DEA Agreement No. DEA-04-P0007

# Final Report

**Alvin M. Matsumoto, M.D.**

**and Brett T. Marck**

Geriatric Research, Education and Clinical Center, Veterans
Administration Puget Sound Health Care System, Seattle, USA.

Analysis of the Androgenic and Anabolic Activities of
1,4-Androstadien-3,17-Dione (Boldione) and
19-Nor-4,9(10)-Androstadienedione in Male Sprague Dawley Rats.

## Background and Rationale 

The classical bioassay to assess the anabolic (muscle growth-promoting)
and androgenic effects of an agent are to examine the stimulation of
levator ani muscle, and prostate and seminal vesicle growth,
respectively, in a castrated male rat \[1, 2\]. Administration of an
agent with both anabolic and androgenic actions prevents the decrease in
weight of the levator ani muscle, and prostate and seminal vesicles,
respectively, induced by castration of a male rat \[3, 4\]. In castrated
animals, loss of negative feedback by the testicular androgen,
testosterone \[T\], and its active metabolite, estradiol
(1,3,5-estratrien-3,17β-diol), a potent estrogen, results in an increase
in serum levels of pituitary gonadotropins, (i.e., luteinizing hormone
\[LH\] and follicle stimulating hormone \[FSH\]) \[5, 6\].
Administration of androgens (and estrogens) in castrated animals
suppresses serum LH and FSH levels . When administered to intact
animals, androgens suppress serum LH and FSH levels resulting in reduced
testicular size, and usually maintain or stimulate growth of the
androgen-sensitive tissues, such as the prostate and seminal vesicles,
and levator ani muscle \[9, 10\].

This study was designed to examine 1,4-androstadien-3,17-dione
(boldione) and 4,9-estradien-3,17-dione
(19-nor-4,9(10)-androstadienedione) using the classical bioassay for
anabolic and androgenic activity, and to investigate the effects of
these steroids on serum gonadotropin levels in castrate and intact rats
and serum T concentrations and testis size in intact male rats.

## Specific Aims and Methods

**Methods:**

**Animals**

Male Sprague Dawley rats (280-310 g) were purchased (Harlan Sprague
Dawley, Inc., Indianapolis, IN) and housed in an American Association
for Accreditation of Laboratory Animal Care-accredited facility at the
Veteran's Administration Puget Sound Health Care System (Seattle, WA).
Animals were individually housed in polycarbonate rat cages containing
corncob bedding in a light- and temperature-controlled room on a 12-h
light, 12-h dark cycle (lights off from 1800--0600 h). Animals had *ad
libitum* access to Purina rodent chow (5001, Ralston Purina Co., St.
Louis, MO) and tap water. All animal experiments were conducted in
accordance with the principles and procedures outlined in the NIH Guide
for the Care and Use of Laboratory Animals, and were approved by the
Veteran's Administration Puget Sound Health Care System Institutional
Animal Care and Use Committee.

**Hormone Delivery**

Silastic capsules were made using medical grade SILASTIC brand tubing
(Dow Corning Corporation. Midland, MI, 0.078 in ID x 0.125 in OD),
filled with 1,4-androstadien-3,17-dione or 4,9-estradien-3,17-dione both
ends plugged with silicone adhesive (Dow Corning Corp., no. 891), and
incubated overnight in sterile saline at 4 °C before implantation.
Capsules were filled to 4 cm in length for each steroid.
1,4-Androstadien-3,17-dione (A0100-000, batch B0178) and
4,9-estradien-3,17-dione (E0160-000, batch H989) were obtained from
Steraloids Inc (Newport, RI).

**Procedures**

Bilateral castration with sparing of the epididymal fat pad, or sham
operation (a 1- to 2-cm abdominal incision without removal of testes)
was performed using aseptic procedures under isoflurane anesthesia. At
the time of surgery, steroid-filled or empty silastic capsules were
implanted subcutaneously in the intrascapular region.

**Hormone Assays**

Serum was stored at -30° C until hormone assays were performed in
duplicate. All samples from each experiment were analyzed together for
each assay. T was measured by fluroimmunoassay (Delfia, PerkinElmer,
Waltham, MA). The limit of detection for the T assay was 0.35 ng/ml. The
mean intraassay and interassay co-efficients of variation for T were
6.7% and 5.3% respectively. The cross-reactivity of
1,4-androstadien-3,17-dione in the testosterone assay is 0.0024%. Rat
follicle-stimulating hormone (FSH) was measured using an IRMA C.T. kit
(American Laboratory Products Company, Windham, NH). The limit of
detection for FSH was 0.2 ng/ml. The mean intraassay and interassay
co-efficients of variation for FSH were 2.3% and 7.8% respectively, as
reported by the manufacturer. Rat luteinizing hormone (LH) was measured
using a modified, sensitive immunoflurometric assay described by
Haavisto \[11\]. The rat LH standard HIDDK rLH RP-3 was obtained from
the National Institutes of Health (Bethesda, MD), capture anti-body 518
B7 kindly provided by Dr. J. F. Roser (Department of Animal Science,
University of California, Davis California), tracer antibody anti-hLH
#5303 obtained from Medix Biochemica (Kauniainen, Finland) and labeled
with europium using a Delfia labeling kit (PerkinElmer, Waltham, MA).
The assay buffer, and streptavidin labeled plates were obtained from
PerkinElmer (Waltham, MA). The lower limit of detection and quantitation
for the LH assay was 0.02 ng/ml. The mean intraassay and interassay
coefficients of variation were 4.6% and 3.8% respectively. Estradiol was
measured using a kit from Diagnostic Systems Laboratories (DSL-4800,
Webster, TX). The cross-reactivity of 4,9-estradien-3,17-dione in the
estradiol assay is 0.0015%. The lower limit of detection and
quantitation for the estradiol assay was 5 pg/ml. The mean intraassay
and interassay coefficients of variation are 6.5% and 9.7% respectively,
as reported by the manufacturer.

**Statistical Analyses**

Hormone levels, food intake, body weight and tissue weights were
compared among treatment groups in each study by two-way ANOVA. Posthoc
testing by Fisher's PLSD test was performed for between-treatment
differences separately for each experimental group. Values for the lower
limit of quantitation were used in calculating mean values when results
were below this level. Results are reported as the mean ± SEM, and P \<
0.05 was considered significant. The statistical software package used
was StatView version 4.57 for Windows (Abacus Concepts, Inc., Berkeley,
CA; and SAS Institute, Inc., Cary, NC).

**Specific Aim 1:** To determine the androgenic and anabolic effects of
4,9-estradien-3,17-dione and 1,4-androstadien-3,17-dione on the
androgenic tissues (prostate and seminal vesicles), anabolic effects on
the levator ani muscle, pituitary hormones (gonadotropins: LH and FSH)
and the testicular hormones (T and estradiol) in adult rats.

For each test steroid, a group of (n=10) castrated male Sprague Dawley
(SD) rats were implanted with four 4 cm silastic capsules filled with
test steroid. Each test group was compared to a control group of (n=10)
castrated male SD rats implanted with four 4 cm empty silastic capsules.
After eight weeks of treatment, the rats were sacrificed by decapitation
and the prostate, seminal vesicles, and levator ani muscle were
dissected and weighed. Trunk blood was collected and serum was stored
for subsequent analysis of the reproductive hormones (LH, FSH, T, and
estradiol). Castration and silastic capsule implantation were verified
by visual inspection after sacrifice.

**Specific Aim 2:** To determine the androgenic and anabolic effects of
4,9-estradien-3,17-dione and 1,4-androstadien-3,17-dione on testicular
size, the androgenic tissues (prostate and seminal vesicles), anabolic
effects on the levator ani muscle, pituitary hormones (gonadotropins: LH
and FSH) and the testicular hormones (T and estradiol) in adult rats.

For each test steroid, a group of (n=10) intact male SD rats were
implanted with four 4 cm silastic capsules filled with test steroid.
Each test group was compared to a control group of (n=10) SD rats
implanted with four 4 cm empty silastic capsules. After eight weeks of
treatment, the rats were sacrificed by decapitation and the testes,
prostate, seminal vesicles, and levator ani muscle were dissected and
weighed. Trunk blood was collected and serum was stored for subsequent
analysis of the reproductive hormones (LH, FSH, T, and estradiol).
Silastic capsule implantation was verified by visual inspection after
sacrifice.

**Results**

Compared to untreated sham-operated control rats (Sham/Sham), untreated
castrated rats (Cx/Sham) demonstrated a decrease in serum T levels to
the limit of detection (\< 0.32 nmol/l), and an increase in serum LH
levels to the upper limit of detection (\> 10 mg/ml) and an increase in
serum FSH levels (all, p\<0.05). Serum estradiol levels were not
affected by castration. \[See Table 1 and Figure 1\].

In castrated rats, 1,4-androstadien-3,17-dione administration reduced
serum LH levels to below detectable levels (\< 0.02 mg/ml), suppressed
serum FSH levels, and increased serum T levels, in both castrated
(p\<0.001 vs. Cx/Sham) and sham-operated rats (p\<0.05 vs. Sham/Sham).
\[See Table 1 and Figure 1; where 1,4-androstadien-3,17-dione is
designated as A0100\].

4,9-Estradien-3,17-dione administration reduced serum LH and FSH levels
in both the castrated (p\<0.001 vs. Cx/Sham) and sham-operated rats
(p\<0.05 vs. Sham/Sham), and reduced serum T levels to below the limit
of detection in sham-operated rats (p\<0.05 vs. Sham/Sham) \[See Table 1
and Figure 1; where 4,9-estradien-3,17-dione is designated as E0160\].
The limit of detection for the LH assay is 10 times lower than the serum
values measured in the sham-operated animals implanted with empty
capsules (Sham/Sham). Serum estradiol was increased in both castrated
(p\<0.001 versus Cx/Sham) and sham-operated rats implanted with
4,9-estradien-3,17-dione (p\<0.05 versus Sham/Sham).

+-----------------+------------+-----------+------------+------------+
|                 | Te         | Estradiol | LH         | rFSH       |
|                 | stosterone | (pg/ml)   |            |            |
|                 | (nmol/l)   |           | (ng/ml)    | (ng/ml)    |
+-----------------+------------+-----------+------------+------------+
| Cx/Sham         | 0.32 ±     | 8.5 ± 0.4 | 10.0 ±     | 58.1 ±     |
|                 | 0.0^+^     |           | 0.00^+^    | 3.2^+^     |
+-----------------+------------+-----------+------------+------------+
| Cx/A0100        | 46.2 ±     | 13.3 ±    | 0.02 ±     | 7.7 ±      |
|                 | 3.5^\*^    | 0.7       | 0.00^\*^   | 0.7^\*^    |
+-----------------+------------+-----------+------------+------------+
| Cx/E0160        | 0.32 ± 0.0 | 166.7 ±   | 0.02 ±     | 8.0 ±      |
|                 |            | 14.2^\*^  | 0.00^\*^   | 0.4^\*^    |
+-----------------+------------+-----------+------------+------------+
| Sham/Sham       | 12.0 ± 2.0 | 9.4 ± 0.4 | 0.39 ±     | 11.1 ± 0.7 |
|                 |            |           | 0.09       |            |
+-----------------+------------+-----------+------------+------------+
| Sham/A0100      | 41.5 ±     | 13.2 ±    | 0.02 ±     | 5.6 ±      |
|                 | 3.6^+^     | 0.7       | 0.00^+^    | 0.3^+^     |
+-----------------+------------+-----------+------------+------------+
| Sham/E0160      | 0.9 ±      | 195.8 ±   | 0.08 ±     | 6.8 ±      |
|                 | 0.6^+^     | 62.4^+^   | 0.06^+^    | 0.6^+^     |
+-----------------+------------+-----------+------------+------------+

Table 1. Serum levels of reproductive hormones in sham-operated (Sham)
and castrated male rats (Cx) implanted with empty (Sham) silastic
capsules or silastic capsules containing either
1,4-androstadien-3,17-dione (A0100) or 4,9-estradien-3,17-dione (E0160).
^\*^ p \< 0.001 vs. Cx/Sham; ^+^ p \< 0.05 vs. Sham/Sham.

![](media/image1.wmf){width="6.435416666666667in"
height="4.604861111111111in"}

Figure 1. Serum levels of reproductive hormones in sham-operated (Sham)
and castrated rats (Cx) implanted with an empty (Sham) capsule or a
silastic capsule containing either 1,4-androstadien-3,17-dione (A0100)
or 4,9-estradien-3,17-dione (E0160). ^\*^ p \< 0.001 vs. Cx/Sham; ^+^ p
\< 0.05 vs. Sham/Sham.

Both sham-operated and castrated rats implanted with
4,9-estradien-3,17-dione (E0160) showed significant reductions in body
weight and body weight gain after eight weeks of treatment, compared to
the sham-operated or castrated rats implanted with empty capsules
(Figure 2 and Table 3). The reduction in rate of growth in
4,9-estradien-3,17-dione-treated animals was associated with a decrease
in food intake (Table 2). There were no significant differences in body
weight and food intake between the sham-operated and castrated animals
implanted with empty silastic capsules, and between sham-operated and
castrated rats implanted with silastic capsules containing
1,4-androstadien-3,17-dione (A0100), respectively, in this eight-week
study.

+---------------+---------------------+---------------+---------------+
|               | 7 Day Cumulative    | Food Intake   | Food intake   |
|               |                     | per Day (g)   | corrected for |
|               | Food Intake         |               | body weight   |
|               |                     |               | (g/Kg)        |
|               | \(g\)               |               |               |
+---------------+---------------------+---------------+---------------+
| Cx/Sham       | 184 ± 5             | 26.1 ± 0.5    | 75 ± 1        |
+---------------+---------------------+---------------+---------------+
| Cx/A0100      | 172 ± 3             | 24.9 ± 0.3    | 71 ± 1        |
+---------------+---------------------+---------------+---------------+
| Cx/E0160      | 141 ± 5^\*^         | 21.2 ±        | 67 ± 2^\*^    |
|               |                     | 0.6^\*^       |               |
+---------------+---------------------+---------------+---------------+
| Sham/Sham     | 182 ± 3             | 26.3 ± 0.7    | 76 ± 2        |
+---------------+---------------------+---------------+---------------+
| Sham/A0100    | 175 ± 5             | 24.6 ± 0.5    | 72 ± 1        |
+---------------+---------------------+---------------+---------------+
| Sham/E0160    | 149 ± 5^+^          | 20.1 ± 0.7^+^ | 71 ± 3^+^     |
+---------------+---------------------+---------------+---------------+

Table 2. Cumulative food intake measured for a seven-day period during
the study in sham-operated (Sham) and castrated animals (Cx) implanted
with empty silastic capsules (Sham) or silastic capsules containing
either 1,4-androstadien-3,17-dione (A0100) or 4,9-estradien-3,17-dione
(E0160). ^\*^ p \< 0.05 vs. Cx/Sham; ^+^ p \< 0.001 vs. Sham/Sham.

![](media/image2.wmf){width="3.420138888888889in"
height="5.559027777777778in"}

Figure 2. Final body weight and change in body weight (∆ Wt) after eight
weeks of treatment in sham-operated (Sham) and castrated animals (Cx)
implanted with empty silastic capsule (Sham) or a silastic capsule
containing either 1,4-androstadien-3,17-dione (A0100) or
4,9-estradien-3,17-dione (E0160). ^\*^ p \< 0.001 vs. Cx/Sham; ^+^ p \<
0.05 vs. Sham/Sham.

Castration reduced the mass of the androgenic tissues (seminal vesicles
and prostate) and the levator ani muscle (a marker of anabolic activity)
in animals implanted with empty silastic capsules (p\<0.05 vs.
Sham/Sham). Administration of both 1,4-androstadien-3,17-dione
(designated as A0100) and 4,9-estradien-3,17-dione (designated as E0160)
prevented the loss of mass associated with castration in all three
tissues, seminal vesicles, prostate, and levator ani \[See Table 3 and
Figure 3\]. Compared to castrated rats implanted with empty capsules
(Cx/Sham), castrated rats implanted with 1,4-androstadien-3,17-dione
(A0100) had higher levator ani muscle, prostate, and seminal vesicle
weights (relative to total body weight, g/Kg; p\<0.001 vs. Cx/Sham).
Weights of tissues were comparable in both the sham-operated and
castrated rats treated with 1,4-androstadien-3,17-dione. Only the weight
of the levator ani muscle was increased in sham-operated animals treated
with 1,4-androstadien-3,17-dione compared to sham-operated controls
implanted with empty silastic capsules (p\<0.05 vs. Sham/Sham), while
the weight of the seminal vesicles was decreased (p\<0.05 vs.
Sham/Sham).

Both castrated and sham-operated rats implanted with
4,9-estradien-3,17-dione (E0160) had higher levator ani muscle, prostate
and seminal vesicle weights compared to animals implanted with empty
silastic capsules (p \< 0.001 vs. Cx/Sham; p\<0.05 vs. Sham/Sham).
Again, the weights of these tissues were similar in both the
sham-operated and castrated animals treated with
4,9-estradien-3,17-dione. Compared to sham-operated rats implanted with
empty capsules, both sham-operated rats implanted with
1,4-androstadien-3,17-dione and 4,9-estradien-3,17-dione had lower
absolute testes weights. However, when testes weight was corrected for
body weight, sham-operated animals treated with 1,4-androstadien-3,
17-dione had lower (p\<0.05 vs. Sham/Sham) and 4,9-estradien-3,17-dione
had higher (p\<0.05 vs. Sham/Sham) testes weights \[See Table 3 and
Figure 3\].

+-------------+------+------+---------+---------+---------+---------+
|             | We   | ∆ wt | Testes  | Levator | P       | Seminal |
|             | ight |      |         | ani     | rostate | Vesicle |
|             |      | \    | (g/Kg)  | muscle  |         |         |
|             | \    | (g\) |         |         | (g/Kg)  | (g/Kg)  |
|             | (g\) |      |         | (g/Kg)  |         |         |
+-------------+------+------+---------+---------+---------+---------+
| Cx/Sham     | 3    | 89±3 |         | 0.86±   | 0.26±   | 0.31±   |
|             | 87±4 |      |         | 0.04^+^ | 0.01^+^ | 0.01^+^ |
+-------------+------+------+---------+---------+---------+---------+
| Cx/A0100    | 3    | 95±6 |         | 3.64±0  | 2.47±0  | 2.18±0  |
|             | 95±7 |      |         | .09^\*^ | .16^\*^ | .13^\*^ |
+-------------+------+------+---------+---------+---------+---------+
| Cx/E0160    | 3    | 24±3 |         | 4.24±0  | 4.48±0  | 6.46±0  |
|             | 24±5 | ^\*^ |         | .09^\*^ | .18^\*^ | .30^\*^ |
|             | ^\*^ |      |         |         |         |         |
+-------------+------+------+---------+---------+---------+---------+
| Sham/Sham   | 3    | 98±6 | 9.      | 2.      | 2.      | 3.      |
|             | 98±6 |      | 37±0.23 | 87±0.08 | 84±0.23 | 43±0.19 |
+-------------+------+------+---------+---------+---------+---------+
| Sham/A0100  | 3    | 84±  | 8.18±   | 3.48±   | 2.      | 2.26±   |
|             | 92±4 | 4^+^ | 0.19^+^ | 0.07^+^ | 49±0.10 | 0.11^+^ |
+-------------+------+------+---------+---------+---------+---------+
| Sham/E0160  | 321± | 17±  | 11.0±   | 4.43±   | 5.02±   | 7.01±   |
|             | 4^+^ | 2^+^ | 0.25^+^ | 0.16^+^ | 0.14^+^ | 0.29^+^ |
+-------------+------+------+---------+---------+---------+---------+

Table 3. Final body weight, change in body weight from baseline after
eight weeks of treatment (∆ wt) and weights of the testes, androgenic
tissues (prostate and seminal vesicle) and levator ani muscle corrected
for body weight in sham-operated (Sham) and castrated (Cx) animals
implanted with an empty silastic capsule (Sham) or silastic capsules
containing either 1,4-androstadien-3,17-dione (A0100) or
4,9-estradien-3,17-dione (E0160). ^\*^ p \< 0.001 vs. Cx/Sham; ^+^ p \<
0.05 vs. Sham/Sham.

![](media/image3.wmf){width="6.308333333333334in"
height="4.565277777777778in"}.

## Figure 3. Weights of the testis, androgenic tissues (prostate and seminal vesicle) and levator ani muscle corrected for body weight in sham-operated (Sham) and castrated (Cx) animals implanted with empty (Sham) or either 1,4-androstadien-3,17-dione (A0100) or 4,9-estradien-3,17-dione (E0160) filled silastic capsules. ^\*^ p \< 0.001 vs. Cx/Sham; ^+^ p \< 0.05 vs. Sham/Sham.

## 

## Summary and Conclusion

In summary, we found that, 1,4-androstadien-3,17-dione (A0100) and
4,9-estradien-3,17-dione (E0160) demonstrated both androgenic activity,
as evidenced by stimulation of the androgenic tissues (prostate and
seminal vesicle) and anabolic activity, as evidenced by stimulation of
the levator ani muscle growth in castrated male rats.

Both LH and FSH levels were suppressed in both
1,4-androstadien-3,17-dione and 4,9-estradien-3,17-dione treated
castrated and sham-operated animals. Only sham-operated animals
implanted with 1,4-androstadien-3,17-dione demonstrated a slight
decrease in testes size as a result of LH and FSH suppression.
4,9-Estradien-3,17-dione demonstrated a slight decrease in absolute
testes size, but an increase in testes size when corrected for body
weight. Suppression of LH concentrations in rats administered
4,9-estradien-3,17-dione resulted in a decrease in serum T levels. In
contrast, high serum concentrations of T were detected in both the
castrated (46.2 ± 3.5 nmol/l) and sham-operated (41.5 ± 3.6 nmol/l) rats
treated with 1,4-androstadien-3,17-dione. 1,4-Androstadien-3,17-dione
did not cross-react significantly in the T assay. It is possible that
measurement of high serum T levels with administration of
1,4-androstadien-3,17-dione could have resulted from the
cross-reactivity of a metabolite of 1,4-androstadien-3,17-dione. High
serum concentrations of estradiol were detected in both the castrated
(166.7 ± 14.2 ng/ml) and sham-operated (195.8 ± 62.4 ng/ml) rats treated
with 4,9-estradien-3,17-dione. 4,9-Estradien-3,17-dione did not
cross-react significantly in the estradiol assay. It is possible that
measurement of high serum estradiol levels with administration of
4,9-estradien-3,17-dione could have resulted from the cross-reactivity
of a metabolite of 4,9-estradien-3,17-dione. A reduction in food intake
in 4,9-estradien-3,17-dione-treated animals contributed to a reduction
in weight gain during treatment, reducing body weight compared to
untreated animals. The loss of body weight and reduction in food intake
associated with 4,9-estradien-3,17-dione treatment is consistent with
the known effects of high doses of androgens aromatizable to estrogens
or estradiol treatment in male rats \[12-18\]. Growth and final body
weight was not effect by administration of 1,4-androstadien-3,17-dione.

4,9-Estradien-3,17-dione stimulated growth of the androgenic tissues,
the prostate and seminal vesicles, and stimulated the growth of the
levator ani muscle in sham-operated rats. The direct androgenic and
anabolic activity of 1,4-androstadien-3,17-dione in sham-operated rats
is less clear because of the measured increases in serum T levels that
could mediate the androgenic and anabolic activities of
1,4-androstadien-3,17-dione. The androgenic and anabolic response to
administration of 1,4-androstadien-3,17-dione did not differ in
sham-operated and castrate rats, consistent with a chemical castration
in sham-operated rats, as a result of severe suppression of serum LH
levels induced by 1,4-androstadien-3,17-dione.

We conclude that both 1,4-androstadien-3,17-dione and
4,9-estradien-3,17-dione are potent androgenic and anabolic steroids, as
determined by the classical bioassays.

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