Metabolism of formestane in humans: Identification of urinary biomarkers for antidoping analysis.

Formestane (4-hydroxyandrost-4-ene-3,17-dione, 4OH-AED) is an aromatase inhibitor prohibited in sports. In recent years, it has been demonstrated that it can also originate endogenously by the hydroxylation in C4 position of androstenedione. Thus, the use of isotope ratio mass spectrometry (IRMS) is mandatory according to the World Antidoping Agency (WADA) to discriminate endogenous from synthetic origin. In a previous work and after oral administrations of formestane (4OH-AED), the ratio between the main formestane metabolite (4α-hydroxyepiandrosterone; 4OH-EA) and formestane parent compound could help to identify the endogenous origin, avoiding unnecessary and costly IRMS confirmations. In the present work, we investigated whether the same criteria could also be applied after transdermal applications. Six volunteers were transdermally treated once with formestane. Urine samples were collected for 120 h postadministration and analyzed by gas chromatography coupled to mass spectrometry (GC-MS and GC-MS/MS). Formestane and its major metabolites were monitored. The kinetic profile of formestane and its main metabolites was found different between oral and transdermal application. A shift on the excretion of the metabolites compared to formestane itself that can be observed after the oral administration, is absent after the transdermal one. This makes that a simple criteria cannot be applied to differentiate the endogenous from the synthetic origin based on metabolic ratios. The ratio between 4-hydroxyepiandrosterone and 4-hydroxyandrosterone (4OH-A) can be used to differentiate the route of administration. Ratios higher than one (4OH-EA/4OH-A > 1) are diagnostic of an oral administration. This allows to correctly interpret the 4OH-EA/4OH-AED ratio as proposed in our previous investigation. The results of this work demonstrate that the use of appropriate biomarkers (metabolic ratios) helps to reach correct conclusions without using complex and costly instrumentation approaches.

Acute developmental exposure to 4-hydroxyandrostenedione has a long-term effect on visually-guided behaviors.

Estrogenic and anti-estrogenic endocrine disrupting compounds (EDCs) are recognized as critical modulators of neural development, including sensory system development. Using the zebrafish model, we tested the effect of transient developmental exposure to a known anti-estrogenic EDC on adult visually-guided behavior. In particular, we exposed zebrafish aged 24-hour post-fertilization (hpf), 72 hpf, or 7-days post-fertilization (dpf) to the aromatase inhibitor 4-hydroxyandrostenedione (4-OH-A) for 24h. After this time, the fish were removed from treatment, placed into control conditions, and reared until adulthood (3-4months) when visually-guided optomotor responses (OMR) were assessed. Our results show significant decreases in positive OMR in adults exposed to 4-OH-A at 72 hpf and 7 dpf. These deficits were not accompanied by changes in overall swimming behaviors and startle responses, suggesting 4-OH-A specifically effected the visual system. Overall, this study identified long-term, quantifiable effects in visually-guided adult behaviors resulting from transient developmental exposure to the anti-estrogenic EDC, 4-OH-A. Further, these effects were noted when 4-OH-A exposure occurred after hatching, suggesting estrogen signaling is important for visual system maturation.

Determination and confirmation of selective estrogen receptor modulators (SERMs), anti-estrogens and aromatase inhibitors in bovine and porcine urine using UHPLC-MS/MS.

Selective estrogen receptor modulators (SERMs), anti-estrogens and aromatase inhibitors are prohibited in human sports doping. However, they also present a risk of being used illegally in animal husbandry for fattening purposes. A method was developed and validated using UHPLC-MS/MS for the determination and confirmation of SERMs, anti-estrogens and aromatase inhibiters in bovine and porcine urine. This method was used in a survey of more than 200 bovine and porcine urine samples from Dutch farms. In 18 out of 103 porcine urine samples (17%) and two out of 114 bovine samples (2%) formestane, an aromatase inhibitor, was detected. None of the other compounds was detected. From human doping control it is known that formestane can, in some cases, be of natural origin. Analyses of reference samples from untreated bovine and porcine animals demonstrated the presence of formestane in bovine animals, but not yet in porcine animals. Future research will focus on whether the detected formestane in porcine and bovine urine is from endogenous or exogenous origin, using GC-c-IRMS.

Effects of acute androstenedione supplementation on testosterone levels in older men.

The purpose of the study was to examine the effects of acute androstenedione supplementation on hormone levels in older men at rest and during exercise. Men (n = 11) between the ages of 58 and 69 were divided into an experimental (n = 6; 62.33 ± 2.57 y) and control (n = 5; 60.2 ± 1.02 y) groups. Each participant received an oral 300 mg dose of either androstenedione (experimental) or a cellulose placebo (control) for 7 d. Pre- and post-supplementation participants completed two separate, 20-min strength tasks consisting of leg extension and leg curls at different percentages of their 10-RM. Researchers collected blood samples pre-, during, and post-exercise. Blood samples were analyzed for testosterone, androstenedione, and estradiol levels. The researchers found a significant difference between pre- (4.36 ± 56 ng/mL) and post- (5.51 ± 0.35 ng/mL) testosterone levels, as well as pre- (0.88 ± 0.20) and post- (7.46 ± 1.25) androstenedione levels, but no significant differences between pre- and post-estradiol levels for either group. It appears that short-term androstenedione supplementation augmented acute testosterone responses to resistance exercise in older men. However, further study of this supplement is needed to determine any potential it may have in mitigating andropause.

Metabolic studies of formestane in horses.

Formestane (4-hydroxyandrost-4-ene-3,17-dione) is an irreversible steroidal aromatase inhibitor with reported abuse in human sports. In 2011, our laboratory identified the presence of formestane in a horse urine sample from an overseas jurisdiction. This was the first reported case of formestane in a racehorse. The metabolism of formestane in humans has been reported previously; however, little is known about its metabolic fate in horses. This paper describes the in vitro and in vivo metabolic studies of formestane in horses, with the objective of identifying the target metabolite with the longest detection time for controlling formestane abuse. In vitro metabolic studies of formestane were performed using homogenized horse liver. Seven in vitro metabolites, namely 4-hydroxytestosterone (M1), 3ß,4α-dihydroxy-5ß-androstan-17-one (M2a), 3ß,4ß-dihydroxy-5ß-androstan-17-one (M2b), 3ß,4α-dihydroxy-5α-androstan-17-one (M2c), androst-4-ene-3α,4,17ß-triol (M3a), androst-4-ene-3ß,4,17ß-triol (M3b), and 5ß-androstane-3ß,4ß,17ß-triol (M4) were identified. For the in vivo studies, two thoroughbred geldings were each administered with 800 mg of formestane (32 capsules of Formadex) by stomach tubing. The results revealed that the parent drug and seven metabolites were detected in post-administration urine. The six in vitro metabolites (M1, M2a, M2b, M2c, M3a, and M3b) identified earlier were all detected in post-administration urine samples. In addition, 3α,4α-dihydroxy-5α-androstan-17-one (M2d), a stereoisomer of M2a/M2b/M2c, was also identified. This study has shown that the detection of formestane administration would be best achieved by monitoring 4-hydroxytestosterone (M1) in the glucuronide-conjugated fraction. M1 could be detected for up to 34 h post-administration. In blood samples, the parent drug could be detected for up to 34 h post administration.

Profiling of urinary steroids by gas chromatography-mass spectrometry detection and confirmation of androstenedione administration using isotope ratio mass spectrometry.

Androstenedione (4-androstene-3,17-dione) is banned by the World Anti-Doping Agency (WADA) as an endogenous steroid. The official method to confirm androstenedione abuse is isotope ratio mass spectrometry (IRMS). According to the guidance published by WADA, atypical steroid profiles are required to trigger IRMS analysis. However, in some situations, steroid profile parameters are not effective enough to suspect the misuse of endogenous steroids. The aim of this study was to investigate the atypical steroid profile induced by androstenedione administration and the detection of androstenedione doping using IRMS. Ingestion of androstenedione resulted in changes in urinary steroid profile, including increased concentrations of androsterone (An), etiocholanolone (Etio), 5α-androstane-3α,17ß-diol (5α-diol), and 5ß-androstane-3α,17ß-diol (5ß-diol) in all of the subjects. Nevertheless, the testosterone/epitestosterone (T/E) ratio was elevated only in some of the subjects. The rapid increases in the concentrations of An and Etio, as well as in T/E ratio for some subjects could provide indicators for initiating IRMS analysis only for a short time period, 2-22h post-administration. However, IRMS could provide positive determinations for up to 55h post-administration. This study demonstrated that, 5ß-diol concentration or Etio/An ratio could be utilized as useful indicators for initiating IRMS analysis during 2-36h post-administration. Lastly, Etio, with slower clearance, could be more effectively used than An for the confirmation of androstenedione doping using IRMS.

A new reliable sample preparation for high throughput focused steroid profiling by gas chromatography-mass spectrometry.

The use of steroid hormones as growth promoters in cattle has been banned within the European Union since 1988 but can still be fraudulently employed in animal breeding farms for anabolic purposes. If an efficient monitoring of synthetic compounds (screening and confirmation) is ensured today by many laboratories, pointing out suspicious samples from a natural steroids abuse remains a tricky challenge due to the difficulty to set relevant threshold levels for these endogenous compounds. The development of focused profiling or untargeted metabolomic approaches is then emerging in this context, with the objective to reveal potential biomarkers signing an exogenous administration of such natural steroids. This study aimed to assess sample preparation procedures based on microextraction and adapt them to high throughput urinary profiling or metabolomic analyses based on gas chromatography-mass spectrometry measurement. Two techniques have been tested and optimised, namely solid phase microextraction (SPME) and microextraction by packed sorbent (MEPS), using five model steroid metabolites (16α-hydroxyandrosterone, 2α-hydroxytestosterone, 11-keto,5ß-androstanedione, 6α-hydroxyestradiol and 7ß-hydroxypregnenolone). The considered performance criteria included not only the absolute response of the targeted compounds but also the robustness of the materials, and the global aspect of the diagnostic ion chromatograms obtained. After only five successive urinary extractions, a clear degradation of the SPME fiber was observed which led to discard this method as a relevant technique for profiling, whereas no degradation was observed on MEPS sorbent. Repeatability and recovery yields were calculated from urine samples fortified at 500 µg L⁻¹ and extracted by MEPS. They were found respectively below 11% and above 60% for all model compounds. Detection limits were in the 5-15 µg L⁻¹ range depending on the compounds, and a good linearity was observed on the 10-75 µg L⁻¹ range (R² > 0.99). This methodology was applied on urine samples collected from control versus androstenedione-treated bovines, revealing a significant concentration increase for several well-known metabolites such as etiocholanolone, 5α-androstane-3ß,17α-diol, 5ß-androstane-3α,17α-diol and 5-androstene-3ß,17α-diol. Finally, these results allowed to confirm the suitability of the developed strategy and give to this new MEPS application a promising interest in the field of GC-MS based steroid profiling and metabolomic.

Transferrin-conjugated lipid-coated PLGA nanoparticles for targeted delivery of aromatase inhibitor 7alpha-APTADD to breast cancer cells.

Transferrin (Tf)-conjugated lipid-coated poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles carrying the aromatase inhibitor, 7alpha-(4'-amino)phenylthio-1,4-androstadiene-3,17-dione (7alpha-APTADD), were synthesized by a solvent injection method. Formulation parameters including PLGA-to-lipid, egg PC-to-TPGS, and drug-to-PLGA ratios and aqueous-to-organic phase ratio at the point of synthesis were optimized to obtain nanoparticles with desired sizes and drug loading efficiency. The optimal formulation had a drug loading efficiency of 36.3+/-3.4%, mean diameter of 170.3+/-7.6nm and zeta potential of -18.9+/-1.5mV. The aromatase inhibition activity of the nanoparticles was evaluated in SKBR-3 breast cancer cells. IC(50) value of the Tf-nanoparticles was ranging from 0.77 to 1.21nM, and IC(50) value of the nanoparticles was ranging from 1.90 to 3.41nM (n=3). The former is significantly lower than the latter (p<0.05). These results suggested that the aromatase inhibition activity of the Tf-nanoparticles was enhanced relative to that of the non-targeted nanoparticles, which was attributable to Tf receptor (TfR) mediated uptake. In conclusion, Tf-conjugated lipid-coated PLGA nanoparticles are potential vehicles for improving the efficiency and specificity of therapeutic delivery of aromatase inhibitors.

Influence of delivery mode on the urinary excretion of nandrolone metabolites.

INTRODUCTION: This study examined the influence of a supplement matrix on the excretion pattern of nandrolone metabolites in response to ingestion of a trace amount of 19-norandrostenedione. METHODS: Ten male and nine female volunteers (mean ± SD: age = 26 ± 3 yr, height = 1.71 ± 0.09 m, body mass = 70.9 ± 13.2 kg) were recruited. On two occasions, subjects entered the laboratory in the morning after an overnight fast. After an initial urine collection, subjects ingested either 500 mL of plain water or a commercially available energy bar; 10 µg of 19-norandrostenedione was added to each. The volume of each urine sample passed during the next 24 h was measured, and an aliquot was retained for analysis. All samples were analyzed for the metabolites 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE) by gas chromatography-mass spectrometry. RESULTS: The total volume of urine passed was higher in the water trial (2.10 ± 0.52 L) than in the bar trial (1.85 ± 0.55 L; P = 0.040). Baseline urinary 19-NA concentrations were all below the limit of quantification for the assay. Peak urinary 19-NA was lower (P = 0.002) in the water trial (4.80 ± 2.84 ng·mL(-1)) than in the bar trial (8.46 ± 4.44 ng·mL(-1)). The time elapsed between ingestion of the supplement and the peak urinary 19-NA concentration was longer (P = 0.023) on the bar trial (4.6 ± 2.4 h) than on the water trial (2.8 ± 1.9 h). There was no difference in the total recovery of 19-NA + 19-NE between the liquid and solid supplements (water 30 ± 10%; bar 28 ± 12%; P < 0.140). CONCLUSIONS: Peak 19-NA concentrations were higher, and occurred later, when the 19-norandrostenedione was added to a solid supplement. This may be due to a slower rate of absorption and/or a reduced diuresis, resulting in a longer period for the metabolites to accumulate in the urine.

The uPA(+/+)-SCID mouse with humanized liver as a model for in vivo metabolism of 4-androstene-3,17-dione.

The metabolism in primary human hepatocyte cultures often deviates from that in clinical studies, which in turn are hampered by ethical constraints. Here the use of urokinase-type plasminogen activator-severe combined immunodeficiency [uPA(+/+)-SCID] mice transplanted with human hepatocytes was investigated as a model for in vivo metabolic studies. The urinary excretion profile after oral administration of 4-androstene-3,17-dione (AD) in chimeric mice was investigated by using gas chromatography-mass spectrometry detection and was compared with previously reported metabolites of AD in humans and cell cultures. Chimeric mice exhibited an AD metabolic profile similar to that of humans, showing androsterone and etiocholanolone as major metabolites. Several hydroxylated steroids were detected as minor metabolites in the chimeric mice compared with hepatocyte cultures. A significant correlation between the extent of liver replacement and the relative abundances of human-type metabolites was established. The results for AD showed that humanized liver uPA-SCID mice can serve as an alternative model for in vivo metabolism studies in humans. In the future, this model could possibly be used for other steroids or pharmaceutical compounds.

Anabolic and androgenic activity of 19-norandrostenedione after oral and subcutaneous administration--analysis of side effects and metabolism.

One of the most frequently misused steroid precursors (prohormones) is 19-norandrostenedione (estr-4-ene-3,17-dione, NOR). Recently we have show that NOR stimulates skeletal muscle growth after s.c. administration in a highly selective manner but exhibits only weak androgenic activity in rats. Because most abusers take NOR orally, the aim of this study was to compare the anabolic and androgenic potency of NOR between s.c. and oral application. Orchiectomised rats were treated with NOR either s.c. (1 mg/kg BW/day) or orally (0.1, 1 and 10 mg/kg BW/day). The tissue weights of the levator ani, the seminal vesicle and the prostate were analysed to determine the anabolic and androgenic activity. Heart and liver wet weights were examined to identify side effects. Serum concentrations of NOR and its metabolite nandrolone (NT) were determined. GCMC analysis revealed that free and glucuronidated NOR and NT were detectable in the serum after oral and s.c. administration and that NOR was converted to NT in comparable amounts independent of the route of administration. In agreement to our previous study s.c. application of NOR stimulates skeletal muscle growth but has only weak androgenic effects. In contrast, after oral administration of NOR neither stimulation of the prostate nor the levator ani could be observed in the doses administered in this study. Interestingly, and in contrast to s.c. treatment, oral administration of NOR resulted in a dose-dependent decrease of body weight. In summary, oral administration of NOR, at least in the rat, seems to be a very ineffective strategy for stimulating skeletal muscle mass increases but may be associated with side effects.

Urinary nandrolone metabolite detection after ingestion of a nandrolone precursor.

INTRODUCTION: Quantities of various anabolic/androgenic steroids have been found in dietary supplements without their presence being disclosed on the label. The aim of this study was to quantify the excretion patterns of the diagnostic metabolites, 19-norandrosterone (19-NA), and 19-noretiocholanolone (19-NE) after ingestion of small doses of 19-nor-4-androstene-3,17-dione (19-norandrostenedione). METHODS: Eleven males and nine females entered the laboratory in the morning after an overnight fast. An initial urine sample was collected, and volunteers then ingested 500 mL of water containing 5 g of creatine monohydrate and 1.0, 2.5, or 5.0 microg of 19-norandrostenedione. The volume of each urine void was measured, and an aliquot was taken. Samples were analyzed for the metabolites 19-NA and 19-NE by GCMS. RESULTS: Baseline urinary 19-NA concentrations were 0.19 +/- 0.14 ng x mL. Ingestion of the supplement resulted in peak mean urinary 19-NA concentrations of 0.68 +/- 0.36, 1.56 +/- 0.86, and 3.89 +/- 3.11 ng.mL in the 1.0-, 2.5-, or 5.0-microg trials, respectively. Under current WADA regulations, ingestion of the 1.0-microg dose produced 0 positive doping tests, 5 subjects (20%) tested positive in the 2.5-microg trial, and 15 subjects (75%) had urinary 19-NA concentrations exceeding 2 ng x mL after ingesting creatine containing 5.0 microg of the steroid. The recovery of the ingested dose was highly variable between individuals, with values ranging from 11% to 84% (mean +/- SD = 47% +/- 18%). CONCLUSIONS: Ingestion of trace amounts of 19-norandrostenedione can result in transient elevations of urinary 19-NA and 19-NE concentrations. The addition of as little as 2.5 microg of 19-norandrostenedione to a supplement (0.00005% contamination) appears sufficient to result in a doping violation in some individuals.

Yolk androgens and the development of avian immunity: an experiment in jackdaws (Corvus monedula).

Maternally derived androgens have been shown to influence offspring phenotype in various ways. In birds, the benefits of prenatal androgen exposure, such as increased competitiveness and accelerated growth in nestlings, have been suggested to be balanced by costs, such as reduced immune function. In this study, we used an integrative approach to examine the influence of yolk androgens on the development of immune function in jackdaw (Corvus monedula) nestlings. Specifically, we tested whether the effects of yolk androgens on offspring immunity may extend over the first few days of life and be detectable even after several weeks. We manipulated yolk androgen concentrations in jackdaw eggs and estimated immune responsiveness by challenging the young with different pathogens at different stages of the nestling period. Six-day-old chicks hatched from eggs with elevated yolk androgen levels had lower pre-challenge antibody titres against lipopolysaccharide (LPS) than control chicks. However, antibody titres against LPS did not differ between treatment groups eight days after a challenge with LPS. During the late nestling phase, both humoral (towards diphtheria/tetanus antigens) and cell-mediated (towards phytohaemagglutinin) immune responsiveness were lower in chicks from yolk androgen-treated eggs compared with control chicks. Our experimental study on jackdaw chicks shows that elevated yolk androgen levels result in a general immunosuppression in offspring; this conclusion was based on results for several immunological tests of both humoral and cell-mediated immunity conducted at 1-2 and 3-4 weeks of age.

Elimination kinetic of 17beta-estradiol 3-benzoate and 17beta-nandrolone laureate ester metabolites in calves' urine.

Efficient control of the illegal use of anabolic steroids must both take into account metabolic patterns and associated kinetics of elimination; in this context, an extensive animal experiment involving 24 calves and consisting of three administrations of 17beta-estradiol 3-benzoate and 17beta-nandrolone laureate esters was carried out over 50 days. Urine samples were regularly collected during the experiment from all treated and non-treated calves. For sample preparation, a single step high throughput protocol based on 96-well C(18) SPE was developed and validated according to the European Decision 2002/657/EC requirements. Decision limits (CCalpha) for steroids were below 0.1 microg L(-1), except for 19-norandrosterone (CCalpha=0.7 microg L(-1)) and estrone (CCalpha=0.3 microg L(-1)). Kinetics of elimination of the administered 17beta-estradiol 3-benzoate and 17beta-nandrolone laureate were established by monitoring 17beta-estradiol, 17alpha-estradiol, estrone and 17beta-nandrolone, 17alpha-nandrolone, 19-noretiocholanolone, 19-norandrostenedione, respectively. All animals demonstrated homogeneous patterns of elimination both from a qualitative (metabolite profile) and quantitative point of view (elimination kinetics in urine). Most abundant metabolites were 17alpha-estradiol and 17alpha-nandrolone (>20 and 2 mg L(-1), respectively after 17beta-estradiol 3-benzoate and 17beta-nandrolone laureate administration) whereas 17beta-estradiol, estrone, 17beta-nandrolone, 19-noretiocholanolone and 19-norandrostenedione were found as secondary metabolites at concentration values up to the microg L(-1) level. No significant difference was observed between male and female animals. The effect of several consecutive injections on elimination profiles was studied and revealed a tendency toward a decrease in the biotransformation of administered steroid 17beta form.

The prohormone 19-norandrostenedione displays selective androgen receptor modulator (SARM) like properties after subcutaneous administration.

One of the most frequently misused steroid precursors (prohormones) is 19-norandrostenedione (4-estrene-3,17-dione, NOR), which is, after oral administration, readily metabolised to nortestosterone, also known as nandrolone (durabolin). In this study we have characterised molecular mechanisms of its action determined its tissue specific androgenic and anabolic potency after subcutaneous (s.c.) administration and investigated potential adverse effects. Receptor binding tests demonstrate that NOR binds with high selectivity to the AR. The potency of NOR to transactivate androgen receptor (AR) dependent reporter gene expression was 10 times lower as compared to dihydrotestosterone (DHT). In vivo experiments in orchiectomised rats demonstrated that s.c. treatment with NOR resulted only in a stimulation of the weight of the levator ani muscle; the prostate and seminal vesicle weights remained completely unaffected. Like testosterone, administration of NOR resulted in a stimulation of AR and myostatin mRNA expression in the gastrocnemius muscle. NOR does not affect prostate proliferation, the liver weight and the expression of the tyrosine aminotransferase gene (TAT) in the liver. Summarizing these data it is obvious that NOR, if administrated s.c. and in contrast to its metabolite nandrolone, highly selectively stimulates the growth of the skeletal muscle but has only weak androgenic properties. This observation may have relevance with respect to therapeutic aspects but also doping prevention.

Supplements and the endocrine system in athletes.

In the world of athletes' nutrition, there are many ethical concerns, because there is the suspicion that in practice, large doses of supplements in athletes are not taken for nutritional purposes. It is beyond the scope of this article to highlight the possible roles of supplements or methods of supplementation in the improvement of athletic performance in elite athletes. Instead, the author briefly reviews some of the substances taken by athletes, with particular attention to their mechanisms of action and the pathways involved. Very often, the effects of many supplements are hormone-related, or supplements influence hormone secretion. Examples of possible links between "supplements or ergogenic compounds" and the endocrine/metabolic system are addressed.

Dopaminergic modulation of reproductive behavior and activity in male zebra finches.

We previously demonstrated that hormone treatments which stimulate female-directed singing increased levels and turnover of dopamine (DA) in brain areas controlling the motor patterning of song. To help determine how DA affects singing, we quantified the effects of treating adult male finches with the D1/D2 receptor antagonist cis-flupenthixol. Adult males were given subcutaneous silastic implants of androgen, in case drug treatment interfered with androgen secretion. One week later, they were tested with females. Males were divided into three groups matched for levels of courtship singing. Males were then subcutaneously implanted with osmotic minipumps containing either saline, a low, or a high dose of cis-flupenthixol. Each male was tested with a different female 5 and 10 days after implantation to determine how this D1/D2 receptor antagonist affected behavior. Both drug doses affected female-directed singing 5 days after initiation of treatment. High-dose males sang to females significantly less often than males in the other two groups. Low-dose males showed fewer high-intensity courtship displays in which males dance towards females as they sing. These effects on courtship singing were not seen at day 10, though other behavioral effects were seen at this time. Male beak wipes, rocks, following females and female withdrawals from males were also affected by drug treatment. General activity in the home cage was decreased by day 11. These data demonstrate that singing and several other female-directed behaviors are sensitive to perturbations in DA receptor function.

Toremifene-atamestane; alone or in combination: predictions from the preclinical intratumoral aromatase model.

Since most breast cancers occur in postmenopausal women and are hormone dependent, we developed a model system that mimics this situation. In this model, tumors of human estrogen receptor (ER) positive breast cancer cells stably transfected with aromatase (Ac-1) are grown in immune-compromised mice. Using this model we have explored a number of therapeutic strategies to maximize the antitumor efficacy of antiestrogens (AEs) and aromatase inhibitors (AIs). This intratumoral aromatase xenograft model has proved accurate in predicting the outcome of several clinical trials. In this current study we compared the effect of an AE toremifene and steroidal AI atamestane, alone or in combination, on growth of hormone-dependent human breast cancer. We have also compared toremifene plus atamestane combination with tamoxifen in this study. The growth of Ac-1 cells was inhibited by tamoxifen, toremifene and atamestane in vitro with IC(50) values of 1.8+/-1.3 microM, 1+/-0.3 microM and 60.4+/-17.2 microM, respectively. The combination of toremifene plus atamestane was found to be better than toremifene or atamestane alone in vitro. The effect of this combination was then studied in vivo using Ac-1 xenografts grown in ovariectomized female SCID mice. The mice were injected with toremifene (1000 microg/day), atamestane (1000 microg/day), tamoxifen (100 microg/day), or the combination of toremifene plus atamestane. In this study, our results indicate that the combination of toremifene plus atamestane was as effective as toremifene or tamoxifen alone but may not provide any additional benefit over toremifene alone or tamoxifen alone.

Phase III, double-blind, controlled trial of atamestane plus toremifene compared with letrozole in postmenopausal women with advanced receptor-positive breast cancer.

PURPOSE: To compare time to progression (TTP) with a steroidal aromatase inhibitor (AI) atamestane (ATA) combined with toremifene (TOR; complete estrogen blockade) versus letrozole (LET) in receptor-positive advanced breast cancer (ABC). PATIENTS AND METHODS: Eligibility included postmenopausal receptor-positive ABC and adjuvant hormonal therapy completed more than 12 months prior to study entry. Participants received daily ATA 500 mg with TOR 60 mg (ATA + TOR), or letrozole 2.5 mg (LET). The primary end point was TTP, whereas secondary objectives included objective response (OR), overall survival (OS), and time to treatment failure (TTF). The study had 80% power to detect a 25% increase in TTP assuming a TTP of 9.4 months in the LET population. RESULTS: A total of 865 patients were randomly assigned (434 to ATA + TOR and 431 to LET) in 60 centers in the United States, Canada, Russia, and Ukraine. Baseline characteristics were balanced. Median TTP was identical in the two arms at 11.2 months (P < .92). Median TTF was similar at 9.24 months (ATA + TOR) versus 10.44 months (LET). The hazard ratios (LET/ATA + TOR) were 1.00 (95% CI, 0.92 to 1.08) for TTP, 0.99 (95% CI, 0.92 to 1.06) for TTF, and 0.98 (95% CI, 0.87 to 1.11) for OS. OR occurred in 30% of patients receiving ATA + TOR and in 36% of patients receiving LET (P < .1). Adverse events (AEs) were similar for patients receiving ATA + TOR versus LET, and serious AEs were 10% v 11%, respectively. CONCLUSION: TTP for patients receiving ATA + TOR was identical to that for patients receiving LET, representing the first endocrine therapy comparable to LET in ABC. Unlike in the Anastrozole, Tamoxifen, and Combined trial, addition of an antiestrogen did not decrease efficacy of the AI. Future studies of AIs in combination with more effective selective estrogen receptor modulators or selective receptor downregulators is warranted.

The effects of androstenedione on renal tubule epithelial cells.

Androstendione (AED) occurs naturally in the body and is a direct precursor to the hormone testosterone. Androstenedione can be purchased over the counter without a prescription and has the potential to be converted to testosterone endogenously increasing strength and muscle mass. The conversion of androstendione to testosterone occurs via the 17B hydroxysteroid dehydrogenase (17 B HSD), which is found to be most active in the liver. Kidney epithelial cells possess 17B HSD and the metabolism of AED to testosterone has not been fully evaluated. In this study, renal epithelial cells were treated with 10, 100 or 300 mg/mL AED for periods of 24, 48 and 72 hours and the cells were evaluated for viability, damage, cellular morphology, and testosterone production. The results show increases in cell number at 48 and 72 hours in the 300 mg/mL treatment group as well as increased cellular protein levels. Over time, all treatment groups showed a dose dependent increase in testosterone production by the kidney epithelial cells. Overall, AED did not result in alterations in cell morphology or induce cellular damage for the duration of the study. The results of this study suggest more research is needed to determine the effects of increased testosterone production by the kidney and overall health implications that may rise as a result.