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Endocrinology, doi:10.1210/en.2005-1524
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Endocrinology Vol. 147, No. 6 3016-3026
Copyright © 2006 by The Endocrine Society

Dimethandrolone Undecanoate: A New Potent Orally Active Androgen with Progestational Activity

Barbara J. Attardi, Sheri A. Hild and Jerry R. Reel

BIOQUAL, Inc., Rockville, Maryland 20850

Address all correspondence and requests for reprints to: Dr. Barbara J. Attardi, Molecular Endocrinology Laboratory, BIOQUAL, Inc., 9600 Medical Center Drive, Rockville, Maryland 20850. E-mail: bjattardi{at}bioqual.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Dimethandrolone (DMA), the 17ß-undecanoic acid ester of dimethandrolone (DMAU; 7{alpha},11ß-dimethyl-19-nortestosterone) is a potent androgen currently in development for therapeutic uses in men. Cleavage of the 17ß-ester bond liberates the biologically active DMA. In this study we investigated the activity of DMAU and DMA both in vivo and in vitro. DMAU was active orally in castrate rat bioassays, and when administered sc, a single dose produced prolonged androgenic activity and suppression of LH with sustained circulating levels of DMA. DMA, other 19-norandrogens, and C-19 androgens bound to recombinant rat androgen receptor with high affinity and were equipotent in stimulating luciferase activity (EC50, 10–10–10–9 M) in CV-1 cells cotransfected with a human androgen receptor expression vector and a luciferase reporter plasmid with three hormone response elements. Because various 19-norandrogens are also known to bind to progestin receptors (PR) and to possess progestational activity in vivo, we evaluated the binding affinity of DMA for rabbit PR and recombinant human PR-A and PR-B and its ability to induce PR-mediated transcription and endogenous alkaline phosphatase activity in T47DCO human breast cancer cells. DMA and related 19-norandrogens bound with high affinity to both rabbit and human PR, whereas the less active 11{alpha}-methyl stereoisomer of DMA and C-19 androgens showed low or negligible binding to PR. In T47DCO cells, 10–8 M DMA and other 19-norandrogens stimulated transcription of a progestin/glucocorticoid/androgen response element-thymidine kinase-luciferase reporter plasmid to the same extent as R5020, the potent progestin promegestone (EC50, ~10–9 M), but C-19 androgens had no effect. Antiprogestins were potent inhibitors of transactivation and alkaline phosphatase activity induced by DMA and other 19-norandrogens in T47DCO cells, whereas antiandrogens were weak inhibitors. DMA and DMAU also exhibited dose-dependent progestational activity in the estrogen-primed immature female rabbit, as assessed by induction of endometrial gland arborization. The dual androgenic and progestational activities of DMA make it a potential candidate for a single-agent male contraceptive as well as for androgen therapy in men, pending a successful outcome of pharmacokinetic and toxicity studies currently in progress.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE CIRCADIAN PATTERN of testosterone (T) secretion (1) and circulating T levels (total and free) tend to decrease in aging males (1, 2, 3, 4). The most important clinical symptoms of age-related androgen deficiency are decreased muscle mass, decreased libido, erectile dysfunction, osteoporosis, decreased penile length or volume, reduced hemopoiesis, disturbances in well-being and mood, hot flushes, myocardial and circulatory problems, disturbances in lipid and glucose metabolism, abdominal obesity, insulin receptor resistance, and development of type 2 diabetes (2). Many of these symptoms also occur in younger men diagnosed with hypergonadotropic hypogonadism due to chromosomal abnormalities (e.g. Klinefelter’s syndrome), multiorgan diseases (e.g. AIDS), receptor defects (e.g. LH), or defects in the synthesis of T (5) or in hypogonadotropic hypogonadism due to disorders at the hypothalamic or pituitary level (6). In men who are hypogonadal due to aging or other causes, any regimen of hormonal therapy should be aimed at treating symptomatology. Current therapies involve the use of the natural hormone, T. When pure T is administered orally, however, only a small amount reaches the circulation due to absorption from the gastrointestinal tract into the portal blood and degradation by the liver (first pass effect) (7). Thus, for oral administration, T has been esterified with fatty acids (e.g. T undecanoate), alkylated at the 17{alpha}-position (e.g. 17{alpha}-methyltestosterone), or applied buccally or sublingually. Alternatively, T can be implanted as a subdermal pellet, administered transdermally as a patch or gel, or injected im as a fatty acid ester (e.g. T enanthate). Hypogonadal men treated with various forms of T replacement have experienced improved sexual function; increased lean body mass, muscle strength, and bone mineral density; as well as decreased fat mass (2).

Dimethandrolone (DMA: 7{alpha},11ß-dimethyl-19-nortestosterone) is a potent synthetic androgen (8). We are currently assessing the 17ß-undecanoic acid ester of DMA (DMAU) as a potential orally active agent for androgen therapy in men. Cleavage of the 17ß-ester bond in the body liberates biologically active DMA. In this study we describe the activity of DMA and DMAU in vivo in castrate rat bioassays (stimulation of ventral prostate (VP), seminal vesicles (SV), and levator ani (LA) muscle weights and suppression of gonadotropins) and in vitro [binding to the recombinant rat androgen receptor (AR) ligand-binding domain (ARLBD) and induction of AR-mediated transcription]. These assays indicate that DMAU is potent and bioavailable after oral administration, and, therefore, could be used at relatively low doses.

Other 19-norandrogens (e.g. 19-nortestosterone, R1881, and 7{alpha}-methyl-19-nortestosterone) are known to possess progestational activity in vivo and to bind to progestin receptors (PR) as well as to AR (9, 10, 11). Thus, we also examined the putative progestational activity of DMA both in vitro and in vivo. We determined the binding affinities of DMA, related 19-norandrogens, and C-19 androgens for rabbit uterine PR and recombinant human (rh) PR-A and rhPR-B. In addition, we evaluated the functional activity of these androgens in inducing PR-mediated transcription of a progestin/glucocorticoid/androgen response element (PRE2)-thymidine kinase (tk)-luciferase (LUC) reporter plasmid and endogenous alkaline phosphatase activity in T47DCO human breast cancer cells. We also performed bioassays to determine the progestational activity of DMA and DMAU in vivo (stimulation of endometrial gland arborization in estrogen-primed immature rabbits and stimulation of endometrial and myometrial width and uterine luminal cell height).

Hormonal male contraceptive methods based on androgens alone have not produced the desired azoospermia in about one third of Caucasian men (12). Recently developed regimens for hormonal contraception in men involve combinations of long-acting T esters or implants with progestins. Initial indications are that these combinations are highly effective in suppressing sperm production and are fully reversible (12, 13). As demonstrated here, DMA exhibits potency in both androgenic and progestational assays, and thus, it offers potential as a single-agent antifertility drug for men.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
T, 5{alpha}-dihydrotestosterone (DHT), 17ß-estradiol (E2), progesterone, androstenedione, and 19-nortestosterone (19-NT) were purchased from Steraloids (Newport, RI); mifepristone and 17{alpha}-methyltestosterone were obtained from Sigma-Aldrich Corp. (St. Louis, MO); methyltrienolone (R1881) and promegestone (R5020), were purchased from PerkinElmer Life Sciences, Inc. (Boston, MA); the antiandrogen nilutamide was obtained from Tocris-Cookson, Ltd. (Bristol, UK); and the antiandrogen bicalutamide was purchased from Toronto Research Chemicals, Inc. (Toronto, Canada). DMA (CDB-1321), DMAU (CDB-4521), and the 11{alpha}-methyl stereoisomer of DMA, CDB-4415 (7{alpha},11{alpha}-dimethyl-19-NT), were synthesized by Dr. P. N. Rao (Southwest Foundation for Biomedical Research, San Antonio, TX) and were 100% pure by HPLC and nuclear magnetic resonance. Dr. Rao’s laboratory also synthesized 5{alpha}-dihydro-DMA, 7{alpha}-methyl-19-nortestosterone (MENT), 7{alpha}-methyl-17a-ß-hydroxy-D-homo-19-noradrost-4,16-diene-3-one (7{alpha}-methyl, D-homo analog), 11ß-methyl-19-NT, the antiandrogen 2-hydroxyflutamide (OH flutamide), and the antiprogestins CDB-2914 (17{alpha}-acetoxy-11ß-[4-N,N-dimethylaminophenyl]-19-norpregna-4,9-diene-3,20-dione) and CDB-4124 (17{alpha}-acetoxy-21-methoxy-11ß-[4-N,N-dimethylaminophenyl]-19-norpregna-4,9-diene-3,20-dione). These compounds were more than 98% to 100% pure as determined by HPLC. CDB-2914 is also known variously as RTI 3021–012, RU 44675, and HRP 2000. The structures of the C-19 and 19-norandrogens used in this study are depicted in Fig. 1Go.


Figure 1
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  		FIG. 1. Structures of the androgens used in this study. The 19-norandrogens include DMA, DMAU, 5{alpha}-dihydro-DMA, MENT, 11ß-methyl-19-NT, 7{alpha}-methyl-17a-ß-hydroxy-D-homo-19-norandrost-4,16-dien-3-one (the 7{alpha}-methyl, D-homo analog), R1881, and 19-NT. The 11{alpha}-methyl stereoisomer of DMA (CDB-4415: 11{alpha}-methyl-DMA) is considerably less active than DMA in vivo. The C-19 androgens include T, DHT, 17{alpha}-methyltestosterone, and androstenedione.

 
Animals
New Zealand White rabbits (HsdOkd:NZW), purchased from Harlan (Oxford, MI), were fed Teklad (Indianapolis, IN) laboratory rabbit diet 2030 and fresh kale daily as a fiber supplement. Sprague Dawley CD rats [Crl:CD(SD)] were purchased from Charles River Laboratories (Kingston, NY, or Raleigh, NC). Rat pups arrived with lactating dams (10/dam) and were weaned on the day of castration (d 22 of age). For the LH suppression assay, male rats were castrated as adults (~300 g). All rats were fed Purina laboratory rodent diet 5001 (Ralston Purina Co., St. Louis, MO) ad libitum. All animals received tap water ad libitum. The photoperiod for the rabbit rooms was a 12-h light, 12-h dark period; that for the rat rooms was a 14-h light, 10-h dark period. The environmental conditions of the animal rooms were maintained as recommended in the Guide for the Care and Use of Laboratory Animals to the maximum extent possible (National Research Council, 1996). All study protocols were approved by BIOQUAL’s institutional animal care and use committee.

In vivo androgenic activity and duration assays
For the androgenic assay, immature male rats (22 d of age) were castrated and treated orally with vehicle, DMA, or DMAU for 7 consecutive days. Twenty-four hours after the final dose, the rats were killed, and body weights were determined. The VP, SV, and LA muscle were excised, trimmed, blotted, and weighed (14). For the duration of androgenic activity bioassay, the immature castrated male rats received a single sc injection of DMAU in aqueous suspending vehicle (ASV). Rats (five per time point) were killed by exsanguination at specified times after treatment, and VP and SV were excised and weighed as described above. Blood was collected in serum separator tubes, and sera were harvested by centrifugation and assayed for levels of DMA by RIA.

In vivo LH suppression assay
On d 0 (wk 0), castrated adult male rats (eight per group) were randomized into treatment groups based on body weight. Rats either received a single 12 mg/kg sc dose of DMAU or 12 mg/kg·d DMAU orally for 14 d. Rats were bled from the tail vein on wk –1, on d 0 immediately before treatment, on d 4, and weekly thereafter until wk 9 (oral group) or wk 14 (sc group). The latter group was also bled at wk 16, 18, 20, 22, 26, and 30. Serum was assayed for levels of LH and DMA by RIA.

LH RIA
LH was measured in rat serum using reagents from the National Hormone and Peptide Program (supplied by Dr. A. F. Parlow) following the procedures received with the reagents. The standard was National Institute of Diabetes and Digestive and Kidney Disease rat LH-reference preparation-3. The limit of detection was 0.13–0.22 ng/ml based on 200 µl serum. The intra- and interassay coefficients of variation were 5–6% and 10%, respectively, for pools from castrate and intact rats.

RIA for DMA and putative immunoreactive metabolites
Serum levels of DMA and putative immunoreactive metabolites were determined using a specific RIA developed at BIOQUAL, Inc. Polyclonal antisera to DMA were generated in rabbits by immunization with the 3-(carboxymethyl)oxime-BSA conjugate of DMA following the methods of Vaitukaitis et al. (15) and Larner et al. (16). The 3-(carboxymethyl)oxime-histamine conjugate of DMA was iodinated with 1 mCi Na[125I] (PerkinElmer Life Sciences, Inc.) using chloramine-T as the catalyst. The iodinated conjugate was extracted with benzene and purified by reverse phase HPLC. For the RIA, a DMA standard curve, suitable serum dilutions, PBS-0.1% BSA, and rabbit antiserum 75042, bleed 5 (final dilution, 1:2–8 x 106), were preincubated in the assay tubes for 1 h at room temperature. Radioligand was added, and the tubes were incubated overnight at 2–6 C. Bound and free radioligands were separated by centrifugation after addition of second antibody (goat antirabbit {gamma}-globulin) and polyethylene glycol. The resulting pellets were counted in a Packard Cobra II {gamma}-counter, and the raw data were exported to the RiaSmart data reduction program (PerkinElmer Life Sciences, Inc.). A four-parameter logistic curve fit was used to generate the standard curve and interpolate the serum concentrations of DMA. The EC50 values of the standard curves for the RIAs reported varied from 3.8–9.2 pg/tube. The limit of detection varied from 112–384 pg/ml for 100 µl serum, calculated from the mean ± 3 SD of serum samples collected before treatment or from vehicle-treated controls. The intra- and interassay coefficients of variation were 5% and 15%, respectively.

Because the metabolism of DMA has not been studied, we do not know whether it is converted in vivo to other products that would cross-react in the RIA. However, the antiserum used in this RIA (from rabbit 75042, bleed 5) has been tested extensively for cross-reactivity with compounds closely related to DMA, and it has been shown to be very specific for DMA itself. We have determined that there is negligible (1–2%) cross-reactivity between DMAU and rabbit antiserum 75042; therefore, levels of DMA in serum of animals administered DMAU are only detectable after cleavage of the 17ß-undecanoic acid moiety. Cross-reactivity with endogenous hormones, i.e. T, DHT, E2, progesterone, and corticosterone, was less than 0.1%.

In vivo progestational agonist activity
For assessing in vivo progestational activity, immature female rabbits (five per group) were primed with E2 (5 µg/rabbit·d, sc) for 6 d, then treated orally with vehicle (10% ethanol/90% sesame oil), DMA (0.25, 0.50, 1.0, or 2.0 mg/d), or an equimolar quantity of DMAU (0.4, 0.8, 1.6, or 3.2 mg/d) or injected sc with progesterone (0.16 mg/d) for 5 d (17). Twenty-four hours after the final dose, rabbits were killed, and their uteri were excised, trimmed of extraneous tissue, blotted, weighed, fixed, and stained. Paraffin sections (5 µm) of fixed uteri were evaluated for endometrial gland arborization based on the scoring system of McPhail (18). Photographs of uterine sections were obtained using the SPOT Insight QE digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI) and software attached to a light microscope (Nikon Corp., Melville, NY). Image Pro Plus, version 4.5 (Media Cybernetics, Inc., Silver Spring, MD), was used to determine endometrial and myometrial widths and luminal epithelial cell heights in uterine sections.

Steroid hormone receptor assays
Competitive binding assays for PR were performed using cytosolic preparations from tissues or cells as described previously (19). Cytosols containing PR were prepared from the uterus of E2-primed immature rabbits. rhPR-A or rhPR-B were assayed in cytosols from Sf9 insect cells infected with recombinant baculovirus expressing either hPR-A or hPR-B obtained from Dr. Dean Edwards (University of Colorado Health Sciences Center, Denver, CO). The assay for wild-type AR employed purified ARLBD) purchased from Invitrogen Life Technologies, Inc. (Carlsbad, CA) (20). Progesterone and R1881 were the standards for the PR and AR assays, respectively.

Cell culture and transfection of plasmid DNAs
Cell culture reagents were obtained from Invitrogen Life Technologies, Inc. (Grand Island, NY) unless otherwise specified. T47DCO human breast cancer cells (a gift from Dr. Kathryn Horwitz, University of Colorado Health Sciences Center), which express approximately equimolar concentrations of constitutively produced hPR-A and hPR-B (21), were used to assess progestational agonist or antagonist activities in an estrogen-free environment. They were grown routinely in monolayer culture in phenol red-free DMEM supplemented with 10% fetal bovine serum, 6 ng/ml bovine insulin, 100 U/ml penicillin G, and 100 µg/ml streptomycin sulfate. CV-1 cells were purchased from American Type Culture Collection (Manassas, VA) and grown in the same medium without insulin. For transient transfections assays, cells were plated in six-well dishes at 0.5–1.0 x 106 cells/well in the corresponding medium containing 10% dextran-coated charcoal-stripped fetal bovine serum (HyClone Laboratories, Inc., Logan, UT) and used 24 or 48 h later at 60–80% confluence. T47DCO cells were transfected with the PRE2-tk-LUC reporter plasmid, containing two copies of a PRE upstream of the tk promoter and the firefly LUC reporter gene (provided by Dr. Dean Edwards) using FuGene 6 transfection reagent (Roche, Indianapolis, IN) as described previously (17). CV-1 cells were cotransfected with the pG3XHRE3 FFR-tk-LUC[3XHRE-LUC] reporter plasmid, containing three copies of a PRE (equivalent to HRE), and an hAR expression plasmid (pCMV5 hAR3.1), both gifts from Dr. Diane Robins (University of Michigan Medical School, Ann Arbor, MI), using the same conditions. Cell lysates were prepared in passive lysis buffer (Promega Corp., Madison, WI) and analyzed for protein content (Bio-Rad Laboratories, Inc., Hercules, CA) and LUC activity using Promega’s luciferase assay system. Light emission was measured in a microplate luminometer (Fluoroskan Ascent FL, Labsystems, Franklin, MA) and expressed as relative light units. Data were normalized for differences in protein content per well.

Alkaline phosphatase assay
Induction or inhibition of endogenous alkaline phosphatase activity in T47DCO cells was assessed as described previously (19) using the procedure of Markiewicz and Gurpide (22).

Analysis of data
Data are expressed as the mean ± SEM (n ≥ 3) or the mean ± SD (n = 2). PRISM, versions 2.0–4.0 (GraphPad, Inc., San Diego, CA), was used for graphics and determination of IC50 and EC50 values for inhibition or stimulation, respectively, of transactivation or alkaline phosphatase activity by antagonists or agonists of PR or AR. In all steroid receptor binding assays, counts per minute were entered into RiaSmart for calculation of EC50 values using a four-parameter logistic curve fit. Relative binding affinities (percentage) for each compound were calculated as follows: EC50 of standard/EC50 of competitor x 100. Uterine weights in the in vivo progestational assay were compared with one another by one-way ANOVA of log10-transformed data followed by the Holm-Sidak multiple comparison test using SigmaStat, version 3.0 (Jandel Scientific, San Rafael, CA). Endometrial and myometrial widths and luminal epithelial cell height in uterine sections from treated rabbits were compared with those in sections from control rabbits by ANOVA, followed by the Holm-Sidak test. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Androgenic activity of DMA and DMAU in vivo
A 7-d bioassay was carried out to assess the oral androgenic potencies of DMA and DMAU (Fig. 2Go). Compared with the oral reference standard, methyltestosterone, potency ratios (based on VP weights) were 0.98 ± 0.33 (n = 2) for DMA and 2.72 ± 0.33 (n = 4) for DMAU. The oral anabolic potency ratios (based on LA muscle weights) were 5.09 for DMA and 22.39 for DMAU (see Fig. 2CGo). The duration of androgenic activity after a single sc dose of 0.6 or 1.2 mg/rat DMAU to 22-d-old castrate male rats is illustrated in Fig. 3Go. There were increases in VP (Fig. 3AGo) and SV (Fig. 3BGo) weights during the first 6 wk of the study, which were greater at the 1.2-mg dose; thereafter, VP and SV weights declined slowly over 21 wk, but these tissues did not involute to the degree observed in immature castrate male rats treated with vehicle. Serum levels of DMA and its putative immunoreactive metabolites were measured by RIA. In rats treated with 1.2 mg DMAU, serum levels of DMA were maximal at wk 3 and declined gradually to undetectable levels by about wk 18. In rats treated with 0.6 mg DMAU, serum levels of DMA were barely detectable over the 21-wk period.


Figure 2
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  		FIG. 2. Oral potency of DMA and DMAU in the 7-d androgenic assay. Castrate 22-d-old rats (eight per group) were treated daily for 7 d with vehicle (ASV), DMA (0.4, 1.6, or 6.4 mg/rat total dose), DMAU (0.63, 2.5, or 10.0 mg/rat total dose), or the oral standard, 17{alpha}-methyltestosterone (1.0, 4.0, or 16.0 mg/rat total dose). The doses of DMAU were equivalent, on a molar basis, to those of DMA. The rats were killed 24 h after the final treatment, and VP (A), SV (B), and LA muscle (C) weights were determined. Values represent the mean ± SE.

 

Figure 3
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  		FIG. 3. Duration of action of a single sc dose of DMAU on VP and SV weights. Castrate 22-d-old rats received a single sc injection of DMAU (0.6 or 1.2 mg) in ASV or ASV alone on the day of castration. Rats (five per time point) were killed by exsanguination at the indicated times after treatment, and VP (A) and SV (B) were excised and weighed. Groups of vehicle-treated rats were killed at the beginning and end of the experiment (wk 3 and 21). VP (A) and SV (B) weights of the vehicle-treated rats were much lower than those of the DMAU-treated animals. Serum was assayed for DMA and its immunoreactive metabolites by RIA (C). Serum interference was unusually high in the assays used to measure DMA in this experiment. The limit of detection (dashed line), calculated as the mean ± 3 SD for 100 µl castrate rat serum, ranged from 350–384 pg/ml. Data points represent the mean ± SE.

 
Another experiment was designed to examine the duration of action of DMAU in suppressing serum LH in castrate adult male rats after a single sc injection or 14 daily oral doses of 12 mg/kg. This dose was approximately equivalent to the 1.2 mg/rat dose administered to immature castrated male rats in the androgenic assay described above. In rats that received DMAU sc, serum LH decreased to levels at or close to undetectable by wk 1, remained at this level for up to 18 wk, and increased gradually over the next 12 wk (Fig. 4Go). Serum DMA levels peaked on wk 1 at about 1000 pg/ml after a single sc injection of DMAU and declined very gradually to the limit of detection (<112 pg/ml) at 30 wk (Fig. 4Go). In those rats that received DMAU orally, serum LH levels were suppressed to close to undetectable during the treatment interval, but they rose to pretreatment levels by wk 3. Serum DMA levels were 300–400 pg/ml at wk 1 and 2, but fell precipitously to the limit of detection when treatment was terminated.


Figure 4
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  		FIG. 4. Suppression of LH by DMAU administered orally or sc. Castrate adult male rats (eight per group) received a single 12 mg/kg sc injection or daily 12 mg/kg oral doses of DMAU for 14 d. Blood was collected from the tail vein at the indicated times, and serum levels of LH and DMA were measured by RIA. The limit of detection of LH was 0.13–0.22 ng/ml, and that of DMA was 112 pg/ml. Data points represent the mean ± SE.

 
Binding of C-19 and 19-norandrogens to PR and ARLBD
Table 1Go summarizes the EC50 values and relative binding affinities of the androgens used in this study for ARLBD, rabbit uterine PR (PR-B) (23), and rhPR-A or rhPR-B. The C-19 androgens showed uniformly low affinity for PR and, with the exception of androstenedione, higher affinity for AR. DMA, MENT, 11ß-methyl-19-NT, 19-NT, and especially the 7{alpha}-methyl, D-homo analog bound relatively well to PR and AR. DMAU as well as the 11{alpha}-methyl stereoisomer of DMA, which is much less active in vivo, demonstrated very low affinity for both PR and AR. The results for DMAU indicated that the ester moiety was not cleaved during incubation of DMAU with AR- or PR-containing cytosols.


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  		TABLE 1. Binding of C-19 and 19-norandrogens to PR and AR

 
Stimulation of androgenic transcriptional activity by C-19 or 19-norandrogens in CV-1 cells
The relative potencies of various androgens was compared by cotransfecting CV-1 cells with a human AR expression vector and the 3XHRE-LUC reporter plasmid (Table 2Go). After incubation of transfected CV-1 cells with various concentrations of C-19 or 19-norandrogens, dose-dependent increases in LUC activity were observed. Methyltestosterone and R1881 were the most potent inducers of LUC activity (EC50, ~10–10 M). All other androgens tested had EC50 values for transactivation of approximately 10–9 M, with the exception of the 11{alpha}-methyl stereoisomer of DMA, which was a much weaker agonist (EC50, ~10–7 M).


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  		TABLE 2. Stimulation of transcription by various androgens in CV-1 cells cotransfected with 3XHRE-tk-LUC and a hAR expression vector (pCMV5hAR3.1)

 
Stimulation of progestational transcriptional activity by C-19 or 19-norandrogens in T47DCO cells
We showed previously (17) that in T47DCO cells transiently transfected with PRE2-tk-LUC, transcriptional activity was induced by progestins, but not by other classes of steroid hormones (i.e. T, E2, and glucocorticoids). Figure 5Go shows that DMA and other 19-norandrogens, at 10–8 M, increased transcriptional activity to the same extent as the progestin standard, R5020, at 10–8 M. This was also observed for 5{alpha}-dihydro-DMA, 11ß-methyl-19-NT, and 19-NT in another experiment (not shown). An appreciable stimulation was obtained with DMAU, suggesting that the esterified parent compound was converted to the free androgen during overnight incubation with T47DCO cells at 37 C. Transiently transfected T47DCO cells were incubated with several 19-norandrogens at concentrations from 10–11–10–6 M. DMA, MENT, 5{alpha}-dihydro-DMA, and 11ß-methyl-19-NT were all very potent inducers of LUC activity with EC50 values of 2.54 ± 0.13, 2.68 ± 0.36, 10.0 ± 4.27, and 2.84 ± 1.03 x 10–9 M, respectively (data not shown). DMA and the other 19-norandrogens tested were comparable in potency to progesterone and were about 10-fold less potent than R5020 (19). In contrast, transactivation by the C-19 androgens shown in Fig. 5Go, by another C-19 androgen, 16{alpha}-hydroxyandrostenedione, as well as by the 11{alpha}-methyl stereoisomer of DMA, all at 10–8 M, was negligible. The failure of C-19 androgens to stimulate transcription of PRE2-tk-LUC indicates that T47DCO cells do not contain functional AR, and therefore, that the transactivation of PRE2-tk-LUC was mediated by PR.


Figure 5
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  		FIG. 5. Effect of 19-norandrogens or C-19 androgens on transcriptional activity in T47DCO cells transiently transfected with PRE2-tk-LUC. Transfected T47DCO cells were treated with the indicated compounds at 10–8 M for 20 h and lysed. LUC activity was normalized for differences in protein content per well. Data points represent the mean ± SD of duplicate values. Comparable results were obtained in a second experiment.

 
Additional confirmation of this notion was obtained by treating transfected T47DCO cells with 10–8 M DMA, DMAU, MENT, R1881, or the 7{alpha}-methyl, D-homo analog in the presence of antiprogestins (mifepristone or CDB-2914) or an antiandrogen, OH flutamide, at a 100-fold excess (Fig. 6Go). Although transactivation by all 19-norandrogens was completely inhibited by both antiprogestins at 10–6 M, OH flutamide showed incomplete and variable inhibition (16–70%). This was investigated in more detail by determining the dose-dependent inhibition of 10–8 M DMA-stimulated transcription by antiprogestins or antiandrogens. T47Dco cells transfected with PRE2-tk-LUC were incubated with the antagonists at concentrations from 10–11–10–5 M (data not shown). In fact, antiprogestins were extremely efficacious at blocking transactivation by DMA with IC50 values of 0.98 ± 0.09 x 10–10 M for mifepristone, 3.21 ± 0.45 x 10–10 M for CDB-2914, and 2.94 ± 1.01 x 10–10 M for another antiprogestin, CDB-4124, whereas androgen antagonists were at least 4 orders of magnitude less potent (IC50, 1.15 ± 0.42 x 10–6 M for OH flutamide, >10–5 M for bicalutamide, and 6.94 ± 4.33 x 10–6 M for nilutamide). The slight effect of the antiandrogens may be due to a weak interaction with PR.


Figure 6
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  		FIG. 6. Effects of antiprogestins or antiandrogens on transcriptional activity stimulated by 19-norandrogens. T47DCO cells were transiently transfected with PRE2-tk-LUC and treated with the indicated compounds at 10–8 M in the presence or absence of mifepristone, CDB-2914, or OH flutamide (10–6 M) for 20 h. LUC activity was normalized for differences in protein content per well. Data points represent the mean ± SD of duplicate values.

 
Stimulation of endogenous alkaline phosphatase activity by C-19 or 19-norandrogens in T47DCO cells
The results for regulation of endogenous alkaline phosphatase activity were in good agreement with those for regulation of the exogenous PRE2-tk-LUC reporter gene in T47DCO cells. We showed previously (19) that alkaline phosphatase was stimulated by progestins, but not by other steroid hormones (T, E2, corticosterone, and dexamethasone). However, at 10–8 M, DMA, MENT, R1881, the 7{alpha}-methyl, D-homo analog, and 19-NT all produced substantial increases in alkaline phosphatase activity (Fig. 7Go). Alkaline phosphatase activity was not induced in cells treated with C-19 androgens or the 11{alpha}-methyl stereoisomer of DMA at 10–8 M. T47DCO cells were incubated with selected 19-norandrogens at concentrations ranging from 10–11–10–5 M to estimate EC50 values. The values obtained for DMA (6.3 ± 0.4 x 10–9 M), MENT (7.9 ± 3.8 x 10–9 M), 5{alpha}-dihydro-DMA (2.7 ± 1.0 x 10–9 M), and 11ß-methyl-19-NT (5.5 ± 2.1 x 10–9 M; data not shown) were similar to that for progesterone (2.3 ± 1.7 x 10–9 M), whereas, as reported previously, R5020 was about 10-fold more potent (19). Thus, the observation that the 19-norandrogens at 10–8 M did not induce alkaline phosphatase to the same extent as 10–8 M R5020 (Fig. 7Go) was presumably due to the difference in their EC50 values. Induction of alkaline phosphatase activity by DMA, like transactivation, was strongly inhibited by antiprogestins (IC50, 9.8 ± 1.1 x 10–10 M for mifepristone, 7.4 ± 4.4 x 10–10 M for CDB-2914, and 1.3 ± 0.8 x 10–9 M for CDB-4124), but not by antiandrogens (IC50, >10–5 M for OH flutamide and nilutamide), indicating that it is mediated by PR (Fig. 8Go).


Figure 7
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  		FIG. 7. Effects of 19-norandrogens or C-19 androgens on endogenous alkaline phosphatase activity in T47DCO cells. T47DCO cells were treated with the indicated compounds at 10–8 M for 72 h, washed, and lysed for measurement of alkaline phosphatase activity. Data points represent the mean ± SE of quadruplicate values.

 

Figure 8
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  		FIG. 8. Dose response of inhibition of DMA (10–8 M)-stimulated endogenous alkaline phosphatase activity by antiprogestins or antiandrogens. T47DCO cells were treated with DMA (10–8 M) in the presence or absence of increasing concentrations of antiprogestins (mifepristone and CDB-2914) or antiandrogens (OH flutamide and nilutamide) for 72 h, washed, and lysed, and alkaline phosphatase activity was measured. Data points represent the mean ± SE of quadruplicate values.

 
Progestational activity of DMA and DMAU in vivo
Because DMA binds PR and stimulates PR-mediated transcription and alkaline phosphatase activity in vitro, DMA and DMAU were tested for oral progestational activity in E2-primed immature female rabbits. The doses of DMAU used in this assay were equivalent to those of DMA on a molar basis. After oral administration in 10% ethanol/90% sesame oil, DMA and DMAU demonstrated dose-dependent stimulation of endometrial gland arborization and uterine wet weight (Table 3Go). This response reached a plateau at doses of 1 and 1.6 mg/rabbit·d for DMA and DMAU, respectively. However, a maximal response (McPhail index score, 4.0), as obtained for the progesterone standard and R5020 (not shown), was not achieved, even at the highest dose levels. Endometrial cross-sections from rats treated for 5 d with vehicle (A), DMA (B), or progesterone (C) are shown in Fig. 9Go. The increase in uterine wet weight could be attributed to significant (P < 0.05) increases in both endometrial and myometrial widths compared with uteri from vehicle-treated rabbits (Table 3Go). In a second similar experiment (not shown), we observed that both luminal epithelial cell height and endometrial width were significantly (P < 0.05) increased in progesterone- or DMA-treated rabbits compared with those in animals treated with vehicle; however, only DMA increased (P < 0.05) myometrial width.


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  		TABLE 3. Stimulation of endometrial gland arborization (McPhail index) and uterine weight in immature E2-primed female rabbits by DMA or DMAU

 

Figure 9
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  		FIG. 9. Progestational activity of DMA in vivo: representative sections showing stimulation of endometrial gland arborization. Immature female rabbits (five per group) were primed with E2 for 6 d, then treated orally with either vehicle (A; 10% ethanol/90% sesame oil) or DMA (B; 1.0 mg/d) or sc with progesterone (C; 0.16 mg/d) for 5 d. Rabbits were killed 24 h after the final dose, and their uteri were weighed, fixed, and stained. Paraffin sections (5 µm) of fixed uteri were evaluated for endometrial gland arborization based on the scoring system of McPhail (18 ). The McPhail scores for vehicle, DMA, and progesterone in this assay were 0, 2.3, and 4.0, respectively. Similar results were obtained for DMAU at 1.6 or 3.2 mg/d (Table 3Go).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
T is the principal androgen secreted by Leydig cells. It exerts both androgenic effects involving growth stimulation and functional maintenance of the male reproductive tract and anabolic effects involving growth stimulation of nonreproductive organs, such as muscle, kidney, liver, and submaxillary salivary glands (24). This natural androgen is both 5{alpha}-reduced to DHT, a potent androgen that binds with high affinity to AR, and aromatized to E2. In hypogonadal or aging men with clinical symptoms suggestive of androgen deficiency and reduced serum T levels, T replacement therapy has successfully alleviated symptoms. To what extent these effects are mediated directly through T or through conversion to DHT or E2 has not been clearly delineated.

Alternative androgens, that may demonstrate greater potency, duration of action, and oral efficacy than T, are being developed for hormonal therapy. However, the relative androgenic, anabolic, and estrogenic activities of these compounds have to be taken into account based on their metabolism in various tissues. In addition, as we have shown here, 19-norandrogens exhibit considerable progestational activity both in vitro and in vivo, which may or may not be a desirable property for therapeutic androgens. In the immature rabbit model, DMA and DMAU demonstrated dose-dependent stimulation of endometrial gland arborization. A McPhail index score of 2.3–2.6 was obtained for these compounds compared with a maximal response of 4.0 obtained for the progesterone standard. This result suggests that these 19-norandrogens should be classified as impeded progestin agonists in vivo (25). MENT was also shown to have progestational activity in immature rabbits, as measured by the McPhail index (26).

As reported here, DMA and other 19-norandrogens exhibited high potency in the in vitro assays for progestational activity in T47DCO cells. Although the PRE2-tk-LUC reporter plasmid is responsive to androgens and glucocorticoids as well as to progestins, we have clearly shown that the actions of 19-norandrogens in T47DCO cells are mediated by PR. Antiprogestins, but not antiandrogens, were potent inhibitors of DMA-induced transactivation and alkaline phosphatase activity, and C-19 androgens showed little or no induction of transcription or alkaline phosphatase activity. In contrast, in CV-1 cells cotransfected with a human AR expression vector and 3XHRE-LUC, 19-norandrogens and C-19 androgens were equipotent in stimulating luciferase activity. The order of affinity of the various androgens for the ARLBD was 11ß-methyl-19-NT>7{alpha}-methyl, D-homo analog>DMA, 5{alpha}-dihydro-DMA>MENT, 19-NT>DHT>T> 17{alpha}-methyltestosterone>7{alpha},11{alpha}-dimethyl-19-NT>androstenedione. The order of binding of selected androgens to AR in rat VP cytosols and their potency in stimulation of an androgen-inducible reporter plasmid reported by Kumar et al. (11) are in reasonable agreement with the present results.

The effect of introducing methyl groups at both the 7{alpha}- and 11ß-positions in the 19-NT backbone (to produce DMA) was initially examined by Cook and Kepler (8). They concluded from studies involving AR binding and stimulation of VP, SV, and LA weights after sc administration for 7 d that the combination of 7{alpha} and 11ß substituents in 19-NT led to a more potent and selective anabolic to androgenic ratio than observed for either monomethylated 19-NT derivative. As shown here, the ratio of anabolic potency (stimulation of LA weights) to androgenic potency (stimulation of VP weights) of DMA and DMAU was also favorable when they were administered orally. Furthermore, DMAU demonstrated prolonged duration of action after a single sc injection; in castrate 22-d-old rats, VP and SV growth were maintained above control levels for over 20 wk, and in castrate adult rats, circulating LH was suppressed for at least 18 wk, corresponding to sustained serum levels of DMA. Oral administration of DMAU was also effective in reducing LH secretion as long as treatment was continued, and serum levels of DMA remained elevated.

MENT, the 7{alpha}-monomethylated derivative of 19-NT, has been extensively evaluated for long-term clinical use either as androgen replacement therapy for hypogonadal men or as part of a male hormonal contraceptive regimen. Sundaram et al. (24) found that MENT was not 5{alpha}-reduced by rat liver or prostate homogenates, which they attributed to steric hindrance from the 7{alpha}-methyl group. The lack of 5{alpha}-reduction was used to support the idea that a suitable dose of MENT could be chosen for hormonal therapy that would maintain muscle mass, but not stimulate prostate growth. Although we have not directly examined the ability of DMA to be 5{alpha}-reduced in vivo, this possibility seems unlikely because of the presence of the 7{alpha}-methyl group. It is interesting to note that synthetic 5{alpha}-dihydro-DMA demonstrated androgenic and progestational potencies comparable to those of DMA in our in vitro assays.

The pharmacokinetic properties of MENT make it unsuitable for once-daily oral treatment or long-term injection; thus, administration by sc implant or by patch or gel is required (27). MENT showed a more rapid metabolic clearance rate than T in men and monkeys, probably due in part to its failure to bind SHBG (28). In monkeys, MENT acetate in subdermal implants was 10 times as potent as T in suppression of gonadotropin secretion and anabolic effects, but was only twice as potent in stimulating prostate growth (29). Clinical studies carried out using MENT acetate implants in men (30) demonstrated dose-dependent decreases in LH, FSH, T, DHT, and SHBG, but no change in prostate-specific antigen or IGF-I. A recent study in rats showed that MENT had protective effects on bone and muscle at concentrations that did not cause prostate hypertrophy (31). Clinical testing of DMA, DMAU, or other esters of DMA, which could be developed either for oral administration or as a long-acting injectable for hormonal therapy in men, will depend on a successful outcome of the pharmacokinetic and toxicity testing currently in progress.

An important goal of research involving hormonal control of testicular function is the development of safe, effective, and reversible male hormonal contraceptives. Hormonal male contraception methods based on androgens alone have not produced the desired azoospermia in about one third of Caucasian men (12). Although progesterone and synthetic progestins also suppress spermatogenesis, they are not good candidates for single-agent male contraception, because they result in decreased T secretion and iatrogenic hypogonadism (32). Therefore, recently developed regimens involve combinations of progestins or GnRH antagonists with T implants or long-acting T esters (12, 32, 33). Although these regimens may involve the inconvenience of weekly im injections and produce undesirable side effects, such as weight gain or suppression of high-density lipoprotein (32, 33), initial indications are that these combinations are highly effective in suppressing sperm production and are fully reversible (12, 13, 32). Continuous administration of a GnRH agonist together with MENT in rhesus monkeys resulted in an extended period of azoospermia, followed by complete recovery after cessation of treatment (34). In a clinical trial to assess the potential of MENT as a hormonal male contraceptive, 82% of the subjects who received four subdermal MENT acetate implants achieved azoospermia (35). Because DMA exhibits potency in both androgenic and progestational assays, it also offers potential as a single-agent antifertility drug for men. DMAU resulted in sustained suppression of serum LH in castrated adult male rats after a single sc injection, suggesting that it may be an effective antispermatogenic agent. The possible antispermatogenic activity of DMAU is currently being investigated in our laboratory in a rabbit model. In fact, our preliminary results indicate that DMAU is very effective in inducing severe oligospermia in the adult male rabbit, while not affecting libido or ejaculation, and the antispermatogenic effect appears to be completely reversible. Future work will involve elucidation of the time course, dose dependency, and specificity of this antispermatogenic effect.


   Acknowledgments
 
We thank Dr. Richard Blye (Contraception and Reproductive Health Branch, National Institute of Child Health and Human Development) for his input into the design of these studies, his continued support, and his review of this manuscript. We are grateful to Janet Burgenson, Lisa Radler, Trung Pham, Margaret Krol, Bruce Till, Eileen Curreri, David Gropp, and Jessica Luke for their excellent technical assistance, and to Dr. Sailaja Koduri for reviewing this manuscript.


   Footnotes
 
This work was supported by National Institutes of Health, National Institute of Child Health and Human Development Contract NO1-HD-2-3338 (to BIOQUAL, Inc.) and Contract NO1-HD-6-3255 (to Dr. P. N. Rao). A portion of this work was presented at the 86th Annual Meeting of The Endocrine Society, June 2004.

B.J.A., S.A.H., and J.R.R. have nothing to declare.

First Published Online February 23, 2006

Abbreviations: AR, Androgen receptor; ARLBD, androgen receptor ligand-binding domain; ASV, aqueous suspending vehicle; DHT, 5{alpha}-dihydrotestosterone; DMA, dimethandrolone; DMAU, 17ß-undecanoic acid ester of dimethandrolone (7{alpha},11ß-dimethyl-19-nortestosterone); E2, 17ß-estradiol; h, human; LA, levator ani; LUC, luciferase; MENT, 7{alpha}-methyl-19-nortestosterone; 19-NT, 19-nortestosterone; OH flutamide, 2-hydroxyflutamide; PR, progestin receptor; PRE, progestin/glucocorticoid/androgen response element; SV, seminal vesicle; T, testosterone; tk, thymidine kinase; VP, ventral prostate.

Received December 1, 2005.

Accepted for publication February 15, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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