Relative binding affinity of novel steroids to androgen receptors in hamster prostate

The in vivo and in vitro antiandrogenic activity of four aromatic esters 10a-10d, one aliphatic ester 10e based on the pregna-4,16-diene-6, 20-dione structure and two aromatic 17c, 17d and two aliphatic valeroyloxy esters 17a, 17b based on the more saturated 4-pregnene-6,20-dione skeleton was examined. The biological activity of steroids 9, 10a-10e and 17a-17d, was determined using prostate glands from gonadectomized adult male golden hamsters. In the in vitro studies, the relative binding affinity of these steroids to cytoplasmic androgen receptor (AR) of hamster prostate was determined from, the corresponding IC50 values obtained from the competitive binding plots. The standards dihydrotestosterone (DHT) and cyproterone (CA) acetate used have displaced [3H]DHT from the AR with an IC50 value of 3.2 and 4.4 nM respectively. All steroidal compounds synthesized in this study showed a binding affinity for the androgen receptor, present in the cytosol from prostate hamster; compounds 10a-10c showed the highest affinities for this receptor. The in vivo experiments showed that all steroidal derivatives were subcutaneously active, since they decreased the weight of the prostate gland in gonadectomized hamsters treated with DHT, and are antagonists for the androgen receptor since they block the DHT-induced prostate weight gain. The derivatives having the more conjugated 4,16-pregnadiene-6, 20-dione system (10a-10c) exhibited a higher antiandrogenic activity than the corresponding steroids (17a-17d) based on the more saturated 4-pregnene-6,20-dione system.     

Identification of a lead pharmacophore for the development of potent nuclear receptor modulators as anticancer and X syndrome disease therapeutic agents

A series of tetrahydroisoquinoline-N-phenylamide derivatives were designed, synthesized, and tested for their relative binding affinity and antagonistic activity against androgen receptor (AR). Compound 1b (relative binding affinity, RBA = 6.4) and 1h (RBA = 12.6) showed higher binding affinity than flutamide (RBA = 1), a potent AR antagonist. These two compounds also exerted optimal antagonistic activity against AR in reporter assays. The derivatives were also tested for their activities against another nuclear receptor, farnesoid x receptor (FXR), with most compounds acting as weak antagonists, however, compound 1h behaved as a FXR agonist with activity slightly less than that of chenodeoxycholic acid (CDCA), a natural FXR agonist.     

In vivo and in vitro effect of novel 4,16-pregnadiene-6,20-dione derivatives, as 5alpha-reductase inhibitors

In this study, we report the synthesis and biological evaluation of several new 3-substituted pregna-4,16-diene-6,20-dione derivatives (11a-11d). These compounds were prepared from the commercially available 16-dehydropregnenolone acetate. The biological effect of these steroids was demonstrated in in vivo and in vitro experiments. In the in vivo experiments, we measured the activity of the 11a-11d on the weight of the prostate gland of gonadectomized hamsters treated with testosterone plus finasteride or with the new steroids. For the studies in vitro, we determined the IC50 values by measuring the steroid concentration that inhibits 50% of the activity of 5alpha-reductase present in human prostate. In order to study the mechanism of action of 11a-11d, we also determined the capacity of these steroids to bind to the androgen receptor (AR) present in the rat prostate cytosol using labeled mibolerone as a tracer. The results from this work indicated that compounds 11a-11d significantly decreased the weight of the prostate as compared to testosterone treated animals and this reduction of the weight of the prostate was comparable to that produced by the finasteride. On the other hand 11a-11d exhibited a high inhibitory activity for the human 5alpha-reductase enzyme with IC50 values of 1.4 x 10(-8), 1.8 x 10(-9), 1.0 x 10(-8) and 4 x 10(-5) respectively. However the IC50 value of 11a (1.8 x 10(-9)) was the only one lower than that of finasteride (8.5 x 10(-9)). Nevertheless this compound did not show a higher potency in vivo as compared to that of compounds 11b-11d. The competition analysis for the androgen receptor indicated that the IC50 value of non-labeled mibolerone used in this experiment was 1nM, whereas steroids 10, 11a-11d did not inhibit the labeled mibolerone binding to the androgen receptor. On the other hand, steroid 10 did not show any activities in vitro or in vivo, and for this reason these steroidal derivatives (11a-11d) cannot be considered as prodrugs of compound 10. In conclusion, the compounds containing chlorine 11a, bromine 11b, iodine 11c atoms, and 11d (without any substituent in the ester moiety) at C-3 produce a significant decrease of the prostate weight in castrated animals treated with T and inhibits the activity of the 5alpha-reductase. Apparently the presence of the halogen atoms in compounds 11a-11c enhances the inhibitory activity for the 5alpha-reductase enzyme.     

Regulation of androgen receptor protein and mRNA concentrations by androgens in rat ventral prostate and seminal vesicles and in human hepatoma cells

The effects of androgen withdrawal and replacement on the concentrations of androgen receptor (AR) protein and AR mRNA were investigated in rat ventral prostate and seminal vesicles and in cultured human hepatoma (HepG2) cells. AR mRNA concentrations were determined by Northern blotting with single stranded AR cRNA as the hybridization probe, whereas antibodies raised against two synthetic 17-amino acid long peptides corresponding to the N-terminal and steroid-binding regions of the AR were employed in immunological receptor assays. AR mRNA levels in both prostate and seminal vesicles increased about 2-fold within 24 h after castration and continued to rise within the next 48 h to values that were 9- to 11-fold higher than those in intact controls. Administration of pharmacological doses of testosterone (400 micrograms steroid/day) to 1-day castrated animals for 24-48 h brought about a decrease in AR mRNA levels in accessory sex organs to levels in intact controls. Similar results were obtained in cultured HepG2 cells where a switch to serum- and steroid-free medium elicited a rapid increase (approximately 4-fold in 10 h) in the AR mRNA level, which was prevented by inclusion of 10(-7) M testosterone in culture medium. Similar, but quantitatively less marked, changes occurred in the AR protein concentration in prostate, seminal vesicles, and HepG2 cells, as determined by immunoblotting using antibodies against AR peptides. In addition, immunohistochemical studies showed that AR is a nuclear protein of the prostatic epithelial cells in both intact and castrated rats, and suggested that short term castration increases the concentration of nuclear AR in the prostate. Taken together, these data indicate that androgens down-regulate the concentration of AR protein and AR mRNA in a variety of target tissues.     

Effects of steroidal and non-steroidal antiandrogens on the androgen binding properties of the rat ventral prostate androgen receptor

Steroidal (cyproterone acetate) and non-steroidal (RU23908 and hydroxyflutamide) antiandrogens are able to block testosterone-induced increases in nuclear androgen receptor (AR) in the prostate of 1-day orchidectomized rats, but when given alone, RU23908 and hydroxyflutamide increase nuclear AR (RU23908 greater than hydroxyflutamide) in the same animal model. The increases in nuclear AR induced by antiandrogen alone or with testosterone alone are blocked by cycloheximide 1 h after administration, suggesting that androgen or antiandrogens induce de novo AR synthesis. Concomitant to nuclear AR accumulation, testosterone is able to induce depletion of cytosol and microsomal AR. Blockade of testosterone-induced depletion of microsomal AR, but not of cytosol AR, occurs in the presence of antiandrogens. Cyproterone acetate has a higher relative binding affinity (RBA) for microsomal AR and cytosol AR than RU23908 or hydroxyflutamide. This phenomenon is in good agreement with the degree of inhibition by these compounds of the association rate of androgen for the microsomal AR. This correlation between RBA and inhibition of the initial rate of hormone binding to the receptor is not found for cytosol AR. The results show that antiandrogens are not 'pure' antagonists of androgen action and they are potent agonists in the absence of testosterone. Furthermore, testosterone alone or antiandrogens per se regulate AR levels acutely by protein-synthesis dependent mechanisms of action, in rat ventral prostate.     

Prostate androgen receptor: immunohistological localization and mRNA characterization

Four androgen receptor (AR) specific monoclonal antibodies were used for the immunohistochemical localization of AR in the human prostate tissue. The prostate tissue consisted of alveoli embedded in fibromuscular stroma and lined with a single layer of columnar secretory epithelial cells. The immunoreactive ARs were found predominantly in the nuclei of epithelial cell, suggesting ARs, like estrogen receptors and progesterone receptors, are mainly nuclear proteins. Northern blot hybridization showed that AR mRNA is about 9 kilobases (kb) and relative abundant in the androgen-sensitive organs, such as ventral prostate, dorsolateral prostate and seminal vesicle.     

A nuclear binding assay for measurement of biologically active androgen receptors in animal tissues and human prostate cancer

A nuclear binding (NB) assay has been developed for the measurement in intact viable cells of biologically active (functional) estrogen and progesterone receptors, i.e. those capable of binding to nuclear acceptor sites [Spelsberg et al., Endocrinology 121: 631 (1987)]. This paper describes the application of this assay to analyses of androgen receptors in the guinea pig seminal vesicle and in human prostatic carcinoma. Cells from fresh animal seminal vesicles or human prostate carcinoma are isolated using collagenase and are incubated with [3H]R1881 for 1 h at 22 degrees C, after which nuclei are isolated at 4 degrees C and assayed for DNA and radioactivity. This NB assay demonstrates a saturable, temperature dependent, steroid and tissue specific nuclear binding of [3H]R1881 for the guinea pig-seminal vesicle system. The nuclear binding is of high affinity and low capacity. The NB assay reveals several important aspects of the androgen and estrogen receptors in target tissues: (1) the nuclear acceptor sites for androgen receptor (AR) are steroid receptor specific; (2) there are different concentrations of the androgen and estrogen receptors between the epithelium and the fibromuscular components of the guinea pig seminal vesicle; and finally (3) some biopsies of human prostate cancer appear to contain biologically inactive AR. This assay may be useful in the analyses of functional receptors in biopsies of human cancer cells.     

Subcellular location of androgen receptor in rat prostate, seminal vesicle and human osteosarcoma MG-63 cells

Location of the androgen receptor (AR) before and after dihydrotestosterone (DHT) administration was studied in 6 castrated and 2 normal male rats, as well as in MG-63 human osteosarcoma cell culture. Two days after castration, rats were injected with DHT and sacrificed 0, 6 and 24 h later. Cryosections of ventral prostate and seminal vesicle were stained with a polyclonal anti-AR antibody. Cultured MG-63 cells were also stained similarly. The intensity of immunoreaction was measured semiquantitatively by computer-assisted image analysis. In both normal and castrated rats, a positive reaction was seen mainly in the nuclei of epithelial cells and stromal cells of the prostate and seminal vesicle, as well as in those of smooth muscle cells of the seminal vesicle. AR immunoreactivity was up-regulated by DHT, it decreased clearly in both organs after castration. Nuclear AR and its up-regulation by androgen were also seen in MG-63 cells. At the immunoelectron microscopy, silver enhanced gold particles were predominantly found in the heterochromatin of cell nuclei. Treatment with DHT caused a decondensation of the heterochromatin and AR was more dispersed. Thus, AR appears to be nuclear independently of the ligand.     

Anabolic-androgenic steroid interaction with rat androgen receptor in vivo and in vitro: a comparative study

Anabolic steroids are synthetic derivatives of testosterone and are characterized by their ability to cause nitrogen retention and positive protein metabolism, thereby leading to increased protein synthesis and muscle mass. There are disagreements in the literature in regards to the interaction of anabolic steroids with the androgen receptor (AR) as revealed by competitive ligand binding assays in vitro using cytosolic preparations from prostate and skeletal muscle. By use of tissue extracts, it has been shown that some anabolic steroids have binding affinities for the AR that are higher than that of the natural androgen testosterone, while others such as stanozolol and methanedienone have significantly lower affinities as compared with testosterone. In this study we show that stanozolol and methanedienone are low affinity ligands of the rat recombinant AR as revealed by a ligand binding assay in vitro, however, based on a cell-based AR-dependent transactivation assay, they are potent activators of the AR. We also show that a single injection of stanozolol and methanedienone causes a rapid cytosolic depletion of AR in rat skeletal muscle. Based on these results, we conclude that anabolic steroids with low affinity to AR in vitro, can in fact in vivo act on the AR to cause biological responses.     

Studies on the mechanism of action of Win 49596: a steroidal androgen receptor antagonist

Win 49596 is an orally active antiandrogen in the rat. This report describes a series of in vitro and in vivo studies which were performed to characterize the mechanism of action of this compound. In vitro competition and Lineweaver-Burk analyses indicate that Win 49596 binds competitively to the rat ventral prostate androgen receptor with a Ki of 2.2 +/- 0.4 microM. Similar to other androgen antagonists, the relative binding affinity (RBA) of Win 49596 was greater after 1 h of incubation with androgen receptor than after an 18 h incubation (RBA of 2.2 versus 0.05, respectively). Win 49596 did not bind to rat cytosolic uterine estrogen or progesterone receptors or thymus glucocorticoid receptors. Furthermore, Win 49596 did not inhibit rat ventral prostate 5 alpha-reductase or 3 alpha-oxidoreductase, rat adrenal 3 beta-hydroxysteroid dehydrogenase or human placental aromatase activity in vitro at concentrations as high as 10 microM. A series of in vivo studies demonstrated that Win 49596 inhibited the uptake of [3H]testosterone as well as testosterone-induced nuclear accumulation of androgen receptor in the rat ventral prostate. Collectively, these results support direct androgen receptor antagonism as the mechanism for the antiandrogenic effects of Win 49596.     

Autologous down-regulation of androgen receptor messenger ribonucleic acid

Autoregulation of androgen receptor (AR) mRNA was investigated using Northern blot analysis with AR cDNA fragments as probes. The amount of AR mRNA increased 2- to 10-fold with androgen withdrawal and decreased below control levels after androgen stimulation in rat ventral prostate, coagulating gland, epididymis, seminal vesicle, kidney, and brain, and in a human prostate cancer cell line, LNCaP. In rat ventral prostate, AR mRNA increased 2- to 3-fold within 24 h after castration and remained elevated for 4 days. Treatment with testosterone propionate beginning 24 h after castration reduced ventral prostate AR mRNA 4-fold within 8 h of androgen replacement. Administration of estradiol 24 h after castration had no significant effect on prostatic AR mRNA. Androgens, including testosterone and the synthetic androgen methyltrienolone (R1881), or the antiandrogen cyproterone acetate down-regulated AR mRNA in vitro in LNCaP cells, whereas estradiol was without effect. Administration of testosterone propionate to rats with androgen insensitivity did not decrease AR mRNA. Down-regulation of AR mRNA by androgen is therefore a receptor-mediated process which occurs in vivo in rat tissues that differ in androgen responsiveness and in cultured human prostate cells.     

Antiandrogenic and apoptotic effects of RU-486 on animal prostate

Mifepristone (RU-486) is a potent antagonist of steroid hormone receptors such as glucocoticoid and progesterone receptors. This compound also is a very strong inducer of the interaction between androgen receptors and corepressors NCoR and SMRT and therefore could be used as selective receptor modulator.

In this study we determined the relative binding affinity of RU-486 to androgen receptors (AR) obtained from rat prostate cytosol as well as the in vivo effect of different doses of RU-486 on the prostate weight of hamsters treated with dihydrotestosterone and/or RU-486. We determined also the prostate cell death (apoptosis) in hamster treated with, dihydrotestosterone (DHT) and/or RU-486.

The results of this study indicated that the relative binding affinity of RU-486 for AR is 4.3%. The data from the in vivo experiments also showed that RU-486 inhibited the prostate weight significantly in the highest doses thus indicating the antagonistic action of this compound on hamster prostate.

The immunohistochemistry analysis showed that after 1 month of castration, the hamster prostate was atrophic. Treatment with DHT produced epithelial cell activity (measured by the increase in the prostate weight) and very low rate of apoptosis. When DHT was administered together with RU-486 (10 mg/kg) no change was observed. On the other hand, DHT plus higher doses of RU-486 (40, 80 mg/kg) resulted in an increase of apoptosis in stromal and secretory epithelial cells. In addition to the increase of the prostate cell apoptosis produced by the treatment with high dose of RU-486, other factors could contribute to the decrease of the prostate weight observed. Another possibility could be a reduction in the ductal fluid due to poor epithelial cell secretory activity more than apoptosis itself. Furthermore, in this experiment, RU-486 could have inhibited the growth of the prostate gland produced by DHT in a greater extent than the induction of atrophy and cell death. This fact could depend on the doses used, due to the low affinity of this compound for the androgen receptors.     

Identification of Anabolic Selective Androgen Receptor Modulators with Reduced Activities in Reproductive Tissues and Sebaceous Glands

Androgen replacement therapy is a promising strategy for the treatment of frailty; however, androgens pose risks for unwanted effects including virilization and hypertrophy of reproductive organs. Selective Androgen Receptor Modulators (SARMs) retain the anabolic properties of androgens in bone and muscle while having reduced effects in other tissues. We describe two structurally similar 4-aza-steroidal androgen receptor (AR) ligands, Cl-4AS-1, a full agonist, and TFM-4AS-1, which is a SARM. TFM-4AS-1 is a potent AR ligand (IC₅₀, 38 nm) that partially activates an AR-dependent MMTV promoter (55% of maximal response) while antagonizing the N-terminal/C-terminal interaction within AR that is required for full receptor activation. Microarray analyses of MDA-MB-453 cells show that whereas Cl-4AS-1 behaves like 5α-dihydrotestosterone (DHT), TFM-4AS-1 acts as a gene-selective agonist, inducing some genes as effectively as DHT and others to a lesser extent or not at all. This gene-selective agonism manifests as tissue-selectivity: in ovariectomized rats, Cl-4AS-1 mimics DHT while TFM-4AS-1 promotes the accrual of bone and muscle mass while having reduced effects on reproductive organs and sebaceous glands. Moreover, TFM-4AS-1 does not promote prostate growth and antagonizes DHT in seminal vesicles. To confirm that the biochemical properties of TFM-4AS-1 confer tissue selectivity, we identified a structurally unrelated compound, FTBU-1, with partial agonist activity coupled with antagonism of the N-terminal/C-terminal interaction and found that it also behaves as a SARM. TFM-4AS-1 and FTBU-1 represent two new classes of SARMs and will allow for comparative studies aimed at understanding the biophysical and physiological basis of tissue-selective effects of nuclear receptor ligands.     

Farnesyl diphosphate synthase is abundantly expressed and regulated by androgen in rat prostatic epithelial cells

Farnesyl diphosphate synthase (FPPS) has been identified as an androgen-response gene in the rat ventral prostate using a highly sensitive PCR-based cDNA subtraction technique. FPPS is an essential enzyme that catalyzes the synthesis of farnesyl diphosphate (FPP), which is required for cholesterol biosynthesis as well as protein prenylation. We have characterized the expression of FPPS in the rat prostate in response to androgen manipulation. Northern blot analysis showed that castration induced a 10-fold down-regulation of FPPS mRNA within 24 h in the ventral prostate and androgen replacement up-regulated FPPS mRNA rapidly in the regressed ventral prostate of a castrated rat. The expression of FPPS was also regulated by androgen in the lateral and dorsal prostate, indicating that FPPS is important to androgen action in all three lobes of the prostate. Western blot analysis showed that FPPS protein level was also regulated by androgen in the prostate. Northern blot analysis of tissue specificity indicated that FPPS was most abundantly expressed in the ventral prostate of a mature rat and was responsive to androgen manipulation in the prostate and seminal vesicles, but not in other tissues. In situ hybridization study showed that FPPS mRNA was localized to the prostatic epithelium. Interestingly, the expression of FPPS was elevated in Dunning rat prostate tumor cell lines. The above findings suggest that FPPS has the potential to play an important role in androgen action and prostate cancer progression.     

Synthesis of the 7alpha-cyano-(17alpha,20E/Z)-[125I]iodovinyl-19-nortestosterones: potential radioligands for androgen and progesterone receptors

We report the preparation of the 7alpha-cyano derivative of the isomeric (17alpha,20E/Z)-[125I]iodovinyl-19-nortestosterones (IVNT) together with their binding affinity for the androgen receptor (AR) and their biodistribution in two different animal models. The cyano group was introduced at the 7alpha-position by hydrocyanation of 4,6-estradien-17beta-ol-3-one with diethylaluminum cyanide. Selective protection of the A-ring enone system as the dienol ether followed by ethynylation and deprotection under base and acid hydrolysis condition gave 7alpha-cyano-17alpha-ethynyl-19-nortestosterone. The stannyl derivatives were prepared by addition of tri-n-butylstannyl hydride and converted stereospecifically to the corresponding [125I]iodovinyl analog using [125I]NaI and H2O2. The [125I]iodovinylsteroids were intravenously administered to male rats and estrogen-primed immature female rats and tissue uptake was measured up to 6h post-injection. Co-administration of NLP-004 or ORG-2058, highly selective ligands for the progesterone receptor, to the female rats did not affect uterus uptake of the 125I-ligands. However co-injection of testosterone to DES-primed male rats induced a marked increase in prostate uptake of the 20Z-isomer of 7alpha-cyano-[125I]-IVNT. The relative binding affinity (RBA) of either 7alpha-cyano-(17alpha,20E/Z)-IVNT isomer for the AR is low (RBA=4 and 3, respectively, versus 100 for 5alpha-dihydrotestosterone (DHT)), suggesting the absence of a possible role of the AR in the localization process. These findings contrast previously reported data for the analogous 7alpha-methyl-[125I]-IVNT where co-administration of testosterone was shown to result in a 50% drop in prostate uptake. These data indicate that the addition of an electron withdrawing 7alpha-cyano group to 123I-labeled nortestosterone derivatives does not improve their potential to serve as SPECT agents for the imaging of AR densities in the prostate.