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
  • G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
  • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
• • 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
  • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
  • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • 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
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57407—Specifically defined cancers
• • 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
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57407—Specifically defined cancers
  • G01N33/57442—Specifically defined cancers of the uterus and endometrial
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/136—Screening for pharmacological compounds

Definitions

• the present invention relates to a method for qualitatively and/or quantitatively determining endometrial proliferation in a sample.
• the invention also relates to compositions or kits and arrays for qualitatively and/or quantitatively determining endometrial proliferation. Further, the invention relates to a method for the diagnosis of a disorder that is associated with a dysfunction of endometrial proliferation or a predisposition therefore in a subject.
• the endometrium is the mucous membrane of the cavity that lines the uterus.
• the endometrium thickens under hormonal control and, if pregnancy does not occur, is shed in menstruation and regenerates each menstrual cycle throughout the reproductive life of a female.
• the endometrium shows proliferation of its stroma and glands, the latter elongating.
• the cells lining the glands are cuboidal with definite limiting membranes and the stroma cells are thin and spindly, indicating the early proliferative phase.
• the glands are very large and are now dilated, indicating the late proliferative phase.
• the blood vessels are also prominent and capillaries are dilated.
• disordered endometrial proliferation may be associated with several gynecological diseases including endometriosis, adenomyosis, endometrial hyperplasia and gynecological cancer.
• the invention provides a method for identifying the stage of endometrial proliferation in a subject. This object is achieved according to the invention by providing a method for qualitatively and/or quantitatively determining endometrial proliferation in a sample, comprising determining the level of expression and/or activity of at least one gene which is modulated by estrogens and/or Selective Estrogen Receptor Modulators
• SERMs wherein said modulation of said genes correlates with the different potential of estrogens and/or SERMs to induce endometrial proliferation.
• Estrogens are steroid hormones essential for normal sexual development and functioning of female reproductive organs such as the endometrium. Estrogens also have important non-reproductive effects on bones and the heart. Estrogens comprise a group of natural and synthetic substances. Natural estrogens include estradiol (i.e. 17/?-estradiol), estrone and estriol. Particularly under the influence of 17/?- estradiol, the proliferation of the endometrium takes place. 17yff-estradiol induces the synthesis of its own receptors as well as the progesterone receptors.
• the endometrium proliferates in the first half of the cycle, i.e. in the proliferation phase, resulting in a certain thickness of the endometrium which reaches a maximum of 8 to 10 mm.
• estrogens are sometimes given therapeutically in the form of a conjugate, such as ethinyl estradiol for example, conjugated estrogens or diethylstilbestrol.
• Tissues in the body that are responsive to estrogens are called estrogen- sensitive” or ,,estrogen-responsive” tissues and include cells of the urogenital tract, cardiovascular system and skeletal system.
• the cells that comprise estrogen - sensitive tissues contain estrogen receptors (ER).
• ER can be of the alpha type or beta type.
• Estrogens enter cells and bind to ER in the cytoplasm of such cells and an estrogen-ER complex is formed.
• a molecule such as estrogen that binds to a receptor is termed a Jigand".
• the estrogen-ER complex migrates to the nucleus of the cell and binds to specific sequences of DNA within the cellular genome called ..estrogen response elements". Such estrogen response elements are located in the promoters of the specific genes in the cell nucleus.
• Binding of the estrogen-ER complex to estrogen-responsive elements causes activation or suppression of the transcription of the specific genes.
• the activation or suppression of specific gene transcription is one type of molecular and/or cellular response that can result from the formation of a ligand-receptor complex. When such a response occurs, the receptor is said to have been ..activated".
• Estrogen-ER complexes therefore, act as transcription factors to regulate the expression of these genes.
• a ligand binds to a receptor and a molecular and/or cellular response (e.g. transcriptional regulation of genes) occurs, such ligands are referred to as ..agonists" and the response produced is called M agonism".
• M agonism M agonism
• SERMs bind to the ER and prevent the molecular and/or cellular responses caused by estrogens.
• SERMs can also be called ,,anti-estrogens”.
• SERMs encompass ER ligands that produce different responses.
• one particular SERM may be an antagonist.
• Another particular SERM may be a partial agonist.
• Still another particular SERM may bind to ER and produce a molecular and/or cellular response that is only slightly less in magnitude than the response produced by estrogens.
• Such a SERM would result in a molecular and/or cellular response of a greater magnitude than the response to produced by a partial agonist, but would not be referred to as an agonist because the molecular and/or cellular response is less than that produced by an agonist-like estrogen.
• SERMs can be classified into three groups.
• the first group comprises triphenylethylene derivatives, of which tamoxifen, an anti-cancer agent, is one.
• Other substances that are triphenylethylene derivatives are toremifene, droloxifene, (3-hydroxytamoxifen), idoxifene, TAT-59 (a phosphorylated derivative of 4- hydroxytamoxifen) and GW5638 (a carboxylic acid derivative of tamoxifen).
• the second group of SERMs comprises other non-steroidal compounds. This group comprises EM-
• SERMs comprises steroidal compounds that have a better ability to inhibit the response produced by estrogens.
• ICE-182,780 ICE is a member of this third group.
• SERMs that correlated with the different potential of said estrogens and SERMs to induce endometrial proliferation. Therefore, measuring the levels of expression and/or activity of said certain genes provides a method for qualitatively and/or quantitatively determining endometrial proliferation in a sample.
• the estrogen is estradiol and the SERMs are tamoxifene and/or raloxifene.
• the genes which are modulated by estrogens and/or SERMs are mammalian genes, preferably human genes.
• the method of the present invention is contemplated for application in human, veterinary and/or diagnostic medicine.
• SERMs is selected from the group consisting of the genes anterior gradient 2 homolog
• the at least one gene is collagen, type I, alpha 1 (Gene ID NO: 1)
• the at least one gene is prostaglandin D2 synthase 21kDa
• the level of gene expression and/or activity of the at least one gene which is modulated by estrogens and/or SERMs is determined by measuring the transcript level of said genes. [26] Surprisingly, it was found that the transcript level of the transcripts (see example 1 ) is associated with the different estrogenic potency of estrogens and SERMs to induce endometrial proliferation.
• the determination of significance may be carried out as described in Example 1.
• the determination of significance may be carried out by a gene expression analysis of a sufficient number, e.g. of at least 500, preferably 500 to 700 probe sets (genes).
• the tissue from which the transcripts are obtained is preferably pituitary and uterus tissue.
• Methods for measuring the level of gene transcription comprise measuring the level of mRNA. RNA can be isolated from the samples by methods well known to those skilled in the art, for example as described in Molecular Cloning, A Laboratory Manual, Volume 1 , Chapter 7, pp 7.4 to 7.12, Cold Spring Harbor Laboratory Press, New York, 3 rd Edition (2001 ).
• the determination of the transcript level is carried out using a hybridization reaction.
• the hybridization reaction involves the hybridization of at least one oligonucleotide or polynucleotide to a transcript product, an mRNA.
• the determination further comprises an elongation and/or amplification reaction.
• the amplification comprises at least one technique selected from the group consisting of reverse transcriptase PCR and/or real time PCR.
• the oligonucleotide or polynucleotide are preferably of sufficient length to specifically hybridize to complementary transcripts of the above genes, according to the invention.
• the terms "olignucleotide” or “polynucleotide” refer to a single-stranded nucleic acid.
• the olignucleotide or polynucleotide will be at least 16 to 20 nucleotides in length, although in some cases longer probes of at least 20 to 25 nucleotides will be desirable.
• the olignucleotide or polynucleotide can be labelled with one or more labelling moieties to permit detection of the hybridized probe/target polynucleotide complexes.
• Labelling moieties can include compositions that can be detected by spectroscopic, biochemical, photochemical, bioelectronic, immunochemical, electrical, optical or chemical means. Examples of labelling moieties include, but are not limited to, radioisotopes, e.g. 32 P, 33 P, 35 S, chemiluminescent compounds, labelled binding proteins, heavy metal atoms, spectroscopic markers such as fluorescent markers and dyes, linked enzymes, mass spectrometry tags and magnetic labels.
• the level of gene expression is determined by measuring the level of a protein product of the at least one gene being modulated by estrogens and/or SERMs.
• the protein product is selected from the group consisting of the protein products with the corresponding accession numbers anterior gradient 2 homolog (NP 006399), chondroitin sulfate proteoglycan 2 (versican) (NP 004376), alpha 1 type I collagen preproprotein (NP_000079), alpha 2 type I collagen (NP_000080), alpha 1 type III collagen (NP_000081), alpha 3 type IV collagen isoform 1 , precursor (NP_000082), alpha 4 type IV collagen precursor (NP_000083), alpha 1 type IV collagen preproprotein (NP_001836), alpha 3 type IV collagen isoform 1 precursor (NP_004360), brain creatine kinase (NP 001814), cytoplasmatic dynein light polypeptide (NP_003737), glycer
• the protein product is alpha 1 type I collagen preproprotein
• NP_000079 alpha 2 type I collagen
• NP_000081 alpha 1 type III collagen
• NP_00181 brain creatine kinase
• NP_000945 prostaglandin D2 synthase 21kDa
• the protein product is prostaglandin D2 synthase 21kDa
• the protein product level is determined by enzymatic binding, immunological and/or physical methods, which are suitable for protein determination such as for example immunoassays or mass spectrometry.
• Expression of the at least one protein or a fragment thereof, e.g. catalytic domain, can be detected by a probe which is detectably labelled, or which can be subsequently labelled.
• the probe can be an antibody, an antibody derivative or an antibody fragment which is able to recognise the expressed protein.
• the term ..antibody includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, humanized or chimeric antibodies and biologically functional antibody fragments, which are those fragments sufficient for binding of the antibody fragment to the protein or a fragment thereof.
• various host animals may be immunized by injection with the polypeptide or a portion thereof.
• host animals may include, but are not limited to, rabbits, mice and rats, etc.
• adjuvants may be used to increase the immunological response, depending on the host species, including, but not limited to, Freund's (complete and incomplete), mineral gels such as aluminium hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol and potentially useful human adjuvants such as BCG (bacille Calnett-Guerfin) and Corinebacterium parvum.
• Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, such as a target product, or an antigenic functional derivative thereof.
• host animals such as those described above, may be immunized by injection with the encoded protein, or a portion thereof, supplemented with adjuvants as is also described above.
• Monoclonal antibodies which are homogenous populations of antibodies to a particular antigen, may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique of Kohler and Milstein (Nature, Vol. 256, pp. 495-497 (1975); and U.S. Patent No. 4,376,110), the human B-cell hybridoma technique (Kosbor et al., Immunology Today, Vol. 4, p. 72 (1983); Cole at al. Proc. Natl. Acad. Sci. USA, Vol. 80, pp.
• Such antibodies may be of any immunoglobulin class, including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
• the hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production.
• techniques developed for the production of ..chimeric antibodies (Morrison et al., Proc. Natl. Acad. Sci.
• a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable or hypervariable region derived from a murine mAb and a human immunoglobulin constant region.
• Antibody fragments which recognize specific epitopes may be generated by known techniques.
• such fragments include, but are not limited to, the F(ab') 2 fragments, which can be produced by pepsin digestion of the antibody molecule, and the Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
• Fab expression libraries may be constructed (Huse et al., Science, Vol. 246, pp. 1275-1281 (1989)) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
• the level of protein (fragment) expressed in a biological sample may then be determined by immunoassay methods which utilize the antibodies, antibody derivatives, or antibody fragments described above.
• immunoassay methods include, but are not limited to, Western blotting, fluorescence-activated cell sorting (FACS), immunohistochemistry, enzyme-linked immunosorbant assays (ELISA), enzyme linked immuno-spot assay (ELISPOT), dot blotting, competitive and noncompetitive protein binding assays, and other methods commonly used and widely described in scientific and patent literature, and many employed commercially.
• sandwich ELISA of which a number of variations exist, all of which are intended to be encompassed by the present invention.
• unlabelled antibody, antibody derivative or antibody fragment is immobilized on a solid substrate and the sample to be tested is brought into contact with the bound molecule and incubated for a period of time sufficient to allow formation of an antibody-antigen binary complex.
• a second antibody, antibody derivative, or antibody fragment labelled with a molecule capable of inducing a detectable signal is then added and incubated, allowing time sufficient for the formation of a ternary complex of antibody-antigen-labelled antibody.
• any unreacted material is washed away and the presence of the antigen is determined by observation of a signal or may be quantified by comparing with a control sample containing known amounts of antigen.
• Variations on the forward assay include the simultaneous assay in which both sample and antibody are added simultaneously to the bound antibody or a reverse assay in which the labelled antibody and sample to be tested are first combined, incubated and added to the unlabelled surface-bound antibody.
• reporter molecules for labelling an antibody, antibody fragment or derivative in this type of assay are either enzymes, fluorophore- or radionuclide-containing molecules.
• EIA enzyme immunoassay
• an enzyme is conjugated to the second antibody, usually by means of glutaraldehyde or periodate.
• glutaraldehyde or periodate an enzyme conjugated to the second antibody, usually by means of glutaraldehyde or periodate.
• Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, among others.
• the substrates to be used with the specific enzymes are generally chosen for the production of a detectable color change upon hydrolysis by the corresponding enzyme.
• p-nitrophenyl phosphate is suitable for use with alkaline phosphatase conjugates; for peroxidase conjugates, 1 ,2-phenylenediamine or toluidine are commonly used.
• fluorogenic substrates which yield a fluorescent product, rather than the chromogenic substrates noted above.
• a solution containing the appropriate substrate is then added to the tertiary complex.
• the substrate reacts with the enzyme linked to the second antibody giving a qualitative visual signal which may be further quantified, usually spectrophotometrically, to give an evaluation of the amount of protein or fragment thereof.
• fluorescent compounds such as fluorescein and rhodamine may be chemically coupled to antibodies without altering their binding capacity.
• the fluorochrome-labeled antibody When activated by illumination with light of a particular wavelength, the fluorochrome-labeled antibody absorbs the light energy, inducing a state of excitability in the molecule, followed by emission of the light at a characteristic longer wavelength. The emission appears as a characteristic color visually detectable with a light microscope.
• Immunofluorescence and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotopes, chemiluminescent or bioluminescent molecules may also be employed. It will be readily apparent to the skilled artisan how to vary the procedure to suit the required use.
• the sample which is subjected to the method according to the invention is a body fluid or tissue sample.
• the body fluid is blood, plasma, urine and/or se ⁇ jm.
• a further aspect of the invention relates to a composition or kit for qualitatively and/or quantitatively determining endometrial proliferation in a sample, comprising reagents for determining the level of gene expression and/or activity of at least one gene being modulated by estrogens and/or SERMs.
• the gene expression and/or activity of the at least one gene being modulated by estrogens and/or SERMs is selected from the group consisting of the genes as defined above.
• the composition or kit comprises reagents for determining the transcript level of at least one gene being modulated by estrogens and/or SERMs.
• said reagents for determining the transcript level comprise a hybridization probe and, optionally, primers for elongation and/or amplification.
• the hybdrization probe and primers are able to bind to a transcript of the above-mentioned genes.
• Another preferred embodiment of the invention comprises a composition or kit for qualitatively and/or quantitatively determining endometrial proliferation in a sample, comprising reagents for determining the level of a protein product encoded by at least one gene as defined above.
• the composition or kit comprises reagents for enzymatic, binding, physiological and/or immunological determination in order to determine the level of a protein product encoded by at least one gene as defined above. More preferably, the composition or kit comprises at least one antibody, antibody derivative or antibody fragment able to bind at least one protein product encoded by at least one gene as defined above. Preferably, said composition or kit comprises a monoclonal antibody as described in detail above.
• composition or kit further comprising a device for collecting a biological sample. More preferably, the composition or kit of the invention further comprises a device for collecting a biological sample of a subject and, in addition, may also comprise instructions for use of the kit and interpretation of the determined level of gene expression and/or activity and/or level of protein product.
• Still a further aspect of the invention relates to an array for qualitatively and/or quantitatively determining endometrial proliferation in a sample, comprising a carrier and a probe for determining the level of expression and/or activity and/or the transcript level of at least one gene being modulated by estrogens and/or SERMs in said sample.
• An array is a particularly useful method for detecting the level of mRNA transcripts obtained from a plurality of genes which involves hybridization of labelled mRNA to an ordered array of olignucleotides or polynucleotides. Typically, the olignucleotides or polynucleotides utilized in this hybridization method are bound to a solid support.
• solid supports include, but are not limited to, membranes, filters, slides, paper, nylon, wafers, fibres, magnetic or non-magnetic beds, gels, tubing, polymers, polyvinyl chloride dishes, etc. Any solid surface to which the olignucleotides or polynucleotides can be bound, either directly or indirectly, either covalently or non- covalently, can be used.
• probe arrays for expression monitoring can be prepared and used and techniques which are well known to those skilled in the art as described in the manuals of the Affymetrix company, for example. Such a method allows the level of transcription of a plurality of genes to be measured simultaneously to generate gene expression profiles or patterns.
• an array is a preferred method for determining the presence of at least one polymorphism in the genes as defined above.
• Such an array preferably comprises a carrier having immobilized thereto at least one probe for determining the presence of at least one polymorphism of a gene being modulated by estrogens and/or SERMs.
• the array carrier e.g. a planar carrier or a microchannel device, has immobilized thereto a plurality of different probes located at different areas of the carrier which are designed such that they can bind nucleic acid molecules, e.g. RNA molecules or DNA molecules, amplification products, primer elongation products, etc, containing the sequence in which the polymorphism to be tested is located.
• nucleic acid molecules e.g. RNA molecules or DNA molecules, amplification products, primer elongation products, etc.
• a further aspect of the invention relates to a method for the diagnosis of a disorder and/or condition that is associated with endometrial proliferation or a predisposition therefor in a subject, comprising determining the level of expression and/or activity of at least one gene as defined above in said sample, wherein a level of expression and/or activity substantially deviating from the reference value for a healthy subject is indicative of a dysfunction of endometrial proliferation or a predisposition therefor.
• disorder is defined as a pathological state of a mammal, preferably a human
• condition is defined as a non-pathological state of a mammal, preferably a human.
• a condition which is associated with endometrial proliferation is, for example, pregnancy or ovulation
• a disorder that is associated with endometrial proliferation is, for example, endometriosis or endometritis.
• a method for the diagnosis of a disorder according to the invention is associated with a dysfunction of endometrial proliferation.
• the present invention discloses for the first time genes which are differentially expressed in subjects having a dysfunction of endometrial proliferation or a predisposition therefor.
• the gene expression profile derived from the biological sample obtained from the subject can be compared with a gene expression profile derived from the sample obtained from a subject or subject population not affected by the dysfunction of endometrial proliferation or a predisposition therefor. It can thereby be determined whether the subject is affected or is at risk of being affected by a dysfunction of endometrial proliferation.
• the subject is a mammal, more preferably a human.
• the level of gene expression and/or activity is determined by measuring the transcript level of the at least one gene and/or the level of a protein product.
• the proteins codified by the above-mentioned genes can be used as biomarkers for endometrial proliferation, if measurable in body fluids.
• the disorder and/or condition associated with endometrial proliferation or a predisposition therefor is selected from the group consisting of endometrial atrophy, underdeveloped endometrium amenorrhea, endometritis, enhanced endometrial proliferation, endometriosis, disordered menstrual cycle, pregnancy, ovulation, infertility, endometrial hyperplasia, endometrial polyps and/or gynecological tumours.
• Endometrial atrophy is a disorder wherein the endometrium resists proper development.
• a typical cause for endometrial atrophy is long-term treatment with gestagens, causing a strong depletion of estrogen in the organism.
• the endometrium may be underdeveloped as a consequence of a failure of uterine development.
• menstrual abnormalities such as amenorrhea are associated with a dysfunction of endometrial proliferation.
• Amenorrhoea is defined as the absence of menstruation under a wide variety of circumstances. Under physiological conditions, amenorrhea occurs at four distinct phases of life: the early stage of the menarche, during pregnancy, lactation and following the menopause. Under pathological conditions, amenorrhea occurs as a cause of endometrial atrophy or an underdeveloped endometrium, for example. Then, conditions relating to the menstrual cycle such as pregnancy and ovulation are associated with endometrial proliferation.
• a further disorder associated with a dysfunction of endometrial proliferation is infertility, e.g. as a result of endometriosis or endometritis, which are further explained below.
• Another important disease associated with a dysfunction of endometrial proliferation according to the invention is endometriosis.
• Endometriosis is the second most common disease in women and is defined as the occurrence of endometrial cells outside the uterus. Endometriosis affects about 1 in 5 women of reproductive age and as many as 1 in 2 women with fertility problems. [65] Under normal circumstances, the endometrium is only found in the uterus. In endometriosis, tissue with a histological appearance resembling the endometrium is found outside the uterus, for example externally on the uterus, on the intestine or even in the pancreas or the lung. Although these endometriotic foci are located outside the uterus, they also bleed during menstruation, thus they are influenced by hormones of the female cycle.
• endometriotic foci like the endometrium, go through volume changes during the cycle, these changes may cause pain depending on location. Moreover, the body reacts to endometriotic cells and inflammatory response which again causes pain. Furthermore, inflammation leads to adhesions in the area of the ovaries and Fallopian tubes and, as a result, is responsible for the so-called mechanical sterility of affected women. Hence, however, in endometriosis, messengers are also released (e.g. cytokines, prostaglandins) which can reduce the fertility of affected women even in the absence of adhesions.
• messengers e.g. cytokines, prostaglandins
• endometriotic cells could be classified as being between normal cells and tumour cells: on the one hand they show no neoplastic behaviour, on the other hand, however, like metastasising tumour cells, they are capable of moving across organ boundaries in the organism and of growing into other organs, i.e. they show invasive behaviour. For this reason, endometriotic cells are defined as ..benign tumour cells" in the literature, although up until now no tumour- specific mutations in proto-oncogenes have been found in cells of this type.
• a further disorder associated with a dysfunction of endometrial proliferation or a predisposition therefor is endometritis. This disorder is defined as the acute inflammation of the endometrium, which may develop in response to infection following childbirth or abortion or in response to the insertion of a contraceptive device or as part of a gonorrheal infection.
• a further associated disorder according to the invention is the endometrial hyperplasia which is defined as an enhanced endometrial proliferation, particularly of the glandular epithelium.
• the disorder is sub-divided into grades I to III (atypical hyperplasia). Firstly, grade III is a precancerous condition of the endometrial carcinoma. Endometrial hyperplasia develops under the protracted influence of estrogens in the absence of gestagens (..unopposed estrogens"), mostly in the climacterium and the post-menopause.
• endometrial polyps occur mainly in climacterium in 10% of all women. Most polyps grow as circumscribed hyperplasia in the basalis of the endometrium. In patients with endometrial polyps the incidence of myoma is greater than that of the normal population. This is an indication of general proliferative activity in the uterus. Only in less than 1% of cases is a carcinoma found in an endometrial polyp. In endometrial polyps irregular or ongoing bleeding from the uterus can occur.
• Endometrial cancer is one of the most common gynecological cancers. It is usually found in post-menopausal women (75%), most frequently between the ages of 55 and 65 years and the majority, 60%, in the latter part of that span. The majority of tumours (60%) are pure adenocarcinomas. They can be divided into three groups according to the degree of glandular differentiation. In general, this cancer is slow to spread from the uterine cavity, probably because the endometrium lacks lymphatics.
• a further aspect of the invention relates to a method for screening for a modulator for a disorder that is associated with a dysfunction of endometrial proliferation or a predisposition therefor, comprising determining the level of expression and/or activity of at least one gene as defined above in a sample of cells in the presence of a candidate agent, the effect of which on endometrial proliferation is to be examined, compared to said level of expression and/or activity in the absence of said candidate agent.
• Such methods include in vivo or cell-based and/or cell-free assays to identify compounds which are capable of interfering with the expression and/or activity of at least one gene as defined above which is modulated by estrogens and/or SERMs.
• the invention also relates to the use of at least one gene and/or protein product as defined above as target for the development of a drug which is active against a disorder and/or condition that is associated with endometrial proliferation.
• Drug targeting is a strategy aiming at the delivery of a compound to a particular tissue of the body.
• the drugability of a given target is defined either by how well a compound, such as small molecule drugs or antibodies, can excess the target, or by the efficiency with which a compound can actually achieve the target.
• a long list of parameter influences drugability of a given target; these include cellular location, development of resistance, transport mechanisms such as export pumps, side effects, toxicity and others.
• a pharmaceutical composition preferably contains the active ingredient in combination with one or more pharmaceutically acceptable carriers, excipients and/or additives.
• Such a composition may be administered alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including but not limited to, saline, buffered saline, dextrose and water.
• the composition may be administered alone or in combination with other agents, drugs or hormones to a patient, wherein the level of the at least one gene and/or the level of the at least one protein product is substantially deviating from the reference for a healthy subject which is indicative for a dysfunction of endometrial proliferation or a predisposition therefor.
• the pharmaceutical compositions may be administered by any number of routes.
• compositions may be administered by any number of routes, including, but not limited to, oral, sublingual, intravenous, intramuscular, intraarticular, intraarterial, intramedullary, intrathecal, intraventricular, intraoccular, intrathecal, intracereberal, intracranial, respiratoral, intratracheal, nasopharyngeal, transdermal, intradermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, or via rectal means, infusion or implant.
• routes of administration is oral.
• the pharmaceutical composition of the present invention is administered in pharmaceutically acceptable preparations.
• pharmaceutically-acceptable carrier means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a mammals including humans.
• carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
• pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients.
• Such preparations may routinely contain pharmaceutically acceptable concentrations of salts, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents, such as chemotherapeutic agents.
• salts When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention.
• the pharmaceutical compositions may contain suitable buffering agents, including acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt.
• suitable buffering agents including acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt.
• compositions also may contain, optionally, suitable preservatives, such as benzalkonium chloride; chlorobutanol; parabens and thiomerosal.
• suitable preservatives such as benzalkonium chloride; chlorobutanol; parabens and thiomerosal.
• compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing the active agent into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
• compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the active compound.
• Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir or an emulsion.
• compositions suitable for parenteral administration conveniently comprise a sterile aqueous or non-aqueous preparation of a polypeptide or nucleic acid encoding the polypeptide, which is preferably isotonic with the blood of the recipient.
• This preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents.
• the sterile injectable preparation also may be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butane diol.
• acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
• sterile, fixed oils are conventionally employed as a solvent or suspending medium.
• any bland fixed oil may be employed including synthetic mono-or di-glycerides.
• fatty acids such as oleic acid may be used in the preparation of injectables.
• Carrier formulation suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington's Pharmaceutical
• a further aspect of the invention encompasses the use of a modulator of at least one gene as defined above for the manufacture of a medicament for the prevention and/or treatment of a disorder and/or condition that is associated with endometrial proliferation.
• the modulator of the pharmaceutical composition is an estrogen and/or a Selective Estrogen Receptor Modulator (SERM), an antagonist and/or agonist thereof such as an antibody or an antibody fragment, a siRNA (small interfering RNA), a miRNA (microRNA), an antisense molecule and/or a ribozyme.
• SERM Selective Estrogen Receptor Modulator
• Agonists of proteins or hormones are substances such as enzymes, coenzymes, membrane receptors etc., which increase the activity of the respective protein or hormone.
• antibodies which are specific for the protein products of the invention and homologous protein products may be used directly as a modulator, e.g. an antagonist or an agonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which express the protein.
• the antibodies may be generated using methods that are well-known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric single chain, Fab fragments and fragments produced by a Fab expression library. Neutralizing antibodies (i.e. those which inhibit dimer formation) are especially preferred for therapeutic use.
• peptides, fragments or oligopeptides used to induce antibodies to the protein products have an amino acid sequence consisting of at least five amino acids, and more preferably at least 10 amino acids.
• Monoclonal antibodies to the protein products may be prepared using any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (K ⁇ hler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81 :31-42; Cote, R. J. et al. Proc. Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al. (1984) MoI. Cell Biol. 62:109-120).
• Antibodies with related specificity, but of distinct idiotypic composition may be generated by chain shuffling from random combinatorial immunoglobulin libraries (Burton, D. R. (1991) Proc. Natl. Acad. Sci. 88:11120-11123). Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).
• Antibody fragments which contain specific binding sites for the protein products may also be generated.
• fragments include, but are not limited to, the F(ab') 2 fragments which can be produced by Pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of F(ab') 2 fragments.
• Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281 ).
• Various immunoassays may be used for screening to identify antibodies having the desired specificity.
• polynucleotides or fragments thereof or nucleic acid modulator molecules such as antisense molecules, aptamers, siRNA molecules, miRNA molecules or ribozymes may be used for therapeutic purposes, in one aspect, aptamers, i.e. nucleic acid molecules, which are capable of binding to a protein product of the invention and modulating its activity, may be generated by a screening and selection procedure involving the use of combinatorial nucleic acid libraries.
• antisense molecules may be used in situations in which it would be desirable to block the transcription of the mRNA.
• cells may be transformed with sequences complementary to polynucleotides encoding the protein products as defined above and homologous protein products.
• antisense molecules may be used to modulate protein product activity or to achieve regulation of gene function.
• sense or antisense oligomers or larger fragments can be designed from various locations along the coding or control regions of sequences encoding the protein products.
• Expression vectors derived from retroviruses, adenovirus, herpes or vaccinia viruses or from various bacterial plasmids may be used for delivery of nucleotide sequences to the targeted organ, tissue or cell population. Methods, which are well known to those skilled in the art, can be used to construct recombinant vectors, which will express antisense molecules complementary to the polynucleotides of the genes encoding the proteins of the invention and homologous protein products. These techniques are described both in Sambrook et al. (supra) and in Ausubel et al. (supra).
• Genes encoding the protein products of the invention and homologous protein products can be turned off by transforming a cell or tissue with expression vectors, which express high levels of polynucleotides that encode the protein products of the invention and homologous protein products or fragments thereof.
• Such constructs may be used to introduce untranslatable sense or antisense sequences into a cell. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until they are disabled by endogenous nucleases. Transient expression may last for a month or more with a non-replicating vector and even longer if appropriate replication elements are part of the vector system.
• modifications of gene expression can be obtained by designing antisense molecules, e.g. DNA, RNA or nucleic acid analogues such as PNA, to the control regions of the genes encoding the above defined protein products and homologous protein products, i.e., the promoters, enhancers, and introns.
• Oligonucleotides derived from the transcription initiation site e.g., between positions -10 and +10 from the start site, are preferred.
• inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it cause inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors or regulatory molecules.
• the antisense molecules may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
• Ribozymes enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
• Examples which may be used, include engineered hammerhead motif ribozyme molecules that can be specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding the protein products of the invention and homologous protein products.
• Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for secondary structural features which may render the oligonucleotide inoperable.
• the suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
• Nucleic acid modulator molecules e.g. antisense molecules and ribozymes may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis.
• RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences. Such DNA sequences may be incorporated into a variety of vectors with suitable RNA polymerase promoters such as T7 or SP6.
• these cDNA constructs that synthesize antisense RNA constitutively or inducibly can be introduced into cell lines, cells or tissues. RNA molecules may be modified to increase intracellular stability and half-iife.
• flanking sequences at the 5' and/or 3' ends of the molecule or modifications in the nucleobase, sugar and/or phosphate moieties, e.g. the use of phosphorothioate or 2 1 O-methyl rather than phosphodiesterase linkages within the backbone of the molecule.
• vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection and by liposome injections may be achieved using methods, which are well known in the art. Any of the therapeutic methods described above may be applied to any suitable subject including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
• EXAMPLE EXAMPLE
• OWM pelleted diet Dietex France, SDS, Saint Gratien, France
• Ovariectomy was performed by standard surgical protocols at CIT (Evreux, France) 10 weeks prior to the first day of treatment. Ovaries were removed following anesthesia with a combined intramuscular injection of xylazine (Rompun®: 0.4 mL/animal, Bayer Pharma Division Sante Animale, Puteaux, France) and ketamine hydrochloride (Imalgene®: 0.6 ml_/kg, Merial, Lyon, France). Sham-operated animals were subjected to the same surgical procedure, except for the removal of ovaries.
• Estradiol serum levels were determined in each animal approximately 2 weeks after surgery to verify the effect of ovariectomy. Venous blood samples (approximately 1.5 mL) of non-fasted animals were collected in tubes without anticoagulant and analyzed using a radioimmunoassay (Sorin, autoimmune Nationale Veterinaire de Lyon, France). In addition, an estradiol assay was performed at approproximately 2 and 4 weeks after initiating drug treatments to determine if any animals had developed ectopic ovarian tissue growth; any such animals were removed from the study and subsequent analysis.
• Ovariectomized and sham-operated animals were divided into groups of 4. Sham-operated and one group of OVX animals received only vehicle, while the remaining groups of OVX monkeys received estradiol (Sigma No. E8875), or tamoxifen (Tamofene®, Aventis Pharma), or raloxifene (Evista®, Lilly France SA).
• the test items were prepared as suspensions in vehicle and administered daily by oral gavage. On the final day of treatment, animals were sacrificed by deep anesthesia induced by intramuscular ketamine hydrochloride and intravenous sodium pentothal followed by exsanguination.
• RNA was obtained by acid guanidinium isothiocyanate-phenol- chloroform extraction (Trizol; Invitrogen Life Technologies, San Diego, CA, USA) and purified on an affinity resin column (RNeasy; Quiagen, Hilden, Germany) according to manufacturer instructions. DNA microarray experiments were conducted as recommended by the manufacturer of the GeneChip system (Affymetrix, Inc. 2002). Prior microarray studies have proven the validity of cross-species DNA chip analysis, therefore the human gene expression probe arrays HGU133A (Affymetrix Inc., Santa Clara, CA, USA) containing 22,283 probe sets interrogating primarily annotated human genes were used. One GeneChip was used per tissue, per animal.
• the resulting image files (.dat files) were processed using the Microarray Analysis Suite 5 (MAS5) software (Affymetrix, Inc.). Tab-delimited files were obtained containing data regarding signal intensity (Signal) and categorical expression level measurement (Absolute Call).
• MAS5 Microarray Analysis Suite 5
• Tab-delimited files were obtained containing data regarding signal intensity (Signal) and categorical expression level measurement (Absolute Call).
• transcripts which were found to be differentially expressed are indicated by reference by the Affymetrix Probe Set IDs 209173_at, 221731_x_at, 202310_s_at, 202403_s_at, 201852_x_at, 215076_s_at, 211161_s_at, 211980_at, 201438_at, 200884_at, 200703_at, 217398_x_at, 213453_x_at, 212581_x_at, 201841_s_at, 221011_s_at, 203570_at, 209014_at, 202593_s_at, 202465_at, 211663_x_at, 204051_s_at, 207714_s_at, 206116_s_at and/or 211959_at.
• OVX group the ovariectomized animals
• OVX-EE estradiol-treated OVX
• the covariance matrix of the Iog10-transformed gene expression (GEP) data of the OVX and OVX-EE animals were subjected to a principal component analysis (PCA), a statistical dimensional reduction method that has been used previously for describing microarray gene expression data.
• PCA principal component analysis
• the PCA algorithm calculates the so- called "loadings” and "scores".
• the loadings are the weights in the linear combinations of the variables (genes), which define the axes (Principal Components, PCs) of the coordinate system.
• the scores represent the coordinates of the individual animals based on their GEP data in the same coordinate system.

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