Estimating Potency in High-Throughput Screening Experiments by Maximizing the Rate of Change in Weighted Shannon Entropy.

High-throughput in vitro screening experiments can be used to generate concentration-response data for large chemical libraries. It is often desirable to estimate the concentration needed to achieve a particular effect, or potency, for each chemical tested in an assay. Potency estimates can be used to directly compare chemical profiles and prioritize compounds for confirmation studies, or employed as input data for prediction modeling and association mapping. The concentration for half-maximal activity derived from the Hill equation model (i.e., AC50) is the most common potency measure applied in pharmacological research and toxicity testing. However, the AC50 parameter is subject to large uncertainty for many concentration-response relationships. In this study we introduce a new measure of potency based on a weighted Shannon entropy measure termed the weighted entropy score (WES). Our potency estimator (Point of Departure, PODWES) is defined as the concentration producing the maximum rate of change in weighted entropy along a concentration-response profile. This approach provides a new tool for potency estimation that does not depend on the assumption of monotonicity or any other pre-specified concentration-response relationship. PODWES estimates potency with greater precision and less bias compared to the conventional AC50 assessed across a range of simulated conditions.

Gestrinone inhibits growth of human uterine leiomyoma may relate to activity regulation of ERα, Src and P38 MAPK.

The study was to investigate the effect of gestrinone on the growth of human uterine leiomyoma cells and on the levels and activity of p38, Src and estrogen receptor alpha (ERα). Human uterine leiomyoma cells were cultured and treated with dimethylsulfoxide (DMSO) or a gestrinone concentration gradient. Morphological changes were observed and apoptosis was evaluated. Levels of p38 and phosphorylated-p38 (pp38) were assayed by enzyme-linked immunosorbent assay (ELISA). Levels of ERα and Src were analyzed using real-time RT-PCR and Western blotting. The result showed that gestrinone significantly inhibited the growth of cultured human uterine leiomyoma cells in a concentration- and time-dependent manner, with a 50% inhibitory concentration (IC(50)) value and corresponding 95% confidence intervals (CI) of 43.67 (23.46∼81.32), 27.78 (12.51∼61.68) and 15.25 (7.17∼32.43) µmol/L at 20, 40 and 60h, respectively. Compared with control-treated leiomyoma cells, gestrinone significantly reduced both the expression of ERα (P<0.05) and the levels of phospho-Ser167-ERα (P<0.05). Gestrinone also markedly suppressed the level of phospho-Tyr416-Src (P<0.05). Moreover, gestrinone significantly increased the ratio of phospho-p38/p38 mitogen-activated protein kinase (MAPK) (P<0.05). However, no significant increase in apoptosis or cell cycle arrest was observed (P>0.05) in response to the tested concentrations of 0.1 to 3.0µmol/L. As a conclusion, gestrinone suppresses the proliferation of uterine leiomyoma cells mainly by regulating the activity of ERα/Src/p38 MAPK in a concentration-dependent manner at a low concentration of 0.1∼3.0µM, but not significantly regulating apoptosis. Gestrinone opposes the growth of uterine leiomyoma through multiple genes.

The androgenic anabolic steroid tetrahydrogestrinone produces dioxin-like effects via the aryl hydrocarbon receptor.

For a long time, athletes have used androgenic anabolic steroids (AASs) in an inappropriate and veiled manner with the aim of improving exercise performance or for cosmetic purposes. Abuse of AASs triggers adverse effects such as hepatocarcinogenesis, heart attacks, and aggressive behavior. However, AAS-induced toxicity is not completely understood at the molecular level. In the present study, we showed, by performing a dioxin response element (DRE)-luciferase reporter gene assay, that tetrahydrogestrinone (THG), a popular and potent androgen receptor agonist, has dioxin-like effects. In addition, we showed that THG increased cytochrome P-450 1A1 (CYP1A1) mRNA and protein levels, and enzyme activity. The gene encoding CYP1A1 is involved in phase 1 xenobiotic metabolism and a target gene of the aryl hydrocarbon receptor (AhR). Using the AhR antagonist CH-223191, we also examined whether the effects of THG on DRE activation depended on AhR. Our results suggest that synthetic anabolic steroids may have dioxin-like side effects that can disturb endocrine systems and may cause other side effects including cancer through AhR.

Structure-activity relationships of synthetic progestins in a yeast-based in vitro androgen bioassay.

The recent identification of tetrahydrogestrinone (THG), a non-marketed designer androgen used for sports doping but previously undetectable by established mass spectrometry-based urine drug screens, and its production by a facile chemical modification of gestrinone has raised concerns about the risks of developing designer androgens from numerous marketed progestins. We therefore have used yeast-based in vitro androgen and progesterone bioassays to conduct a structure-activity study assessing the intrinsic androgenic potential of commercially available progestins and their derivatives, to identify those compounds or structures with the highest risk of forming a basis for such misapplication. Progestins had a wide range of androgenic bioactivity that was not reliably predicted for individual steroids by their progestin bioactivity. 17alpha-Hydroxyprogesterone and 19-norprogesterone derivatives with their bulky 17beta-substituents were strong progestins but generally weak androgens. 17alpha-Ethynylated derivatives of testosterone, 19-nortestosterone and 18-methyl-19-nortestosterone such as gestrinone, ethisterone, norethisterone and norgestrel had the most significant intrinsic androgenicity of all the commercially marketed progestins. Facile chemical modification of the 17alpha-ethynyl group of each of these progestins produces 17alpha-methyl, ethyl and allyl derivatives, including THG and norbolethone, which further enhanced androgenic bioactivity. Thus by using the rapid and sensitive yeast bioassay we have screened a comprehensive set of progestins and associated structures and identified the ethynylated testosterone, 19-nortestosterone and 18-methyl-19-nortestosterone derivatives as possessing the highest risk for abuse and potential for conversion to still more potent androgens. By contrast, modern progestins such as progesterone, 17alpha-hydroxyprogesterone and 19-norprogesterone derivatives had minimal androgenic bioactivity and pose low risk.

Mechanism of emergency contraception with gestrinone: a preliminary investigation.

BACKGROUND: A previous investigation showed that among 120 healthy women treated with a single oral dose of gestrinone for emergency contraception (EC), there was only one pregnancy. The effect of a single oral dose of gestrinone given for EC on ovarian function and endometrial development was studied. STUDY DESIGN: Healthy fertile women were randomly assigned to Group A (n=8) or Group B (n=7). Gestrinone 5 mg was orally administered to each woman before (Group A) or after (Group B) ovulation. The day of ovulation was determined by transvaginal ultrasound and by urinary luteinizing hormone (LH) measured by enzyme immunoassay (One Step LH Ovulation Test). An endometrial biopsy was performed during implantation. Endometrial maturation and expression of markers of endometrial receptivity were analyzed. The tested markers were integrins alpha(1)beta(1), alpha(4)beta(1) and beta(3). Serum estradiol (E(2)) and progesterone (P) levels in serum were determined by radioimmunoassay, and estradiol receptors and progesterone receptors (PRs) in the endometrium were assessed by immunohistochemistry. RESULTS: Gestrinone administered during the periovulatory period did not affect follicular development, ovulation, menstrual cycle length and E(2) and P levels but decreased the expression of PR in the endometrium. Integrin alpha(4)beta(1) tended to increase after treatment with gestrinone without reaching statistical significance. CONCLUSION: The mode of action of gestrinone used for EC is probably inhibition of implantation by acting on the endometrium rather than inhibition of ovulation.

Effects of gestrinone on uterine leiomyoma and expression of c-Src in a guinea pig model.

AIM: To investigate the effect of gestrinone on uterine leiomyomas and the expression of c-Src, estradiol receptors (ER), and progesterone receptors (PR) in a guinea pig model. METHODS: After being oophorectomized, the guinea pigs were allocated into random groups. The model group was treated with estradiol benzoate (E2) for 16 weeks. In the gestrinone-treated groups, the animals were treated with E2 for 6 weeks in advance, and then in combination with gestrinone for 10 weeks. Histological examination was performed to evaluate whether there were leiomyoma features in the animals. The protein levels of c-Src, phospho-( 416)Src, ER, and PR were assayed by Western blotting and an immunohistochemical method. RESULTS: Morphological changes were observed in the myometrium of the guinea pig model, including an increase of uterine weights, proliferation of uterine smooth muscles, and the formation of nodules. High protein levels of c-Src, phospho- 416Src, ER, and PR were observed in the myometrium of the guinea pig model. In the gestrinone-treated group, there were no nodules observed. The histological features of the myometrium were similar to that of the control group. Low protein levels of c-Src, phospho-(416 )Src, ER, and PR were observed in the gestrinonetreated group. CONCLUSION: The upregulation of c-Src and phospho-(416 )Src indicated that the activity of c-Src is augmented in the uterine leiomyoma model. c-Src was associated with the formation of uterine leiomyomas in the model, and gestrinone markedly suppressed the growth of uterine leiomyomas in the model. Gestrinone inhibited not only the protein expression of ER and PR, but also c-Src and the autophosphorylation of c-Src in the guinea pig leiomyoma model.

Tetrahydrogestrinone is a potent but unselective binding steroid and affects glucocorticoid signalling in the liver.

Tetrahydrogestrinone (THG) is a steroid recently identified to be misused as doping agent. However, the knowledge on functions of this substance in humans or animal models is rather limited. Therefore, it was our aim to further characterize the pharmacological profile of THG and identify potential adverse side effects. THG was synthesized, the purity was confirmed and its biological activity was tested. The potency of THG to transactivate AR dependent reporter gene expression was two orders of magnitude lower compared to dihydrotestosterone. THG binds with high affinity but unselective to the androgen (AR), progesterone (PR), glucocorticoid (GR) and mineralocorticoid (MR) receptor. Treatment of orchiectomised rats with THG resulted in a stimulation of prostate, seminal vesicle and levator ani muscle, indicating androgenic and anabolic properties. In the liver THG, in contrast to testosteronepropionate (TP), down regulates the expression of the GR dependent tyrosine aminotransferase gene (TAT). In summary, our results demonstrate that THG is not a specific AR agonist. THG exhibits a high binding affinity to all tested steroid hormone receptors and binds with highest affinity to the GR. Our in vivo data are indicative of an anabolic and androgenic potency of THG, but the repression of TAT demonstrates that THG also interferes with the glucocorticoid hormone system. Therefore, it is conceivable that an intake will result in adverse side effects.

Cardiovascular toxicities of performance-enhancing substances in sports.

Athletes commonly use drugs and dietary supplements to improve athletic performance or to assist with weight loss. Some of these substances are obtainable by prescription or by illegal means; others are marketed as supplements, vitamins, or minerals. Nutritional supplements are protected from Food and Drug Administration regulation by the 1994 US Dietary Supplement Health and Education Act, and manufacturers are not required to demonstrate proof of efficacy or safety. Furthermore, the Food and Drug Administration lacks a regulatory body to evaluate such products for purity. Existing scientific data, which consist of case reports and clinical observations, describe serious cardiovascular adverse effects from use of performance-enhancing substances, including sudden death. Although mounting evidence led to the recent ban of ephedra (ma huang), other performance-enhancing substances continue to be used frequently at all levels, from elementary school children to professional athletes. Thus, although the potential for cardiovascular injury is great, few appropriately designed studies have been conducted to assess the benefits and risks of using performance-enhancing substances. We performed an exhaustive OVID MEDLINE search to Identify all existing scientific data, review articles, case reports, and clinical observations that address this subject. In this review, we examine the current evidence regarding cardiovascular risk for persons using anabolic-androgenic steroids including 2 synthetic substances, tetrahydrogestrinone and androstenedione (andro), stimulants such as ephedra, and nonsteroidal agents such as recombinant human erythropoietin, human growth hormone, creatine, and beta-hydroxy-beta-methylbutyrate.

Tetrahydrogestrinone is an androgenic steroid that stimulates androgen receptor-mediated, myogenic differentiation in C3H10T1/2 multipotent mesenchymal cells and promotes muscle accretion in orchidectomized male rats.

The discovery of tetrahydrogestrinone (THG) abuse by several elite athletes led the U.S. Congress to declare it a controlled substance, although conclusive evidence of its anabolic/androgenic activity is lacking. We determined whether THG affects myogenic differentiation and androgen receptor (AR)-mediated signaling, whether it binds to AR, and whether it has androgenic and anabolic effects in vivo. Accordingly, we measured the dissociation constant for THG with a fluorescence anisotropy assay using recombinant AR-ligand binding domain. The AR nuclear translocation and myogenic activity of androstenedione were evaluated in mesenchymal, multipotent C3H10T1/2 cells. We performed molecular modeling of the THG:AR interaction. The androgenic/anabolic activity was evaluated in orchidectomized rats. THG bound to AR with an affinity similar to that of dihydrotestosterone. In multipotent C3H10T1/2 cells, THG upregulated AR expression, induced AR nuclear translocation, dose dependently increased the area of myosin heavy chain type II-positive myotubes, and up-regulated myogenic determination and myosin heavy chain type II protein expression. The interaction between AR and the A ring of THG was similar to that between AR and the A ring of dihydrotestosterone, but the C17 and C18 substituents in THG had a unique stabilizing interaction with AR. THG administration prevented the castration-induced atrophy of levator ani, prostate gland, and seminal vesicles and loss of fat-free mass in orchidectomized rats. We conclude that THG is an anabolic steroid that binds to AR, activates AR-mediated signaling, promotes myogenesis in mesenchymal multipotent cells, and has anabolic and androgenic activity in vivo. This mechanism-based approach should be useful for rapid screening of anabolic/androgenic agents.

[Effect of gestrinone on growth and apoptosis in isolated ectopic endometrium cells in vitro].

OBJECTIVE: To investigate the effects of gestrinone on growth and apoptosis, as well as the expression of phosphatase and tension homologue deleted on chromosome 10 (PTEN) in isolated ectopic endometrium cells in vitro and the underlying mechanisms. METHODS: Ectopic endometrium cells were cultured and exposed to gestrinone of different doses of 0, 10(-6) and 10(-4) mol/L respectively. The inhibition of the cells during 48 hours was determined by methylthiazolyl tetrazolium (MTT) assay, and the cell growth curve was made. Gestrinone was administered to the cells and at 24 hours the morphological changes were observed by transmission electron microscopy and the apoptosis rate, cell cycle and PTEN expression were monitored by flow cytometry (FCM) at the same time. RESULTS: Gestrinone at different concentrations could inhibit the growth and proliferation of ectopic endometrium cells in a dose- and time-dependent manner. The inhibition rate of cell growth after exposed to gestrinone for 8, 16, 24, 32, 40 and 48 h was 99.6%, 87.3%, 79.8%, 62.3%, 51.7% and 44.2% in the 10(-6) mol/L group, and 99.2%, 77.1%, 69.6%, 51.1%, 33.7% and 23.6% in the 10(-4) mol/L group (P < 0.05), and cell growth curve was changed accordingly. After 24 hour exposure to gestrinone from 10(-6) to 10(-4) mol/l, apoptotic changes of cells were observed under transmission electron microscope. FCM showed that after the exposure to gestrinone, the apoptotic rate of ectopic endometrium cells was 1.3% in 10(-6) mol/L group and 15.0% in 10(4) mol/L group. It was significantly increased when compared with the 0 mol/L group, the apoptotic rate of which was 0% (P < 0.05). The level of PTEN expression of the ectopic endometrium cells was 60.6% after treated with 0 mol/L gestrinone, while in 10(-6) and 10(-4) mol/l groups the level of PTEN expression was increased to 75.3% and 85.7%, significantly higher than that of the 0 mol/L group (P < 0.05). CONCLUSION: Gestrinone can significantly inhibit the growth and proliferation of ectopic endometrium cells, and this effect was related to increase of PTEN expression.

Anabolic-androgenic steroids and testosterone precursors: ergogenic aids and sport.

This article reviews the recent literature on the use of anabolic-androgenic steroids (AAS) for performance enhancement. Recent studies utilizing supraphysiologic doses of testosterone have demonstrated increases in strength and improvements in body composition, despite earlier assertions by the medical community that steroids were ineffective as ergogenic aids. Although data that support the theory of conversion of prohormones, such as androstenediol, to testosterone in the body is available, support for testosterone precursors alone as ergogenic aids is lacking. Drug testing laboratories are utilizing new techniques that analyze carbon-13 levels of urinary steroids to detect exogenously administered steroids as well as the use of urine-manipulating agents. Investigations that seek to refute athletes' various claims for positive drug tests are ongoing. The recent discovery, characterization, and development of a urine test for tetra-hydro-gestrinone, a designer steroid, has brought the issue of performance enhancement once again into the public spotlight. Increasing attention is also being paid to the long-term effects of AAS abuse, as more authors characterize the changes to hematologic, hepatic, lipid, and hormone profiles as a result of years of steroid use. Although the understanding of AAS and testosterone precursors as performance-enhancing drugs continues to advance, there are likely to be more revelations as scientific investigations continue.

Tetrahydrogestrinone is a potent androgen and progestin.

Tetrahydrogestrinone (THG) was recently identified as a novel steroid used illicitly to improve athletic performance. Although its structure is closely related to gestrinone, a 19-nor progestin, and resembles that of trenbolone, THG was never marketed, so information on its hormonal properties is not known. In this study, we demonstrate that THG is a highly potent androgen and progestin in a yeast-based in vitro bioassay system expressing human androgen and progesterone receptors. It has no estrogenic activity and no antagonism for any of the three steroid receptor classes.

Avaliaçäo dos parâmetros de resposta clínica, hormonal e ultra-sonográfica do tratamento do leiomioma uterino com gestrinona / Clinical, hormonal and ultrasound evaluation after leiomyoma clinical treatment with gestrinone

OBJETIVOS: Avaliar a resposta clínica de pacientes com miomatose tratadas com gestrinona. PACIENTES E MÉTODOS: Foram 14 mulheres com idade entre 24 e 42 anos, com miomatose sintomática, tratadas com gestrinona 15mg/sem durante 6 meses. RESULTADOS: O volume uterino médio, determinado por US, foi de 454,57cmü antes e 264,66cmü depois do tratamento. Em 5 pacientes o volume uterino diminuiu mais de 30 por cento (média de 699,2cmü antes para 369,4cmü depois do tratamento). Em 6 pacientes o volume uterino foi reduzido entre 10 e 30 por cento e em 3 pacientes o volume foi reduzido em menos de 10 por cento. Houve uma relaçäo positiva entre volume uterino pré tratamento e porcentagem de reduçäo. Os níveis de LH e FSH näo diferiram significativamente antes ou depois do tratamento, enquanto os níveis de PRL e E2 reduziram-se significativamente. Esta reduçäo foi maior nas pacientes que exibiram uma melhor resposta clínica ao tratamento. CONCLUSOES: A gestrinona pode ser usada no tratamento clínico da miomatose uterina em pacientes na pré menopausa, como alternativa clínica ao tratamento cirúrgico. Os níveis de estradiol säo um bom fator preditivo de boa resposta de reduçäo uterina

Clinical, endocrine, and metabolic effects of two doses of gestrinone in treatment of pelvic endometriosis.

OBJECTIVE: Our purpose was to determine and compare the efficacy and hormonal and metabolic effects of 1.25 mg with 2.5 mg of gestrinone given twice a week in the treatment of mild and moderate pelvic endometriosis. STUDY DESIGN: A phase II, prospective, randomized, double-blind study involving 11 patients given gestrinone 1.25 mg (five patients) or 2.5 mg (six patients) orally twice a week for 24 weeks was performed. Revised American Fertility Society scores were determined by laparoscopy before and at the end of treatment. Serum hormone (free thyroxine, free testosterone, estradiol, progesterone, follicle-stimulating hormone, luteinizing hormone), sex hormone binding globulin, and lipid concentrations were measured before, throughout, and for 6 months after treatment. Quantitated computerized tomography of thoracic 12 through lumbar 4 vertebral bodies were determined before, at the end of, and 6 months after treatment. RESULTS: Gestrinone 2.5 mg significantly reduced the endometriosis implant score from 10.3 +/- 2.8 to 3.8 +/- 0.8 (p = 0.05). Both doses significantly reduced serum progesterone and sex hormone binding globulin levels. Estradiol, free testosterone, free thyroxine, follicle-stimulating hormone, and luteinizing hormone levels were not significantly affected. Spinal bone increased significantly by 7.1% with 2.5 mg but lost significantly by 7.1% with 1.25 mg gestrinone; these changes had not reversed completely 6 months after stopping treatment. CONCLUSIONS: In mild to moderate pelvic endometriosis 2.5 mg of gestrinone twice a week was more effective and had a more positive effect on bone mass than did 1.25 mg of gestrinone.

Comparative effect of the calcium antagonist verapamil and the synthetic steroids gestrinone and danazol on human monocyte phagocytosis in vitro.

Recent evidence suggested that periovulatory treatment with an immunomodulatory agent such as verapamil might be an effective alternative to conventional treatment for endometriosis-associated subfertility. In particular, it has been reported that the drug might reduce the accentuated macrophage peritoneal activation demonstrated in patients with endometriosis. In this study, we compared the effect of the calcium antagonist verapamil with those of gestrinone, danazol and testosterone on human monocyte phagocytosis in an attempt to evaluate any significant differences in their ability to influence a parameter of cell inflammatory activation. Peripheral blood monocytes were isolated from 37 healthy women. Monocyte function was determined by phagocytosis of fluorescent microspheres after an overnight incubation in the presence or absence of the various agents. This study indicates that verapamil at the pharmacological concentration of 0.4 micrograms/ml, the systemic level in patients taking 40-80 mg/8 h p.o., significantly inhibits monocyte function. A lower immunosuppressive but still significant effect was achieved in this assay system with gestrinone at a concentration of 3 x 10(-8) M). The pharmacological concentration of danazol (10(-6) M) and the physiologic concentration of testosterone (10(-8) M) did not significantly affect this immunologic test system. These results provide evidence that verapamil is able to exert a slightly greater immunosuppressive effect than steroidal drugs on monocyte phagocytosis. However, due to the small differences observed, further studies on the biological mechanism of the drug seem to be necessary to completely elucidate its potential role in endometriosis-associated subfertility.

Gestrinone inhibits macrophage function and mitogen-stimulated lymphocyte proliferation in vitro.

Clinical and experimental evidence supports the hypothesis that some steroidal drugs with androgenic effects might influence the immune system. The present study investigated whether gestrinone is able to affect macrophage and lymphocyte activity in vitro. Macrophage function was determined by phagocytosis of fluorescent microspheres, whilst lymphocyte proliferation was assessed by cell counting. Macrophage phagocytosis was evaluated after an overnight incubation in the presence or absence of gestrinone at serial dilutions; lymphocyte proliferation was detected in basal conditions and after stimulation with Concanavalin A (Con A) in the presence or absence of gestrinone. The results of this study showed that gestrinone significantly inhibited macrophage phagocytosis at the concentrations of 10(-8), 3 x 10(-8) and 10(-7) M. Furthermore, a significant suppression of lymphocyte blastogenesis was observed when lymphocytes were incubated with gestrinone at the concentration of 10(-7) M for 6 days. The biological significance of gestrinone as an inhibitor of immune functions under experimentally defined conditions is discussed in relation to its potential mechanism for fertility enhancement.

Endometriosis: medical therapy.

The management of women with endometriosis is complex and necessitates individualization of patient care. The most commonly used medical therapies are danazol, GnRH agonists, medroxyprogesterone acetate and gestrinone. Studies to date have shown these drugs to have equal efficacy in terms of reduction in laparoscopic score and relief of symptoms. However, their side-effects make them unsuitable for long-term use. The addition of low dose hormone replacement therapy to GnRH agonist regimens may allow prolonged use but the current cost of these agents is high. Low dose oral contraceptive pills deserve further investigation. The role of medical treatment for women with endometriosis and infertility is controversial. There is no place for hormonal therapy in such women with stage I or II disease. When expectant management fails, gamete intrafallopian transfer offers excellent results. For those with stage III or IV disease, surgery is preferable with adjunctive medical therapy in selected cases. If pregnancy does not ensue, in vitro fertilization and embryo transfer are the next line of management, and results are optimized by prior medical therapy and aspiration of endometriomas. Major advances have been made in the medical management of endometriosis. However, current treatment strategies are ineffective in eliminating the disease in most women. New approaches are required in both basic and clinical research in order to finally eradicate this often devastating disease.