MXPA02012573A - Cell lines and cell-based assays for identification of androgen receptor modulators. - Google Patents

Abstract

Stable muscle cell lines comprising an androgen receptor and methods of using these cells in functional transactivation assays to assess the efficacy of compounds as androgen receptor modulators in a muscle cell background are provided.

Description

CELL LINES AND CELL BASE TESTS FOR THE IDENTIFICATION OF ANDROGEN RECEIVER MODULATORS Field of the Invention The invention relates to cell lines and methods for using these cell lines in the identification of compounds having biological activity. In particular, the invention relates to muscle cell lines transfected stably with an androgen receptor and a reporter gene useful in the identification of compounds, which are modulators of the androgen receptor.

BACKGROUND OF THE INVENTION The androgen receptor (AR) is a member of the superfamily of nuclear receptors, steroids of ligand-dependent transcription factors and is widely distributed between reproductive and non-reproductive tissues, which include the prostate and seminal vesicles, male and female genitalia, skin, testis, ovary, cartilage, sebaceous glands, hair follicles, sweat glands, cardiac muscle, skeletal and smooth muscle, gastrointestinal vesicle cells, thyroid follicular cells, adrenal cortex, liver , pineal gland, and numerous regions REF: 143144 cortical and subcortical of the brain, which include the motor, spinal neurons (Negro-Vilar, A. JCE &M 1999 54 (10): 3459-62). As with the other members of the steroid receptor family, RA has several functional domains that include a DNA binding domain (DBD) and a ligand binding domain of 261 residues (LBD, its acronym in English) (Pm = 30,245 Da) which contains the androgen binding site and is responsible for switching in androgen function. The cDNA and amino acid sequences of the androgen receptors of human and rat have already been described (Proc Nati Acad Sci U.A. 1988 85: 7211-7215). AR is an important goal in multiple areas of drug discovery and patient therapy. In oncology, for example, inhibitors (antagonists or partial antagonists) of androgen receptor function are useful for the treatment of androgen-dependent prostate cancer while AR agonists or partial agonists are applicable to the treatment of cancer. of chest For metabolic and endocrine disorders, for example, agonists or partial agonists of androgen receptor function are useful for the treatment of age-related diseases and cachexia conditions in severe disease states including, but not limited to, they are limited a, AIDS. Functional RA has also been identified in several bone cells and androgen administration has beneficial effects on skeletal development and maintenance in men and women. The progress of androgen therapy has been limited by the inability to separate desirable androgenic activities from the undesirable or dose-limiting side effects. However, recent advances in the development of selective modulators of estrogen receptors (SERM ?, with its acronym in English) with a high degree of tissue selectivity in the choice of estrogen receptor target, while eliminating the effects undesirable collaterals, have resulted in the suggestion of MRSAs, selective modulators of androgen receptors (for its acronym in English) (Negro-Vilar, A. JCE &M 1999 54 (10): 3459-62; Reid et al. Investigational New Drugs 1999 17: 271-284). The assays and general methods for detecting the transcpptional activity of a cell receptor when exposed to a known ligand or unknown compound have already been described. For example, U.S. Patent No. 5,071,773 discloses an assay for identifying intracellular hormone receptors, ligands for these receptors and proteins capable of transcriptionally activating the intracellular hormone receptors. The trials involve the use of a cell containing DNA encoding a hormone response element, such as a promoter linked to a reporter, operative and DNA gene encoding the intracellular receptor protein. When the cell is exposed to a hormone or ligand, a complex of the hormone and the intracellular receptor is formed and delivered to an appropriate DNA binding region, to activate thereby the hormone response element, which leads turn to the expression of the product encoded by the reporter gene. The activation of the reporter gene is detected according to the known procedures for the detection of the reporter gene. U.S. Patent No. 6,017,924 discloses non-steroidal compounds characterized as high-affinity, high-affinity agonists, partial agonists (ie, tissue-specific partial activators and / or activators) and antagonists for androgen receptors based on a vvcis test. trans "or of * co-transfection". Non-steroidal compounds characterized as agonists of high specificity, high affinity, partial agonists (ie, partial activators and / or tissue-specific activators) and antagonists for androgen receptors by means of the cis-trans "or w co-assay. -transfection "are also described in patents WO 01/16108, WO 01/16133 and WO 01/16139. It is suggested that this co-transfection assay (Evans et al., Science 1988 240: 889-95) provides a method for identifying functional agonists and partial agonists, which mimic, or antagonists, which inhibit, the effect of native hormones, and to quantitate their activity for proteins. responsive of intracellular receptors. In this assay, CV-1 cells (African green monkey kidney fibroblasts) are transiently transfected with the plasmid pRShAR containing the human RA under the constitutive control of the SV40 promoter and a reporter plasmid MTV-LUC containing the Firefly luciferase cDNA under the control of a long terminal repeat of the mouse mammary tumor virus (MTV, for its acronym in English). Plasmid pRS-β-Gal, which encodes the constitutive expression of β-galactosidase from E. coli, is included as an internal control for the evaluation of the transfection efficiency and the toxicity of the compound. It has also been suggested that hydroxyflutamide, a known AR antagonist in most tissues, functions as a selective AR modulator (? ARM) for its effects on the production of IL-6 by osteoblasts (Hofbauer et al. J Bone Miner, Res. 1999 14: 1330-1337). The selectivity of hydroxyflutamide was tested by evaluating the proliferation and differentiation of a human fetus osteoblast cell line (HFOB / AR-6) that It expresses a mature osteoblast phenotype and a physiological number of androgen receptors in the presence of this compound. Hydroxyflutamide and Casodex, both of which are known to be full AR antagonists in most tissues, have also been shown to activate the MAP kinases Erk-1 and Erk-2 in PC3 transfected cells with a RA-dependent manner. AR as dihydrotestosterone (DHT; Peterziel et al., Oncogene 18, 6322-6329 (1999)). The compound LGD2226, a non-steroidal AR agonist, has also been characterized as a selective modulator of androgen receptors for use in the treatment of androgen-related diseases such as osteoporosis, male hormone replacement, male and female sexual dysfunction and cachexia based on its activity in the CV-1 assay described supra (SCRIP - World Pharmaceutical New FILED May 12, 2000, WO 01/16108, WO 01/16133 and WO 01/16139). U.S. Patent No. 5,952,488 describes a bioassay for androgen materials in a cell culture, wherein HeLa cells or PC-3 cells are transiently transfected or stably integrated to a DNA sequence cloned from the promoter region of the cell. probasin gene (PB) coupled to a CAT reporter gene. U.S. Patent No. 5,506,102 describes methods and assays useful for selecting compounds for potential transcriptional antagonists mediated by intracellular steroid receptors, wherein the cells are transfected with a first vector encoding the intracellular receptor, a second vector encoding the PR-A isoform of the progesterone receptor of human and a third vector that encodes a reporter gene. Applicants have now developed, for the first time, cell lines and assays in which an AR and a reporter have been stably transfected into muscle tissue cells.

Brief Description of the Invention One objective of the present invention is to provide muscle cell lines, comprising a mammalian androgen receptor stably introduced into muscle cells. These cell lines are useful in functional transactivation assays to assess the efficacy of compounds as modulators of androgen receptors in a muscle cell background. Another objective of the present invention is to provide functional transactivation assays for use in the assessment of the efficacy of compounds as modulators of androgen receptors in a muscle cell background by means of these cell lines? C2C12 mouse skeletal muscle comprising the mammalian androgen receptor. Yet another objective of the present invention is to provide modulators of androgen receptors, and in particular selective modulators of androgen receptors, identified by means of functional transactivation assays with Stable mouse muscle cell lines C2C12 Stable comprising an androgen receptor. of a mammal Detailed Description of the Invention The present invention relates to muscle cell lines to which a mammalian androgen receptor and a reporter gene were stably introduced. Various muscle cells can be used in the present invention. In a preferred embodiment, the muscle cell line comprises the Stable cells of mouse skeletal muscle C2C12. However, other exemplary muscle cells useful in the present invention include, but are not limited to, mouse cells G-7, G-8, P19 and Sol ?, rat cells H9c2 (2-1), L6 and L8 , and human cells? JRH30 (RMS13). The muscle cell lines of the present invention further comprise an androgen receptor of mammal, which is introduced stably into muscle cells. The androgen receptors useful in the present invention have been isolated from various mammalian species. These receptors and their sequences have been described in detail in the prior art. See, for example, U.S. Patent No. 5,614,620. In addition, rat androgen receptors are set forth in Genbank Accession No. M23264 and J05454, as well as by Chang et al. (Science 1988 240 (4850): 324-326). Mouse androgen receptors are set forth in Genbank Accession No. M37890 and by Gaspar et al. (Proc.

Nati Acad. ? ci, USA 1991 88: 8606-8610) and He and collaborators (Biochem Biophys, Res. Commun, 1990 171 (2): 697-704).

Also, He and co-workers have described a guinea pig androgen receptor (Biochem Biophys, Res. Commun. 1990 171 (2): 697-704). He and co-workers (Biochem. Biophys., Res. Commun. 1990 171 (2): 697-704) also describe a dog androgen receptor as the Genbank Accession No. AF197950. Shiba et al. Describe a hamster androgen receptor (J. Dermatol, Sci. 2001 26 (3): 163-8). In addition, human androgen receptors are disclosed in Genbank Accession No. M34233 and by Trapman et al (Biochem Biophys, Res. Commun. 1988 153 (1): 241-248) and Tilley et al. (Proc. Nati Acad.? Ci. USA 1988 86 (1): 327-331). In a preferred embodiment, the cell lines of the present invention comprise a rat androgen receptor. The cell lines of the present invention are useful in the evaluation of the activity of compounds as modulators of androgen receptors in a muscle cell pool. In a modality, the cell line comprises the stable, skeletal C2C12 mouse cells that contain a full length rat androgen receptor such as that set forth in Genbank Accession No. M23264. This cell line is referred to herein as stable 1. To generate the? 1 cell line containing the full-length rat androgen receptor (rAR), the mouse skeletal cell line C2C12 (Yaffe D. and Saxel, O. Nature 1977 270: 725-727) were transfected with a plasmid, pIRESneo / rAR, which encodes a bicistronic message containing a full-length rAR and the neomycin resistance gene (Jackson and collaborators, Trends Biochem, Sci. 1990 15: 477-483; Jang et al., J. Virol. 1988 62: 2636-2643). Specifically, 50 μq of pIREα neo / rAR was transfected into C2C12 cells using the LipofectAmine PlusMK reagent (Gibco BRL) with 250 μl of plus reagent and 375 μl of lipofectamine reagent in 10 milliliters of optiMEM media (Gibco BRL) according to the manufacturer's instructions. Cells (0.75 x 10) in 10 milliliters of growth media (Dulbecco's modified Eagle medium (DMEM) with high glucose content supplemented with 10% FBS, sodium pyruvate IX and 0.5X antibiotic-antimycotic agent (all of Gibco BRL)), later referred to as stable growth media 1, were placed on each of five 10 cm culture plates. The next day, the media was removed from each culture cell and replaced with 4.5 milliliters of optiMEM. Then two milliliters of the transfection mixture was added to each culture cell. After three hours of incubation, the transfection media was removed and replaced with 6.5 milliliters of growth media. The cells were allowed to grow for 24 hours in nonselection media. To select the stably transfected cells with neo / rAR, the cells were split 1:15 in stable culture media 1 supplemented with 800 μg / L of G418 and allowed to propagate as separate clonal cell lines. After fourteen days, a total of 80 resistant clones were isolated. Clones exhibiting normal growth characteristics were transiently transfected with the enhancer / promoter / reporter construct, pGL3 / 2XDR-1 / luciferase. The? Table 1 cells were identified as clones that showed a significant increase in luciferase activity, measured via the Steady-GloMR luciferase assay system (Promega), with the addition of 0.1 μM dihydrotestosterone (DHT). In a preferred embodiment, the Stable 1 cell line exhibits an increase of about 12-fold or more in luciferase activity with the addition of DHT. Stable 1 cells of the present invention comprising a line of stable, skeletal C2C12 mouse cells containing a full-length rat androgen receptor were sent for deposit on June 12, 2001 to the American Type Culture Collection (ATCC ), 10801 Umversity Boulevard, Manassas, VA USA 20110-2209. Twenty-five flasks of Stable 1 cells, with an activity of approximately 30,000 units of relative luminescence, specific (RULs) in the presence of 100 nM DTH in the transactivation assay described infra, were shipped to the ATCC. The ATCC deposit number for cell line? Table 1 is PTA-3458. In another embodiment, the stable, skeletal mouse C2C12 cell line contains a full-length rat androgen receptor plus an enhancer / promoter / reporter construct. This cell line is referred to herein as stable 2.

Several enhancer / promoter constructs can be used in the construction of the Stable 2 cell line. In a preferred embodiment, the enhancer comprises an androgen response element (ARE). The exemplary AREs used in these constructions include, but are not limited to, C3-1, PB-ARE, and DR1. C3-1 is a consensual ARE / GRE (glucocorticoid receptor response element) isolated from the C3 subunit promoter of the gene for the rat prosthetic binding protein. 2XC3, which contains two C3-1 elements, comprises a consensual intensifier sequence for RR and GR (Claessens et al., J. Biol. Chem. 1996 271: 19013-19016). PB-ARE is a specific response element for androgen receptors, isolated from the promoter of the rat probasin gene (Claessens et al., J. Biol. Chem. 1996 271: 19013-19016). The DR1 response element is also specific for androgen receptors, however, it was synthetically derived from a pool of degenerate oligonucleotides containing a consensual ARE / GRE using a random method of sequence selection and amplification. The IX DR-1, which contains 1 DR-1 and 2X DR-1 element, which contains two DR-1 elements, both have been reported as specific for AR (Zhou et al., J. Biol. Chem. 1997 272: 8227 -8235). Each DR1 element consists of two AR core union sites oriented or overlapping direct repeats (Zhou et al., J. Biol. Chem. 1997 272: 8227-7235). Several promoters can also be used in these constructions. Exemplary promoters include, but are not limited to, SV40, CMV, beta-globin and HSVtk. However, as will be well understood by those skilled in the art, upon reading this description, other promoters useful in the present invention can be routinely selected. In a preferred embodiment, the enhancer / promoter construct of the Stable 2 cell line comprises pGL3 / 2XDR-1, which carries the strongest SV40 promoter. It was reported that 2X DR-1 is a specific response element for AR in CV-1 cells (Zhou et al. J. Biol. Chem. 1997 272: 8227-8235). This was developed by the random utagénesis of a sequence of consensual intensifiers AR / GR. The experiments described in detail in Example 2 showed that 2X DR-1 exhibits better stimulation and selectivity with the addition of DHT compared to the enhancer / promoter constructions comprising AREs C3, IX DR-1, as well as PB-ARE . However, alternative enhancer / promoter constructs that can be used to construct the Stable 2 cell line of the present invention include, but are not limited to, pGL3 / 2XC3, PGL3 / 1XDR-1, pGL3 / PB-ARE, H? Vtk / 2XC3, H? Vtk / lXDR-1, HSVtk / 2XDR-1 and H? Vtk / PB-ARE. The construction of these enhancer / promoter constructs is described in detail in this document in Example 1. Various reporter genes can also be used in the construction. Examples include, but are not limited to, luciferase, beta-galactosidase, secretory alkaline phosphatase, beta-lactamase, numerous green fluorescent proteins and chloramphenicol acetyltransferase. In a preferred embodiment, the reporter gene is luciferase. To generate the? 2 cell line containing the full-length rat androgen receptor plus the enhancer / promoter / reporter construct, pGL3 / 2XDR-1 / luciferase, the cells of the Stable 1 cell line are transfected with a plasmid containing the enhancer / promoter / reporter construct and a plasmid conferring resistance to hygromycin B (pcDNA3, Hygro, Invitrogen, Carlsbad, CA). Specifically, 60 pq of pGL3 / 2XDR-1 luciferase and 15 pq of pCDNA3 were transfected. l- / Hygro within Stable 1 cells using the LipofectAMINE Plus FV reagent (Gibco BRL) with 300 μl of plus reagent and 450 μl of lipofectamine reagent in 12 milliliters of optiMEM media (Gibco BRL) according to the manufacturer's instructions . The cells (6.0 x 105) in 10 milliliters of media stable growth 1 supplemented with 800 g / ml of G418 were placed in each of six 10 cm culture plates. The next day, the media was removed from each culture cell and replaced with 4.5 milliliters of optiMEM. Then two milliliters of the transfection mixture were added to each culture cell. After a three-hour incubation, the transfection media was removed and replaced with 6.5 milliliters of stable growth media 1. The cells were allowed to grow overnight and then divided 1:18 and 1:24 into media stable growth 1 supplemented with 800 μq of hygromycin B to select the individual cells stably transfected with the enhancer / promoter / reporter construct as well as the resistance to hygromycin B and then allowed to propagate as clonal, separate lines. After fourteen days, resistant clones were isolated and clones exhibiting normal growth characteristics were tested for luciferase activity in the presence of 0.1 μM DHT. Clones with activities that varied from an increase of 3 to 12 times over the background expanded. Further characterization using the standard reference compounds DHT, fluoxi estrone, ox ndrolone and medroxyprogesterone acetate was performed in several of these clones. In a preferred embodiment, the cell line Stable 2 exhibits an increase of 12X or greater on the background in luciferase activity with the addition of DHT with an EC50 value in the sub-nanomolar range. The expected activity was exhibited when the Stable 2 cells were exposed to other reference compounds. The β2 cells of the present invention comprising a line of stable, skeletal C2C12 mouse cells, containing a full length rat androgen receptor and pGL3 / 2XDR-1 / reporter of stably transfected luciferase, were sent for deposit on June 12, 2001 to the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA USA 20110-2209. Twenty-five flasks of Stable 2 cells, with an approximate activity of 30,000 relative luminescence, specific (URLs) in the presence of 100 nM DHT in the transactivation assay described infra, were shipped to the ATCC. The deposit number in ATCC for cell line? Table 2 is PTA-3459. The present invention also relates to functional transactivation assays developed to assess the activity of the compounds as modulators of androgen receptors in a muscle cell background by means of detecting the expression of a reporter gene. '"Modulator", for purposes of the present invention, means that it is inclusive of agonists, partial agonists, antagonists and / or partial antagonists of AR. In these trials, the efficacy of a compound as an agonist. or partial androgen receptor agonist is assessed by contacting either the transiently transfected transfected cells with an androgen response element, a promoter and a reporter gene or Stable 2 cells with a compound and detecting the expression of the reporter gene in the cells in the presence of the compound. An increase in the expression of the reporter gene in the cells in the presence of the compound, compared to the control cells without contact with or exposed to the compound, is indicative that the compound is an agonist or partial agonist of androgen receptors in the cells of muscle. The effectiveness of a compound as a partial antagonist or antagonist of androgen receptors is assessed by a competition test wherein the ability of a compound to prevent the induction of the expression of a reporter gene by DHT in the cells is determined. Table 1 or stable 2. In a preferred embodiment, approximately 1 nM DHT is used in the assay to induce the expression of the reporter gene. A decrease in reporter gene expression in the presence of the compound compared to control cells exposed to DHT, but in contact with the compound, is indicative that the compound is a partial antagonist or antagonist of androgen receptors in muscle cells. These assays can be used to determine the concentration at which the compound inhibits the induction of DHT by 50%, also referred to as IC50 value. More specifically, a first assay of the present invention, referred to herein as a stable androgen receptor transactivation assay (ARTA), 1 uses the Stable 1 cell line, which stably expresses the full-length rat androgen receptor but requires transient transfection of an enhancer / promoter / reporter construct. In this assay, the? 1 cells are plated, preferably in a 96-well format, at about 5, COÜ at 10,000 cells / well, preferably 6,000 cells / well, in a high glucose DMEM without red. phenol (Gibco BRL, Cat. No .: 21063-029) containing 10% charcoal and FB? treated with dextran (HyClone Cat. No .: SH30068.02), 50 mM HEPES buffer (Gibco BRL, Cat. No .: 15630-080), MEM Na IX pyruvate (Gibco BRL, Cat. No .: 11360-070 ), antibiotic-antimicotic agent 0.5X, and 800 μg / ml of 0.5X Geneticin (Gibco BRL, Cat. No .: 10131-035). Once the cells have been adhered and acclimated, and have reached the optimal confluence for transfection, approximately twenty-four hours after plating, the cells are transfected with a construct of enhancer / promoter / reporter such as pGL3 / 2XDR-1 / luciferase. Preferably, transfection is performed using the LipofectAMINE Plus ™ reagent (Gibco BRL, Cat. No .: 10964-013). In this preferred embodiment, pGL3 / 2XDR-1 / luciferase DNA (approximately 5 ng / well) and a carrier, such as salmon sperm DNA (50 ng / well) or a generic plasmid DNA, are diluted with 5 μl / Opti-MEM media well (Gibco BRL, Cat. No .: 31985-070). To this is added 0.5 μl / well of the plus reagent. This mixture is incubated for 15 minutes at room temperature. In a separate container, 0.385 μl / well of LipofectAMINE reagent is diluted with 5 μl / well of Opti-MEM. The DNA mixture is then combined with the LipofectAMINE mixture and incubated for an additional 15 minutes at room temperature. During this time, the cell mediae are removed and replaced with 60 μl / well Opti-MEM. To this is added 10 μl / well of the DNA / LipofectAMINE transfection mixture. The cells are incubated for 4 hours. The transfection mixture is removed from the cells and replaced with 90 μl of the DMEM with high glucose content described above. Other methods of tranefection which can be used in the present invention include, but are not limited to, DEAE-dextran, calcium phosphate, microinjection direct, electroporation and supply of biolistic particles. The compounds to be tested by activity in this assay are then placed in each well. In a preferred embodiment, 10 μl of an appropriate dilution of the compound is placed in each well. It is preferred that a range of compound concentrations be tested, that is, from about 0.001 nM to 3000 nM. It is also preferred that the initial dilutions of the compounds be made in dimethyl sulfoxide or ethanol and that the subsequent dilutions be made in assay media. Twenty-four hours later, the activity of the compound is detected by means of a detection system such as the luciferase assay system? Teady-GloMR (Promega, Cat. No .: E2520), or by means of other luciferin substrates (Tropix or Packard Bioeciencee) according to the manufacturer's instructions. A second assay of the present invention, referred to herein as stable ARTA 2, uses the cell line β2, derived from stable 1, which stably expresses both the rat androgen receptor and an enhancer construct. promoter / reporter. The intercept / promoter / reporter construct used in this system preferably comprises pGL3 / 2XDR-1 / luciferase.

In the stable ARTA 2 assay, Stable 2 cells are plated, preferably in a 96-well format, at approximately 5,000 to 10,000 cells / well, preferably 6,000 cells / well, in high glucose DMEM without phenol red (Bibco BRL, Cat. No .: 21063-029) containing 10% charcoal and FBS treated with dextran (HyClone Cat. No .:? H30068.02), HEPE buffer? 50 mM (Bibco BRL, Cat. No .: 15630-080), MEM pyruvate Na IX (Bibco BRL, Cat. No .: 11360-070), 0.5X antibiotic-antifungal agent, 800 μg / ml Geneticin (Bibco) BRL, Cat. No .: 10131-035) and 800 μg / ml hygromycin B (Bibco BRL, Cat. No .: 10687-010). Approximately 24 hours later, the media in the cells is removed and replaced with 90 μl of new assay media. The compounds to be tested by activity in this assay are then plated in each well. In a preferred embodiment, an aliquot of 10 μl of the composition is placed in each well at a concentration ranging from about 0.001 nM to 3000 nM. It is preferred that the initial dilutions of a compound be made in dimethyl sulfoxide or ethanol and subsequent dilutions be made in assay media. 24 hours later, the activity is detected by means of the luciferase assay system? Teady-GloMR (Promega, Cat. No .: E2520) or by means of other substrates of luciferin (Tropix or Packard Biosciencee) according to the manufacturer's instructions. An agonist or partial agonist, for purposes of the present invention, is defined as any compound that achieves 50% of the maximum activity of DHT at a concentration less than or equal to 3000 nM (3 μM) in the transactivation assay of the present invention. A partial antagonist or antagonist, for the purpose of the present invention, is defined as any compound that is capable of inhibiting 50% of the maximum activity of 1 nM DHT at a concentration less than or equal to 3000 nM in the transactivation assay of the present invention. The assays of the present invention are particularly useful in the identification of specific or selective modulators of androgen receptors or? ARMs (by sue acronyms in English). By "ARM" is meant an androgen receptor modulator that exhibits a different kind of modulation effected on a type of tissue, i.e. tumors, that contains the androgen receptor in relation to the modulation effected in other tissues, is said non-tumor tissues containing the androgen receptor In this embodiment, the agonist or antagonist activity of a potential MRSA is measured in an assay of the present invention to discover the activity of the compound in the muscle cell background. of MRSA potential can also be measured in other non-tumor cell lines such as epithelial and stromal cells, rat primary prostate, guinea pig smooth primary cells, immature rat penis smooth muscle primary cells (IP? MC) to adult (A-PSMC), rabbit primary smooth muscle cell line, PS-1 lung prostatic muscle cell line, PSMC1 smooth muscle prosthetic cell line, mouse and osteoblast cell cultures and primary cell lines from rat seminal vesicle AVC-1 and? VC-2. These cell lines are described in the following exemplary references and the references contained therein: Nemeth et al. J. Andrology 19, 718-724 (1998), Zhuang et al., J.? Teroid Biochem. Mol. Biol. 41, 693-696 (1992), Zhang et al., Prostate 30, 117-129 (1997), Ricciardelli et al., J. Endocrinol. 140, 373-383 (1994), Gonzalez-Cadavid and collaborators. Mol. Cell.

Endocrinol 90, 219-229 (1993), Sadeghi-Nej ad et al.

Int. J. Impotence Res. 10, 165-169 (1998), Gerdes et al., Endocrinology 139, 3569-3577 (1998), Sarah et al. J. Cell Physiol. 185, 416-424 (2000), Chen et al., FEB? Letters 491, 91-93 (2001) and Tajana et al. EMBO J. 3, 637-644 (1984). They can use varioe methods to identify the ARMs that have antagonist activity against tumors dependent on hormones while exhibiting no activity, or more preferably agonist activity against other non-tumor tissues containing the androgen receptor. The potential ARM agonist or antagonist activity is then discovered in hormone-dependent tumors by selection to inhibit growth in the hormone-dependent tumor cell lines. Examples of hormone-dependent tumor cell lines that can be used to screen for potential ARMs include, but are not limited to, human MDA MB453 breast tumor cell line, human ZR breast tumor cell line -75-1, Shionogi murine breast line, Dunning R-3327 rat prostate adenocarcinoma line, human prostate tumor cell line MDA Pea 2a and Pea 2b, LNCap human prostate cell line, human prostate tumor cells CWR22, human prostate tumor cell line LuCaP 35 and LuCaP 23.12, human prostate cell line LAPC-4 and LAPC-9, human prostate tumor cell line PC- 295, PC-310 human prostate tumor cell line and MG-63 human osteosarcoma cell line. These experimental and human prostate and breast cell lines are well accepted by those skilled in the art as indicative of the pharmacology of human hormone-dependent tumors, such as prostate cancer. Examples of the relationship of such models with the human disease state can be found in, but are not limited to, the following references and the references contained therein, Jacques et al., Endocrinology 140, 416-421 (1999); Yeap et al., Endocrinology 140, 3282-3291 (1999), Sharma et al., Oncogene 18, 5349-5355 (1999), Isaacs, J. T. Urol. Oncol. 2, 115-116 (1996), Bentei et al., In Vitro Cell Dev. Biol. 35, 655-662 (1999), Suzuki et al., J.? Teroid Biochem. Mol. Biol. 37, 559-567 (1990), Peehl, D.M. Urol. Oncol. 2, 100-102 (1996), Wytske et al., Urol. Oncol. 2, 122-125 (1996), Leland, C.W.K. Urol. Oncol. 2, 126-128 (1996), Buhler et al., The Proetate 43, 63-70 (2000), Navone and collaborator Clin. Cancer Res. 6, 1190-1197 (2000), Etreby et al., The Prostate 42, 99-106 (2000), Jongsma et al. Cancer Res. 60, 741-748 (2000), Jongsma et al., Amer. J. Path. 154, 543-551 (1999), Ye et al., Clin. Cancer Res. 5, 2171-2177 (1999), Navone et al., Clin. Cancer Res. 3, 2493-2500 (1997), Klein et al., Nature Medicine 3, 402-408 (1997), Chen et al., Cancer Res. 58, 2777-2783 (1998) and Craft et al. Cancer Ree. 59, 5030-5036 (1999). The "preferred ARMs, identified by means of the assays of the present invention, are those that they exhibit antagonistic activity in tumors against agonist activity in others, non-malignant tissues that contain the androgen receptor. The? ARMs identified according to these assays as androgen receptor agonists in muscle tissue are useful in inhibiting muscle wasting and cachexia sometimes observed in patients suffering from cancer or AIDS. The following non-limiting examples are provided to further illustrate the present invention.

EXAMPLES Example 1: Construction of Plasmids Androgen Receptor Plasmid pIRESneo / rAR The rat androgen receptor (accession no.

GenBank M23264) was subcloned as a NotI fragment in the NotI eitio from pIRESneo (Clontech Laboratoriee, Palo Alto, CA).

Plasmids d? Lucifersfera ARE / Reporter A series of luciferase reporter constructs containing elements of response to androgen receptors (AREs), C3, DR-1 and PB-ARE were prepared in the vector pGL3-Promoter ( Promega Corporation, Madison, Wl). pGL3 / lXDR-1 / Lucyloserase Equimolar quantities of the complementary oligonucleotide DR-1 (F) and DR-1 (R) were hybridized and then ligated into the pGL3-Xhol-digested promoter plasmid (Promega Corporation). The oligonucleotide DR-1 (F) has the sequence: 5'-TCGAGTCCTGAAGGAACGGAACAGACTGA-3 '(SEQ ID NO: 1). The oligonucleotide DR-1 (R) has the sequence: 5 '-TCGATCAGTCTGTTCCGTTCCTTCAGGAC-3' (SEQ ID NO: 2). pGL3 / 2XDR-1 Luciferase A second response element to DR-1 was inserted upstream of the DR-1 element in pGL3 / lXDR-1 / luciferaea by hybridizing equimolar amounts of the complementary oligonucleotide? XDR-I (F) and 1XDR- 1 (R) and then ligated into plasmid pGL3 / lXDR-1 / luciferase digested with Sacl / Xhol. The oligonucleotide? XDR-I (F) has the sequence: 5'-CGTCCTGAAGGAACGGAACAGACTGA-3 '(SEQ ID NO: 3). Oligonucleotide 1XDR-1 (R) has the sequence: 5'-TCGATCAGTCTGTTCCGTTTTTCCTTCAGGACGAGCT-3 '(? EC ID NO J). pGL3 / 2XC3-1 / Luci fera sa Equimolar amounts of the complementary oligonucleotides C3-1 (F) and C3-1 (R) were hybridized and then ligated together. The gel-purified dimers are then ligated into the plasmid pGL3-promoter digested with Xhol (Promega Corporation). Oligonucleotide C3-1 (F) has the sequence: 5 '-TCGAGTACATAGTACGTGATGTTCTCAA-3' (SEQ ID NO: 5). Oligonucleotide C3-1 (R) has the sequence: 5'-TCGATTGAGAACATCACGTACTATGTAC-3 '(SEQ ID NO: 6). pGL3 / 2XPB-ARE / Lucif erasa Equimolar amounts of the complementary oligonucleotides PB-ARE-2F and PB-ARE-2R were hybridized and then ligated together. The gel-purified dimers were then ligated into the plasmid pGL3-Xhol-digested promoter (Promega Corporation). The oligonucleotide sequence of PB-ARE-2F has the sequence: 5 '-TCGAGTAATAGGTTCTTGGAGTACTTTACGG-3' (? EC ID NO: 7). The oligonucleotide sequence of PB-ARE-2R has the sequence: 5'-TCGACCGTAAAGTAACTCCAAGAACCTATTAC-3 '(? EC ID NO: 8). pGL3 / HSVtk This vector was prepared by replacing the promoter ? V40 in the pGL3-promoter plasmid (Promega Corporation) with HSVtk (herpes simplex virus thymidine kinase) promoter from pRL-TK (Promega Corporation). Both promoters are contained within the Bgl II / Hind III fragments and are they easily exchanged by ligating the H? Vtk fragment of pRL-TK in the pGL3-promoter vector digested with Bgl II / Hind II. pGL3 / HSVtk / lXDR-l / Lucif erasa, pGL3 / HSVtk / 2XDR-l / Lucifersfera, pGL3 / HSVtk / 2XC3-l / Luciferase and pGL3 / HSVtk / 2XPB-ARE / Lucifsrase Those constructs containing the HSVtk promoter in place of the? V40 promoter were prepared by replacing the SV40 promoter in the parent progenitor plasmid, respectively, by the HSVtk promoter of pRL-TK as described for pGL3 / H? Vtk.

Example 2: Intensifier / Reporter Construction Selection Two sets of luciferase reporter vectors were constructed and tested in the mouse skeletal muscle cell line C2C12. The first variant of the luciferase reporter construct led to a strong promoter, in this specific example the SV40 promoter. The second variant of the reporter construct of luciferaea carried a basic promoter, in this specific example the HSVtk promoter. To select the most effective androgen response element (ARE) to boost the expression of the luciferase gene, both SV40 and HSVtk promoters were coupled to four different AREs, C3, DR-1 (IX and 2X) and PB-ARE. He C3 enhancer is a strong, androgen-dependent regulatory element with a cross-activity with the glucocorticoid receptor (GR). Both DR-1 and PB-ARE are considered elements of response to the specific androgen. A transient transactivation experiment was performed, in which CMVrAR was co-transfected with the aforementioned enhancer / promoter / reporter construct (receptor to intensifier / promoter / reporter 10: 1) in the C2C12 cells using the LipofectAMINE PlusMR reagent (GibcoBRL ) according to the manufacturer's instructions, was used to compare the activities of the intensifier / promoter / reporter constructions. Specifically, 10,000 cells / well in growth media were plated (Dulbecco's Modified Eagle Medium (DMEM) with high glucose content supplemented with 10% FB, sodium pyruvate IX and 0.5X antibiotic-antimycotic agent. (all from Bibco BRL). The next day, the media was removed and replaced with optiMEM media (Gibco BRL). The transfection mixture was prepared so that 10 μl was added to each well resulting in 0.05 μg / well of the receptor, 0.005 μg / well of enhancer / promoter / reporter construct and 0.385 μl / well of lipofectamine. After three hours of incubation, the transfection mixture was removed and replaced with media from growth which had been done using FB charcoal / dextran? (Hyclone, Logan UT). The detection method used was the Steady-GloMR luciferase assay system (Promega Corporation), with the count performed in a Packard TopCount (Packard Instrument Co. Downers Grove, IL). The results showed that C3, DR-1 and the constructs of PB-ARE / H? Vtk-luciferase reporter had a lower background signal compared to C3, DR-1 and PB-ARE / SV40-luciferase reporters. In addition, 1 ßA of testosterone gave an increase 3.5 times over the background with C3 / HSVtk / luciferase and an increase 2.0, 2.0 and 6.5 times with 2XPB-ARE-H? Vtk / luciferase, IXDR-1 / HSVtk / luciferase and 2XDR- 1 / HSVtk / luciferase, respectively. Constructs with the largest stimulation times range, C3 and DR-1 / HSVtk / luciferase, both showed a minimum 100-fold selectivity of testosterone over dexamethasone when tested in dose response experiments. Thus, for reasons of interval of stimulation and selectivity times, the 2XDR-1 construction was selected. Although in the system of transiently transfected receptors, the HSVtk promoter appeared to be preferable due to the lower background, when tested on the Stable 1 cell line, the signal decreased greatly. Therefore, the construction used in the production of cell line? 2 was pGL3 / 2XDR-l / luciferase which carries the strongest SV40 promoter.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, which is clear from the preeente decription of the invention.

Claims (20)

1. Claims Having described the invention as above, the content of the following claims is claimed as property: 1. A line of muscle? Table cells, characterized in that it comprises muscle cells and a mammalian androgen receptor stably introduced into the lae. muscle cell.
2. 2. The muscle Stable cell line according to claim 1, characterized in that the muscle cells comprise the mouse skeletal muscle cells C2C12.
3. 3. The muscle cell line according to claim 1, characterized in that the mammalian androgen receptor comprises a rat androgen receptor.
4. 4. The muscle Stable cell line according to claim 1, characterized in that it further comprises a stably transfected reporter / promoter / promoter construct.
5. 5. The muscle Stable cell line according to claim 4, characterized in that the enhancer comprises an androgen response element.
6. 6. The muscle Stable cell line according to claim 5, characterized in that the enhancer comprises C3-1, DR-1 or PB-ARE.
7. 7. The stable cell line of muscle according to claim 4, characterized in that the promoter comprises? V40.
8. 8. The Stable cell line of muscle according to claim 4, characterized in that the promoter comprises H? Vtk.
9. 9. The muscle Stable cell line according to claim 4, characterized in that the reporter gene comprises luciferase.
10. 10. The muscle Stable cell line according to claim 4, characterized in that the enhancer / promoter / reporter construct comprises PGL3 / 2XDR-1 / luciferase.
11. 11. A muscle cell-table line, characterized in that it comprises the deposit number in ATCC PTA-3458.
12. 12. A muscle cell-table line, characterized in that it comprises the deposit number in ATCC PTA-3459.
13. 13. A functional transactivation assay to assess the efficacy of a compound as an agonist or partial agonist of androgen receptors, characterized because it comprises: (a) Transiently transfecting the cell line according to claim 1 with a plasmid containing an androgen response element, a promoter and a reporter gene; (b) contacting the transfected cell line transiently with a compound; and (c) detecting the expression of the reporter gene in the line of transiently transfected cells, wherein an increase in the expression of the reporter gene in the transiently transfected cell line in the presence of the compound is indicative that the compound is an agonist or partial agonist of androgen receptors.
14. 14. An androgen receptor modulator, characterized in that it comprises a compound identified with the method according to claim 13.
15. 15. A functional transactivation assay to assess the efficacy of a compound as a partial antagonist or antagonist of androgen receptors, characterized in that it comprises: (a) Transiently transfecting the cell line according to claim 1 with a plamidid containing an androgen response element, a promoter and a reporter gene; (b) contacting the cell line transiently transiently with a compound and dihydrotestoeterone; and (c) detecting the expression of the reporter gene in the line of transiently transfected cells, wherein a decrease in the expression of the reporter gene in the line of cells transiently transfected in the presence of the compound and dihydrotestosterone compared to the cells exposed only to dihydrotestosterone ee indicative that the compound is a partial antagonist or antagonist of androgen receptors.
16. 16. An androgen receptor modulator, characterized in that it comprises a compound identified with the method according to claim 15.
17. 17. A functional transactivation assay to assess the efficacy of a compound as an agonist or partial agonist of androgen receptors, characterized in that it comprises: (a) contacting the cell line according to claim 4 with a compound; and (b) detecting the expression of the reporter gene in the cell line, wherein an increase in reporter gene expression in the cell line in the presence of the compound is indicative that the compound is an agonist or partial receptor agonist. of androgen.
18. 18. An androgen receptor modulator, characterized in that it comprises a composition identified with the method according to claim 17,
19. 19. A functional transactivation assay for assessing the efficacy of a compound as a partial antagonist or antagonist of androgen receptors, characterized in that it comprises: (a) putting into contacting the cell line according to claim 4 with a compound and dihydrotestosterone; and (b) detecting the expression of the reporter gene in the cell line, wherein a decrease in reporter gene expression in the presence of the compound and dihydrotestosterone compared to cells exposed only to dihydrotestosterone ee indicative that the compound is a antagonist or partial antagonist of androgen receptors.
20. 20. An androgen receptor modulator, characterized in that it comprises a compound identified with the method according to claim 19.