WO1993007290A1 - An in vitro method of evaluating the effects of a substance - Google Patents

An in vitro method of evaluating the effects of a substance Download PDF

Info

Publication number
WO1993007290A1
WO1993007290A1 PCT/SE1992/000698 SE9200698W WO9307290A1 WO 1993007290 A1 WO1993007290 A1 WO 1993007290A1 SE 9200698 W SE9200698 W SE 9200698W WO 9307290 A1 WO9307290 A1 WO 9307290A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
hormone
solvent
containers
medium
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/SE1992/000698
Other languages
French (fr)
Inventor
Stefan Nilsson
Mårten ÖSTERLUND
Karin Heeroma
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Karo Healthcare AB
Original Assignee
Karo Bio AB
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=20383927&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=WO1993007290(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Priority to US08/211,487 priority Critical patent/US5578445A/en
Priority to JP5506843A priority patent/JP2698476B2/en
Priority to DE69221629T priority patent/DE69221629T2/en
Priority to CA002120604A priority patent/CA2120604C/en
Priority to EP92921468A priority patent/EP0607268B1/en
Application filed by Karo Bio AB filed Critical Karo Bio AB
Priority to AU27735/92A priority patent/AU657171B2/en
Publication of WO1993007290A1 publication Critical patent/WO1993007290A1/en
Priority to NO19941246A priority patent/NO310439B1/en
Priority to FI941611A priority patent/FI103672B/en
Anticipated expiration legal-status Critical
Priority to GR970402995T priority patent/GR3025354T3/en
Ceased legal-status Critical Current

Links

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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • 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
    • G01N33/5044—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
    • 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

Definitions

  • the present invention relates to an in vitro method of evalu ⁇ ating the effects of a substance, especially the antagonistic versus agonistic effects of a receptor-binding substance, on a selected type of cells containing endogenous intra-cellular hormone receptors.
  • the present invention provides a tool for the prediction of the antagonistic versus agonistic effects of a receptor- binding substance on different kinds of tissues in a selected species. Description of the invention
  • the present invention is directed to an in vitro method of evaluating the antagonistic versus agonistic effects of a receptor-binding test substance on a selected type of cells containing endogenous intra-cellular hormone receptors. The method is carried out so
  • a sample of said cells, in a defined hormone-depleted first medium is distributed into several separate culture containers, such as microtiter wells,
  • d 1 is added said test substance, dissolved in a first solvent, at a known concentration
  • d 2 is added a reference substance, known to be either an antagonist or an agonist, dissolved in a second solvent, at a concentration known to result in a distinct cellular response selected to be analyzed,
  • d 3 is added said first solvent and said second solvent.
  • d 4 is added said test substance, dissolved in said first solvent, at the same concentration as used for d 1 , and said reference substance, dissolved in said second solvent, at the same concentration as used for d 2 ,
  • the first solvent and the second solvent being the same or different, and the amount of the first solvent and the amount of the second solvent not exceeding a level known to be harmful to the cells.
  • the method is directed to the evaluation of a test substance which is known to bind to intra-cellular hormone receptors.
  • a test substance which is known to bind to intra-cellular hormone receptors.
  • the binding of the test substance to the intra- cellular hormone receptors of a selective type of cells may be confirmed by well-known methods in the art, e.g. binding studies.
  • the expression "cells containing endogenous intra-cellular hormone receptors" is intended to mean that intra-cellular hormone receptors are encoded by the unmanipulated genome of the cells, contrary to the case where the genes for the hormone receptors have been transfected into cells.
  • the first solvent and the second solvent used are both added to each of the containers d 1 to d 4 , in the amounts used for the containers of the group d ⁇ and the group d 2 , respectively. In this case the possible effect of different amounts of solvents in the containers are eliminated.
  • the cells of the selected type are anchorage dependent cells. Such cells have been used in the experimental part of this specification.
  • the containers d of the group d ⁇ comprise increasing concentrations of said test substance. In this case it will be possible to simul ⁇ taneously evaluate the effects of the test substance at different concentrations.
  • the containers d of the group d 2 comprise increasing concentrations of said reference substance. In this case it will be possible to evaluate the effects of the reference substance at different concentrations.
  • the containers d 4 of the group d 4 comprise increasing concentrations of said test substance, and comprise said reference substance at the same concentration as used for the containers d 2 . In this case it will be possible to eliminate the possibility that the effect of the test substance is masked.
  • the cells of the selected type derive from mammalian bone, heart, breast or liver. In a preferred embodiment the cells of the selected type derive from human bone, heart, breast, liver or endo- metrium.
  • the method is per ⁇ formed on at least two selected types of cells which derive from different kinds of tissues, thus enabling the evaluation of the pattern of antagonistic versus agonistic effects of the selected test substance on said kinds of tissues.
  • the kinds of tissues derive from at least two members of the group consisting of mamma ⁇ lian and especially human bone, heart, breast, liver and endo etrium.
  • the cells of the selected type contain receptors which are members of the group consisting of steroid hormone receptors, thyroid hormone receptors and vitamin D receptors.
  • the steroid hormone receptors are selected from estrogen receptors and glucocorticoid recep ⁇ tors.
  • the magnitude of the cellular response selected to be analyzed is the amount of a specific protein product, the gene expression of which is regulated by hormone/receptor interaction.
  • the specific protein product is preferably an extra-cellular protein product, and is in one embodiment an endogenous protein product.
  • the selected cellular response resulting from hormone/recep ⁇ tor interaction need not be the expressed amount of a protein product, but may be e.g. the increase or decrease of the cell proliferation rate.
  • the amount of an expressed protein product can be analyzed with the aid of an enzymatic assay or a commercial immunological assay using antibodies specifically directed to the selected protein product.
  • enzymatic assay Enzyme Linked Immunosorbent Assay
  • RIA Radio Immunological Assay
  • Steroid and thyroid hormones have a common mechanism of action at the cellular level.
  • Most of the unliganded recep ⁇ tors are nuclear proteins, except the glucocorticoid receptor which is considered to be cytoplasmic.
  • the receptors can be divided into three domains: one domain binds to the hormone, one domain binds to DNA and the third domain seems to be important for interaction with other proteins.
  • the mechanism of action is as follows: the hormone enters the cell either by passive diffusion or by other mechanisms and binds to the receptor. Upon binding of the ligand the receptor undergoes a conformational change, and becomes activated. This enables the receptor to bind to specific DNA sequences, so called hormone response elements which are located in the vicinity of hormonally regulated genes.
  • the receptor interacts with transcription factors in order to regulate the expression of genes.
  • transcription factors There are certain transcription factors which are found in all cells and in addition there are transcription factors which are cell type specific.
  • the regulation can be negative or positive depending on the promotor context and the collection of transcription factors found in the cell. What determines whether a hormonally regulated gene should be expressed in a cell is thus not only the presence of hormone and receptor but also cell specific transcription factors.
  • the present invention relies on cells having endogenous intra-cellular receptors, as opposed to the EP-A-0 287 653, where the genes for hormone receptors are transiently trans- fected into hormone receptor negative cells.
  • hormone agonists and antagonists in a cell line derived from a tissue of interest for the specific indication it is possible to predict the effect in vivo at the cellular level.
  • tamoxifen used in the treatment of breast cancer. Many breast cancers are dependent on estrogens for growth and by blocking the receptor with antiestrogens, the tumor growth is inhibited. The use of tamoxifen is however associated with an incidence of new primary tumors at other sites such as the endometrium and the liver. This results from estrogenic activity of tamoxifen at these sites.
  • Hormone replacement therapy which means administration of estrogens to post enopausal women prevents osteoporosis.
  • this therapy is not accepted in some countries because of the increased risk of breast cancer.
  • a bone selec ⁇ tive estrogen analogue would be a substancial improvement of osteoporosis therapy.
  • genes which express gene products that can be measured to determine the antagonistic versus agonistic effects of a test compound in the method of the invention are listed below. These genes may be either positively or negatively regulated by hormones and this may occur only in certain celltypes.
  • EGF receptor jun transcription factor
  • fos transcription factor
  • tyrosine amine transferase collagen osteocalcin NaKATPase collagenase urokinase
  • NaKATPase beta-adrenergic receptors growth hormone (Yaffe and Samuels, 1984, JBC 260, 6284-6291)
  • TSH thyroid stimulating hormone
  • beta myosin heavy chain (Izumo et al. , 1984, Science 231,
  • estradiol (E 2 ) (Sigma, product No E 1132)
  • Partial antagonist tamoxifen (Tam) (a gift from Orion
  • Antagonist LY 117018 (a gift from Eli Lilly, USA; a non-steroidal benzothiophene, Endocrinology ill (1983) 611-617)
  • Second medium (B) Coon's medium without phenol red
  • First medium (C) Ham's F12 without phenol red (manufactu ⁇ red by Statens Veterinarmedicinska Anstalt, Uppsala, Sweden, in accordance with instructions given in the product catalogue of GIBCO-BRL)
  • the cells were suspended in the first medium (A) .
  • Four dishes per cell line were used. These petridishes were incubated at 37°C and 5% C0 2 in a temperature and humidity controlled chamber.
  • the first medium was replaced by the second medium.
  • the following hormone additions were made:
  • one petridish/cell line - no hormone added one petridish/cell line - + 10 nM estradiol (E 2 ) one petridish/cell line - + 100 nM tamoxifen (Tarn) one petridish/cell line - + 10 nM estradiol + 100 nM tamoxifen
  • the petridishes were incubated in the temperature and humidi ⁇ ty controlled chamber under the same conditions as above.
  • the relative amount of DS2 secreted into the medium fthe amount of pS2 secreted from the respective cell lines without d t 1.
  • pS2 can be induced by E 2 in the breast cancer cell lines MCF7 and ZR-75-1. Further it can be concluded that 10" 7 M tamoxifen functions as agonist in both the cell lines MCF7 and ZR-75-1 in the absence of E 2 . Furthermore, 10" 7 M tamoxifen functions as an antagonist in the presence of 10" 8 M E 2 .
  • the cells were suspended in the first medium (A) without 1% non-essential amino acids, but with 50 ⁇ g/ml gentamycin (from Gibco-BRL) .
  • the wells were washed with 100 ⁇ l Coon's medium/well.
  • the first medium was replaced by the second medium (B) without 1% FCS.
  • the following hormone additions were made:
  • Experiment 1 The relative amount of pS2 secreted into the medium in the presence of different concentration of E 2 .
  • the amount of pS2 secreted from their respective cell lines without added hormone is set at 1.
  • Experiment 2 The relative amount of pS2 secreted into the medium in the presence of different concentrations of Tam, and different concentrations of Tam in the presence of 0.1 nM E 2 , respectively.
  • the amount of pS2 secreted from the respec ⁇ tive cell lines without added hormone is set at 1.
  • pS2 can be induced by E 2 in the breast cancer cell lines MCF7 and ZR 75-1. Maximal induction is seen at 1 nM E 2 . Further, it can be concluded that 10 "6 M Tamoxifen functions as an antagonist in the presence of 10 "10 M E 2 . Furthermore, 10" 8 M LY 117018 functions as an antagonist in the presence of 10" - l 1 ⁇ 0 M E 2 *
  • the experiment was conducted using the human breast cancer cell line MCF7 (ATCC HTB 22).
  • the selected cellular response to be analyzed was the amount of expressed cathepsin D (Cath D), the expression of which is regulated by the estrogen receptor.
  • the agonist estradiol was used as a reference substance and the effects of the partial antagonist tamoxifen or the antagonist LY 117018, respectively, were evaluated. Furthermore, the effects in the presence of both estradiol and tamoxifen, as well as the effects of both estradiol and LY 117018 were evaluated.
  • the amount of Cath D secreted into the medium was determined with the aid of a commercial RIA (ELSA-CATH-D from CIS Bioindustries, Gif-sur-Yvette, France). The performance of the method is as follows:
  • a sample of the cell line MCF7 (4xl0 5 cells) was distributed into plastic petridishes (suitable for the culturing of mammalian cells). The cells were suspended in the first medium (C) .
  • the petridishes were incubated at 37°C and 5% C0 2 in a tempe ⁇ rature and humidity controlled chamber.
  • the first medium (C) was replaced by the second medium, which consisted of fresh first medium (C).
  • the following hormone additions were made:
  • the relative amount of Cath D secreted into the medium (the amount of Cath D secreted from cells without added hormone is set at 1)
  • Tamoxifen is previously known to be a partial agonist in breast, especially at low concentrations ( ⁇ 10 "6 M) and in the absence of E 2 or phenol red in the culture medium (as already stated in the experiment with pS2) .
  • the reason for Tam not to function as an antagonist in the presence of E 2 in this experiment as compared to the pS2 experiment may be due to the fact that the set up of the two experiments are not the same.
  • Cath D can be induced by E 2 in the breast cancer cell line MCF7, and that 10" 7 M tamoxifen functions as a weak agonist in the absence of E 2 . Further, 10 " M tamoxifen does not function as an antagonist in the presence of 10" 8 M E 2 . Finally 10" 7 M LY 117018 acts as an estrogen antagonist both in the absence and in the presence of 10 "8 M E 2 .
  • cathepsin D (Cath D) expression is regulated by estrogen in the human breast cancer cell lines MCF7 (ATCC HTB 22) and ZR 75-1 (ATCC CRL 1500), respectively, in the presence of the agonist estradiol, or the antagonists tamoxifen and LY 117018, respectively, or in the presence of both estradiol and tamoxifen, and estradiol and LY 117018, were tested.
  • the amount of Cath D secreted into the medium was determined with the aid of a commercial RIA (ELSA-CATD-D from CIS Bio- industries, Gif-sur-Yvette, France).
  • the cells were suspended in the first medium (A) without 1% non-essential amino acids, but with 50 ⁇ g/ml gentamycin (from Gibco-BRL) .
  • Day 5 ;
  • the wells were washed with 100 ⁇ l Coon's medium/well.
  • the first medium was replaced by the second medium (B) without 1% FCS.
  • the following hormone additions were made:
  • Experiment 1 The relative amount of Cath D secreted into the medium in the presence of different concentrations of E 2 .
  • the amount of Cath D secreted from the cells without added hormo ⁇ ne is set at 1.
  • LY 117018 functions as a stronger antagonist than Tamoxifen. In the presence of both 10 nM LY 117018 and 0.1 nM E 2 in the culture medium the ex ⁇ pression of Cath D is inhibited down to same level as in the absence of added hormone in the ZR 75-1 and also MCF7 cells.
  • Cath D can be induced by E 2 in the breast cancer cell lines MCF7 and ZR 75-1. Maximal induction is seen at 1 nM E 2 . Further, 10" 6 M Tamoxifen functions as an antagonist in the presence of 10" 10 M E 2 in the ZR 75-1 cells. Furthermore, 10 "8 M LY 117018 functions as an antagonist in the presence of 10 "10 M E 2*
  • the first medium was replaced by a second medium (Ham's without phenol red + 5% FCS (2xDCC) + 50 ⁇ g/ml gentamycin + 1% non-essential amino acids).
  • the following hormone additions were made:
  • the medium was replaced by fresh medium of the same composi ⁇ tion as on day 6, and hormone additions as on day 6.
  • Experiment 1 The relative amount of cells measured as the amount of ATP after culturing for 6 days in the presence of different concentrations of Tam, and different concentrations of Tam in the presence of 0.1 nM E 2 , respectively.
  • the amount of ATP without added hormone is set at 1.
  • MCF7 increase their growth dramatically in the presence of 0.1 nM E 2 .
  • Tamoxifen has no effect on the growth of the MCF7 cells, but a weak agonistic effect of tamoxifen on ZR 75-1 can be seen at 10' 7 M.
  • the growth of MCF7 cells is inhibited at a Tamoxifen concen ⁇ tration exceeding 10" 7 M.
  • Tamoxifen is a weaker antagonist in ZR 75-1, and not until at 10" 6 M Tamoxifen an antagonistic effect can be seen.
  • LY 117018 functions as a stronger anta ⁇ gonist than Tamoxifen.
  • the growth of both the cells MCF7 and ZR 75-1 are inhibited down to the same level as in the absence of added hormone.
  • 10" 7 M Tamoxifen functions as a weak agonist in ZR 75-1 cells.
  • 10 -7 M LY 117018 functions as an antagonist in the presence of 10" 10 M E 2 in MCF7 and ZR 75-1 cells.
  • the experiment was conducted using a reporter cell line (ZR 75-AF) which had been constructed by a stable transfection of the human breast cancer cell line ZR 75-1 (ATCC CRL 1500) with a plasmid comprising estrogen responsive element (ERE) and alkaline phosphatase (Alk. phos.) as reporter protein.
  • the selected cellular response to be analyzed was thus the amount of expressed Alk. phos., the expression of which is regulated by the estrogen receptor.
  • the expression of Alk. phos. is studied in the presence of various concentrations of the agonist estradiol, the antagonist tamoxifen and in the presence of both estradiol and tamoxifen. The amount of Alk. phos.
  • the method is conducted in the following way:
  • a sample of the cells ZR 75-AF was distributed into plastic petridishes (suitable for the culturing of mammalian cells).
  • the cells were suspended in the first medium (A) .
  • These petri- dishes were incubated at 37°C and 5% C0 2 in a temperature and humidity controlled chamber.
  • a sample of the cells ZR 75-AF (4xl0 4 cells/well) was dis ⁇ tributed into a micro titer plate having 96 wells.
  • the cells were suspended in fresh first medium (A) .
  • the plate was incubated at 37 ⁇ C and 5% C0 2 in a temperature and humidity controlled chamber.
  • the first medium (A) was replaced by the second medium (B)
  • the following hormone additions were made: Experiment 1: 3 wells/hormone no hormone added
  • Ex p eriment 2 The relative amount of Alk. phos. secreted into the medium in the presence of various concentrations of Tam and various concentrations of Tam in the presence of InM E 2 (the amount of Alk. phos. secreted without hormone addition is set at 1.
  • HepG2:1 Two different variants of HepG2 were used in this study designated. HepG2:1 and HepG2:2.
  • Experimental design Two different variants of HepG2 were used in this study designated. HepG2:1 and HepG2:2.
  • Approximately lxlO 6 cells were seeded in 6-well plastic petri ⁇ dishes (suitable for growth of mammalian cells) in 2 ml of the first medium (A). Six wells per cell line were used. These culture plates were incubated at 37 C C and 5% C0 2 in a temperature and humidity controlled cell incubator.
  • the first medium was aspirated and the cells were rinsed once with Coon's (without any additions) and then refed with 1 ml of the second medium (B) [without 1% FCS (2xDCC)].
  • the follo ⁇ wing hormone additions were made: two wells/cell line - no hormone added
  • the culture plates were incubated in the temperature and humidity controlled cell incubator under the same conditions as above.
  • the relative amount of SHBG expressed and secreted by the HepG2 cell lines in the presence or absence of T3 (the level of SHBG secreted from the cells in the absence of added hormone is set at 1)
  • T3 can induce the expression of SHBG in both HepG2:l and HepG2:2.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Toxicology (AREA)
  • Endocrinology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

An in vitro method of evaluating the antagonistic versus agonistic effects of a receptor-binding test substance on a selected type of cells containing endogenous intra-cellular hormone receptors, is disclosed. Said test substance, and separately a reference substance, known to be either an antagonist or an agonist, are incubated with said selected type of cells, and the magnitude of the selected cellular response resulting from hormone/receptor interaction, is analyzed. In case the method is performed on at least two selected types of cells which derive from different kinds of tissues, it is possible to evaluate the pattern of antagonistic versus agonistic effects of the selected test substance on said kinds of tissues.

Description

An in vitro method of evaluating the effects of a substance.
The present invention relates to an in vitro method of evalu¬ ating the effects of a substance, especially the antagonistic versus agonistic effects of a receptor-binding substance, on a selected type of cells containing endogenous intra-cellular hormone receptors.
Background
Efforts are made in the medical industry to reduce animal tests for the evaluation of the effects of substances which are candidates for incorporation into medicines. The reasons for this is i.a. the high costs and the comparatively long time needed for the experiments. Moreover, the extrapolation of the results from animal studies to humans does not always reflect the actual effects in humans.
There is an obvious need for screening methods which give species and tissue specific, comparatively reliable predic- tion of the effects of drug candidates.
In cases where the mechanism of action of drug candidates is known at the cellular level, it is possible to study the effects of the candidates in vitro. In such cases it should be possible to study the effects on cell lines derived from different types of tissues, and thus get a pattern of the effects in the studied types of tissues.
The present invention provides a tool for the prediction of the antagonistic versus agonistic effects of a receptor- binding substance on different kinds of tissues in a selected species. Description of the invention
The present invention is directed to an in vitro method of evaluating the antagonistic versus agonistic effects of a receptor-binding test substance on a selected type of cells containing endogenous intra-cellular hormone receptors. The method is carried out so
a) that a sample of said cells, in a defined hormone-depleted first medium, is distributed into several separate culture containers, such as microtiter wells,
b) that the containers of a) are incubated in a temperature and humidity controlled chamber for an appropriate time for the establishment of stable cell growth,
c) that following b) , the spent first medium is replaced by a defined hormone-depleted second medium,
d) that the equally treated containers of b) are divided into four groups, d- to d4 , each comprising at least one container, d1 to d4, respectively, and each container being treated in the subsequent steps,
e) that to the container:
d1 is added said test substance, dissolved in a first solvent, at a known concentration,
d2 is added a reference substance, known to be either an antagonist or an agonist, dissolved in a second solvent, at a concentration known to result in a distinct cellular response selected to be analyzed,
d3 is added said first solvent and said second solvent. d4 is added said test substance, dissolved in said first solvent, at the same concentration as used for d1, and said reference substance, dissolved in said second solvent, at the same concentration as used for d2,
the first solvent and the second solvent being the same or different, and the amount of the first solvent and the amount of the second solvent not exceeding a level known to be harmful to the cells.
f) that all the containers d1 to d4 are incubated in a tempe¬ rature and humidity controlled chamber for a period of time sufficient for the substances to affect the cells to such a degree that a distinct cellular response selected to be analyzed is reached,
g) that the incubated containers of f) are all analyzed with regard to the magnitude of the selected cellular response resulting from hormone/receptor interaction, and
h) that the antagonistic versus agonistic effects of said test substance on said selected type of cells are evaluated from a comparison of the analyzed magnitudes of the selected cellular response obtained for said groups dj to d4.
Thus, the method is directed to the evaluation of a test substance which is known to bind to intra-cellular hormone receptors. There are two possibilities, namely that the effects of the test substance are unknown or that the properties of the intra-cellular hormone receptors are unknown. The binding of the test substance to the intra- cellular hormone receptors of a selective type of cells may be confirmed by well-known methods in the art, e.g. binding studies. The expression "cells containing endogenous intra-cellular hormone receptors" is intended to mean that intra-cellular hormone receptors are encoded by the unmanipulated genome of the cells, contrary to the case where the genes for the hormone receptors have been transfected into cells.
In one embodiment of the invention the first solvent and the second solvent used are both added to each of the containers d1 to d4, in the amounts used for the containers of the group dχ and the group d2, respectively. In this case the possible effect of different amounts of solvents in the containers are eliminated.
In another embodiment of the invention the cells of the selected type are anchorage dependent cells. Such cells have been used in the experimental part of this specification.
In yet another embodiment of the invention the containers d of the group dχ comprise increasing concentrations of said test substance. In this case it will be possible to simul¬ taneously evaluate the effects of the test substance at different concentrations.
In still another embodiment of the invention the containers d of the group d2 comprise increasing concentrations of said reference substance. In this case it will be possible to evaluate the effects of the reference substance at different concentrations.
In an additional embodiment of the invention the containers d4 of the group d4 comprise increasing concentrations of said test substance, and comprise said reference substance at the same concentration as used for the containers d2. In this case it will be possible to eliminate the possibility that the effect of the test substance is masked. In yet another embodiment of the invention the cells of the selected type derive from mammalian bone, heart, breast or liver. In a preferred embodiment the cells of the selected type derive from human bone, heart, breast, liver or endo- metrium.
In a further embodiment of the invention the method is per¬ formed on at least two selected types of cells which derive from different kinds of tissues, thus enabling the evaluation of the pattern of antagonistic versus agonistic effects of the selected test substance on said kinds of tissues. In drug development it is of utmost importance to be able to evaluate the pattern of the antagonistic versus agonistic effects of a selected test substance on different kinds of tissues. In a preferred case of this embodiment the kinds of tissues derive from at least two members of the group consisting of mamma¬ lian and especially human bone, heart, breast, liver and endo etrium.
In a preferred embodiment of the invention the cells of the selected type contain receptors which are members of the group consisting of steroid hormone receptors, thyroid hormone receptors and vitamin D receptors.
In a further embodiment, the steroid hormone receptors are selected from estrogen receptors and glucocorticoid recep¬ tors.
In still another embodiment of the invention the magnitude of the cellular response selected to be analyzed is the amount of a specific protein product, the gene expression of which is regulated by hormone/receptor interaction. The specific protein product is preferably an extra-cellular protein product, and is in one embodiment an endogenous protein product. The selected cellular response resulting from hormone/recep¬ tor interaction need not be the expressed amount of a protein product, but may be e.g. the increase or decrease of the cell proliferation rate.
Further, the amount of an expressed protein product can be analyzed with the aid of an enzymatic assay or a commercial immunological assay using antibodies specifically directed to the selected protein product. Examples of conventional assays are ELISA (Enzyme Linked Immunosorbent Assay) and RIA (Radio Immunological Assay) , but other assays are also known in the art.
Application of the invention on cells containing endogenous intra-cellular steroid and thyroid receptors.
Steroid and thyroid hormones have a common mechanism of action at the cellular level. Most of the unliganded recep¬ tors are nuclear proteins, except the glucocorticoid receptor which is considered to be cytoplasmic. The receptors can be divided into three domains: one domain binds to the hormone, one domain binds to DNA and the third domain seems to be important for interaction with other proteins. The mechanism of action is as follows: the hormone enters the cell either by passive diffusion or by other mechanisms and binds to the receptor. Upon binding of the ligand the receptor undergoes a conformational change, and becomes activated. This enables the receptor to bind to specific DNA sequences, so called hormone response elements which are located in the vicinity of hormonally regulated genes. The receptor interacts with transcription factors in order to regulate the expression of genes. There are certain transcription factors which are found in all cells and in addition there are transcription factors which are cell type specific. The regulation can be negative or positive depending on the promotor context and the collection of transcription factors found in the cell. What determines whether a hormonally regulated gene should be expressed in a cell is thus not only the presence of hormone and receptor but also cell specific transcription factors.
The present invention relies on cells having endogenous intra-cellular receptors, as opposed to the EP-A-0 287 653, where the genes for hormone receptors are transiently trans- fected into hormone receptor negative cells.
Thus, by testing hormone agonists and antagonists in a cell line derived from a tissue of interest for the specific indication it is possible to predict the effect in vivo at the cellular level.
Steroid and thyroid hormone analogs have been used in medicine for a long time. The use of these kinds of compounds frequently give rise to side effets because of their lack of tissue selectivity. There is a need for improved compounds with selective effect. Antiestrogens such as tamoxifen are used in the treatment of breast cancer. Many breast cancers are dependent on estrogens for growth and by blocking the receptor with antiestrogens, the tumor growth is inhibited. The use of tamoxifen is however associated with an incidence of new primary tumors at other sites such as the endometrium and the liver. This results from estrogenic activity of tamoxifen at these sites.
Hormone replacement therapy which means administration of estrogens to post enopausal women prevents osteoporosis. However, this therapy is not accepted in some countries because of the increased risk of breast cancer. A bone selec¬ tive estrogen analogue would be a substancial improvement of osteoporosis therapy.
Examples of such genes which express gene products that can be measured to determine the antagonistic versus agonistic effects of a test compound in the method of the invention are listed below. These genes may be either positively or negatively regulated by hormones and this may occur only in certain celltypes.
Genes which are regulated by estrogens:
progesterone receptor cathepsin D
TPA pS2 urokinase
LDL receptor alkaline phosphatase
IGF I
IGF II TGF alpha
EGF receptor jun (transcription factor) fos (transcription factor) myc
Genes which are regulated by glucocorticoids:
tyrosine amine transferase (tat) collagen osteocalcin NaKATPase collagenase urokinase
Gene which is regulated by vitamin D3:
osteocalcin
Genes which are regulated by thyroid hormones!
NaKATPase beta-adrenergic receptors growth hormone (Yaffe and Samuels, 1984, JBC 260, 6284-6291) TSH (thyroid stimulating hormone) (Shupnik et al., 1985,
JBC 260, 2900-2903) malic enzyme (Towle et al., 1981, Biochemistry 20, 3486-3492) S14("sρotl4") (Liaw and Towle, 1984, JBC 259, 7253-7260) alpha myosin heavy chain (Izumo et al., 1984, Science 231,
591-600) beta myosin heavy chain (Izumo et al. , 1984, Science 231,
597-600)
Experiments performed making use of the method of the invention
Test and reference substances
Agonist: estradiol (E2) (Sigma, product No E 1132)
Partial antagonist: tamoxifen (Tam) (a gift from Orion
Corporation Farmos, Turku, Finland)
Antagonist: LY 117018 (a gift from Eli Lilly, USA; a non-steroidal benzothiophene, Endocrinology ill (1983) 611-617)
Solvents:
0,01% - 0,5% in ethanol for estradiol
0,01% - 0,5% in ethanol for tamoxifen
0,01% - 0,5% in ethanol for LY 117018
Media:
First medium (A) Coon's medium without phenol red (manufactured by Statens Veterinar- medicinska Anstalt, Uppsala, Sweden, in accordance with instructions given in the SIGMA catalogue) + 10% fetal calf serum (FCS) [twice hormone-depleted with dextran coated charcoal (2xDCC)]
+ 1% non-essential amino acids (from Gibco-BRL)
Second medium (B) : Coon's medium without phenol red
+ 1% FCS (2xDCC)
+ 5% serum substitute (purchased from Dr . Alan Preston, Med . Vet. supplies limited, Botolth Clayton, Buckingham, MK 18 2LR, U .K. )
First medium (C) : Ham's F12 without phenol red (manufactu¬ red by Statens Veterinarmedicinska Anstalt, Uppsala, Sweden, in accordance with instructions given in the product catalogue of GIBCO-BRL)
+ 1% FCS (2xDCC)
+ 5% serum substitute (from Dr. Alan Preston)
Two experiments were conducted using the human breast cancer cell lines MCF7 (ATCC HTB 22) and ZR 75-1(ATCC CRL 1500) respectively. The selected cellular response to be analyzed was the amount of expressed protein pS2, the expression of which is regulated by the estrogene receptor. The agonist estradiol was used as a reference substance and the effects of the partial antagonist tamoxifen was evaluated. The amount of pS2 secreted into the medium was determined with the aid of a commercial RIA (ELSA PS2 from CIS Bioindustries, Gif-sur-Yvette, France) .
The performance of the method is explained simultaneous for the two cell lines, for convenience.
Day 1:
Samples of the cells ZR75-1 (8xl05 cells) and MCF7 (4xl05 cells), respectively, were distributed into plastic petri¬ dishes (suitable for the culturing of mammalian cells). The cells were suspended in the first medium (A) . Four dishes per cell line were used. These petridishes were incubated at 37°C and 5% C02 in a temperature and humidity controlled chamber.
Dav 5:
The first medium was replaced by the second medium. The following hormone additions were made:
one petridish/cell line - no hormone added one petridish/cell line - + 10 nM estradiol (E2) one petridish/cell line - + 100 nM tamoxifen (Tarn) one petridish/cell line - + 10 nM estradiol + 100 nM tamoxifen
The petridishes were incubated in the temperature and humidi¬ ty controlled chamber under the same conditions as above.
Day 7:
48 hours after the hormone addition the number of cells and their morphology were investigated. Medium from each petridish was transferred to Eppendorf tubes and centrifuged 7 minutes at lOOOrpm (Eppendorf centrifuge). The amount of pS2 in the medium samples was analyzed with the aid of a commercial pS2 RIA (ELSA PS2). Results :
The relative amount of DS2 secreted into the medium fthe amount of pS2 secreted from the respective cell lines without d t 1.
ZR-75-1 1
3.73 3.78
Figure imgf000014_0001
1
It can be seen that the degree of induction of pS2 expression from the ZR-75-1 cells is higher than for the MCF7 cells, in the presence of E2 alone (3.7x and 1.4x, respectively) or Tarn alone (3.8x and 1.4x, respectively). The fact that tamoxifen can function as an agonist in human breast cancer cells, especially at low concentrations (< 10"6 M) and in the absence of E2 or phenol red in the culture medium, is previously known (see Br. J. Cancer (1989), 5L: 727-738, and Cancer Res. (1988), 48.: 3693-3697). This known fact is confirmed by the method of the invention. However, in the presence of both E2 and Tarn in the culture medium the expression of pS2 is inhibited to the same level (or lower) as in the absence of added hormone. This applies for both the ZR-75-1 and MCF7 cells.
It can be concluded that pS2 can be induced by E2 in the breast cancer cell lines MCF7 and ZR-75-1. Further it can be concluded that 10"7 M tamoxifen functions as agonist in both the cell lines MCF7 and ZR-75-1 in the absence of E2. Furthermore, 10"7 M tamoxifen functions as an antagonist in the presence of 10"8 M E2.
Additional set of Experiments: E£2
How the pS2 expression is regulated by estrogen in the human breast cancer cell lines MCF7 (ATCC HTB 22) and ZR 75-1 (ATCC CRL 1500), respectively, in the presence of the agonist estradiol, or the antagonists tamoxifen or LY 117019; or in the presence of both estradiol and tamoxifen, and estradiol and LY 117018, respectively, were tested. The amount of pS2 secreted into the medium was determined with the aid of a commercial RIA (ELSA pS2 from CIS Bioindustries, Gif-sur- Yvette, France) .
Performance
Samples of the cells ZR 75-1 and MCF7, respectively, were distributed into plastic petridishes (suitable for the cultu- ring of mammalian cells). The cells were suspended in the first medium (A) without 1% non-essential amino acids, but with 50 μg/ml gentamycin (from Gibco-BRL) .
Day 5
Samples of the cells ZR 75-1 (4xl04 cells/well) and MCF7 (2.5xl04 cells/well), respectively, were distributed in
96-well trays. The cells were distributed in the same medium as used on day 1.
Dav 6:
The wells were washed with 100 μl Coon's medium/well. The first medium was replaced by the second medium (B) without 1% FCS. The following hormone additions were made:
Experiment 1:
3 wells/hormone - No hormone added
+ 10"11 M estradiol (E2)
+ 10'10 M E2
+ 10"9 M E2 " + 10"8 M E2
+ 10-7 M E2
+ 10"6 M E2 Experiment 2: 3 wells/hormone - No hormone added
+ 10"9 M Tamoxifen (Tarn)
+ 10"8 M Tam
+ 10"7 M Tam
+ 10-6 M Tam
+ 10-10 M E2
+ 10-10 M E2 + 10"9 M Tam
+ 10"10 M E2 + 10"8 M Tam
+ 10"10 M E2 + 10-7 M Tam
+ 10"10 M E2 + 10*6 M Tam
Experiment 3: 3 wells/hormone - No hormone added
+ 10"9 M LY 117018
+ 10"8 M LY 117018
+ 10"7 M LY 117018
+ 10-6 M LY 117018
+ 10'10 M E2
+ 10"10 M E2 + 10"9 M LY 117018
+ 10-10 M E2 + 10"8 M LY 117018
+ 10"10 M E2 + 10-7 M LY 117018
+ 10-10 M E2 + 10"6 M LY 117018
Day 8:
48 hours after the hormone addition the number of cells and their morphology were investigated. Medium from each well was transferred to Eppendorf tubes. The possible amount of pS2 in the medium samples was analyzed with the aid of a commercial pS2 RIA (ELSA pS2) .
Results:
Experiment 1: The relative amount of pS2 secreted into the medium in the presence of different concentration of E2. The amount of pS2 secreted from their respective cell lines without added hormone is set at 1.
relative amount of pS2
ZR 75-1 MCF7
No hormone added 1 1 + 10"11 M E2 1.15 1.34 + 10-10 M E, 2.96 1.98
+ lO"9 M E2 4.39 2.02 + 10"8 M E2 5.10 1.98
+ 10-7 M E2 4.73 2.05
+ 10"6 M E, 4.41 1.76
Experiment 2: The relative amount of pS2 secreted into the medium in the presence of different concentrations of Tam, and different concentrations of Tam in the presence of 0.1 nM E2, respectively. The amount of pS2 secreted from the respec¬ tive cell lines without added hormone is set at 1.
relative amount of 2
No Tam added + 10"9 M Tam + 10"8 M Tam + 10"7 M Tam + 10'6 M Tam
Figure imgf000017_0001
Experiment 3: The relative amount of pS2 secreted into the medium in the presence of different concentrations of LY 117018, and different concentrations of LY 117018 in the presence of 0.1 nM E2, respectively. The amount of pS2 secre¬ ted from the respective cell lines without added hormone is set at 1. S2
Figure imgf000018_0001
It can be seen that the degree of induction of pS2 expression from the ZR 75-1 cells and MCF7 cells is dependent on the concentration of E2. An increased secretion of pS2 can be seen at 0.1 nK E2. Maximal induction is reached at 1 nM. Tamoxifen and LY 117018, respectively, do not have an influence on the pS2 secretion. In the presence of both Tamoxifen and E2 (0.1 nM) the expression of pS2 is inhibited at concentrations of Tam exceeding 100 nM. Maximal inhibition is seen at 1 μM Tamoxifen. LY 117018 functions as a stronger antagonist than Tamoxifen. In the presence of both 10 nM LY 117018 and 0.1 nM E2 in the culture medium the expression of pS2 is inhibited down to the same level as in the absence of added hormone in ZR 75-1 and also MCF7 cells.
It can be concluded that pS2 can be induced by E2 in the breast cancer cell lines MCF7 and ZR 75-1. Maximal induction is seen at 1 nM E2. Further, it can be concluded that 10"6 M Tamoxifen functions as an antagonist in the presence of 10"10 M E2. Furthermore, 10"8 M LY 117018 functions as an antagonist in the presence of 10" -l1ϋ0 M E 2*
Cathepsin D
The experiment was conducted using the human breast cancer cell line MCF7 (ATCC HTB 22). The selected cellular response to be analyzed was the amount of expressed cathepsin D (Cath D), the expression of which is regulated by the estrogen receptor. The agonist estradiol was used as a reference substance and the effects of the partial antagonist tamoxifen or the antagonist LY 117018, respectively, were evaluated. Furthermore, the effects in the presence of both estradiol and tamoxifen, as well as the effects of both estradiol and LY 117018 were evaluated. The amount of Cath D secreted into the medium was determined with the aid of a commercial RIA (ELSA-CATH-D from CIS Bioindustries, Gif-sur-Yvette, France). The performance of the method is as follows:
Day 1:
A sample of the cell line MCF7 (4xl05 cells) was distributed into plastic petridishes (suitable for the culturing of mammalian cells). The cells were suspended in the first medium (C) .
The petridishes were incubated at 37°C and 5% C02 in a tempe¬ rature and humidity controlled chamber.
Day 2:
The first medium (C) was replaced by the second medium, which consisted of fresh first medium (C). The following hormone additions were made:
two petridishes/hormone addition - no hormone added
- + 10 nM estradiol (E2)
" - + 100 nM tamoxifen (Tam)
- + 100 nM LY 117018 " - + 10 nM estradiol + 100 nM tamoxifen
- + 10 nM estradiol + 100 nM
LY 117018
The petridishes were incubated in the temperature and humidity controlled chamber under the same conditions as above. Day ? :
72 hours after the hormone addition visual inspection of the number of cells and their morphology was performed. Medium from each petridish was transferred to Eppendorf tubes and centrifuged 7 minutes at lOOOrpm (Eppendorf centrifuge) . The amount of Cath D in the medium samples was analyzed with the aid of the commercial Cath D RIA (ELSA-CATH D)
Results:
The relative amount of Cath D secreted into the medium (the amount of Cath D secreted from cells without added hormone is set at 1)
relative amount of Cath D
1. no hormone added 1
2. + E2 (10 nM) 2.22
3. + Tam (100 nM) 1.23
4. + LY 117018 (100 nM) 0.88 5. + E2 (10 nM) + Tam (100 nM) 2.43
6. + E2 (10 nM) + LY 117018 (100 nM) 0.76
It can be seen that LY 117018 functions as an antagonist both in the absence and in the presence of E2 (compare line 1, 4 and 6), whereas Tam shows weak agonism in the absence of E2
(compare line 1 and 3) and no antagonism in the presence of E2 (compare line 2 and 5). Tamoxifen is previously known to be a partial agonist in breast, especially at low concentrations (< 10"6 M) and in the absence of E2 or phenol red in the culture medium (as already stated in the experiment with pS2) . The reason for Tam not to function as an antagonist in the presence of E2 in this experiment as compared to the pS2 experiment may be due to the fact that the set up of the two experiments are not the same. A more interesting explanation may be that the mechanism underlying the ability of the estrogen receptor to regulate the expression of the genes Cath D and pS2, respectively, differs considerably, which may explain why 100 nM Tam (in the presence of 10 nM E2) was enough for the down-regulation of pS2 but not Cath D (see Br.J. Cancer (1989), 5!_ 727-738).
It can be concluded that Cath D can be induced by E2 in the breast cancer cell line MCF7, and that 10"7 M tamoxifen functions as a weak agonist in the absence of E2. Further, 10" M tamoxifen does not function as an antagonist in the presence of 10"8 M E2. Finally 10"7 M LY 117018 acts as an estrogen antagonist both in the absence and in the presence of 10"8 M E2.
Additional set of experiments: Cathepsin D
How cathepsin D (Cath D) expression is regulated by estrogen in the human breast cancer cell lines MCF7 (ATCC HTB 22) and ZR 75-1 (ATCC CRL 1500), respectively, in the presence of the agonist estradiol, or the antagonists tamoxifen and LY 117018, respectively, or in the presence of both estradiol and tamoxifen, and estradiol and LY 117018, were tested. The amount of Cath D secreted into the medium was determined with the aid of a commercial RIA (ELSA-CATD-D from CIS Bio- industries, Gif-sur-Yvette, France).
Performance
Day 1:
Samples of the cells ZR 75-1 and MCF7, respectively, were distributed into plastic petridishes (suitable for the cultu- ring of mammalian cells). The cells were suspended in the first medium (A) without 1% non-essential amino acids, but with 50 μg/ml gentamycin (from Gibco-BRL) . Day 5 ;
Samples of the cells ZR 75-1 (4xl04 cells/well) and MCF7 (2.5xl04 cells/well), respectively, were distributed in 96- well trays. The cells were distributed in the same medium as used on day 1.
Day 6:
The wells were washed with 100 μl Coon's medium/well. The first medium was replaced by the second medium (B) without 1% FCS. The following hormone additions were made:
Experiment 1: 3 wells/hormone - No hormone added
- + 10"11 M estradiol (E2)
Figure imgf000022_0001
Experiment 2: 3 wells/hormone - No hormone added
- + 10"9 M Tamoxifen (Tam)
- + 10"8 M Tam
- + 10'7 M Tam
- + 10-6 M Tam
- + 10"10 M E2
- + 10"10 M E2 + 10"9 M Tam
- + 10"10 M E2 + 10"8 M Tam
- + 10"10 M E2 + 10"7 M Tam
- + 10"10 M E2 + 10"6 M Tam Experiment 3: 3 wells/hormone - No hormone added
- + 10"9 M LY 117018
- + 10"8 M LY 117018
- + 10"7 M LY 117018
- + 10"6 M LY 117018
Figure imgf000023_0001
- + 10"10 M E2 + 10"9 M LY 117018
- + 10"10 M E2 + 10"8 M LY 117018
- + 1010 M E2 + 10"7 M LY 117018 - + 10"10 M E2 + 10"6 M LY 117018
Day 8:
48 hours after the hormone addition the number of cells and their morphology were investigated. Medium from each well was transferred to Eppendorf tubes. The possible amount of Cath D in the medium samples was analyzed with the aid of a commer¬ cial Cath D RIA (ELSA-CATH-D) .
Results:
Experiment 1: The relative amount of Cath D secreted into the medium in the presence of different concentrations of E2. The amount of Cath D secreted from the cells without added hormo¬ ne is set at 1.
relative amount of Cath D
ZR 75-1 MCF7
No hormone added 1 1
+ 10"11 M E2 0.93 1.20
+ 10"10 M E2 1.92 1.61
+ 10"9 M E 2 2.76 1.66
+ 10"8 M E2 2.84 1.66
+ 10"7 M E2 3.19 1.78
+ 10"6 M E, 2.98 1.56 Experiment 2: The relative amount of Cath D secreted into the medium in the presence of different concentrations of Tam, and different concentrations of Tam in the presence of 0.1 nM E2, respectively. The amount of Cath D secreted from the cells without added hormone is set at 1.
re a iv ath D
No Tam added
+ 10"9 M Tam
+ 10"8 M Tam
+ 10-7 M Tam
+ 10"6 M Tam
Figure imgf000024_0001
Experiment 3: The relative amount of Cath D secreted into the medium in the presence of different concentrations of LY 117018, and different concentrations of LY 117018 in the presence of 0.1 nM E2, respectively. The amount of Cath D secreted from the cells without added hormone is set at 1.
relative m u t f ath D
No LY 117018 added
+ 10"9 M LY 117018
+ 10"8 M LY 117018
+ 10-7 M LY 117018
+ 10"6 M LY 117018
Figure imgf000024_0002
It can be seen that the degree of induction of Cath D in ZR 75-1 and MCF7 is dependent on the concentration of E2. An increased secretion of Cath D can be seen at 0.1 nM E2. Maxi¬ mal induction is reached at 1 nM. Tamoxifen and LY 117018, respectively, do not have an influence on the Cath D secre¬ tion. In the presence of both Tamoxifen and E2 (0.1 nM) the expression of Cath D is inhibited at concentrations of Tam of 1 μM in the ZR 75-1 cells. In MCF7 Tamoxifen functions as a weaker antagonist and at 1 μM a slight decrease in the amount of secreted Cath D can be seen. LY 117018 functions as a stronger antagonist than Tamoxifen. In the presence of both 10 nM LY 117018 and 0.1 nM E2 in the culture medium the ex¬ pression of Cath D is inhibited down to same level as in the absence of added hormone in the ZR 75-1 and also MCF7 cells.
It can be concluded that Cath D can be induced by E2 in the breast cancer cell lines MCF7 and ZR 75-1. Maximal induction is seen at 1 nM E2. Further, 10"6 M Tamoxifen functions as an antagonist in the presence of 10"10 M E2 in the ZR 75-1 cells. Furthermore, 10"8 M LY 117018 functions as an antagonist in the presence of 10"10 M E 2*
Proliferation
How the human breast cancer cell lines MCF7 (ATCC HTB 22) and ZR 75-1 (ATCC CRL 1500), respectively, regulate their growth in the presence of the agonist estradiol, or the antagonists tamoxifen and LY 117018, and in the presence of both estradiol and tamoxifen, and estradiol and LY 117018, respectively, were tested. The growth is determined by measuring the amount of ATP in the cells after incubation with ligands for 6 days. The amount of ATP is determined with the aid of a method based on luciferin-luceferas reaction according to the manufacturer's instructions (LKB, Wallac).
Performance
Day 1:
Samples of the cells ZR 75-1 and MCF7, respectively, were dis¬ tributed into plastic petridishes (suitable for the culturing of mammalian cells). The cells were suspended in the first medium (A) without 1% non-essential amino acids, but with 50 μg/ml gentamycin (from Gibco-BRL) . Dav 5 :
Samples of the cells ZR 75-1 (8000 cells/well) and MCF7 (4000 cells/well), respectively, were distributed into microtiter plates having 96 wells. The cells were suspended in the same medium as on day 1.
Day 6:
The first medium was replaced by a second medium (Ham's without phenol red + 5% FCS (2xDCC) + 50 μg/ml gentamycin + 1% non-essential amino acids). The following hormone additions were made:
Experiment 1: 3 wells/hormone No hormone added
+ 10"9 Tamoxifen (Tam)
+ 10"8 M Tam
+ 10"7 M Tam
+ 10"6 M Tam
+ 10"10 M E2
+ 10"10 M E2 + 10"9 M Tam
- + 10 ,-"110U M E. + 10"° 8 M Tam
- + 10 ,-"1ι0υ M E2 + 10 .-"7' M Tam
- + 10"10 M E, + 10"6 M Tam
Experiment 2: 3 wells/hormone No hormone added
+ 10"9 M LY 117018
+ 10"8 M LY 117018
+ 10"7 M LY 117018
+ 10"6 M LY 117018
+ 10"10 M E2
+ 10"10 M E2 + 10"9 M LY 117018
+ 10"10 M E2 + 10"8 M LY 117018
+ 10"10 M E2 + 10"7 M LY 117018
+ 10"10 M E2 + 10"6 M LY 117018 Day 9 :
The medium was replaced by fresh medium of the same composi¬ tion as on day 6, and hormone additions as on day 6.
Day 12:
6 days after hormone addition the number of cells and their morphology were investigated. Medium from each well was poured off. To each well was added 100 μl 1% TCA (trichloro acetic acid) . After an incubation of 15 min the amount of ATP was measured according to a method based on the luceferin-lucefe- ras reaction (LKB, Wallac).
Results:
Experiment 1: The relative amount of cells measured as the amount of ATP after culturing for 6 days in the presence of different concentrations of Tam, and different concentrations of Tam in the presence of 0.1 nM E2, respectively. The amount of ATP without added hormone is set at 1.
o of ATP
No Tam added
+ 10"9 M Tam
+ 10"8 M Tam
+ 10"7 M Tam + 10"6 M Tam
Figure imgf000027_0001
Experiment 2: The relative amount of cells measured as the amount of ATP after culturing for 6 days in the presence of different concentrations of LY 117018, and different concentra- tions of LY 117018, respectively, in the presence of 0.1 nM E2. The amount of ATP without added hormone is set at 1. TP
No LY 117018 added + 10"9 M LY 117018 + 10"8 M LY 117018 + 10"7 M LY 117018 + 10"6 M LY 117018
Figure imgf000028_0001
It can be seen that the breast cancer cell lines ZR 75-1 and
MCF7 increase their growth dramatically in the presence of 0.1 nM E2. Tamoxifen has no effect on the growth of the MCF7 cells, but a weak agonistic effect of tamoxifen on ZR 75-1 can be seen at 10'7 M. In the presence of both Tamoxifen and 0.1 nM E2 the growth of MCF7 cells is inhibited at a Tamoxifen concen¬ tration exceeding 10"7 M. Tamoxifen is a weaker antagonist in ZR 75-1, and not until at 10"6 M Tamoxifen an antagonistic effect can be seen. LY 117018 functions as a stronger anta¬ gonist than Tamoxifen. In the presence of both 0.1 nM E2 and 10 nM LY 117018 in the culture medium the growth of both the cells MCF7 and ZR 75-1 are inhibited down to the same level as in the absence of added hormone.
It can be concluded that 0.1 nM E2 stimulates the growth of the breast cancer cell lines MCF7 and ZR 75-1. Further, 10"7 M Tamoxifen functions as an antagonist in the presence of 10 -10 M
E2 in the MCF7 cells. Also, 10"7 M Tamoxifen functions as a weak agonist in ZR 75-1 cells. Furthermore, 10-7 M LY 117018 functions as an antagonist in the presence of 10"10 M E2 in MCF7 and ZR 75-1 cells.
Alkaline phosphatase
The experiment was conducted using a reporter cell line (ZR 75-AF) which had been constructed by a stable transfection of the human breast cancer cell line ZR 75-1 (ATCC CRL 1500) with a plasmid comprising estrogen responsive element (ERE) and alkaline phosphatase (Alk. phos.) as reporter protein. The selected cellular response to be analyzed was thus the amount of expressed Alk. phos., the expression of which is regulated by the estrogen receptor. The expression of Alk. phos. is studied in the presence of various concentrations of the agonist estradiol, the antagonist tamoxifen and in the presence of both estradiol and tamoxifen. The amount of Alk. phos. secreted into the medium was determined by a chemi- luminiscence method based on AMPPD (disodium 3-[4-methoxy- sρiro[1,2-dioxithane-3,2'-tricyclo[3.3.1]decan]-4-yl]phenyl phosphate) (Tropix Inc, USA) as substrate.
The method is conducted in the following way:
Day 1:
A sample of the cells ZR 75-AF was distributed into plastic petridishes (suitable for the culturing of mammalian cells). The cells were suspended in the first medium (A) . These petri- dishes were incubated at 37°C and 5% C02 in a temperature and humidity controlled chamber.
Day 4:
A sample of the cells ZR 75-AF (4xl04 cells/well) was dis¬ tributed into a micro titer plate having 96 wells. The cells were suspended in fresh first medium (A) . The plate was incubated at 37βC and 5% C02 in a temperature and humidity controlled chamber.
Day 6:
The first medium (A) was replaced by the second medium (B) The following hormone additions were made: Experiment 1: 3 wells/hormone no hormone added
+ 10"12 M estradiol (E2)
+ 10"11 M E2
+ 10'10 M E2
+ 10"9 M E2
+ 10"8 M E
+ 10"7 M E
Experiment 2: 3 wells/hormone - no hormone added
- + 10"10 M Tamoxifen (Tam)
- + 10"9 M Tam
- + 10"8 M Tam
- + 10'7 M Tam
- + 10"6 M Tam
- + 5xl0*6 M Tam
- + InM E2
- + InM E2 + 10"10 M Tam
- + InM E2 + 10"9 M Tam
- + InM E2 + 10"8 M Tam
- + InM E2 + 10"7 M Tam
- + InM E2 + 10"6 M Tam
- + InM E2 + 5xl0"6 M Tam
Day 81
48 hours after the hormone addition the number of cells and their morphology were investigated. Medium from each well was transferred to Eppendorf tubes and incubated at 65βC for 10 minutes. The relative amount of Alk. phos. was then deter¬ mined by a chemiluminiscence assay (Tropix Inc., USA). Results:
Experiment 1: The relative amount of Alk. phos. secreted into the medium in the presence of various concentrations of E2 (the amount of Alk. phos. secreted without hormone addition is set at 1) .
relative amount of Alk . phos no hormone added 1 + 10"12 M E2 1 .05 + 10"11 M E 1. 12
+ 10"10 M E 3 .26
+ 10"9 M E2 5.67 + 10"8 M E2 6 . 85 + 10"7 M E, 7 . 90
Experiment 2: The relative amount of Alk. phos. secreted into the medium in the presence of various concentrations of Tam and various concentrations of Tam in the presence of InM E2 (the amount of Alk. phos. secreted without hormone addition is set at 1.
relative amount of Alk. phos.
-E2 +lnM E2 no Tam added 1 8.97 + 10"10 M Tam 1.13 9.50
+ 10"9 M Tam 1.08 9.12
+ 10"8 M Tam 1.27 10.28
+ 10"7 M Tam 1.80 7.57
+ 10"6 M Tam 3.11 3.65 + 5xl0"6 M Tam 2.87 2.62
It can be seen that the degree of induction of heat stable Alk. phos. from the reporter cell line ZR 75-AF is dependent on the concentration of E2. An increased secretion of Alk. phos. can be seen at already 10"10 M E2. At 10"7 M E2 the degree of induction of 7.9 is reached. Tamoxifen (10"7 M) alone functions as a weak agonist. A maximum agonistic effect is reached at 10"6 M Tamoxifen. As is earlier stated herein, it is previously known that Tamoxifen may function as an agonist in human breast cancer cells in the absence of E2 or phenol red in the culture medium. In the presence of both Tamoxifen and E2 (1 nM) the expression of Alk. phos. is inhibited by
Tamoxifen concentrations exceeding 10"7 M. Maximum inhibition is reach at 10"6 M Tamoxifen.
It can be concluded that Alk. phos. can be induced by E2 in the reporter cell line ZR 75-AF. Further, it can be concluded that Tamoxifen in a concentration of 10"6 M functions as an agonist in the absence of E2. Moreover, Tamoxifen at a con¬ centration of 10"6 M functions as an antagonist in the presen¬ ce of 10"9 M E2.
In order to study the effects of ligands to the thyroid hormone receptor in liver, the following experiment was conducted:
The effect of T3 on the expression of steroid hormone binding globulin (SHBG) . in human liver cells.
In this experiment we analyze the effect of the hormone 3,3',5-Triiodothyronine (T3) (agonist: purchased from Sigma, cat. no. T 6397) on the expression of sex (steroid) hormone binding globulin (SHBG) in the human liver cell line HepG2 (ATCC HB 8065). The level of SHBG, secreted into the culture medium is determined by a commercially available SHBG Delfia assay (manufactured by Wallac OY, Turku, Finland) .
Two different variants of HepG2 were used in this study designated. HepG2:1 and HepG2:2. Experimental design
Day 1:
Approximately lxlO6 cells were seeded in 6-well plastic petri¬ dishes (suitable for growth of mammalian cells) in 2 ml of the first medium (A). Six wells per cell line were used. These culture plates were incubated at 37CC and 5% C02 in a temperature and humidity controlled cell incubator.
Day 2:
The first medium was aspirated and the cells were rinsed once with Coon's (without any additions) and then refed with 1 ml of the second medium (B) [without 1% FCS (2xDCC)]. The follo¬ wing hormone additions were made: two wells/cell line - no hormone added
+ 10"8 M T3 + 10"7 M T3
The culture plates were incubated in the temperature and humidity controlled cell incubator under the same conditions as above.
Day 4:
48 hours after the addition of hormone the condition of the cells and their morphology were examined by visual inspection in the microscope. Medium from each well was transferred to Eppendorf tubes and centrifuged at 1000 rpm for seven minutes in an Eppendorf centrifuge. The supernatants were transferred to fresh Eppendorf tubes and the amount of SHBG in 25 μl of the conditioned medium was determined by an SHBG Delfia assay (Wallac Oy, Turku, Finland) according to the manufacturer's instructions. Results :
The relative amount of SHBG expressed and secreted by the HepG2 cell lines in the presence or absence of T3 (the level of SHBG secreted from the cells in the absence of added hormone is set at 1)
HepG2:1 HepG2:2 no hormone added 1 1
+ 10"8 M T3 1.6 16 + 10"7 M T3 1.6 18
As shown in the table the inducibility of SHBG is greater in the HepG2:2 variant. The reason for the discrepancy in T3 inducibility of the SHBG expression in HepG2:l cells compared to HepG2:2 cells is not known. However, it is a well known fact that the behaviour of different versions of originally one and the same cell line can differ depending on the condi¬ tions under which they have been cultivated. Whether HepG2:2 represents an isolated clone from the original population of HepG2 cells is not known.
It can be concluded from the experiment that T3 can induce the expression of SHBG in both HepG2:l and HepG2:2.

Claims

1. An in vitro method of evaluating the antagonistic versus agonistic effects of a receptor-binding test substance on a selected type of cells containing endogenous intra-cellular hormone receptors, c h a r a c t e r i s e d in
a) that a sample of said cells, in a defined hormone-deple¬ ted first medium, is distributed into several separate culture containers, such as microtiter wells,
b) that the containers of a) are incubated in a temperature and humidity controlled chamber for an appropriate time for the establishment of stable cell growth,
c) that following b), the spent first medium is replaced by a defined hormone-depleted second medium,
d) that the equally treated containers of b) are divided into four groups, dj to d4 , each comprising at least one container, d1 to d4, respectively, and each container being treated in the subsequent steps.
e) that to the container :
d1 is added said test substance, dissolved in a first sol¬ vent, at a known concentration,
d2 is added a reference substance, known to be either an antagonist or an agonist, dissolved in a second solvent, at a concentration known to result in a distinct cellular response selected to be analyzed. d3 is added said first solvent and said second solvent,
d4 is added said test substance, dissolved in said first solvent, at the same concentration as used for d1, and said reference substance, dissolved in said second solvent, at the same concentration as used for d2,
the first solvent and the second solvent being the same or different, and the amount of the first solvent and the amount of the second solvent not exceeding a level known to be harmful to the cells,
f) that all the containers d1 to d4 are incubated in a tempe¬ rature and humidity controlled chamber for a period of time sufficient for the substances to affect the cells to such a degree that a distinct cellular response selected to be analyzed is reached,
g) that the incubated containers of f) are all analyzed with regard to the magnitude of the selected cellular response resulting from hormone/receptor interaction, and
h) that the antagonistic versus agonistic effects of said test substance on said selected type of cells are evaluated from a comparison of the analyzed magnitudes of the selected cellular response obtained for said groups dj to d4.
2. A method according to claim 1, wherein said first solvent and said second solvent are both added to each of the con¬ tainers d1 to d4, in the amounts used for the containers of the group dχ and the group d2, respectively.
3. A method according to claim 1, wherein the cells of the selected type are anchorage dependent cells.
4. A method according to claim 1, wherein the containers d of the group dj comprise increasing concentrations of said test substance.
5. A method according to claim 1, wherein the containers d of the group d2 comprise increasing concentrations of said reference substance.
6. A method according to claim 1, wherein the containers d4 of the group d4 comprise increasing concentrations of said test substance, and comprise said reference substance at the same concentration as used for the containers d2.
7. A method according to claim 1, wherein the cells of the selected type derive from mammalian bone, heart, breast, liver or endometrium.
8. A method according to claim 7, wherein the cells of the selected type derive from human bone, heart, breast, liver or endometrium.
9. A method according to claim 1, wherein the method is performed on at least two selected types of cells which derive from different kinds of tissues, thus enabling the evaluation of the pattern of antagonistic versus agonistic effects of the selected test substance on said kinds of tissues.
10. A method according to claim 9, wherein said kinds of tissues derive from at least two members of the group consisting of mammalian bone, heart, breast, liver and endometriu .
11. A method according to claim 10, wherein said kinds of tissues derive from at least two members of the group consisting of human bone, heart, breast, liver and endometrium.
12. A method according to claim 1, wherein the cells of the selected type contain receptors which are members of the group consisting of steroid hormone receptors, thyroid hormone receptors and vitamin D receptors.
13. A method according to claim 12, wherein the steroid hormone receptors are selected from estrogen receptors and glucocorticoid receptors.
14. A method according to claim 1, wherein the magnitude of the cellular response selected to be analyzed is the amount of a specific protein product, the gene expression of which is regulated by hormone/receptor interaction.
15. A method according to claim 14, wherein the specific protein product is an extracellular protein product.
16. A method according to claim 14 or 15, wherein the specific protein product is an endogenous protein product.
PCT/SE1992/000698 1991-10-07 1992-10-06 An in vitro method of evaluating the effects of a substance Ceased WO1993007290A1 (en)

Priority Applications (9)

Application Number Priority Date Filing Date Title
AU27735/92A AU657171B2 (en) 1991-10-07 1992-10-06 An (in vitro) method of evaluating the effects of a substance
JP5506843A JP2698476B2 (en) 1991-10-07 1992-10-06 In vitro method to evaluate the effect of a substance
DE69221629T DE69221629T2 (en) 1991-10-07 1992-10-06 IN VITRO METHOD FOR EVALUATING EFFECTS OF A SUBSTANCE
CA002120604A CA2120604C (en) 1991-10-07 1992-10-06 An in vitro method of evaluating the effects of a substance
EP92921468A EP0607268B1 (en) 1991-10-07 1992-10-06 An in vitro method of evaluating the effects of a substance
US08/211,487 US5578445A (en) 1991-10-07 1992-10-06 In vitro method of evaluating the effects of a substance
NO19941246A NO310439B1 (en) 1991-10-07 1994-04-06 In vitro method for evaluating the effects of a substance
FI941611A FI103672B (en) 1991-10-07 1994-04-07 Procedure for in vitro extrusion of the effects of a substance
GR970402995T GR3025354T3 (en) 1991-10-07 1997-11-12 AN -i(IN VITRO) METHOD OF EVALUATING THE EFFECTS OF A SUBSTANCE.

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE9102901A SE500453C2 (en) 1991-10-07 1991-10-07 An in vitro method for the evaluation of a substance's effects
SE9102901-7 1991-10-07

Publications (1)

Publication Number Publication Date
WO1993007290A1 true WO1993007290A1 (en) 1993-04-15

Family

ID=20383927

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/SE1992/000698 Ceased WO1993007290A1 (en) 1991-10-07 1992-10-06 An in vitro method of evaluating the effects of a substance

Country Status (14)

Country Link
US (1) US5578445A (en)
EP (1) EP0607268B1 (en)
JP (1) JP2698476B2 (en)
AT (1) ATE156862T1 (en)
AU (1) AU657171B2 (en)
CA (1) CA2120604C (en)
DE (1) DE69221629T2 (en)
DK (1) DK0607268T3 (en)
ES (1) ES2104945T3 (en)
FI (1) FI103672B (en)
GR (1) GR3025354T3 (en)
NO (1) NO310439B1 (en)
SE (1) SE500453C2 (en)
WO (1) WO1993007290A1 (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0652005A1 (en) * 1993-10-15 1995-05-10 Eli Lilly And Company Methods for inhibiting endometriosis
RU2138261C1 (en) * 1993-12-21 1999-09-27 Эли Лилли Энд Компани Use of 2-phenyl-3-aroylbenzothiophenes for inhibition of central nervous system disorder in women in postmenopause
RU2143897C1 (en) * 1993-12-21 2000-01-10 Эли Лилли Энд Компани Method of suppression of skin and vagina atrophy
US6516294B1 (en) 1999-07-01 2003-02-04 The Regents Of The University Of California Nuclear receptor for 1α,25-dihydroxyvitamin D3 useful for selection of vitamin D3 ligands and a method therefor

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020001797A1 (en) * 2000-06-30 2002-01-03 Mitsuko Ishihara Method for detecting endocrine disrupting action of a test substance
US20080085912A1 (en) * 2006-10-06 2008-04-10 Unitech Pharmaceuticals, Inc. Isoxazole derivatives and methods of treating diseases

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991007488A1 (en) * 1989-11-16 1991-05-30 The Salk Institute For Biological Studies Beta retinoic acid response elements compositions and assays

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4859585A (en) * 1986-04-17 1989-08-22 Trustees Of Tufts College In-vitro methods for identifying compositions which are agonists and antagonists of estrogens
US5071773A (en) * 1986-10-24 1991-12-10 The Salk Institute For Biological Studies Hormone receptor-related bioassays
US5266464A (en) * 1988-02-10 1993-11-30 Ict Pharmaceuticals, Inc. Method of screening for protein inhibitors and activators

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991007488A1 (en) * 1989-11-16 1991-05-30 The Salk Institute For Biological Studies Beta retinoic acid response elements compositions and assays

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Breast Cancer Research and Treatment, Vol. 17, 1990, RICHARD POULIN et al.: "Multiple actions of synthetic "progestins" on the growth of ZR-75-1 human breast cancer cells: An in vitro model for the simultaneous assay of androgen, progestin, estrogen, and glucocorticoid agonistic and antagonistic activities of......", pp. *
Neuroendocrinology, Vol. 50, 1989, STEVEN M. GABRIEL et al.: "Iso Stimulation of GH and cAMP: Comparison of beta-Adrenergic- to GRF-Stimulated GH Release and cAMP Accumulation in Monolayer Cultures of Anterior Pituitary Cells in vitro", pp. 170-176, see especially page 171. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0652005A1 (en) * 1993-10-15 1995-05-10 Eli Lilly And Company Methods for inhibiting endometriosis
AU677700B2 (en) * 1993-10-15 1997-05-01 Eli Lilly And Company Methods for inhibiting endometriosis
RU2138261C1 (en) * 1993-12-21 1999-09-27 Эли Лилли Энд Компани Use of 2-phenyl-3-aroylbenzothiophenes for inhibition of central nervous system disorder in women in postmenopause
RU2143897C1 (en) * 1993-12-21 2000-01-10 Эли Лилли Энд Компани Method of suppression of skin and vagina atrophy
US6516294B1 (en) 1999-07-01 2003-02-04 The Regents Of The University Of California Nuclear receptor for 1α,25-dihydroxyvitamin D3 useful for selection of vitamin D3 ligands and a method therefor

Also Published As

Publication number Publication date
CA2120604A1 (en) 1993-04-15
EP0607268B1 (en) 1997-08-13
AU657171B2 (en) 1995-03-02
NO310439B1 (en) 2001-07-02
GR3025354T3 (en) 1998-02-27
FI103672B1 (en) 1999-08-13
AU2773592A (en) 1993-05-03
FI103672B (en) 1999-08-13
FI941611L (en) 1994-04-07
JPH07500184A (en) 1995-01-05
EP0607268A1 (en) 1994-07-27
NO941246L (en) 1994-04-06
FI941611A0 (en) 1994-04-07
NO941246D0 (en) 1994-04-06
SE9102901L (en) 1993-04-08
CA2120604C (en) 1999-08-17
ES2104945T3 (en) 1997-10-16
DE69221629T2 (en) 1998-01-22
DE69221629D1 (en) 1997-09-18
SE500453C2 (en) 1994-06-27
JP2698476B2 (en) 1998-01-19
US5578445A (en) 1996-11-26
SE9102901D0 (en) 1991-10-07
ATE156862T1 (en) 1997-08-15
DK0607268T3 (en) 1997-09-08

Similar Documents

Publication Publication Date Title
Johnson Follicle selection in the avian ovary
Krysko et al. Life and death of female gametes during oogenesis and folliculogenesis
Gallego et al. Prolactin, growth hormone, and epidermal growth factor activate Stat5 in different compartments of mammary tissue and exert different and overlapping developmental effects
Borton et al. The loss of Smad3 results in a lower rate of bone formation and osteopenia through dysregulation of osteoblast differentiation and apoptosis
Jezierski et al. Neurotrophin expression in the reproductively senescent forebrain is refractory to estrogen stimulation
Chaudhary et al. The helix-loop-helix inhibitor of differentiation (ID) proteins induce post-mitotic terminally differentiated Sertoli cells to re-enter the cell cycle and proliferate
Miller et al. Estrogen modulates spontaneous alternation and the cholinergic phenotype in the basal forebrain
Schwarzkopf et al. Muscle cachexia is regulated by a p53–PW1/Peg3-dependent pathway
Zhu et al. Estrogen and thyroid hormone interaction on regulation of gene expression.
Guo et al. The effects of myostatin on adipogenic differentiation of human bone marrow-derived mesenchymal stem cells are mediated through cross-communication between Smad3 and Wnt/β-catenin signaling pathways
Dobrenis et al. Human and mouse microglia express connexin36, and functional gap junctions are formed between rodent microglia and neurons
Åberg et al. IGF-I has a direct proliferative effect in adult hippocampal progenitor cells
Günther et al. Fhl2 deficiency results in osteopenia due to decreased activity of osteoblasts
Fu et al. Proinflammatory cytokine production and insulin sensitivity regulated by overexpression of resistin in 3T3-L1 adipocytes
Fiorini et al. Expression of RALT, a feedback inhibitor of ErbB receptors, is subjected to an integrated transcriptional and post-translational control
Schiffer Brain tumors: pathology and its biological correlates
Shang et al. Constitutively active signal transducer and activator of transcription 5 can replace the requirement for growth hormone in adipogenesis of 3T3-F442A preadipocytes
Kelvin et al. Growth factors, signaling pathways, and the regulation of proliferation and differentiation in BC3H1 muscle cells. II. Two signaling pathways distinguished by pertussis toxin and a potential role for the ras oncogene.
AU2773592A (en) An (in vitro) method of evaluating the effects of a substance
Swolin-Eide et al. Effects of cortisol on the expression of interleukin-6 and interleukin-1 beta in human osteoblast-like cells
Pederson et al. Evidence of a correlation of estrogen receptor level and avian osteoclast estrogen responsiveness
Volentine et al. Epidermal growth factor in the germinal disc and its potential role in follicular development in the chicken
CA2688480A1 (en) Use of fibroblast growth factor 7 (fgf7) and of the receptor fgfr2b as biomarkers
Shapiro et al. Down regulation of the muscarinic cholinergic receptor of the rat prostate following castration
Bartholomew et al. Decreased tyrosine phosphorylation of focal adhesion kinase after estradiol treatment of MCF-7 human breast carcinoma cells

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA FI JP NO US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL SE

WWE Wipo information: entry into national phase

Ref document number: 1992921468

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2120604

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 08211487

Country of ref document: US

WWE Wipo information: entry into national phase

Ref document number: 941611

Country of ref document: FI

WR Later publication of a revised version of an international search report
WWP Wipo information: published in national office

Ref document number: 1992921468

Country of ref document: EP

WWG Wipo information: grant in national office

Ref document number: 1992921468

Country of ref document: EP

WWG Wipo information: grant in national office

Ref document number: 941611

Country of ref document: FI

WWW Wipo information: withdrawn in national office

Ref document number: 1992921468

Country of ref document: EP