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The Prostate 37:10–18 (1998)
Novel Nonsteroidal Inhibitor of Cytochrome
P45017␣ (17␣-Hydroxylase/C17–20 Lyase),
YM116, Decreased Prostatic Weights by
Reducing Serum Concentrations of Testosterone
and Adrenal Androgens in Rats
Yukitaka Ideyama,1 Masafumi Kudoh,1 Kyoko Tanimoto,1 Yoko Susaki,1
Taiki Nanya,1 Takahito Nakahara,1 Hiroko Ishikawa,1 Toru Yoden,1
Minoru Okada,1 Takashi Fujikura,1 Hideyuki Akaza,2 and Hisataka Shikama1*
1
Metabolic Diseases Research, Pharmacology Laboratories, Institute for Drug Discovery
Research, Yamanouchi Pharmaceutical Co., Ltd., Tsukuba, Ibaraki, Japan
2
Department of Urology, Institute of Clinical Medicine, University of Tsukuba,
Tsukuba, Ibaraki, Japan
BACKGROUND. The purpose of this study was to determine the effects of a nonsteroidal
C17–20 lyase inhibitor, 2-(1H-imidazol-4-ylmethyl)-9H-carbazole (YM116), on serum concentrations of androgens and ventral prostatic weight in rats.
METHODS. Serum concentrations of testosterone and of dehydroepiandrosterone sulfate
and prostatic weights were measured in rats treated with YM116.
RESULTS. YM116 inhibited testicular C17–20 lyase competitively (Ki, 0.38 nM), and decreased the serum testosterone concentration in gonadotropin-releasing hormone-treated rats
(ED50, 0.7 mg/kg), indicating that YM116 was about 21–24 times more potent than other
C17–20 lyase inhibitors such as ketoconazole and liarozole, and was twice as potent as
CB7630. YM116 also reduced dehydroepiandrosterone sulfate levels in ACTH-treated castrated rats (ED50, 11 mg/kg). YM116 (40 mg/kg, p.o., for 2 weeks) was almost comparable to
bilateral orchiectomy with respect to the time course and magnitude of the reduction in
prostatic weight. Each of these two treatments decreased the prostatic weight 3 days following
the treatment. Contrarily, leuprolide transiently increased the prostatic weight and then decreased it. YM116 (100 mg/kg) had no effect on the serum cortisol level in guinea pigs, and
slightly decreased the serum aldosterone level in rats.
CONCLUSIONS. YM116 is a selective C17–20 lyase inhibitor which decreases rat prostatic
weight by reducing androgen production in the testes and adrenal glands. Prostate 37:10–18,
1998. © 1998 Wiley-Liss, Inc.
KEY WORDS:
adrenal gland; orchiectomy; ketoconazole; leuprolide; CB7630; liarozole
INTRODUCTION
Since most prostate cancers show androgendependent growth, several antiandrogenic therapies
have been attempted against this disease. Gonadotropin-releasing hormone (GnRH) agonists are widely
recognized as a medical alternative to orchiectomy in
© 1998 Wiley-Liss, Inc.
the treatment of prostate cancer because of the potent
inhibition of testicular testosterone synthesis by GnRH
*Correspondence to: Hisataka Shikama, Ph.D., Metabolic Diseases
Research, Pharmacology Laboratories, Institute for Drug Discovery
Research, Yamanouchi Pharmaceutical Co., Ltd., 21 Miyukigaoka,
Tsukuba, Ibaraki 305, Japan.
Received 23 September 1997; Accepted 20 March 1998
Inhibition of P45017␣ and Prostatic Weight
agonists [1]. However, neither GnRH agonists nor orchiectomy can modify the production of adrenal androgens such as dehydroepiandrosterone (DHEA), its
sulfate, and androstenedione. Adrenal androgens are
converted to testosterone or dihydrotestosterone in
the peripheral tissues. The prostatic concentration of
testosterone or dihydrotestosterone was reported to
be 25% or 10% of the pretreatment level, even after
treatment with a GnRH agonist for 3 months [2,3]. The
combined usage of a GnRH agonist or orchiectomy
with an antiandrogen (flutamide or nilutamide) improved the median time to progression and survival in
patients compared with those given the GnRH agonist
or orchiectomy alone [4,5]. Recently, it was also reported that adrenal androgens activated mutant androgen receptors expressed in androgen-dependent
human prostate cancer cells [6,7]. The exchange of a
single valine into methionine in the androgen receptor
promoted transactivation not only by testicular but
also by adrenal androgens. This pattern of liganddependent transactivation may have significance in
the process controlling the progression of prostatic
carcinoma. These findings strongly suggest a significant role of adrenal androgens in stimulating cancer
growth.
C17–20 lyase is one of the key enzymes responsible
for the biosynthesis of androgenic hormones [8]. This
enzyme is responsible for production of testosterone
and adrenal androgens in the testes and adrenal
glands. The inhibition of this enzyme may therefore
result in a decrease in the production of androgenic
hormones in both the testes and adrenal cortices, suggesting that an inhibitor of this enzyme may be useful
for the treatment of androgen-dependent diseases [1].
It thus seems likely that a C17–20 lyase inhibitor
would be more effective than agents which only inhibit testicular androgen production, such as GnRH
agonists. Ketoconazole, an antifungal agent, inhibits
C17–20 lyase activity and has been used clinically in
the treatment of advanced prostate cancers [9,10].
These clinical studies showed that ketoconazole could
produce prolonged responses in previously hormonerefractory prostate cancers. Although this agent received favorable evaluation in some studies, it proved
less promising in others [11,12]. Ketoconazole was
withdrawn from clinical use because it caused gastrointestinal intolerance (nausea and vomiting) and/or
hepatic lesions. These combined findings prompted a
search for a more specific and safe inhibitor of C17–20
lyase that could block adrenal androgens [13,14].
In this study we examined the effects of a novel
nonsteroidal compound, YM116 (Fig. 1), on C17–20
lyase activity, on serum concentrations of testosterone
and DHEA sulfate, and on ventral prostatic weight in
rats. In addition, we examined the specific inhibition
11
Fig. 1. Chemical structure of YM116.
of androgen synthesis by YM116, estimated by the
changes in serum concentrations of cortisol in guinea
pigs and aldosterone in rats.
MATERIALS AND METHODS
Materials
The materials used in this study were obtained
from the following sources: gonadotropin-releasing
hormone (GnRH), Peptide Institute, Inc., Osaka, Japan; Leuplin威 (sustained-release microspheres of leuprolide acetate), Takeda Chem. Ind. Co., Ltd., Osaka,
Japan; pregnant mare’s serum gonadotropin (PMSG),
Seikagaku Co., Tokyo, Japan; ACTH (synacthen depot), Ciba-Geigy, Basel, Switzerland; ketoconazole,
Paesel + Lorei GMBH & Co., Frankfurt, Germany; flutamide, Sigma Chemical Co., St. Louis, MO; and 17␣[1,2-3H]-hydroxyprogesterone (1480 GBq/mmol), DuPont NEN, Boston, MA. The radioimmunoassays
(RIA) used in this study were obtained from the following sources: estradiol, Daiichi Radioisotope Lab.,
Tokyo, Japan; aldosterone, Dainabot, Tokyo, Japan;
cortisol, Incstar Co., Stillwater, MN; testosterone, Diagnostic Products Co., Los Angeles, CA; and dehydroepiandrosterone sulfate, ICN Biomedicals, Inc., Costa
Mesa, CA. 2-(1H-imidazol-4-ylmethyl)-9H-carbazole
monohydrochloride monohydrate (YM116) and other
drugs such as CB7630 [15], liarozole [16], and bicalutamide [17] were synthesized at Yamanouchi Pharmaceutical Co., Ltd. (Tokyo, Japan). All other reagents
were of analytical grade commercially available.
Preparation of Rat Testicular Microsomes
Testes were obtained from anesthetized male 10week-old Wistar rats (Japan SLC, Shizuoka, Japan).
Testicular microsomes were prepared according to the
method described by Schatzman et al. [18]. Briefly, the
testes were minced with scissors and homogenized in
4 volumes of 0.25 M sucrose with a polytron homogenizer (Kinematica GMBH, Lucerne, Switzerland).
The homogenate was centrifuged at 12,000g for 10
12
Ideyama et al.
min, and the resulting supernatant was centrifuged at
105,000g for 60 min. After decantation of the supernatant, the microsomal pellets were washed and resuspended in the solution containing 50 mM phosphate
buffer (pH7.4) and glycerol (3:1). All of the above procedures were performed at 4°C. The final protein concentration was 0.5 mg/ml. The protein concentration
was determined with a Bio-Rad protein assay kit (BioRad Laboratories, Inc., Hercules, CA).
Measurement of C17–20 Lyase Activity
C17–20 lyase activity was determined by incubating 50 ␮g of rat testicular microsomes in an incubation
mixture containing, in a final volume of 200 ␮l, 50 mM
phosphate buffer (pH 7.4), 1 ␮M 17␣-[1,2- 3 H]hydroxyprogesterone (9.25 kBq), the NADPH generating system (1 mM NADPH, 40 mM G6P, and 12
IU/ml G6P dehydrogenase), and 2 ␮l of a test compound and/or solvent at 37°C for 60 min. Following
quenching of the incubates by 400 ␮l tetrahydrofuranmethanol (3:2), the mixture was centrifuged at 2,000g
for 5 min and the upper layer was filtered (UltrafreeMC, 0.45 ␮m, Millipore Co., Bedford, MA). Androstenedione and testosterone were separated and quantified from their peak areas relative to the peak of the
internal standard, using high-performance liquid
chromatography (HPLC), a Shimadzu LC-10A system
equipped with an autosampler (Shimadzu, Kyoto, Japan), and a flow-scintillation analyzer (Series A-500,
Flow/bata, Packard Instrument Co., Meriden, CT), according to the method described by Schatzman et al.
[18]. The sum of the radioactivity in the fractions of
androstenedione and testosterone was defined as the
C17–20 lyase activity. Ki and the mode of enzyme inhibition were estimated by double-reciprocal plots.
Serum Concentration of Testosterone in Male
Rats Treated With or Without GnRH
GnRH (60 ng) was administered intramuscularly
(i.m.) to rats which had been pretreated with several
doses of YM116 (0.4–1.5 mg/kg), ketoconazole (5–30
mg/kg), CB7630 (0.75–2 mg/kg), or liarozole (5–40
mg/kg) 1 hr earlier [19]. Blood specimens were obtained 1 hr after GnRH administration in the GnRHtreated rats or 4 hr after the single oral administration
of each test compound in normal rats. The serum concentration of testosterone was measured by a specific
RIA. The experimental protocol was approved by the
local ethics committee for animal studies.
Serum Concentration of Adrenal Androgen in
Castrated Rats Pretreated With ACTH
ACTH (synacthen depot, 1 mg/kg) was administered subcutaneously (s.c.) to the rats which had been
castrated 6 days before. YM116, ketoconazole, or
CB7630 was orally administered 13 hr after ACTH
treatment, and blood specimens were taken 2 hr after
each drug administration. The serum concentration of
DHEA sulfate was measured by RIA. To demonstrate
the accuracy of the assay, the known amount (3 ng/
ml) of DHEA sulfate was added to the assay medium,
including the standard ranging from 0.5–25 ng DHEA
sulfate/ml; the hormone recovered was 96.7 ± 8.2%.
When 3 ng/ml DHEA sulfate were added to five previously evaluated serum samples (6.9–9.1 ng/ml), the
DHEA sulfate recovered was 167.7 ± 4.9%. The results
of the recovery tests indicated that the assay was interfered with serum to a certain extent. Further dilution samples did not improve assay values (data not
shown). We did not know the reasons for this, but one
possible reason may be the high cross-reactivity of the
antiserum used in this study to other adrenal androgens (cross-reaction percentage at 50% displacement
compared to DHEA sulfate standard curve: 58.5% for
DHEA, and 30.5% for androstenedione, according to
the manufacturer’s instructions).
Estrogen Content in Rat Ovaries Treated
by PMSG
The in vivo inhibition of estradiol synthesis was
evaluated by methods described elsewhere [20].
Briefly, 100 IU of PMSG were administered s.c. to female Wistar rats. Three days later, the ovaries were
removed 3 hr after YM116 or CB7630 treatment. Estradiol was extracted with diethyl ether from the ovarian homogenate and then measured by RIA.
Weights of Ventral Prostate and Seminal Vesicles
Male adult Wistar rats were treated with several
inhibitors for C17–20 lyase, antiandrogens, or a vehicle
for 2 weeks. The ventral prostates were removed and
weighed 16–20 hr after the last dose. Male Wistar rats
at age 6 weeks were treated with leuprolide (3 mg/kg,
s.c., once on day 0) [21,22] or YM116 (40 mg/kg, p.o.,
for 20 days). Some of the rats were castrated on day 0.
The animals were sacrificed serially, and the weights
of the ventral prostate and seminal vesicles were measured 2 hr after the last treatment.
Serum Concentrations of Aldosterone in Rats and
of Cortisol in Guinea Pigs
The serum concentrations of aldosterone in male
Wistar rats and of cortisol in guinea pigs were measured by RIA. The rats were pretreated with ACTH
(synacthen depot, 1 mg/kg). All animals were treated
Inhibition of P45017␣ and Prostatic Weight
13
TABLE I. Inhibitory Effects of YM116, Ketoconazole,
CB7630, and Liarozole on Rat Testicular C17–20
Lyase Activities*
Inhibition of C17–20 lyase
activities, IC50 (nM)
YM116
Ketoconazole
CB7630
Liarozole
5.4
587
8.2
356
*Results are expressed as the concentration required to inhibit
the enzyme activity by 50% (IC50).
with either a test compound or a vehicle. The animals
were sacrificed 2 hr after drug treatment.
Analytical Procedures
In order to determine the ED50 value, which was
the dose required to decrease the serum concentration
of each hormone observed in the control group by
50%, the logarithm of the dose of a test compound vs.
the serum concentration of each hormone was linearized by least-squares fitting (SAS Software, SAS Institute Japan, Tokyo, Japan). The regression equation
was used to determine the ED50 value.
Statistical Analyses
Comparisons between experimental groups were
made using the one-way analysis of variance
(ANOVA) test, followed by Dunnett’s multiple range
test. Differences were accepted as significant at the P <
0.05.
RESULTS
Inhibition of C17–20 Lyase Activity in Rat
Testicular Microsomes
YM116 inhibited C17–20 lyase activity in rat testicular microsomes with an IC50 value of 5.4 nM, a value
similar to that for CB7630 (Table I). YM116 was a
much more potent inhibitor of this enzyme than either
ketoconazole or liarozole. The kinetic analysis showed
that YM116 was a competitive inhibitor of C17–20 lyase, with a Ki of 0.38 nM (Fig. 2).
Decreases in Serum Concentrations of
Testosterone in Male Rats Treated With or
Without GnRH
GnRH increased the serum testosterone concentration from a basal value of 0.82 ± 0.14 ng/ml to
Fig. 2. Inhibition of rat testicular C17–20 lyase by YM116; Lineweaver-Burk plots of enzyme activities at varying concentrations
of 17␣-hydroxyprogesterone and YM116. Inset: Replot of the
slope of each reciprocal plot vs. YM116.
9.82 ± 1.01 ng/ml. YM116 dose-dependently reduced
the concentration of serum testosterone with an ED50
value of 0.7 mg/kg in the GnRH-treated rats (Fig. 3).
YM116 was about 21–24 times more potent than ketoconazole and liarozole, and was twice as potent as
CB7630 in decreasing the serum testosterone concentration. In untreated normal rats, YM116 as well as
CB7630 decreased serum testosterone concentrations
in a dose-dependent manner (Table II).
Decrease in Estradiol Content in Rat Ovaries
Stimulated by PMSG
YM116 decreased the estradiol content in the ovaries pretreated with PMSG, with an ED50 of 1.1 mg/kg
(Fig. 4), indicating that YM116 blocked both estrogen
and androgen synthesis; its potency was about twice
that of CB7630.
Decrease in Serum Concentration of DHEA
Sulfate in Castrated Rats Pretreated With ACTH
YM116 reduced the serum concentration of DHEA
sulfate (one of the adrenal androgens) in castrated
rats, with an ED50 value of 11 mg/kg. YM116 was
about 5 times more potent than ketoconazole. CB7630,
at a dose of up to 40 mg/kg, induced no significant
reduction in the DHEA sulfate level (Fig. 5).
Reduction in Weight of the Rat Ventral Prostate
and Seminal Vesicles
Treatment with YM116 for 2 weeks to rats decreased their prostatic weights in a dose-dependent
14
Ideyama et al.
Fig. 3. Dose-dependent decreases in the serum concentrations
of testosterone by YM116, ketoconazole, CB7630, and liarozole in
GnRH-treated male rats. GnRH (60 ng) was administered i.m. to
rats which were pretreated with YM116 (䊉), ketoconazole (䉱),
CB7630 (䊐), or liarozole (䊊) 1 hr earlier. Blood specimens were
obtained 1 hr after GnRH administration from GnRH-treated rats.
Serum concentrations of testosterone were measured by a specific RIA. Each point with a vertical line shows the mean ± SEM of
5–10 rats. The serum testosterone concentration in GnRHtreated rats was 9.82 ± 1.01 ng/ml. ED50 was the dose required to
decrease the serum concentration of testosterone by 50%.
Fig. 4. Dose-dependent inhibition by YM116 of estradiol production stimulated by PMSG in rat ovaries. PMSG (100 IU) was
administered s.c. to female Wistar rats. Three days later, the ovaries were removed 3 hr after YM116 or CB7630 treatment. Estradiol was measured by RIA after the extraction. Each column
represents the mean ± SEM of 4–5 rats. Statistical significance was
analyzed by one-way ANOVA, followed by Dunnett’s multiple
range test. *P < 0.05, significantly different from the controls
treated with PMSG.
TABLE II. Effects of YM116 and CB7630 on Serum
Concentrations of Testosterone in Male Rats*
Serum testosterone (ng/ml)
Dose (mg/kg)
0.0
0.5
0.75
1.0
1.5
2.0
YM116
CB7630
0.82 ± 0.14
0.61 ± 0.11
0.60 ± 0.06
<0.2
<0.2
<0.2
0.82 ± 0.14
0.71 ± 0.13
0.44 ± 0.11
0.40 ± 0.17
<0.2
<0.2
*Results are mean ± SEM of 5 rats. <0.2, below the detection
limit of the assay. The testosterone concentration was determined 4 hr after the single oral administration of YM116 or
CB7630.
manner (Fig. 6). The reduction in organ weight by
YM116 at a dose of 40 mg/kg was almost the same as
that induced by surgical castration. YM116 was about
2 times more potent than CB7630. Liarozole at a dose
of 80 mg/kg produced no significant reduction in
prostatic weight. Although flutamide and bicalutamide dose-dependently decreased prostatic weight,
Fig. 5. Effects of YM116, ketoconazole, and CB7630 on the
serum concentrations of DHEA sulfate in castrated rats pretreated with ACTH. ACTH (1 mg/kg) was administered to rats
which had been castrated 6 days earlier. Thirteen hours later, the
test compound was orally administered, and blood specimens
were taken 2 hr later. Serum concentrations of DHEA sulfate
were measured by RIA, and the mean concentration in the ACTHtreated rats was 6.2 ± 0.9 ng/ml. Each column represents the mean
± SEM of 4–5 rats. Statistical significance was analyzed by one-way
ANOVA, followed by Dunnett’s multiple range test. **P < 0.01, *P
< 0.05, significantly different from control rats treated with ACTH
(100%).
the maximum reduction at a dose of 80 mg/kg did not
reach castration levels. In order to compare the differences in the time courses of the reduction of androgendependent organs induced by surgical castration and
by medical castration, the effects of surgical castration,
YM116, and a GnRH agonist were examined (Fig. 7).
Inhibition of P45017␣ and Prostatic Weight
Fig. 6. Effects of YM116, CB7630, liarozole, and antiandrogens
on the weights of the ventral prostates in rats. Male adult Wistar
rats were treated with YM116 (䊉), CB7630 (䊐), liarozole (䊊),
flutamide (䉱), bicalutamide (䉭), or a vehicle for 2 weeks. The
ventral prostates were removed and their weights were measured
16–20 hr after the last dosing. The weight of the ventral prostate
in the control rats was 0.881 ± 0.048 mg/g body weight. Each point
with a vertical line shows the mean ± SEM of 5–20 rats. The effects
of YM116 or CB7630 at every dose used in this study were significantly different from the control value without treatment (data
not shown).
YM116 and surgical castration significantly reduced
the weights of the prostate and seminal vesicles at 3
days after the start of treatment. There was no difference between these two groups with respect to the
time course and magnitude of the reduction in the
weights of both organs. In contrast, a single subcutaneous injection of leuprolide elicited a rapid increase
in the weights of both organs on day 3, and subsequently decreased them.
Effect on Serum Concentrations of Aldosterone in
Rats and Cortisol in Guinea Pigs
YM116 at a dose of 100 mg/kg had no significant
effect on the serum cortisol concentration in the
guinea pigs, but elicited a 37% decrease in the serum
aldosterone level in the rats (Table III). CB7630 had no
effect on cortisol or aldosterone levels. In contrast, ketoconazole and liarozole significantly decreased the
aldosterone level, with ED50 values of 1.4 and 9.4 mg/
kg, respectively, although these drugs had no significant effect on cortisol levels.
DISCUSSION
Cytochrome P450 17␣ is known as 17␣hydroxylase/C17–20 lyase and plays a key role in the
synthesis of androgens in the testes and of adrenal
15
androgens and cortisol in the adrenal glands in humans [8,13,23]. In rats and mice, however, it was reported that plasma concentrations of cortisol were
near the detection limit of the assay, and that these
low levels of cortisol amounted to less than 4% of the
levels found for corticosterone. These findings could
be attributable to the lack of 17␣-hydroxylase in rat
adrenals [24,25]. In contrast, Touitou et al. [26] showed
the in vitro syntheses of 17␣-hydroxyprogesterone
and cortisol from progesterone in rodent adrenals,
suggesting the presence of 17␣-hydroxylase activity in
this tissue. There is thus a controversy regarding the
role of 17␣-hydroxylase/C17–20 lyase in the synthesis
of adrenal androgens and cortisol in rat adrenal
glands. In this study, we examined the effects of
YM116 on C17–20 lyase activity, on serum concentrations of testosterone and DHEA sulfate (one of the
adrenal androgens), and on ventral prostatic weight in
rats. In addition, we examined the specific inhibition
of androgen production by YM116, as estimated by
changes in the serum concentrations of aldosterone in
rats and cortisol in guinea pigs.
YM116 dose-dependently inhibited C17–20 lyase
activity in rat testicular microsomes, with an IC50
value of 5.4 nM (Table I), and was a competitive inhibitor of this enzyme with a Ki of 0.38 nM, based on
kinetic analysis (Fig. 2). The potency of this agent was
comparable to or even greater than other known steroidal or nonsteroidal inhibitors of C17–20 lyase, i.e.,
ketoconazole [27], CB7630 [13], and liarozole [28].
YM116 decreased serum concentrations of testosterone with an ED50 of 0.7 mg/kg in rats, whose testosterone level was increased about 12-fold by GnRH
treatment (Fig. 3). YM116 was about 21–24 times more
potent than ketoconazole and liarozole, and was twice
as potent as CB7630 in decreasing the serum testosterone concentration. It should be pointed out that
YM116 was one of the most potent inhibitors of C17–
20 lyase and serum testosterone production in rats,
and that the relative potency of the four agents in
inhibiting C17–20 lyase activity was very similar to
that in decreasing serum testosterone in rats following
oral administration. Consistent with these findings,
ketoconazole and liarozole were reported to produce a
drop in the level of circulating testosterone in humans
[28–30].
YM116 and ketoconazole reduced serum concentrations of DHEA sulfate in castrated rats, with ED50 values of 11 and 60 mg/kg, respectively. CB7630 at a dose
of up to 40 mg/kg produced no significant reduction
in the DHEA sulfate level (Fig. 5). This last finding
was unexpected, since the two other inhibitors significantly decreased DHEA sulfate in serum, and CB7630
significantly decreased the serum testosterone concentration. However, no information is available about
16
Ideyama et al.
Fig. 7. Effects of YM116, leuprolide, and surgical castration on
weights of the ventral prostate and seminal vesicles in rats. Male
Wistar rats at age 6 weeks were divided into four groups. They
received YM116 (䊉, 40 mg/kg, p.o., for 20 days), leuprolide (䊏, 3
mg/kg, s.c., once on day 0), surgical castration (䊊, on day 0), or a
vehicle. The animals were sacrificed and the weights of both the
ventral prostate and seminal vesicles were determined. The re-
TABLE III. Effects of YM116, Ketoconazole, CB7630,
and Liarozole on Serum Concentrations of Aldosterone
in Rats and Cortisol in Guinea Pigs
Aldosterone
in rats,
ED50 (mg/kg)
YM116
Ketoconazole
CB7630
Liarozole
37% decreasea
9.4b
No effecta
1.4b
Cortisol
in guinea pigs,
ED50 (mg/kg)
No
No
No
No
effecta
effecta
effecta
effecta
a
Rats or guinea pigs were orally given a test compound at a
dose of up to 100 mg/kg. The number of animals used in each
group was 5 rats and 7 guinea pigs.
b
ED50.
the effect of CB7630 on adrenal androgen levels [31].
An in vivo study of male volunteers reported by
Bruynseels et al. [28] confirmed that ketoconazole (600
mg, b.i.d.) and liarozole (300 mg, b.i.d.) decreased
plasma testosterone levels, and these levels were reduced close to or within castration levels. As to the
suppression of adrenal androgen production, ketoco-
sults are expressed as percentage of control value at the same
time-point. During the 20-day treatment, prostate weight increased from a control value of 0.473 ± 0.024 mg/g to 0.662 ±
0.043 mg/g, and the weight of the seminal vesicles increased from
0.515 ± 0.071 mg/g to 2.095 ± 0.129 mg/g. Each point with a
vertical line shows the mean ± SEM of 5–10 rats. *P < 0.05, significantly different from the control value at the same time-point.
nazole was much more active in comparison with liarozole, indicating that there was a difference between
these two agents in inhibiting adrenal androgen synthesis. In this study, a significant difference between
the ED50 values for serum testosterone and adrenal
androgen was also observed in the YM116- or ketoconazole-treated rats. An about 16-fold higher dose of
YM116 was required to decrease the serum DHEA
sulfate concentration compared to testosterone, and an
about 4-fold higher dose of ketoconazole was required
to do so. The reason for this difference is as yet unknown, but it is in part attributed to the difference in
experimental conditions at which the ED50 values
were determined.
YM116 dose-dependently decreased rat prostatic
weights, which were reduced to close to or within
castration levels when YM116 was administered at a
dose of 40 mg/kg for 2 weeks (Fig. 6). CB7630 at a
dose of 80 mg/kg, but neither flutamide nor bicalutamide, decreased prostatic weights to castration levels. Liarozole produced no significant reduction in
prostatic weight. YM116 significantly reduced the
weights of the prostate and seminal vesicles as quickly
Inhibition of P45017␣ and Prostatic Weight
as did surgical castration (Fig. 7). There was no difference between YM116 and surgical castration with respect to the time course and magnitude of the reduction in the weights of the prostate and seminal
vesicles. A single subcutaneous injection of leuprolide,
however, elicited a rapid increase in the weights of the
prostate and seminal vesicles and subsequently decreased the weights of these organs. YM116 at a dose
of 100 mg/kg had no significant effect on serum cortisol concentration in guinea pigs, but elicited a 37%
decrease in the serum aldosterone level in rats, suggesting that YM116 was a selective inhibitor of androgen production (Table III).
Some limitations of ketoconazole when used clinically were high doses and frequent administration
(400 mg, every 8 hr). Some patients experienced a
moderate rise in serum testosterone levels 1 month
after the start of administration, which might be due
to a rise in LH [9]. These observations may be caused
by gradual escape from C17–20 lyase inhibition and a
short elimination half-life. As shown in Figures 6 and
7, YM116 at a dose of 40 mg/kg decreased prostatic
weight to castration levels until 2 or 3 weeks. Although we have not yet examined the long-term inhibitory effect of this agent on prostatic weight, our
preliminary results demonstrated that such suppression was also observed after 1 month. In addition, our
pharmacokinetics study showed that plasma concentration of YM116, which was administered orally to
rats at a dose of 1 or 3 mg/kg, reached a maximum
between 1–3 hr postdose and subsequently declined,
with an elimination half-life of 2.5 or 10.9 hr (unpublished observations). These values were equal to or
even greater than those (1.5–2.2 hr) of ketoconazole
when administered orally at a dose of 5 mg/kg [32].
Further study will be needed to clarify the long-term
effect of YM116 on prostatic weight in rats and other
species of the animals.
It is well-established that the production of androgens in the testes must be inhibited to effectively treat
hormone-dependent prostate cancer [33]. Surgical castration or treatment with a GnRH agonist is an effective way to decrease the testicular production of androgens in prostate cancer patients. However, these
treatments have no effect on the supply of androgens
and their precursors in the adrenal glands. A GnRH
agonist is used alone or in a combination with an antiandrogen for this purpose [34]. The function of the
antiandrogen in the combined treatment is to counteract the stimulatory action of androgens of adrenal origin on the androgen receptor in prostatic cancer cells.
YM116 is a single agent which may inhibit both testicular and adrenal androgen production, and may
have an advantage over the present combination
therapy in clinical practice.
17
CONCLUSIONS
YM116 is a potent inhibitor of C17–20 lyase, and
produced a significant reduction in the weight of the
rat prostate by decreasing the serum concentrations of
testosterone and adrenal androgen. Further studies
are necessary to determine the efficacy of this agent
for the treatment of hormone-dependent prostate cancer in clinical studies.
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