 t 8'
2500 pg 265 t lob 236 -t 13' 225 t 9'

Ventral Prostate (mg)

Control 342 t 25 394 f 24 490 2 3 1 4w 2 3 1

LHRH 25 pg 359 f 23 415 f 31b 378 t 13'
250 jLg 360 2 26 380 f 38' 370 f 25'
2500 j ~ g 325 f 12b 355 t 31' 373 f 18'

"Ten rats for each control and treated group at time of death.
bp<0.05, experimental versus control of the same week.
'p<O.OI, experimental versus control of the same week.
 LHRH and Analogues in BPH and Cancer 241

1 00

90

80
h

70
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60

50

40
w
e
I 30

20
For personal use only.

10

0 1 2 3
Weeks of Treatment
FIGURE 3. Effect of 1 to 3 weeks' treatment with LHRH on testicular LHhCG receptor levels.

organs after the first week of treatment. However, this high dose also caused an atrophy
of the testes that was observed after the second week of treatment. Chronic daily
administration of LHRH for 3 weeks caused a marked decrease of testicular LH/hCG
receptor levels (Fig. 3). A near maximal 85% inhibition of LH receptors was reached
after the first week of treatment with 25 pg LHRH and no further reduction was
observed with time or higher doses.
All treatments with LHRH caused a marked reduction of plasma testosterone con-
centrations (Fig. 4). Regardless of the dose used, the reduction was already highly
significant ( p < 0.01) after 1 week of treatment and remained suppressed throughout
the duration of the study.

DISCUSSION
Daily subcutaneous injections for 7 days of LHRH or (D-Ala6,des-Gly-NHzlo)LHRH
ethylamide caused a dose-related inhibition of testicular function. The analogue was
at least 35 times more potent than LHRH in effecting down-regulation of LH receptors
and 350 times more active in decreasing circulating levels of testosterone. At the highest
dose used (125 pg daily), the analogue induced a maximal 80% reduction of plasma
 242 C. Auclair, M. Stern and M. L. Givner

O---a LHRH 25 pg
)---I LHRH 2 5 0 p g
LHRH 2500 pg
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For personal use only.

I
0
I
1
I
2
I
3
Weeks of Treatment
FIGURE 4. Effect of 1 to 3 weeks' treatment with LHRH on plasma testosterone concentrations.

testosterone levels while LHRH caused only a 55% decrease despite the fact that in
both cases LH binding sites were reduced by 90%. The difference between LHRH and
its analogue on the androgen levels is further reflected by the lack of inhibitory effect
of LHRH on the testicular and ventral prostatic weight under these experimental con-
ditions.
Chronic daily subcutaneous administration for 3 consecutive weeks of 25 pg and 250
pg LHRH, in addition to causing desensitization of the testicular tissue, can selectively
decrease the weight of seminal vesicles and ventral prostate without affecting the weight
of the testes. Testicular weights were decreased only at the highest dose of 2500 pg
daily. This selective effect of LHRH is of major interest because highly potent analogues
of LHRH such as (D-Ala6,des-Gly-NH2l0)LHRH-EA and (D-Leu6,des-Gly-NH2l0)
LHRH-EA do not show such a selectively [ I , 2, 51.
Peptide and steroid hormones have specific receptor sites located in respective target
tissues. LH receptors have been demonstrated in the gonads of several species, in-
cluding man [7], by autoradiography [ 191, immunochemistry [3], and in vitro radiore-
ceptor assay [4]. To trigger its biological action, the LH molecule has to bind to a
highly specific receptor located on the plasma membrane of testicular Leydig cells [3]
and ovarian granulosa and luteal cells [ 191. The resulting hormone-receptor complex
 LHRH and Analogues In BPH and Cancer 243

activates the CAMP-protein kinase system [201, which then generates the hormonal
response. However, exogenous administration of LH or hCG have been reported to
induce a marked loss of LH/hCG receptors [2, 111 and to impede the steroidogenic
response to gonadotropins [I 11. Such down-regulation of hormone receptors by the
hormone itself has also been described for insulin [8], thyrotropin-releasing hormone
[lo], growth hormone [16], and catecholamines [17]. The concept of receptor down-
regulation is presently under extensive investigation and could prove to be as effective
as a “chemical castration.”
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Chronic treatment with LHRH showing less deleterious effect on the testicular tissue
than its analogue, appears to offer more potential for treating benign prostatic hyper-
plasia. However, a potent LHRH analogue such as (D-Ala6,des-Gly-NH210)LHRH
ethylamide because it effected more rapid and pronounced desensitization of gonadal
functions would appear to be more promising than LHRH itself for the clinical treatment
of prostatic cancer. Potent LHRH analogues could be also very useful for the treatment
of breast cancer since they have been shown to block the ovarian function [15] and to
cause regression of DMBA-induced rat mammary tumors [13].

Acknowledgments: We are grateful to Mr. P. Gauthier, Mrs. M. Goupil-Turcotte, and Miss R. Singh
for their skillful assistance.
For personal use only.

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