Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
  • A61P5/24—Drugs for disorders of the endocrine system of the sex hormones
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
  • A61P5/24—Drugs for disorders of the endocrine system of the sex hormones
  • A61P5/30—Oestrogens
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5082—Supracellular entities, e.g. tissue, organisms
  • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
  • G01N33/743—Steroid hormones
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
  • G01N2333/723—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor

Definitions

• the present invention relates to a method for screening for progesterone receptor ligands having a selectivity for either the progesterone receptor isoform A or the progesterone receptor isoform B.
• the present invention further relates to methods for screening for tissue-selective, in particular breast/uterus-selective, progesterone receptor ligands.
• the present invention further relates to an assay kit for screening for progesterone receptor isoform specific ligands.
• the present invention relates to SK-N-MC cell lines stably transfected with a plasmid expressing the progesterone receptor isoform A or the progesterone receptor isoform B and a progesterone-inducible luciferase reporter gene.
• the present invention also relates to the progesterone receptor isoform specific ligands and to the tissue-selective progesterone receptor ligands identified through the methods of the present invention.
• the present invention also relates to medical uses of the progesterone receptor isoform specific and the tissue-selective progesterone receptor ligands identified through the methods of the present invention.
• Progesterone is a unique reproductive hormone, and it plays a decisive role for tissues of female reproduction. Its principal target organs are uterus, breast, ovary and the hypothalamus-pituitary axis.
• steroids a group of substances
• progestins In addition to the primary use as birth control for women (e.g., oral contraception (OC)), progestins, combined with estrogen, are widely used in hormone replacement therapy (HRT).
• HRT hormone replacement therapy
• Progestins are also used to treat several gynecological disorders, e.g., dysmenorrhea, endometriosis, and dysfunctional uterine bleeding caused by hormonal deficiency or imbalance. Due to undesirable side effects or cross-reactivities with other receptors, the development of new generations of progestins to improve their selectivity profile has been a great challenge. Additionally, the exploration of therapeutic applications such as oncology demands progestins with new activity profiles.
• PR is expressed in two isoforms in certain organisms, including humans (H. Gronemeijer et al., J. Steroid Biochem. 1991, 40, 271- 278; T.T. Ilenchuck et al., Endocrinology 1987, 120, 1449-1456).
• hPR-A and hPR-B have been identified (P. Estes et al., Biochemistry 1987, 26, 6250-6262; L. Klein-Hitpass et al., Nucleic Acid Res. 1991, 19, 1227-1234; K. Christensen et al, Mol. Endocrinol.
• Human PR-A is a truncated form of hPR-B and lacks 164 amino acids at the N-terminus (K.B. Horwitz et al., Endocrinology 1983, 113, 2195-2201). Both isoforms are identical in the DNA-binding and ligand-binding domain and induce progestin-mediated gene transcription. However, they have a different transactivation behavior as outlined below.
• PR-isoforms are expressed in all progesterone target organs tested so far (e.g. breast, uterus).
• PR-A and PR-B function in a tissue- specific manner to mediate responses to progesterone. Isoform-specific knock-out mice show different functions of PR-A and PR-B in the same target organ.
• PR-B seems to be the most responsible receptor for mammary gland proliferation and differentiation, whereas the antiproliferative action of progestins on the uterine epithelium and on ovulation is most likely mediated by PR-A (B. Mulac-Jericevic, Science 2000, 289, 1751-1754; Orla Conneely, Endocrine Society Meeting, Toronto, June 2000).
• the prior art fails to disclose any method used specifically to identify such ligands.
• the prior art furthermore fails to provide a method for identifying tissue-selective PR ligands. Accordingly, the prior art does not provide PR isoform-specific and/or tissue-selective PR ligands.
• a hormone-dependent transcription modulation system was established on the basis of a two-step transfection procedure of the human PR-A and PR-B isoforms and a progesterone receptor-responsive reporter (R. Dijkema et al., J. Steroid. Biochem. Molec.
• transactivation data were compared to relative binding affinities and biopotency estimations with McPhail staging of rabbit endometrium, ovulation and pregnancy interruption in rats (W.G.E.J. Schoonen et al, J. Steroid Biochem. Molec. Biol. 1998, 64, 157-170). It was found that due to the good resemblance of these in vitro and in vivo tests, direct pre-screening of agonists, antagonists and partial antagonists of PR in general is possible. Thus, this transactivation assay can replace binding assays and is a valuable pre-screening tool for in vivo studies involving a particular class of chemical compounds.
• the present invention is based on the new theory that isoform-specific ligands of PR activity may allow tissue-selective modulation of progestin activity in hormonal therapy. Therefore, one approach towards mammary tissue protective PR ligands which, however, maintain their desired effects on other target organs, such as the uterus, could be to identify pure agonists that selectively stimulate PR-A transactivation or pure antagonists that selectively inhibit PR-B transactivation or partial agonists with stronger agonistic activity on PR-A and stronger antagonistic activity on PR-B.
• the present invention is not intended to be limited to this approach. Furthermore, this approach should be applicable to other tissue systems as well. Although many progestins are on the market, no progestin with dissociated antiproliferative potential, preferably on breast tissue, is presently available. Objects of the Invention
• progesterone receptor (PR) ligands with target tissue specificity, i.e. to achieve, for example, dissociation of progestational activity in the reproductive tract from that in the breast and thereby reduce undesired effects of COC's and HRT, such as an increased risk of breast cancer incidence and thus protect both the mammary gland and the uterus.
• PR progesterone receptor
• the method for identifying PR isoform specific ligands should be suitable for identifying ligands that are pure agonists that selectively stimulate PR-A transactivation or pure antagonists that selectively inhibit PR-B transactivation or partial agonists with stronger agonistic activity on PR-A and stronger antagonistic activity on PR-B.
• the desired method for screening for tissue-selective PR ligands should be suitable for screening for PR ligands that exhibit an inhibiting or at least a non- stimulating effect on a first selected target tissue, preferably breast tissue, and a protective effect on a second selected target tissue, preferably uterine tissue.
• the screening methods should be relatively simple, highly selective and efficient, thus possibly allowing high-throughput screening. Furthermore, the screening methods should be applicable for PR pure agonists, pure antagonists and partial agonists/antagonists. It is a further object of the present invention to provide an assay kit suitable for performing the method for screening for isoform-specific PR ligands in vitro.
• PR isoform A or B specific ligands and/or tissue-selective, preferably breast/uterus selective, PR ligands should be suitable for use as medicaments, preferably in fertility control and HRT.
• the present invention provides a method for screening for PR isoform A or B specific ligands as well as a first and a second method for screening for tissue-selective PR ligands.
• Both the method for screening for PR isoform-specific ligands as well as the first method for screening for tissue-selective PR ligands according to the present invention basically comprise selecting PR ligands having a selectivity for either the PR-A or the PR- B isoform.
• the first method for screening for tissue-selective PR ligands additionally comprises subjecting the PR isoform-specific ligands identified by the PR isoform specific in vitro test according to the present invention to in vivo tests in a first and second target tissue and selecting those ligands having the desired activity with respect to said first and said second target tissue.
• the second method for screening for tissue-selective PR ligands comprises subjecting PR ligands to in vivo tests in the desired target tissues and selecting those ligands that selectively modulate PR mediated effects in one of the desired target tissues with respect to the other desired target tissue.
• the first method for screening for tissue-selective PR ligands according to the present invention is based on the new and surprising concept that certain steroidal or non- steroidal PR ligands, which are selective PR-A or PR-B-specific pure agonists, pure antagonists or partial agonists/antagonists as evidenced by the selectivity assay, preferably a transactivation assay according to the present invention, induce tissue-selective progestational effects.
• the second method for screening for tissue-selective PR ligands according to the present invention is based on the different progesterone receptor mediated effects induced by tissue-selective PR ligands in different target organs as identified through in vivo tests.
• the present invention also provides PR isoform A or B specific ligands and/or tissue- selective PR ligands that are identified through the methods according to the present invention.
• the PR isoform A or B specific and/or tissue-selective PR ligands according to the invention are suitable for use as medicaments, e.g. in fertility control or HRT.
• the PR isoform specific ligands identified by the method according to the present invention selectively block or at least do not affect PR-B transactivation, but are still strong agonists for PR-A.
• tissue-selective PR ligands identified by the first and/or second method for screening for tissue-selective PR ligands according to the present invention preferably inhibit differentiation and proliferation of the mammary epithelium, whereas the progestational activity in the reproductive tract is maintained or even enhanced.
• the tissue-selective PR ligands according to the present invention thus exert a protective effect both on the mammary gland as well as the uterus.
• the present invention provides an assay kit suitable for performing the method according to the present invention, i.e. for screening for PR-A or PR-B isoform- specific ligands.
• the present invention provides SK-N-MC cell lines stably transfected with either a plasmid expressing PR-A or PR-B. These cell lines are especially suitable for use in the screening method for PR isoform-specific ligands as well as the first method for screening for tissue-selective PR ligands according to the present invention and as part of the assay kit according to the present invention.
• the present invention provides a method for selectively inhibiting or stimulating PR isoform A or PR isoform B mediated effects.
• the present invention also provides methods for selectively modulating PR-mediated conditions in a selected tissue, preferably for not influencing or inhibiting PR mediated, preferably PR-B mediated, conditions in a first selected tissue, preferably breast tissue, and, at the same time, enhancing PR mediated, preferably PR-A mediated, effects in a second selected tissue, preferably uterine tissue.
• the present invention pertains to a method for screening for progesterone receptor (PR) isoform A or B specific ligands. Basically, the method comprises the steps of:
• the present invention pertains to a first method for identifying tissue-selective progesterone receptor ligands.
• this method comprises the selection of isoform specific progesterone receptor ligands, i.e. ligands having a selectivity for either the progesterone receptor isoform A (PR-A) or the progesterone receptor isoform B (PR-B) according to the first aspect of the present invention as explained above.
• the first method for identifying tissue- selective PR ligands comprises subjecting the selected PR isoform-specific ligands to in vivo tests in a first and second tissue and selecting those ligands having the desired acitivity in said first and second tissue.
• the fist method for identifying tissue- selective PR ligands comprises at least the following steps:
• step (f) selecting a ligand having a selectivity for the progesterone receptor isoform A or the progesterone receptor isoform B; (g) subjecting the ligand selected in step (f) to in vivo tests in a first and a second target tissue; and (h) selecting the ligand having the desired activity with respect to said first and said second target tissue.
• the "selectivity" for one PR isoform, PR-A or PR-B as determined in step (f) of both methods as explained above, i.e. the method for screening for PR isoform-specific ligands and the first method for screening for tissue-selective PR ligands according to the present invention is defined as a difference in transcription efficacy induced by a certain test ligand in said first and said second cells.
• this difference must be above or equal to 10 %, more preferably above or equal 15 % and most preferably even higher, such as above or equal 20 %.
• Transcription efficacy in the context of the present invention is defined as the response achieved with a defined concentration of test ligand relative to a standard in either PR-A or PR-B transfected cells.
• Another potential parameter for evaluating the activity and thus also the selectivity of a test ligand with respect to either PR-A or PR-B is "potency", i.e. the EC 50 (for agonism) or IC 50 (for antagonism) value, determined in vitro in either PR-A or PR-B transfected cells.
• "selectivity" for one PR isoform, PR-A or PR-B as determined in step (f) of both above methods may also - additionally or alternatively - be defined as a difference in potency achieved by a certain test ligand in said first cells with respect to said second cells.
• this potency difference should be in the range of or above a factor of 10, more preferably even larger.
• the "selectivity" defined in step (f) of the method for screening for PR isoform- specific ligands as well as the first method for screening for tissue-selective PR ligands according to the present invention is defined such that the difference in transcription efficacy determined for a potential PR isoform-specific and/or tissue-selective PR ligand in said first and said second cells should preferably be above or equal to 10 %, more preferably above or equal to 15 % and most preferably above or equal to 20 % and/or the potency achieved with said ligand in said first cells should preferably differ by a factor of at least 10, more preferably by an even larger factor, from the potency achieved with said ligand in said second cells.
• both efficacy and potency may be used, either alternatively or in combination, as criterion for determining PR-A PR-B selectivity according to step (f) in both above-described screening methods.
• the present invention pertains to a second method for identifying tissue-selective PR ligands.
• This second method is based on in vivo tests in different selected target tissues and provides PR ligands that selectively modulate PR mediated effects in these target tissues.
• the second method for screening for tissue-selective PR ligands according to the present invention comprises the steps of
• ligand in the present invention is meant to encompass any compound binding to a receptor (in the present invention, preferably the progesterone receptor) and inducing either activation or inhibition or any other potential form of interaction with this receptor.
• ligand is also used for compounds which are potential ligands of a receptor, but whose binding affinity and/or activity at the receptor is not yet known, but has to be tested with methods according to the present invention.
• Plasmid in the present invention is intended to encompass “plasmid vectors”.
• one achievement of the present invention is that for the first time a correlation of PR-A/PR-B isoform specificity of PR ligands with tissue-selectivity of PR ligands, in particular breast/uterus selectivity, is possible. Furthermore, it has become possible by the methods of the present invention to identify tissue-selective, in particular breast/uterus- selective, PR ligands. It is particularly advantageous to design tissue-selective progestins for use in fertility control and hormone (replacement) therapy in order to avoid disadvantages of known therapies, for example an increased incidence of breast cancer.
• Isoform-specific and/or tissue-selective PR ligands identified through the methods according to the present invention only activate the progesterone receptor at a specific target tissue, but not at any other, undesired tissue, thus rendering these treatments well tolerable and less prone to serious side effects or even the induction of further health problems.
• these progestins may be administered in a much smaller dose due to their target specificity than the progestins currently used in (combined) oral contraceptives and HRT.
• the present invention allows for tailored modulation of PR mediated conditions by the use of PR isoform-specific and/or tissue-selective PR ligands and may thus open up a path for further beneficial uses of progestins, in particular progesterone receptor pure agonists, pure antagonists and partial agonists/antagonists, in the treatment of PR related conditions.
• progestins in particular progesterone receptor pure agonists, pure antagonists and partial agonists/antagonists
• Isoform-specific and/or tissue-specific PR ligands identified by the method according to the present invention will even contribute to the further elucidation of biological pathways involved with certain pathological conditions related with intracellular receptors, e.g. progesterone receptors in general and PR isoforms in particular.
• the methods according to the present invention for screening for PR isoform-specific and first method according to the present invention for screening for tissue-selective PR ligands may be applied mutatis mutandis to other receptor/ligand systems involving different forms of a receptor and/or different receptor mediated effects in selected target organs (e.g. the androgen receptor system).
• both methods according to the present invention for screening for tissue-selective PR ligands may also be applied mutatis mutandis to other desired tissues or target organs.
• steps (a) to (f) are identical in their broadest as well as their preferred meanings in the method for screening for PR isoform-specific ligands and in the first method for screening for tissue-selective PR ligands, these steps (a) to (f) will be described in detail only with reference to the method for screening for PR isoform-specific ligands.
• steps (g) and (h) comprised in the first method for screening for tissue-selective PR ligands are explained in detail afterwards.
• the method for screening for PR isoform specific ligands according to the first aspect of the present invention is preferably a transactivation assay, comprising a multiple stable transfection, i.e. steps (a) to (c) as defined above.
• the cells used in this screening method according to the first aspect of the present invention as well as comprised in the assay kit according to the present invention are SK-N-MC cells (human neuroblastoma cells).
• This cell line is especially well suited for the purposes of the present invention, as it comprises monospecific cells, i.e. cells expressing only PR, but no other hormonal receptors, e.g. androgen, glucocorticoid or other steroid receptors.
• the PR isoform A is equally active as the PR isoform B.
• the PR isoform B is equally active as the PR isoform B.
• other cell lines are equally suitable, for example, the Chinese Hamster Ovary (CHO) cells or the CV-1 cells.
• CHO Chinese Hamster Ovary
• the method for screening for PR isoform A or B specific ligands according to the present invention cells as described above are stably transfected with a plasmid expressing PR-A or PR-B, respectively (a detailed description of an exemplary method for obtaining the stably transfected cells according to the present invention is given in Example 5).
• first cells are obtained specifically expressing PR-A (corresponding to step (a)) and “second cells” specifically expressing PR-B (corresponding to step (b)).
• the most preferred cells used in the screening methods according to the first and second aspect of the present invention and the assay kit according to the present invention are SK-N-MC cells.
• transactivation assay it is most preferred that cells are stably transfected with a plasmid expressing human PR-A or PR-B (hPR-A or hPR-B).
• the plasmids expressing PR-A or PR-B, respectively, are preferably plasmids pRSVhPR- neo (the PR-B plasmid; for the cells specifically expressing PR-B) as well as phPR-2 (the PR-B plasmid; for the cells specifically expressing PR-B) as well as phPR-2 (the PR-B plasmid; for the cells specifically expressing PR-B) as well as phPR-2 (the
• PR-A plasmid PR-A plasmid
• pRSV-neo for the cells specifically expressing PR-A
• Example 5 gives a detailed description of how and/or where to obtain these plasmids.
• PR-A or PR-B expressed from the long terminal repeat (LTR) of the rous sarcoma virus (RSV).
• LTR long terminal repeat
• RSV rous sarcoma virus
• the neomycin resistance gene expressed from the SN40 early promoter, allows for neomycin selection of stable transfectants.
• other constructs are also envisaged, e.g., comprising any other antibiotic resistance gene suitable for the purposes of the present invention.
• the first and second cells according to the present invention and suitable for use in the screening methods of the first and second aspect of the present invention and as part of the assay kit according to the present invention are further stably transfected in step (c) with a plasmid comprising a reporter gene linked to a hormonally responsive promoter.
• this plasmid is a pMTV-LUC plasmid, wherein the hormonally responsive promoter is the mouse mammary tumor virus (MTV) promoter, which is linked to the luciferase (LUC) reporter gene (for a detailed description, see again Example 5).
• MTV mouse mammary tumor virus
• LOC luciferase
• the cells stably transfected with the PR-A or PR-B plasmids and the promoter/reporter gene are additionally stably transfected as described e.g. in Example 5 with another antibiotic resistance gene, preferably a puromycin resistance gene (e.g., pS V2pac).
• another antibiotic resistance gene preferably a puromycin resistance gene (e.g., pS V2pac).
• pS V2pac puromycin resistance gene
• antibiotic resistant SK-N-MC clones stably expressing PR-A and MTV-LUC or PR-B and MTV-LUC were selected for transactivation studies, i.e. for the method for screening for PR isoform-specific ligands as well as for the first method for screening for tissue-selective PR ligands according to the present invention.
• step (d) of the method for screening for PR-A or PR-B isoform-selective ligands said first and said second stably transfected cells (i.e. PR-A or PR-B expressing cells, respectively) are contacted with a ligand to be tested, i.e. a compound being a potential PR isoform-specific ligand.
• Ligands to be tested can be of steroidal or non-steroidal nature.
• the method can be performed with compounds having already been tested on PR affinity/activity in general and found to bind to and activate or inhibit PR, but also with compounds of which their PR ligand potential is unknown. In case the compound to be tested has already qualified as PR ligand, it can be a pure agonist, pure antagonist or partial agonist/antagonist of PR.
• the transcription efficacy and/or potency of the potential PR-A or PR-B selective ligand in said first and said second cells is determined in step (e) of the screening method according to the present invention.
• the efficacy and/or the potency of said potential PR isoform-specific ligand as defined above can be determined via the level of expressed reporter gene product (cf. Example 1).
• the reporter gene is a LUC reporter gene as described above as a preferred embodiment of the present invention
• the level of LUC reporter gene product is determined in cell lysates and is measured as RLU (relative light units).
• any other method for determining the transcription efficacy and/or potency in said first and second cells is equally applicable in the screening methods as well as in the assay kit according to the present invention.
• the respective cells are cultured in the presence of increasing concentrations of the ligand to be tested, preferably 0.01 nmol/1 to 1 ⁇ mol/1.
• the same type of cells is treated with a standard progestin, preferably the synthetic progestin R5020 (promegestone), preferably with concentrations of 0.01 nmol/1 to 1 ⁇ mol/1.
• the same type of cells are preferably cultured in 1 % ethanol.
• all obtained data points are calculated relative to a standard progestin, preferably relative to the induction of LUC activity obtained by treating the cells with 10 "7 mol/1 of R5020.
• agonistic potency [nM] the EC 50 value (i.e., the amount [nM] of ligand which provides for half of the maximum agonistic effect of this ligand) is determined graphically (see, e.g., Example
• the respective cells are treated with a standard progestin, preferably with 0.1 nmol/1 R5020 and additionally with increasing amounts of the ligand to be tested, preferably 0.01 nmol/1 to 1 ⁇ mol/1.
• a standard antiprogestin preferably the known antiprogestin mifepristone, preferably in concentrations of 1 pmol/1 to 100 nmol/1.
• IC 50 value i.e., the amount [nM] of ligand which provides for half of the maximum antagonistic effect of this ligand
• ligands having a selectivity for either PR-A or PR-B as defined above have to be selected.
• ligands having purely PR-A agonistic or purely PR-B antagonistic activity or ligands having stronger partial agonistic activity on PR-A and stronger partial antagonistic activity on PR-B are selected.
• exemplary ligands to be selected by these methods of the present invention are ligands having purely PR-A antagonistic and purely PR-B agonistic activity or ligands having partial PR-A antagonistic/PR-B agonistic activity.
• the difference in transcription efficacy induced by a ligand tested in both groups of PR isoform-specific cells, i.e. in said first and said second cells has to be preferably above or equal to 10 %, more preferably above or equal to 15 % and most preferably even higher, such as above or equal to 20 % and/or the potency achieved with such ligand in said first cells should preferably differ by a factor of at least 10 from the potency achieved with such ligand in said second cells.
• One preferred embodiment of the present invention is e.g. the assay according to Example 1, wherein a detailed description of an exemplary method for screening for PR isoform- specific ligands according to the invention is given.
• Example 1 some exemplary results obtained for some PR-A or PR-B isoform-specific ligands identified through the method according to the invention as defined above are summarized. Additionally, in Example 5 a detailed description of the stable transfections is provided.
• the first method for screening for tissue-selective PR ligands according to the second aspect of the present invention comprises identical steps (a) to (f) as the method for screening for PR isoform-specific ligands according to the present invention as described above.
• steps (a) to (f) of the first method for screening for tissue-selective PR ligands are disclosed above with respect to the first aspect of the present invention, i.e. the method for screening for PR isoform specific ligands.
• the first method for screening for tissue-selective PR ligands according to the second aspect of the present invention additionally comprises steps (g) and (h), which will be explained below.
• step (f) of the first method for screening for tissue-selective PR ligands this ligand is subjected in step (g) of this method to in vivo tests in a first and a second selected tissue, preferably in breast and uterine tissue.
• in vivo tests are preferably tests to determine PR mediated effects in said first and said second selected tissue.
• possible in vivo tests may be those tests described in Examples 2, 3 and 4 of the present invention, i.e.
• any other PR mediated effect-determining in vivo test in the selected first and second tissue is equally suitable for the purposes of the present invention and may be chosen according to the nature of the selected tissues and the desired tissue selectivity profile.
• a selection step is performed in the first method for screening for tissue-selective PR ligands according to the second aspect of the present invention, wherein the ligand having the desired activity with respect to the first and second target tissue is selected.
• “desired activity” means that a ligand is selected that does not influence or even inhibit PR-mediated effects in the first target tissue, preferably breast tissue, and enhances or maintains at the same time PR-mediated effects in the second target tissue, preferably uterine tissue.
• a ligand is selected that does not stimulate or that even inhibits differentiation/proliferation in the mammary tissue, but maintains or enhances its beneficial (protective) effects on the reproductive tract, i.e. exerts antiproliferative activity on the uterus.
• Such ligands seem especially suitable for use in fertility control, e.g. as oral contraceptives, and hormone replacement therapy (HRT).
• HRT hormone replacement therapy
• the suitability of the present invention is not restricted to the areas of fertility control or HRT, but that the invention can beneficially be applied in other areas involving receptor-mediated conditions as well.
• the subject matter of the invention is not limited to PR, but can also be used in other receptor/ligand systems.
• the present invention may also be extended to more than two target tissues.
• a PR ligand in step (a) of this method has to be subjected to at least one in vivo test, preferably at least one in vivo test that is directed to determining PR mediated effects, in a first selected tissue.
• the first selected tissue is breast or uterine tissue, but can also be any other tissue of interest wherein PR mediated effects are observed.
• Suitable in vivo tests for the purposes of this third aspect of the present invention are e.g.
• the in vivo tests described in Examples 2, 3 and 4 such as a rodent bioassay on proliferation/differentiation of mammary tissue, a pregnancy maintenance test or an endometrium proliferation/differentiation test in rodents, and an ovulation inhibition test or a superovulation test in rodents.
• the second method for screening for tissue-selective PR ligands according to the present invention may analogously be performed for any other receptor ligands and may comprise in vivo tests in any other desired target tissue. It is within the purview of an average skilled person to perform the necessary and appropriate variations to transfer the method as described herein to other receptor systems and/or target tissues.
• step (b) of the second method for screening for tissue-selective PR ligands the PR ligands already tested in vivo in a first selected tissue as described above are subjected to at least another in vivo test in a second selected tissue.
• the in vivo test should be a test suitable for determining PR mediated effects. Such suitable tests are described above with respect to step (a) for the first selected tissue.
• the second selected tissue according to step (b) is again preferably breast or uterine tissue, whichever of these has not been tested in step (a).
• step (c) of the second method for screening for tissue-selective PR ligands the PR mediated effects induced by the tested ligands in said first and said second tissue are compared and those PR ligands are elected that selectively modulate PR mediated effects in said first selected tissue with respect to said second selected tissue.
• “Selective modulation" of PR mediated effects for the purposes of this aspect of the present invention means that the selected ligand achieves in the first tissue one or more effect(s) which is/are of a different kind (i.e., agonistic, antagonistic or, in other words, inhibiting, stimulating or not affecting) and/or of a different intensity (i.e., weaker or stronger, or even pertaining longer or shorter) compared to the effect(s) induced by said ligand in the second desired tissue.
• a different kind i.e., agonistic, antagonistic or, in other words, inhibiting, stimulating or not affecting
• a different intensity i.e., weaker or stronger, or even pertaining longer or shorter
• the selected ligand inhibits or does not influence PR mediated effects in said first tissue, preferably breast tissue, and enhances or maintains PR mediated effects in said second tissue, preferably uterine tissue.
• the selected ligand inhibits or at least does not influence proliferation/differentiation of the mammary epithelium while it maintains beneficial (i.e., protective) PR mediated effects in the reproductive tract (cf. Examples 2, 3 and 4), e.g. exerts an antiproliferative effect on uterine tissue.
• the type of the effect induced by said ligand i.e., whether the ligand inhibits, does not influence, enhances or maintains the PR mediated effect
• the intensity of the induced effect are measured, preferably relative to the effect induced by a known "standard" PR ligand, such as the standard progestin R5020 (promegestone).
• a known "standard" PR ligand such as the standard progestin R5020 (promegestone).
• those PR test ligands are selected that exhibit the desired selectivity for one target tissue with respect to the other target tissue. It is certainly within the skilled person's knowledge to select and perform suitable in vivo tests as defined above in certain desired target tissues other than the preferred tissues as defined above and to determine the effect induced by a certain test ligand in relation to a suitable standard ligand.
• all methods described above i.e., methods for screening for PR isoform-specific and/or tissue-selective PR ligands
• methods for screening for PR isoform-specific and/or tissue-selective PR ligands are applicable for automated high-throughput screening of a large number of potentially PR isoform-specific and/or tissue-selective PR ligands. It is within the skilled person's knowledge to design automated processes based on the methods according to the present invention suitable for high-throughput screening.
• the isoform-specific PR ligands identified through the method according to the first aspect of the present invention can be pure agonists, pure antagonists or partial agonists/antagonists for PR-A or PR-B.
• the isoform-specific PR ligands identified by the method according to the present invention are selective agonists for PR-A and selective antagonists for PR-B or partial agonists with a stronger agonistic activity on PR-A and a stronger antagonistic activity on PR-B.
• Other exemplary ligands according to the invention are selective PR-A antagonists and selective PR-B agonists or partial PR-A antagonists/PR-B agonists.
• tissue-selective PR ligands identified through the first and second method for screening for tissue-selective PR ligands according to the second and third aspect of the present invention are preferably selective for breast tissue or uterine tissue. More preferably, they have no impact on PR mediated effects or even inhibit PR mediated effects in breast tissue (such as proliferation/differentiation of the mammary gland), but enhance or maintain PR mediated effects in uterine tissue, i.e. exert a protective, such as an antiproliferative, effect in uterine tissue.
• tissue-selective PR ligands and or PR-isoform specific ligands can be obtained having the desired, beneficial effects in the uterus and/or the ovary without inducing unwanted or even dangerous side-effects in the breast, such as e.g. the induction of proliferation/differentiation of the mammary tissue (which is evident by increased formation of terminal end buds in the mammary glands), which may induce breast cancer.
• the benefit of the present invention is not limited to breast/uterine tissue and/or to the progesterone receptor, but can be applied to other target tissues and/or receptor/ligand systems as well.
• the tissue-selective, preferably breast/uterus-selective PR ligands identified by the second method for screening for tissue selective PR ligands according to the third aspect of the present invention are PR-A or PR-B selective ligands and more preferably, they are pure PR-A agonists, pure PR-B antagonists or partial agonists/antagonists with stronger agonistic effects on PR-A and stronger antagonistic effects on PR-B.
• the selected tissue-selective ligands preferably activate or maintain PR-A transactivation in a first tissue and inhibit or do not influence PR-B transactivation in a second tissue.
• these ligands are suitable for use as medicaments, in particular for not affecting or for inhibiting PR mediated effects in a first selected tissue and, at the same time, enhancing or maintaining PR mediated effects in a second target tissue, wherein the first tissue is preferably breast tissue and the second tissue is preferably uterine or ovarian tissue.
• These medicaments may preferably be used in fertility control (e.g. oral contraception) or HRT.
• the PR ligand identified may be used in inhibiting or not influencing proliferation/differentiation in breast tissue, and in enhancing or maintaining protective, such as antiproliferative, effects in uterine tissue while also maintaining the potentially desired effects on ovulation.
• the PR ligands identified through the methods according to the first, second and third aspect of the present invention may also be used for the manufacture of a medicament for not affecting or for inhibiting PR mediated effects (e.g. proliferation/differentiation) in a first target tissue, preferably breast tissue, and enhancing or maintaining beneficial (e.g. antiproliferative) PR mediated effects in a second target tissue, preferably uterine or ovarian tissue (e.g., for use in fertility control or HRT).
• beneficial e.g. antiproliferative
• Another aspect of the present invention is a method for selectively inhibiting or selectively stimulating PR-A or PR-B in a patient, comprising the step of administering a therapeutically effective amount of a PR isoform A or B specific ligand identified through the method of the present invention to a patient in need thereof.
• Another related aspect of the present invention is a method for selectively modulating PR mediated conditions in a selected target tissue comprising the step of administering a therapeutically effective amount of a tissue-selective and/or isoform-specific PR ligand identified through one of the methods of the present invention to a patient in need thereof.
• the modulation of PR mediated conditions comprises inhibiting or not affecting PR mediated, preferably PR-B mediated, effects in a first selected tissue, preferably breast tissue, and enhancing or maintaining PR mediated, preferably PR-A mediated, effects in a second selected tissue, preferably uterine or ovarian tissue.
• protective PR-A mediated effects such as antiproliferative effects
• PR-B mediated effects such as proliferation/differentiation
• PR-A mediated effects are inhibited or not influenced, but PR-B mediated effects are enhanced or maintained, are exemplary methods according to the present invention.
• a further aspect of the present invention is an assay kit for screening for PR isoform A or B selective ligands.
• This kit comprises first cells being stably transfected with a plasmid expressing PR-A and second cells being stably transfected with a plasmid expressing PR- B, said first and second cells being further stably transfected with a plasmid comprising a reporter gene linked to a hormonally responsive promoter, wherein the contacting of said first and second cells with a ligand to be tested yields a level of expressed reporter gene product for said first cells and for said second cells and the comparison of these product levels results in a difference of transcription efficacies and/or potencies induced by said ligand in said first and said second cells, wherein this difference in transcription efficacies and/or potencies is indicative of a selectivity for PR-A or PR-B.
• the cells comprised in the kit are SK-N-MC cells having all the properties as described above for the screening methods according to the first and second aspect of the present invention.
• the determination of the efficacy and/or potency of the ligand to be tested in the assay kit according to the present invention is performed exactly as described above for the respective screening methods.
• potential isoform-specific PR ligands identified by means of the assay kit according to the present invention can be of steroidal or non- steroidal nature.
• the kit is useful for screening for compounds having already been tested on PR affinity/activity in general and found to bind to and activate or inhibit PR, but also with compounds of which their PR ligand potential is unknown.
• the compounds to be tested in the assay have already qualified as PR ligands, they can be pure agonists, pure antagonists or partial agonists/antagonists of PR.
• the isoform-specific PR ligands are pure PR-B antagonists and pure PR-A agonists or partial PR-B antagonists/PR-A agonists and thus exert their beneficial effects in the uterus and/or the ovary whereas unwanted and potentially dangerous side-effects in the breast, e.g., due to differentiation proliferation of the mammary tissue, are prevented.
• isoform- specific PR ligands being pure PR-B agonists and pure PR-A antagonists or partial PR-B agonists/PR-A antagonists are in accordance with the present invention.
• SK-N-MC cell lines per se i.e. the cell lines which are preferably used in the methods according to the first and second aspect of the present invention and the assay kit according to the present invention.
• a detailed description of the SK-N-MC cell lines according to the present invention has already been given with respect to the screening methods according to the first and second aspects of the present invention.
• Example 5 gives a detailed description of how the SK-N-MC cell lines according to the present invention may exemplarily be obtained.
• the assay method and the kit according to the present invention as presented above are not only suitable for screening for isoform-specific PR ligands, but, with the appropriate variations, may in principle also be applied to other receptor systems.
• Such further envisaged receptor systems may be other intracellular receptors existing in two or more different isoforms, e.g. the androgen receptor. It is within the purview of the person skilled in the art to perform the necessary alterations to the methods and the kit as described herein to adjust them to screening for isoform-specific and/or tissue-selective ligands of these further envisaged receptors.
• Example 1 The suitability of the method for screening for PR-A or PR-B specific ligands according to the first aspect of the present invention is demonstrated in Example 1 with respect to some exemplary PR ligands. Examples of in vivo tests are provided in Examples 2, 3 and 4.
• Example 5 pertains to the manufacture of the PR isoform-specific cell lines according to the invention by stable transfections.
• the method for screening for isoform-specific PR ligands according to the present invention is carried out with first and second SK-N-MC cells stably transfected with a plasmid expressing the hPR-A (first cells) or the hPR-B (second cells) and the LUC reporter gene linked to the hormonally responsive MTV promoter.
• a detailed description of how the cell lines according to the invention and used in this Example may be obtained is given in Example 5.
• the cells are cultured in Minimum Essential Medium with Earl's Salts (S-MEM, without L-glutamine; Gibco BRL, no. 21090-022), supplemented with 10 % fetal calf serum (FCS), penicillin lOOU/streptomycin 100 ⁇ g/ml (Biochrom, no. A2213) , L-glutamine 4 mmol/1 (Gibco BRL, no. 25030-024), sodium pyruvate 1 mmol/1 (Biochrom, no. L0473) and lx non-essential amino acids (Biochrom, no. K0293) at a temperature of 37 °C and in an atmosphere of 5 % carbon dioxide.
• S-MEM Minimum Essential Medium with Earl's Salts
• the cells are seeded onto 96- well dishes (2xl0 4 cells/dish) and cultured in a medium as described above, with the exception that the FCS is replaced by a 3 % charcoal stripped FCS. 48 hours later, cells are contacted with prediluted test compounds. For determination of agonistic activity, cells are cultured in the presence of increasing concentrations (10 to 10 "11 mol/1) of test compounds. As a positive control for reporter gene induction cells are treated with 10 "6 to 10 ' " mol/1 R5020 (promegestone). As a negative control for reporter gene induction, cells are cultured in 1 % ethanol.
• cells are treated with 10 "10 mol/1 R5020 and additionally with increasing concentrations (10 " to 10 " " mol/1) of test compounds.
• As a positive control for inhibition of reporter gene transcription cells are cultured in increasing concentrations (10 '7 to 10 "12 mol/1) of the antiprogestin mifepristone.
• As a negative control for inhibition of reporter gene transcription cells are cultured in 1 % ethanol.
• the medium is removed and cells are lysed with 20 ⁇ l of lysis buffer (Luciferase Assay System E 153 A; Promega) and under agitation of the plate for 10 min.
• lysis buffer Luciferase reagent
• the activity of the luciferase reporter gene product is determined in the cell lysates by means of a Microlite ML 3000 microtiter plate luminometer (Dynatech) in cycle mode.
• the LUC activity [%] for the measured data points is calculated as follows:
• the efficacy [%] is determined according to:
• the potency [nM], i.e. the EC 50 , is determined graphically.
• the LUC activity [%] for the measured data points is calculated as follows:
• the potency [nM], i.e. the IC 50 , is determined graphically.
• the selectivity of the tested ligands for one of the PR isoforms, A or B is defined as the difference in transcription efficacies achieved for PR-A and PR-B, which must be above or equal to 10 %, preferably above or equal to 15 % and more preferably even higher, such as above or equal to 20 %, and/or the difference in potency achieved for such ligands, which must be of a factor of at least 10.
• Agonism efficacy (PR-A): 132 %; antagonism efficacy (PR-A): none.
• Agonism efficacy (PR-B): 108 %; antagonism efficacy (PR-B): none.
• Difference of agonism efficacies (PR-A - PR-B): 24 % (indicating PR-A selectivity).
• Agonism efficacy (PR-A): 129 %; antagonism efficacy (PR-A): none.
• Agonism efficacy (PR-B): 108 %; antagonism efficacy (PR-B): none.
• Difference of agonism efficacies (PR-A - PR-B): 21 % (indicating PR-A selectivity)
• Agonism efficacy (PR-A): 131 %; antagonism efficacy (PR-A): none.
• Agonism efficacy (PR-B): 114 %; antagonism efficacy (PR-B): none.
• Difference of agonism efficacies (PR-A - PR-B): 17 % (indicating PR-A selectivity). Pure agonists with selectivity for PR-B:
• Agonism efficacy 35 %; antagonism efficacy (PR-A): none.
• Agonism efficacy PR-B
• 81 %; antagonism efficacy PR-B
• Difference of agonism efficacies PR-B - PR-A: 46 % (indicating PR-B selectivity).
• Agonism efficacy (PR-A): 50 %; antagonism efficacy (PR-A): none.
• Agonism efficacy (PR-B): 90 %; antagonism efficacy (PR-B): none.
• Difference of agonism efficacies (PR-B - PR-A): 40 % (indicating PR-B selectivity).
• Agonism efficacy (PR-A): 81 %; antagonism efficacy (PR-A): none.
• Agonism efficacy (PR-B): 106 %; antagonism efficacy (PR-B): none.
• Agonism efficacy (PR-B): 108 %; antagonism efficacy (PR-B): none.
• Agonism efficacy 39.9 %; antagonism efficacy (PR-A): none.
• Difference of agonism efficacies PR-B - PR-A: 17 % (indicating PR-B selectivity).
• Agonism efficacy 29 %
• antagonism efficacy PR-A: 45 %.
• Agonism efficacy 50 %
• antagonism efficacy PR-B: 30 %.
• Example 2 Bioassay on proliferating/differentiating effects in the rat mammary epithelium
• the object of this test is to evaluate the effect of progestins (PR ligands) on the development of the mammary gland, in particular on the formation of terminal end buds in the mammary gland in estrogen primed rats.
• Premature female rats (Wistar Han, SPF) are ovariectomized at the age of 21 days, 4 to 6 days before treatment start.
• the animals are treated for 6 days with standard estrogen (estrone, 70 ⁇ g/kg) and the test ligand (application volume: 0.1 ml/50g body weight; vehicle: benzylbenzoate/castor oil (1+4 v/v); subcutaneous).
• Control groups are e.g.: vehicle, estradiol without progestin, estradiol together with a known progestin, e.g. R5020.
• After the 6 day treatment the animals are killed with carbon dioxide.
• the preparations are fixed over night in alcohol-formalin according to the method of Tellyesniczky (see below). Then the mammary gland tissue and subcutis adhered thereto are stripped from the cutis and the preparations are again fixed over night.
• the further steps are as follows: ethanol 70 %: 1.5 hours; acetone: 3x1.5 hours; acetone: over night; isopropanol: 1.5 hours; ethanol 96 %: 2 hours; hematoxylin-iron: 3 hours; VE water: first rinse the preparations and then 2x0.5 hours; ethanol 70 %: over night; ethanol 80 %: 1.5 hours; ethanol 96 %: 1.5 hours; isopropanol: 1.5 hours.
• the preparations are then moved to petri dishes and left in toluene for approximately 1 hour, i.e. until they have stopped to swim up. Then the preparations are treated with cedarwood oil (Merck, no. 1.06965).
• cedarwood oil Merk, no. 1.06965.
• the incubation times above are minimum times and can be extended. In particular, incubation in ethanol 70 % after fixation can be extended to at least 2.5 weeks.
• Hematoxylin mother solution Hematoxylin (Merck, no. 1.15938): lOg, ethanol 96 %: 100 ml. The solution must stand for 48 hours at 37 °C before use. It can be kept in a dark place for almost unlimited time.
• Hematoxylin-iron solution for use hematoxylin mother solution (filtered): 15.2 ml, ethanol 96 %: 1374 ml, FeCl 3 x 6H 2 O (s. 4): 91.1 ml, 1 mol/1 HC1: 220 ml (total: 1700 ml); adjustment to a pH of 1.25 with 2mol/l NaOH.
• FeCl 3 x 6 H 2 O solution FeCl 3 x 6H 2 O (Merck, no. 1.03943): 1.07 g, VE water: 90.2 ml, HC1: 37 %: 0.92 ml (total: 91.1 ml).
• the terminal end buds near the nipple in direction of the tail are counted.
• the area to be investigated should be about 1.8 mm 2 .
• this area may be reduced, with at least 250 buds to be counted. After counting, the number of end buds per 1 mm 2 is calculated.
• the progestagenic effect of a test compound is either determined as a threshold value (concentration at which a significant progestagenic effect is recognized for the first time), or the dose that is equi-efficient to 0.3 mg/kg per day R5020 is determined.
• MIB-5 immunohistochemistry (according to C. Gerlach et al, Lab. Invest. 1997, 77(6), 697-698, with modifications):
• Mammary glands are fixed in 4% formaldehyde/PBS for 24 h and embedded in paraffin. 4 ⁇ m sections are spread on slides, deparaffinized, treated with microwaves for 10 min. in citrate buffer pH 6.0 and rinsed with PBS. Slides are then blocked with 3% H 0 2 /methanol for 15 min., Blockingkit (Vektor, no. SP-2001) for 10 min. and rat serum (Sigma, no. S- 7648) diluted 1 :2 in PBS for 30 min to reduce nonspecific staining and rinsed in PBS. Slides are incubated for 1 hour with monoclonal antibody MIB-5 (Dianova, no.
• Dia- 5055 which is specific for the rat Ki-67 antigen (1:200 diluted in PBS/0.2% BSA). Then, slides are washed twice in PBS/0.2% TWEEN 20, incubated with biotinylated rat anti- mouse secondary antibodies (Dianova, no. 425-066-100), diluted 1:200 in PBS/0.2% TWEEN 20 for 1 hour and washed again twice in PBS/0.2% TWEEN 20, following an incubation with avidin-biotin-peroxidase complexes (Vecstain Elite ABC Kit no. PK- 6100) for 1 h. Staining is performed by means of diaminobenzidine (Zymed Substrate Kit). All steps are performed at room temperature.
• the percentage of MIB-5 stained mammary epithelium cells is determined.
• Pregnant rats (Wistar Han, SPF) of 190 to 220 g (5 to 8 animals per dose) are ovariectomized on day 8 of pregnancy, 2 hours after the first substance administration. From day 8 to day 14, rats are daily treated with test compound in combination with a standard dose of E
• test compound is dissolved in benzyl benzoate/castor oil (1+4 v/v) and the daily dose is administered in a volume of 1 ml/kg body weight.
• test compound is suspended in a carrier liquid (85 mg Myrj R in 100 ml 0.9 % w/v NaCl solution) and the daily dose is administered in a volume of 2 ml/kg body weight.
• carrier liquid 85 mg Myrj R in 100 ml 0.9 % w/v NaCl solution
• test compound is dissolved in propylene glycol and charged in miniature osmotic pumps (type 2001, 1.0 ⁇ l/h, 7 days), which are placed in the abdominal cavity of the rat.
• estrone is 0.005 g/kg body weight s.c. and is dissolved in benzyl benzoate/castor oil ( 1.4 v/v).
• test compound is administered for 4 days (day 1 to 4) and the cycle is controlled thereafter.
• test compounds are dissolved in benzyl benzoate/castor oil (1+9 v/v) and the daily dose is administered in a volume of 1 ml/kg body weight.
• test compounds are suspended in a carrier liquid (85 mg Myrj R in 100 ml 0.9 % w/v NaCl solution) and the daily dose is administered in a volume of 2 ml/kg body weight.
• a carrier liquid 85 mg Myrj R in 100 ml 0.9 % w/v NaCl solution
• SK-N-MC cells were seeded onto 6- well dishes at a density of 5 x 10 5 cells per dish. Cells were typically about 80 % confluent after 24 hours. Before transfection, cells were washed twice with 1 ml Opti-MEM (Gibco, BRL, Berlin) per dish. For each dish, 5 ⁇ g DNA (pRSVhPR-neo: PR-B plasmid) or 10 ⁇ g DNA (5 ⁇ g phPR-2: PR-A plasmid and 5 ⁇ g pRSV-neo), respectively, were diluted with 500 ⁇ l Opti-MEM.
• pRSVhPR-neo PR-B plasmid
• 10 ⁇ g DNA 5 ⁇ g phPR-2: PR-A plasmid and 5 ⁇ g pRSV-neo
• the DNA and Lipofectamine Reagent dilutions were combined in a polystyrene snap cap tube to obtain 1 ml of transfection solution per dish, gently mixed, incubated at room temperature for 45 min. and added to the washed cells. After 5 hours, the transfection solution was replaced by 2 ml S-MEM supplemented with 10 % FCS. After 48 hours, the cells were trypsinized and replated onto 100 dishes at a density of 3 x 10 4 and 1.5 x 10 4 , respectively.
• SK-N-MC cell clones either stably transfected with pRSVhPR-neo or phPR-2 and pRSV-neo were selected in medium supplemented with 800 ⁇ g G418.
• Neomycin-resistant clones were stably transfected with pMTV-LUC and pSV2pac and were selected in culture medium supplemented with 400 mg/ml G418 and 0.2 mg/ml puromycin.
• clone SK-N-MC C 23.43 stably expressing PR-A and MTV-LUC
• clone SK-N-MC VIII 1.1 were selected for transactivation studies.
• pRSV-neo a) Construction of pRSV-CAT from SV2-CAT (see Gorman et al., Proc. Natl. Acad. Sci. 1982, 79, 6777-6781). b) Construction of pRSV-neo from RSV-CAT (see Gorman et al., Science 1983, 221, 551-555). pS V2 ⁇ ac: According to P. Artelt, Gene 1988, 68, 213-219.
• pRSV hPR-neo Contract from pRC/RSV (containing the neomycin resistance gene expressed from SV40 early promoter; from Invitrogen, San Diego) and hPR-1 (see Kastner et al., J. Biol. Chem. 1990, 265, 12163-
• the vector pRC/RSV is digested with Not I and Xba I; for the insert, hPR-1 digested with EcoR I, linker ligation with Not I, restriction digestion with Not I and Xba I resulting in 2,8 Kb fragment, which is isolated and ligated into the above vector.
• pMTV-LUC From A. Cato, Institut fur Genetik, Kemabastechnik

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