EP1301195A2 - Pharmaceutical use of fibulin-1 - Google Patents

Pharmaceutical use of fibulin-1

Info

Publication number
EP1301195A2
EP1301195A2 EP01939374A EP01939374A EP1301195A2 EP 1301195 A2 EP1301195 A2 EP 1301195A2 EP 01939374 A EP01939374 A EP 01939374A EP 01939374 A EP01939374 A EP 01939374A EP 1301195 A2 EP1301195 A2 EP 1301195A2
Authority
EP
European Patent Office
Prior art keywords
fibulin
endometriosis
polypeptide
nucleic acid
pharmaceutical composition
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.)
Withdrawn
Application number
EP01939374A
Other languages
German (de)
French (fr)
Inventor
Holger Hess-Stump
Bernard Haendler
Bruce Lessey
Kristof Chwalisz
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.)
Bayer Pharma AG
University of North Carolina at Chapel Hill
University of North Carolina System
Original Assignee
Schering AG
University of North Carolina at Chapel Hill
University of North Carolina System
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
Application filed by Schering AG, University of North Carolina at Chapel Hill, University of North Carolina System filed Critical Schering AG
Publication of EP1301195A2 publication Critical patent/EP1301195A2/en
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the invention relates to the pharmaceutical use of a fibulin-1 polypeptide and a fibulin-1 nucleic acid.
  • Endometriosis is one of the most common gynecological diseases, of which approximately 5-10% of all women of reproductive age are affected (Sillem, M. 1998; Programmed (R) 23, Suppl . 1, 1-28) .
  • IVF in vitro fertilization
  • endometriosis suffer from endometriosis (Adamson, G. D. 1997; Sem. Reprod. Biol. 15, 263-271).
  • the reasons for sterility are unknown despite intensive research (Ryan, I. P.; Taylor, R. N. 1997; Obstet. Gynecol. Surv. 52, 365-371; Haney, A.
  • endometriosis is based on the concept of estrogen removal; a causal therapy is not available.
  • an invasive intervention in which endometriosis foci are removed or obliterated during a laparoscopy or laparotomy, generally a medicinal treatment is associated that by the induction of a hypoestrogenism results in an at least partial inhibition of the proliferation of remaining endometriosis foci that cannot be detected (Sillem, M. 1998; Programmed' 10 23, Suppl. 1, 1-28).
  • Examples of medications that are used are (i) danazol, a testosterone derivative, which is administered at a very high dosage (400-800 mg/day) and based on its androgenism results in numerous side effects, (ii) gestagens, which inhibit the ovarian estrogen synthesis and result in a high rate of breakthrough bleeding, and (iii) GnRH- agonists, which produce an inhibition of the ovarian estrogen synthesis and can result in menopausal symptoms and an osteopenia.
  • the problem is therefore to identify genes that are associated with the image of disease of endometriosis.
  • the association of the fibulin-1 gene with the image of disease of the endometriosis was identified by comparison of various patient groups.
  • PCR ddPCR
  • differential display method the mRNA from the endometrium of three clinical groups was compared to one another :
  • LH plus 4 days 4 days after triggering ovulation by shaking out the luteinizing hormone (LH) , the endometrium is still not receptive for an embryo.
  • This group consisted of patients who participated in an IVF program because of a tubal obstruction or sterility of the male and have a normal endometrium.
  • the mRNA of the fibulin-1 is ramped up at the time of the open implantation window in healthy women (group 2), while in women with endometriosis (group 3) , no ramping up can be measured at the same point of the cycle (FIGS. 2 and 3) .
  • Fibulin-1 is a protein that comes in four different forms that are referred to as fibulin-lA to ID and that represent different splice variants of the fibulin-1 gene.
  • Fibulin-lA is the shortest form.
  • Fibulin-IB, -1C and -ID each have an additional but different C-terminal domain.
  • the DNA- and protein sequences are known (FIGS. 4 to 11 and subsequent references: WO91/02755, Argraves, . S. et al . 1990; J. Cell. Biol. Ill, 3155-3164, Tran, H. et al . 1996; 15, 479-493).
  • Fibulin-1 is known as a component of the extracellular matrix.
  • the invention relates to a pharmaceutical composition that as active components contains a. a fibulin-1 nucleic acid, b. a vector that contains a fibulin-1 nucleic acid, c. a cell that contains a fibulin-1 nucleic acid, d. a fibulin-1-antisense nucleic acid, e. a fibulin-1 polypeptide or f . an antibody against a fibulin-1 polypeptide.
  • Fibulin-1 nucleic acid comprises both DNA, cDNA and RNA of all 4 splice variants of fibulin-1 or parts thereof.
  • the DNA and protein sequences are depicted in FIGS. 4 to 11.
  • the fibulin-1 nucleic acid is preferably a DNA.
  • the fibulin-lC and -ID splice variants are preferred; and the fibulin-lD splice variant is especially preferred.
  • the human sequences are preferred.
  • Vectors can be procaryotic or eucaryotic vectors.
  • Examples of vectors are pPRO (Clontech) , pBAD (invitrogen) , pSG5 (stratagene) , pCl (Promega) , pIRES (Clontech) , pBAC (Clontech) , pMET (invitrogen) , pBlueBac (Invitrogen) .
  • the fibulin-1 nucleic acid can be inserted into these vectors with the methods that are known to one skilled in the art.
  • the fibulin- 1 nucleic acid is preferably connected to the vector with expression signals such as, e.g., promoters and enhancers.
  • cells e.g., E. coli, yeast, Pichia, Sf9, COS, CV-1 or BHK can be used. With commonly used methods, the cells can be transformed with a vector that contains the fibulin-1 nucleic acid.
  • Fibulin-1-antisense nucleic acid is a DNA and/or RNA that is complementary to a fibulin-1 mRNA. It can comprise the entire complementary sequence or partial sequences.
  • Fibulin-1 polypeptides are defined as the entire sequence as well as portions of all four splice variants. Preferred are the polypeptides of fibulin-lC and -ID splice variants; especially preferred is the polypeptide of the fibulin-lD splice variants.
  • An antibody against a fibulin-1 polypeptide can be monoclonal or polyclonal . It can be directed against the entire fibulin-1 polypeptide or against fragments thereof. Obtaining such an antibody is carried out according to standard methods by immunization of test animals.
  • compositions of the invention are produced with commonly used solid or liquid vehicles or diluents and the commonly used pharmaceutical and technical adjuvants that correspond to the desired type of administration with a suitable dosage in a way that is known in the art.
  • Tablets can be obtained, for example, by mixing the active ingredient with known adjuvants, for example inert diluents such as dextrose, sugar, sorbitol, mannitol, polyvinylpyrrolidone, explosives such as corn starch or alginic acid, binders such as starch or gelatin, lubricants such as carboxypolymethylene, carboxymethylcellulose, cellulose acetate phthalate or polyvinyl acetate.
  • Capsules that contain active ingredients can be produced, for example, by the active ingredient being mixed with an inert vehicle such as lactose or sorbitol and encapsulated in gelatin capsules.
  • compositions according to the invention can also be used in suitable solutions, such as, for example, physiological common salt solution.
  • oily solutions such as, for example, solutions in sesame oil, castor oil, and cottonseed oil
  • solubilizers such as, for example, benzylbenzoate or benzyl alcohol, can be added.
  • the pharmaceutical composition according to the invention can be used for the treatment or prevention of endometriosis. This can take place by the use of gene therapy.
  • a vector that contains a fibulin-1 nucleic acid is designed and administered. Examples are vectors that are derived from adenovirus, adenovirus-associated virus, Herpes simplex virus or SV40.
  • the gene therapy can be implemented according to a protocol as described by Gomez-Navarro et al . (Eur. J. Cancer (1999) 35, 867-885) .
  • the administration can be done locally, i.e., directly in the uterus, or systemically, i.e., via the circulation.
  • fibulin-1 polypeptide in the endometrium. As a result, the biological function of the fibulin is restored.
  • Another possibility for the treatment and prevention of endometriosis is the administration of a fibulin-1 polypeptide, either as a complete protein or portions thereof that are biologically active.
  • the fibulin-1 polypeptide can be produced in a recombinant manner.
  • the invention relates to the use of a pharmaceutical composition according to the invention for the diagnosis of endometriosis.
  • blood or endometrial samples are removed from women in whom endometriosis is expected.
  • the amount of fibulin-1 polypeptide can be determined by an immune test.
  • antibodies against a fibulin-1 polypeptide or against fragments thereof are produced, and the latter then are used in an ELISA (enzyme-linked-immunosorbent assay) , in an RIA (radioimmunoassay) or in the immune histochemistry to determine the amount of fibulin-1 polypeptide.
  • RNA from endometrial samples is purified, and the amount of fibulin-1 mRNA is measured by Northern blot, PCR or chip- hybridization.
  • fibulin-1 nucleic acids or fragments are present on special nucleic acid chips.
  • the amount of fibulin-1 nucleic acid can also be determined by in situ hybridization with fibulin-1-antisense RNA.
  • the antisense-RNA can be labeled with digoxigenin, 32 P or 33 P.
  • the pharmaceutical composition according to the invention can also be used for the treatment of female sterility.
  • a blood or endometrial sample is removed from patients who desire a treatment because of their sterility, and the amount of fibulin-1 is determined as described above. If the sterility is associated with a disorder of the fibulin-1 expression, fertility can be increased by increasing the amount of fibulin-1 at the time of the implantation window.
  • An increase of the amount of fibulin-1 can be achieved as in the treatment of endometriosis by gene therapy or by administration of the fibulin-1 polypeptide.
  • An increase of the amount of fibulin-1 is also especially desirable in the method of in vitro fertilization to raise the success rate, i.e., the pregnancy rate .
  • Another subject of this invention is the use of a pharmaceutical composition according to the invention for female birth control.
  • Many endometriosis patients are sterile.
  • This invention shows that in endometriosis, the regulation of the fibulin-1 is disrupted.
  • This effect can also be used in reverse by fibulin-1 being artificially adjusted downward in healthy women who do not wish to become pregnant.
  • This can be carried out by the use of antisense RNA, which inhibits the expression of the fibulin-1 gene, or by the use of antibodies that are directed against a fibulin-1 polypeptide and inhibit the biological function of the fibulin-1.
  • the antibodies can be administered, e.g., in utero, and prevent successful detection by the blocking of fibulin-1 and thus the implantation of the embryo.
  • This invention also relates to the use of a pharmaceutical composition according to the invention for detecting uterine receptivity.
  • Uterine receptivity refers to the probability with which a pre-implantation embryo can become implanted in the endometrium of the uterus. Uterine receptivity is greatest at the time of the open implantation window. Since this invention shows that at the time of the open implantation window the fibulin-1 expression is ramped up, the time of high uterine receptivity can be determined by measuring the amount of fibulin-1 mRNA or fibulin-1 polypeptide. The measurement is carried out as described above in the diagnosis of endometriosis by a Northern blot, PCR or chip-hybridization or by an immune test.
  • the determination of uterine receptivity is especially important in the in vitro fertilization. Here, it is important to administer the fertilized ovocyte at the right time.
  • the invention relates to a method for the detection of uterine receptivity, whereby the amount of fibulin-1 polypeptide and/or the amount of fibulin-1 nucleic acid is determined with the aid of a pharmaceutical composition according to the invention. First, a blood or endometrium sample is removed from the women, and then the amount of fibulin-1 mRNA or fibulin-1 polypeptide is determined.
  • This invention also relates to the use of a pharmaceutical composition according to the invention for identifying agonists and/or antagonists of a fibulin-1 polypeptide.
  • Agonists and antagonists are substances that influence the biological activity of the fibulin-1 polypeptide either by binding to the fibulin-1 polypeptide or by competition with the fibulin-1 polypeptide in its binding to its physiological binding partners.
  • Such agonists or antagonists can be low-molecular substances or else peptides or proteins, such as, e.g., antibodies.
  • a cell that expresses the fibulin-1 polypeptide, with potential agonists or antagonists is treated, and the cell differentiation (Gaetje, R. et al .
  • Fibulin-1 antagonists can be used for, e.g., birth control.
  • Fibulin-1 agonists can be used wherever a high fibulin-1 activity is desired. This is the case in, e.g., the treatment of endometriosis and sterility.
  • a fibulin-1 agonist could be added instead of the fibulin-1 nucleic acid or the fibulin-1 polypeptide.
  • FIG. 1 shows the DNA sequence (SEQ ID NO: 1) of the regulated cDNA clone, which was identified by the ddPCR method and made to coincide with the 3 ' -end of the fibulin-lD.
  • FIG. 2 shows the detection by the "RNAase protection" method of the ramping up of the human fibulin-ID gene in the LH+8/fertile group.
  • a radioactive antisense RNA-probe was generated by the clone described in FIG. 1 and hybridized with RNA from the three groups: LH+4/fertile (early secretory), LH+8/fertile (midsecretory) , and LH+8/endometriosis (midsecretory and endometriosis) .
  • An antisense-actin probe was generated as an internal control . After digestion of the non- hybridized single-strand RNA, the reaction products were separated on a polyacrylamide gel and visualized by autoradiography.
  • FIG. 3 shows the quantification of the results of FIG. 2 (RNAase protection method) .
  • the strips of double-strand fibulin-ID and actin RNA were "scanned, " and the measured amount was normalized to the amount of actin.
  • FIG. 4 shows the DNA sequence (SEQ. ID NO : 2) for the human fibulin-1A.
  • FIG. 5 shows the protein sequence (SEQ ID NO : 3) for the human fibulin-lA.
  • FIG. 6 shows the DNA sequence (SEQ. ID NO: 4) for the human fibulin-IB.
  • FIG. 7 shows the protein sequence (SEQ. ID NO: 5) for the human fibulin-IB.
  • FIG. 8 shows the DNA sequence (SEQ. ID NO: 6) for the human fibulin-1AC.
  • FIG. 9 shows the protein sequence (SEQ ID NO: 7) for the human fibulin-lC.
  • FIG. 10 shows the DNA sequence (SEQ ID NO : 8) for the human fibulin- ID.
  • FIG. 11 shows the protein sequence (SEQ ID NO: 9) for the human fibulin-ID.
  • FIG. 12 shows the tissue distribution of the human fibulin- 1D in a Northern blot .
  • a radioactive DNA probe was generated by the fibulin-ID-specific portion and hybridized with human "Multiple Tissue Northern” (Clontech) .
  • the expression pattern of cyclophilin A was determined as an internal control.
  • FIG. 13 shows the DNA sequences (SEQ ID NO: 10) and the derived protein sequences that code for the C-terminal end of the rat fibulin-ID protein (corresponds to amino acid 576 to 696 in the human fibulin-ID) .
  • FIG. 14 shows the expression of the rat fibulin-1 mRNA in the uterus tissue from various days of the female cycle.
  • a sense primer and an antisense primer which detect all fibulin- 1 forms, or specific primers for the fibulin-IC or the fibulin- 1D isoforms were ' used for the PCR amplification.
  • Cyclophilin A (CyPA) -specific primers were used in the control experiment.
  • first-strand cDNA from the rat uterus tissue from diestrus stage (D) , proestrus stage (P) , estrus stage (0) or metestrus stage (M) was used.
  • the PCR products were separated on an agarose gel and then colored with ethidium bromide. The intensity of the strips was quantified with the GelDoc 2000 device (Hercules, CA, USA) .
  • FIG. 15 shows the expression of the rat fibulin-1 mRNA in the uterus tissue from various days post-coitus.
  • a sense primer and an antisense primer which detect all fibulin-1 forms or specific primers for the fibulin-IC or the fibulin-ID isoforms were used for the PCR amplification.
  • Cyclophilin A (CyPA) -specific primers were used in the control experiment.
  • templates first-strand cDNA from the rat uterus tissue of days 1 to 5 post-coitus (p.c.) was used.
  • the PCR products were separated on an agarose gel and then colored with ethidium bromide. The intensity of the strips was quantified with the GelDoc 2000 device. Examples
  • RNAse protection e.g., isolation of RNA, cloning of DNA, sequencing of DNA, RNAse protection, Northern blot analysis, polymerase-chain reaction (PCR) were performed as described in known textbooks, such as, e.g., in Molecular Cloning, A Laboratory Manual (Sambrook, J. et al . 1989; Cold Spring Harbor Laboratory Press) . Methods for subtraction analyses of the gene expression are described in, e.g., Liang, P. and Pardee, A. B. 1995; Curr. Opin. Immunol. 7, 274-280.
  • Example 1 Isolation of gene transcripts that are regulated during the implantation window by the ddPCR method
  • the RNAmapTM kit of the GenHunter Corporation was used for the ddPCR reactions.
  • the sense primers AP-1 to AP-5 and the antisense primer T 12 MA were used for the amplifications in the presence of ⁇ - [ 35 S] dATP.
  • the products were separated on a polyacrylamide gel and compared. Overall, 24 strips that have a different expression pattern in at least two separate tests were further processed.
  • DNA fragments were amplified once more by PCR (GeneAmpTM kit, Perkin Elmer Cetus) , purified on agarose gel and subcloned in the Srfl position of the pCR-ScriptTM Amp SK(+) plasmid (stratagene) . Then, the plasmid-DNA was produced in larger amounts, the size of the insert was verified, and the sequence was determined. A sequence comparison with bioinformatic processes was then performed to detect homologies with already known sequences .
  • PCR GeneAmpTM kit, Perkin Elmer Cetus
  • Antisense RNA probes were generated from the clones isolated in Example 1.
  • the T7 or T3 promoter was used as a function of the orientation of the insert.
  • the residual double-strand RNA molecules were separated on a polyacrylamide gel and visualized by autoradiography.
  • the expression pattern was compared between the three groups and also with the results of the ddPCR method.
  • the clone, which coded for the fibulin- 1D, showed the most advantageous expression pattern (Figs. 1 to 3) .
  • Example 3 Tissue distribution of the huma fibulin-lD (Northern blot)
  • the following primers were used for the amplification of the C-terminal coding area of the human fibulin-ID: 5'- CTCCAGCAGGAGAAGACAGAC-3 ' (SEQ ID NO: 12) and 5'- GAACCAGTACTCAGAGACGAAG-3 ' (SEQ ID NO: 13).
  • a "template” human liver cDNA (QUICK-CloneTM, Clontech) was used. The PCR- amplification was carried out according to standard conditions. After subcloning in the pCR-TopoTM plasmid (invitrogen) , the insert was sequenced, and one hundred percent correspondence with the already published sequence of fibulin-ID was found
  • a template cDNA from the rat brain (QUICK-CloneTM, Clontech) was used. The PCR-amplification was carried out under standard conditions. After subcloning in the pCR-TopoTM plasmid (invitrogen) , the DNA-insert was sequenced (FIG. 13) . The derived protein sequence showed 87.5% identity with that of the human fibulin-ID. Differences between the rat sequence and the human sequence are emphasized (FIG. 13) .
  • Example 5 Expression of rat fibulin during the female cycle and on days 1-5 post-coitus
  • the expression was analyzed by semi-quantitative PCR. To this end, the following primers were used:
  • Antisense 5 ' -TGCAGAGCGGCGATAGTTC-3 ' (SEQ ID NO: 17).
  • Antisense 5 ' -AGCGGTGATGGCCAGCTG-3 ' (SEQ ID NO: 19).
  • Antisense primer 5 ' -AACTTTGCAATCCTGCTAGAC-3 ' (SEQ ID NO 23).
  • Total-RNA was extracted from uterus tissue using Totally RNA Isolation Kits (Ambion, Austin, TX, USA) .
  • the cDNA was produced with the ProSTAR First-Strand RT-PCR Kit (Stratagene, La Jolla, CA, USA) .
  • the PCR analysis was performed with the Ampli Taq DNA Polymerase PCR kit (Perkin Elmer Cetus, Norwalk, CT, USA) .
  • the reaction conditions were as follows: Initially 94°C for 5 minutes; then 32 (fibulin-1) or 25 (cyclophilin A) cycles with 94°C for 20 seconds, 60°C for 20 seconds and 72°C for 40 seconds, finally 72°C for 10 minutes.
  • FIG. 14 show a cycle-dependent expression of rat fibulin-1.
  • the expression of fibulin-1, but also the IC and ID isoforms are weak during the diestrus and then increase to reach a maximum during the metestrus .
  • FIG. 15 shows that the expression of fibulin-1 post coitus remains unchanged. The largest amount was measured on day 3 p.c. Also here, the IC and ID isoforms show a pattern similar to fibulin-1.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Reproductive Health (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Endocrinology (AREA)
  • Zoology (AREA)
  • Gynecology & Obstetrics (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Epidemiology (AREA)
  • Pregnancy & Childbirth (AREA)
  • Urology & Nephrology (AREA)
  • Toxicology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

This invention relates to the pharmaceutical use of the fibulin-1 polypeptide and the fibulin-1 nucleic acid in female birth control and for the treatment and diagnosis of endometriosis.

Description

Pharmaceutical Use of Fibulin-1
The invention relates to the pharmaceutical use of a fibulin-1 polypeptide and a fibulin-1 nucleic acid.
Endometriosis is one of the most common gynecological diseases, of which approximately 5-10% of all women of reproductive age are affected (Sillem, M. 1998; Programmed(R) 23, Suppl . 1, 1-28) . In addition to pains and numerous other symptoms, many endometriosis patients are sterile, and a large portion of the IVF patients (IVF = in vitro fertilization) suffer from endometriosis (Adamson, G. D. 1997; Sem. Reprod. Biol. 15, 263-271). The reasons for sterility are unknown despite intensive research (Ryan, I. P.; Taylor, R. N. 1997; Obstet. Gynecol. Surv. 52, 365-371; Haney, A. F. 1997; Reprod. Med. Rev. 6, 145-161) . In addition to the unsettled connection between endometriosis and sterility, the etiology of endometriosis is also unknown. There are several theories (Van der Linden, P. J. Q., 1996; Hum. Reprod. 11, Suppl. 3, 53-65), but none is proven. A number of processes could play an important role in the development and/or persistence of the disease, such as, e.g., inflammations. In the literature, there are numerous citations that inflammation mediators are present in the peritoneal fluid of women with endometriosis in significantly altered concentrations (Vinatier, D.; Dufour, P.; Oosterlynck, D. 1996; Hum. Reprod. Update 2, 371-384) . In addition, in many cases an increased angiogenesis is observed, as is known in a similar way from tumors. Indeed, higher concentrations of angiogenetic growth factors were found in the peritoneal fluid of women with endometriosis (Taylor, R. N. et al. 1997; Ann. N. Y. Acad. Sci. 828, 194-207). Another analogy to tumors consists in the fact that endometriosis cells can be invasive, which can be attributed to the loss of a protein that is located on the surface of cells and is important for cell- cell adhesion (Starzinski-Powitz, A. et al . 1998; Hum. Reprod. Update 4, 724-729) . Recently, the number of publications that correspond to a genetic predisposition in the development of an endometriosis was increasing (Kennedy, S., 1997; Sem. Reprod. Biol. 15, 309-318) . The loss of tumor suppressor molecules and family clusters was thus described.
The current treatment of endometriosis is based on the concept of estrogen removal; a causal therapy is not available. According to an invasive intervention, in which endometriosis foci are removed or obliterated during a laparoscopy or laparotomy, generally a medicinal treatment is associated that by the induction of a hypoestrogenism results in an at least partial inhibition of the proliferation of remaining endometriosis foci that cannot be detected (Sillem, M. 1998; Programmed'10 23, Suppl. 1, 1-28). Examples of medications that are used are (i) danazol, a testosterone derivative, which is administered at a very high dosage (400-800 mg/day) and based on its androgenism results in numerous side effects, (ii) gestagens, which inhibit the ovarian estrogen synthesis and result in a high rate of breakthrough bleeding, and (iii) GnRH- agonists, which produce an inhibition of the ovarian estrogen synthesis and can result in menopausal symptoms and an osteopenia.
All three substance groups have proven effective for an initial treatment of endometriosis, but have a whole series of side effects and up until now could not contribute to a reduction in the high recurrence rate.
A need for new treatment methods that are aimed at a causal therapy therefore exists.
The problem is therefore to identify genes that are associated with the image of disease of endometriosis.
This problem is achieved according to the invention by the identification of the fibulin-1 gene as an endometriosis- associated gene.
The association of the fibulin-1 gene with the image of disease of the endometriosis was identified by comparison of various patient groups. Using the PCR (ddPCR) differential display method, the mRNA from the endometrium of three clinical groups was compared to one another :
1. LH plus 4 days: 4 days after triggering ovulation by shaking out the luteinizing hormone (LH) , the endometrium is still not receptive for an embryo. This group consisted of patients who participated in an IVF program because of a tubal obstruction or sterility of the male and have a normal endometrium.
2. LH plus 8 days: At this time, the "implantation window" is open, and the endometrium is receptive. By the opening of the "implantation window, " the expression or inhibition of genes was expected, whose protein products play an essential role in the innidiation of the embryo. These genes are identified by subtraction analysis relative to group 1. This group consisted of patients as described under 1.
3. LH plus 8 days plus endometriosis: At this time, the implantation window in healthy women is open. The patients of this group were sterile for unknown reasons, however, and suffered from endometriosis. In this group, another gene regulation pattern in comparison to group 2 was therefore expected. The term of implantation window describes the phenomenon that an embryo that reaches the uterus lumen cannot innidiate at any time, but rather only at a certain period in the mean secretory phase of the menstrual cycle. This period is referred to as an implantation window (Lessey, B. A. 1998; Hum. Reprod., 13 Supp . 3, 247-258; Tabibzadeh, S. 1998; Hum Reprod. Update, 4, 465-471) .
The mRNA of the fibulin-1 is ramped up at the time of the open implantation window in healthy women (group 2), while in women with endometriosis (group 3) , no ramping up can be measured at the same point of the cycle (FIGS. 2 and 3) .
Fibulin-1 is a protein that comes in four different forms that are referred to as fibulin-lA to ID and that represent different splice variants of the fibulin-1 gene. Fibulin-lA is the shortest form. Fibulin-IB, -1C and -ID each have an additional but different C-terminal domain. The DNA- and protein sequences are known (FIGS. 4 to 11 and subsequent references: WO91/02755, Argraves, . S. et al . 1990; J. Cell. Biol. Ill, 3155-3164, Tran, H. et al . 1996; 15, 479-493). Fibulin-1 is known as a component of the extracellular matrix. It is involved in cell adhesion processes and binds to the fibronectin receptor. WO91/02755 mentions a diagnostic and therapeutic use, but there is no reference for what indication the use is to be carried out . The association with the appearance of the endometriosis is new.
The invention relates to a pharmaceutical composition that as active components contains a. a fibulin-1 nucleic acid, b. a vector that contains a fibulin-1 nucleic acid, c. a cell that contains a fibulin-1 nucleic acid, d. a fibulin-1-antisense nucleic acid, e. a fibulin-1 polypeptide or f . an antibody against a fibulin-1 polypeptide. Fibulin-1 nucleic acid comprises both DNA, cDNA and RNA of all 4 splice variants of fibulin-1 or parts thereof. The DNA and protein sequences are depicted in FIGS. 4 to 11. The fibulin-1 nucleic acid is preferably a DNA. The fibulin-lC and -ID splice variants are preferred; and the fibulin-lD splice variant is especially preferred. The human sequences are preferred.
Vectors can be procaryotic or eucaryotic vectors. Examples of vectors are pPRO (Clontech) , pBAD (invitrogen) , pSG5 (stratagene) , pCl (Promega) , pIRES (Clontech) , pBAC (Clontech) , pMET (invitrogen) , pBlueBac (Invitrogen) . The fibulin-1 nucleic acid can be inserted into these vectors with the methods that are known to one skilled in the art. The fibulin- 1 nucleic acid is preferably connected to the vector with expression signals such as, e.g., promoters and enhancers.
As cells, e.g., E. coli, yeast, Pichia, Sf9, COS, CV-1 or BHK can be used. With commonly used methods, the cells can be transformed with a vector that contains the fibulin-1 nucleic acid.
Fibulin-1-antisense nucleic acid is a DNA and/or RNA that is complementary to a fibulin-1 mRNA. It can comprise the entire complementary sequence or partial sequences.
Fibulin-1 polypeptides are defined as the entire sequence as well as portions of all four splice variants. Preferred are the polypeptides of fibulin-lC and -ID splice variants; especially preferred is the polypeptide of the fibulin-lD splice variants.
An antibody against a fibulin-1 polypeptide can be monoclonal or polyclonal . It can be directed against the entire fibulin-1 polypeptide or against fragments thereof. Obtaining such an antibody is carried out according to standard methods by immunization of test animals.
The pharmaceutical compositions of the invention are produced with commonly used solid or liquid vehicles or diluents and the commonly used pharmaceutical and technical adjuvants that correspond to the desired type of administration with a suitable dosage in a way that is known in the art. Tablets can be obtained, for example, by mixing the active ingredient with known adjuvants, for example inert diluents such as dextrose, sugar, sorbitol, mannitol, polyvinylpyrrolidone, explosives such as corn starch or alginic acid, binders such as starch or gelatin, lubricants such as carboxypolymethylene, carboxymethylcellulose, cellulose acetate phthalate or polyvinyl acetate. Capsules that contain active ingredients can be produced, for example, by the active ingredient being mixed with an inert vehicle such as lactose or sorbitol and encapsulated in gelatin capsules.
The pharmaceutical compositions according to the invention can also be used in suitable solutions, such as, for example, physiological common salt solution.
For parenteral administration, especially oily solutions, such as, for example, solutions in sesame oil, castor oil, and cottonseed oil, are suitable. To increase the solubility, solubilizers, such as, for example, benzylbenzoate or benzyl alcohol, can be added.
The pharmaceutical composition according to the invention can be used for the treatment or prevention of endometriosis. This can take place by the use of gene therapy. In this case, a vector that contains a fibulin-1 nucleic acid is designed and administered. Examples are vectors that are derived from adenovirus, adenovirus-associated virus, Herpes simplex virus or SV40. The gene therapy can be implemented according to a protocol as described by Gomez-Navarro et al . (Eur. J. Cancer (1999) 35, 867-885) . The administration can be done locally, i.e., directly in the uterus, or systemically, i.e., via the circulation. This produces an increased expression of the fibulin-1 polypeptide in the endometrium. As a result, the biological function of the fibulin is restored. Another possibility for the treatment and prevention of endometriosis is the administration of a fibulin-1 polypeptide, either as a complete protein or portions thereof that are biologically active. The fibulin-1 polypeptide can be produced in a recombinant manner.
In addition, the invention relates to the use of a pharmaceutical composition according to the invention for the diagnosis of endometriosis. In this regard, blood or endometrial samples are removed from women in whom endometriosis is expected. In these samples, the amount of fibulin-1 polypeptide can be determined by an immune test. In this regard, antibodies against a fibulin-1 polypeptide or against fragments thereof are produced, and the latter then are used in an ELISA (enzyme-linked-immunosorbent assay) , in an RIA (radioimmunoassay) or in the immune histochemistry to determine the amount of fibulin-1 polypeptide. As an alternative, RNA from endometrial samples is purified, and the amount of fibulin-1 mRNA is measured by Northern blot, PCR or chip- hybridization. In the chip-hybridization, fibulin-1 nucleic acids or fragments are present on special nucleic acid chips. The amount of fibulin-1 nucleic acid can also be determined by in situ hybridization with fibulin-1-antisense RNA. In this case, the antisense-RNA can be labeled with digoxigenin, 32P or 33P.
The pharmaceutical composition according to the invention can also be used for the treatment of female sterility. In this regard, a blood or endometrial sample is removed from patients who desire a treatment because of their sterility, and the amount of fibulin-1 is determined as described above. If the sterility is associated with a disorder of the fibulin-1 expression, fertility can be increased by increasing the amount of fibulin-1 at the time of the implantation window. An increase of the amount of fibulin-1 can be achieved as in the treatment of endometriosis by gene therapy or by administration of the fibulin-1 polypeptide. An increase of the amount of fibulin-1 is also especially desirable in the method of in vitro fertilization to raise the success rate, i.e., the pregnancy rate .
Another subject of this invention is the use of a pharmaceutical composition according to the invention for female birth control. Many endometriosis patients are sterile. This invention shows that in endometriosis, the regulation of the fibulin-1 is disrupted. This effect can also be used in reverse by fibulin-1 being artificially adjusted downward in healthy women who do not wish to become pregnant. This can be carried out by the use of antisense RNA, which inhibits the expression of the fibulin-1 gene, or by the use of antibodies that are directed against a fibulin-1 polypeptide and inhibit the biological function of the fibulin-1. The antibodies can be administered, e.g., in utero, and prevent successful detection by the blocking of fibulin-1 and thus the implantation of the embryo.
Tests with rats also show the important role of fibulin-1 in the implantation. Here it was shown that the expression of fibulin-1 remains unchanged post-coitus (see FIG. 15) .
This invention also relates to the use of a pharmaceutical composition according to the invention for detecting uterine receptivity. Uterine receptivity refers to the probability with which a pre-implantation embryo can become implanted in the endometrium of the uterus. Uterine receptivity is greatest at the time of the open implantation window. Since this invention shows that at the time of the open implantation window the fibulin-1 expression is ramped up, the time of high uterine receptivity can be determined by measuring the amount of fibulin-1 mRNA or fibulin-1 polypeptide. The measurement is carried out as described above in the diagnosis of endometriosis by a Northern blot, PCR or chip-hybridization or by an immune test. The determination of uterine receptivity is especially important in the in vitro fertilization. Here, it is important to administer the fertilized ovocyte at the right time. In addition, the invention relates to a method for the detection of uterine receptivity, whereby the amount of fibulin-1 polypeptide and/or the amount of fibulin-1 nucleic acid is determined with the aid of a pharmaceutical composition according to the invention. First, a blood or endometrium sample is removed from the women, and then the amount of fibulin-1 mRNA or fibulin-1 polypeptide is determined.
This invention also relates to the use of a pharmaceutical composition according to the invention for identifying agonists and/or antagonists of a fibulin-1 polypeptide. Agonists and antagonists are substances that influence the biological activity of the fibulin-1 polypeptide either by binding to the fibulin-1 polypeptide or by competition with the fibulin-1 polypeptide in its binding to its physiological binding partners. Such agonists or antagonists can be low-molecular substances or else peptides or proteins, such as, e.g., antibodies. To identify such agonists or antagonists, a cell that expresses the fibulin-1 polypeptide, with potential agonists or antagonists, is treated, and the cell differentiation (Gaetje, R. et al . 1995; Lancet 346, 1463-1464) and other cell properties are analyzed. Fibulin-1 antagonists can be used for, e.g., birth control. Fibulin-1 agonists can be used wherever a high fibulin-1 activity is desired. This is the case in, e.g., the treatment of endometriosis and sterility. Here, a fibulin-1 agonist could be added instead of the fibulin-1 nucleic acid or the fibulin-1 polypeptide.
Description of the Figures
FIG. 1 shows the DNA sequence (SEQ ID NO: 1) of the regulated cDNA clone, which was identified by the ddPCR method and made to coincide with the 3 ' -end of the fibulin-lD.
FIG. 2 shows the detection by the "RNAase protection" method of the ramping up of the human fibulin-ID gene in the LH+8/fertile group. A radioactive antisense RNA-probe was generated by the clone described in FIG. 1 and hybridized with RNA from the three groups: LH+4/fertile (early secretory), LH+8/fertile (midsecretory) , and LH+8/endometriosis (midsecretory and endometriosis) . An antisense-actin probe was generated as an internal control . After digestion of the non- hybridized single-strand RNA, the reaction products were separated on a polyacrylamide gel and visualized by autoradiography.
FIG. 3 shows the quantification of the results of FIG. 2 (RNAase protection method) . The strips of double-strand fibulin-ID and actin RNA were "scanned, " and the measured amount was normalized to the amount of actin.
FIG. 4 shows the DNA sequence (SEQ. ID NO : 2) for the human fibulin-1A. FIG. 5 shows the protein sequence (SEQ ID NO : 3) for the human fibulin-lA.
FIG. 6 shows the DNA sequence (SEQ. ID NO: 4) for the human fibulin-IB.
FIG. 7 shows the protein sequence (SEQ. ID NO: 5) for the human fibulin-IB.
FIG. 8 shows the DNA sequence (SEQ. ID NO: 6) for the human fibulin-1AC.
FIG. 9 shows the protein sequence (SEQ ID NO: 7) for the human fibulin-lC.
FIG. 10 shows the DNA sequence (SEQ ID NO : 8) for the human fibulin- ID.
FIG. 11 shows the protein sequence (SEQ ID NO: 9) for the human fibulin-ID.
FIG. 12 shows the tissue distribution of the human fibulin- 1D in a Northern blot . A radioactive DNA probe was generated by the fibulin-ID-specific portion and hybridized with human "Multiple Tissue Northern" (Clontech) . The expression pattern of cyclophilin A was determined as an internal control.
FIG. 13 shows the DNA sequences (SEQ ID NO: 10) and the derived protein sequences that code for the C-terminal end of the rat fibulin-ID protein (corresponds to amino acid 576 to 696 in the human fibulin-ID) . FIG. 14 shows the expression of the rat fibulin-1 mRNA in the uterus tissue from various days of the female cycle. A sense primer and an antisense primer, which detect all fibulin- 1 forms, or specific primers for the fibulin-IC or the fibulin- 1D isoforms were' used for the PCR amplification. Cyclophilin A (CyPA) -specific primers were used in the control experiment. As "templates, " first-strand cDNA from the rat uterus tissue from diestrus stage (D) , proestrus stage (P) , estrus stage (0) or metestrus stage (M) was used. The PCR products were separated on an agarose gel and then colored with ethidium bromide. The intensity of the strips was quantified with the GelDoc 2000 device (Hercules, CA, USA) .
FIG. 15 shows the expression of the rat fibulin-1 mRNA in the uterus tissue from various days post-coitus. A sense primer and an antisense primer, which detect all fibulin-1 forms or specific primers for the fibulin-IC or the fibulin-ID isoforms were used for the PCR amplification. Cyclophilin A (CyPA) -specific primers were used in the control experiment. As "templates," first-strand cDNA from the rat uterus tissue of days 1 to 5 post-coitus (p.c.) was used. The PCR products were separated on an agarose gel and then colored with ethidium bromide. The intensity of the strips was quantified with the GelDoc 2000 device. Examples
The molecular-biological methods used in the examples, such as, e.g., isolation of RNA, cloning of DNA, sequencing of DNA, RNAse protection, Northern blot analysis, polymerase-chain reaction (PCR) were performed as described in known textbooks, such as, e.g., in Molecular Cloning, A Laboratory Manual (Sambrook, J. et al . 1989; Cold Spring Harbor Laboratory Press) . Methods for subtraction analyses of the gene expression are described in, e.g., Liang, P. and Pardee, A. B. 1995; Curr. Opin. Immunol. 7, 274-280.
Example 1: Isolation of gene transcripts that are regulated during the implantation window by the ddPCR method
RNA was isolated from the endometrium of patients who belong to three groups: LH+4/fertile, LH+8/fertile, and HL+8/endometriosis . For the ddPCR reactions, the RNAmap™ kit of the GenHunter Corporation was used. The sense primers AP-1 to AP-5 and the antisense primer T12MA were used for the amplifications in the presence of α- [35S] dATP. The products were separated on a polyacrylamide gel and compared. Overall, 24 strips that have a different expression pattern in at least two separate tests were further processed. After isolation from the gel, these DNA fragments were amplified once more by PCR (GeneAmp™ kit, Perkin Elmer Cetus) , purified on agarose gel and subcloned in the Srfl position of the pCR-Script™ Amp SK(+) plasmid (stratagene) . Then, the plasmid-DNA was produced in larger amounts, the size of the insert was verified, and the sequence was determined. A sequence comparison with bioinformatic processes was then performed to detect homologies with already known sequences .
Example 2 : Validation of the results by the "RNAase protection" method
Antisense RNA probes were generated from the clones isolated in Example 1. For this purpose, the T7 or T3 promoter was used as a function of the orientation of the insert. After digestion of the single-strand RNA by RNAse, the residual double-strand RNA molecules were separated on a polyacrylamide gel and visualized by autoradiography. The expression pattern was compared between the three groups and also with the results of the ddPCR method. The clone, which coded for the fibulin- 1D, showed the most advantageous expression pattern (Figs. 1 to 3) . Example 3: Tissue distribution of the huma fibulin-lD (Northern blot)
The following primers were used for the amplification of the C-terminal coding area of the human fibulin-ID: 5'- CTCCAGCAGGAGAAGACAGAC-3 ' (SEQ ID NO: 12) and 5'- GAACCAGTACTCAGAGACGAAG-3 ' (SEQ ID NO: 13). As a "template," human liver cDNA (QUICK-Clone™, Clontech) was used. The PCR- amplification was carried out according to standard conditions. After subcloning in the pCR-Topo™ plasmid (invitrogen) , the insert was sequenced, and one hundred percent correspondence with the already published sequence of fibulin-ID was found
(Argraves, S., 1996, Access Number U01244) . A specific probe was derived with the aid of the Prime-It (E) II random primer kit
(stratagene) in the presence of y- [32P] dCTP of the fibulin-ID insert . This probe was used under stringent conditions for hybridization of the Multiple Tissue Northerns "human muscle MTN" and "human tissue MTN" (Clontech) . The washed filters were analyzed by autoradiography. An antisense probe was generated from the human cyclophilin A (Haendler et al . 1987, EMBO J. 6, 947-950) and used in a control-hybridization under stringent conditions. The result shows that fibulin-lD is expressed in many, but not in all tissues. A high expression can be seen in the uterus (FIG. 12) . Example 4: Isolation of the 3 " -end of the rat fibulin-lD
The following primers, derived from the human sequence, were synthesized: 5 ' -AGGAGAAGACAGACACGGTC-3 ' (SEQ ID NO : 14) and 5'-GAACCAGTACTCAGAGACGAA-3 ' (SEQ ID NO: 15). As a "template," cDNA from the rat brain (QUICK-Clone™, Clontech) was used. The PCR-amplification was carried out under standard conditions. After subcloning in the pCR-Topo™ plasmid (invitrogen) , the DNA-insert was sequenced (FIG. 13) . The derived protein sequence showed 87.5% identity with that of the human fibulin-ID. Differences between the rat sequence and the human sequence are emphasized (FIG. 13) .
Example 5: Expression of rat fibulin during the female cycle and on days 1-5 post-coitus
The expression was analyzed by semi-quantitative PCR. To this end, the following primers were used:
Fibulin-1 :
Sense 5 ' -ACATCTGCTCCTACCGCTG-3 ' (SEQ ID NO: 16),
Antisense 5 ' -TGCAGAGCGGCGATAGTTC-3 ' (SEQ ID NO: 17).
Fibulin-lC:
Sense 5 ' -CTGCAGACACCCGCTGTG-3 ' (SEQ ID NO: 18),
Antisense 5 ' -AGCGGTGATGGCCAGCTG-3 ' (SEQ ID NO: 19).
Fibulin-lD:
Sense 5 ' -CGTCTCCCACACTGTCATC-3 ' (SEQ ID NO: 20), Antisense 5 ■ -GGTGGGAAACCACGCCTC-3 ' (SEQ ID NO: 21).
In the control, specific primers for cyclophilin A were used:
Sense primer 5 ' -TGACTTGCGGGCATTTTACC-3 ' (SEQ ID NO: 22),
Antisense primer 5 ' -AACTTTGCAATCCTGCTAGAC-3 ' (SEQ ID NO 23).
Total-RNA was extracted from uterus tissue using Totally RNA Isolation Kits (Ambion, Austin, TX, USA) . The cDNA was produced with the ProSTAR First-Strand RT-PCR Kit (Stratagene, La Jolla, CA, USA) . The PCR analysis was performed with the Ampli Taq DNA Polymerase PCR kit (Perkin Elmer Cetus, Norwalk, CT, USA) . The reaction conditions were as follows: Initially 94°C for 5 minutes; then 32 (fibulin-1) or 25 (cyclophilin A) cycles with 94°C for 20 seconds, 60°C for 20 seconds and 72°C for 40 seconds, finally 72°C for 10 minutes.
The results in FIG. 14 show a cycle-dependent expression of rat fibulin-1. The expression of fibulin-1, but also the IC and ID isoforms are weak during the diestrus and then increase to reach a maximum during the metestrus .
The terms proestrus, estrus, metestrus and diestrus are explained in Freeman, M. E. The Neuroendocrine Control of the Ovarian Cycle of the Rat. In: Knobil, E., Neill, J. D. (Eds.) The Physiology of Reproduction Volume 2, 2nd Edition, New York, Raven Press 1994, 613-658. FIG. 15 shows that the expression of fibulin-1 post coitus remains unchanged. The largest amount was measured on day 3 p.c. Also here, the IC and ID isoforms show a pattern similar to fibulin-1.

Claims

Claims
1. Pharmaceutical composition, characterized in that as active components, it contains a. a fibulin-1 nucleic acid, b. a vector that contains a fibulin-1 nucleic acid, c. a cell that contains a fibulin-1 nucleic acid, d. a fibulin-1-antisense nucleic acid, e. a fibulin-1 polypeptide or f . an antibody against a fibulin-1 polypeptide.
2. Use of a pharmaceutical composition according to claim 1 for the treatment or the prevention of endometriosis.
3. Use of a pharmaceutical composition according to claim 1 for diagnosis of endometriosis.
4. Use of a pharmaceutical composition according to claim 1 as an agent for gene therapy.
5. Use of a pharmaceutical composition according to claim 1 for the treatment of female sterility.
6. Use of a pharmaceutical composition according to claim 1 for female birth control .
7. Use of a pharmaceutical composition according to claim 1 for the detection of uterine receptivity.
8. Method for detecting uterine receptivity, wherein the amount of fibulin-1 polypeptide and/or the amount of fibulin-1 nucleic acid is determined with the aid of a composition according to claim 1.
9. Use of a pharmaceutical composition according to claim 1 for the identification of agonists and/or antagonists of a fibulin-1 polypeptide.
EP01939374A 2000-05-24 2001-05-24 Pharmaceutical use of fibulin-1 Withdrawn EP1301195A2 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US57749900A 2000-05-24 2000-05-24
US577499 2000-05-24
PCT/US2001/016791 WO2001089548A2 (en) 2000-05-24 2001-05-24 Pharmaceutical use of fibulin-1

Publications (1)

Publication Number Publication Date
EP1301195A2 true EP1301195A2 (en) 2003-04-16

Family

ID=24308988

Family Applications (1)

Application Number Title Priority Date Filing Date
EP01939374A Withdrawn EP1301195A2 (en) 2000-05-24 2001-05-24 Pharmaceutical use of fibulin-1

Country Status (4)

Country Link
EP (1) EP1301195A2 (en)
JP (1) JP2004504275A (en)
AU (1) AU2001264900A1 (en)
WO (1) WO2001089548A2 (en)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2008318288B2 (en) * 2007-10-31 2013-11-14 The University Of Sydney Methods and compositions for regulating airway tissue remodelling
WO2010010201A1 (en) 2008-07-22 2010-01-28 Equipo Ivi Investigacion Sl Gene expression profile as a marker of endometrial receptivity
EP4012023B1 (en) 2014-06-17 2024-05-15 Asherman Therapy, S.L. Stem cell therapy in endometrial pathologies
CN113621699A (en) * 2021-08-10 2021-11-09 张存泰 Fibulin-1 protein as vascular aging molecular marker and application thereof

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU6299390A (en) * 1989-08-18 1991-04-03 American National Red Cross, The Fibulin
WO1997038014A1 (en) * 1996-04-04 1997-10-16 Amgen Inc. Fibulin pharmaceutical compositions and related methods

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0189548A2 *

Also Published As

Publication number Publication date
JP2004504275A (en) 2004-02-12
WO2001089548A2 (en) 2001-11-29
WO2001089548A3 (en) 2003-01-23
AU2001264900A1 (en) 2001-12-03

Similar Documents

Publication Publication Date Title
Matsuzaki et al. DNA microarray analysis of gene expression in eutopic endometrium from patients with deep endometriosis using laser capture microdissection
Ledbetter et al. Tissue distribution of ryanodine receptor isoforms and alleles determined by reverse transcription polymerase chain reaction.
US6720472B2 (en) HMGI proteins in cancer and obesity
US20030005471A1 (en) PAR, a novel marker gene for breast and prostate cancers
Yotsumoto et al. Expression of adrenomedullin, a hypotensive peptide, in the trophoblast giant cells at the embryo implantation site in mouse
US6756355B1 (en) HMGI proteins in cancer and obesity
US6323329B1 (en) Nucleic acid sequences of genes encoding high mobility group proteins
Azuma et al. Steroid hormones induce macrophage colony-stimulating factor (MCSF) and MCSF receptor mRNAs in the human endometrium
US6235879B1 (en) Apoptosis modulators that interact with the Huntington's disease gene
JPWO2004096851A1 (en) Galectin 9 inducer
WO2004080479A1 (en) Gonadal function improving agents
EP1301195A2 (en) Pharmaceutical use of fibulin-1
US20070020266A1 (en) Gpr54 receptor agonist and antagonist useful for the treatment of gonadotropin related diseases
WO1999055902A1 (en) Diagnostic markers of human female infertility
EP0977993A1 (en) Cadherin-11 as an indicator of viable pregnancy
US6586581B1 (en) Prolactin regulatory element binding protein and uses thereof
US7125684B2 (en) Human mater proteins
JP4663968B2 (en) EG-VEGF / prokineticin 2-receptor antagonist
US20090068698A1 (en) Pregnancy-related enzyme activity
WO1998010078A2 (en) CLONING OF FULL-LENGTH HUMAN PEX cDNA
Azuma et al. The gene expressions of macrophage colony-stimulating factor (MCSF) and MCSF receptor in the human myometrium during pregnancy: regulation by sex steroid hormones
US20040029158A1 (en) HOP - a novel cardiac-restricted transcriptional factor potentially useful for cardiac regeneration and specification
Lee et al. Identification of estrogen‐regulated genes in the mouse uterus using a delayed‐implantation model
US20040019006A1 (en) Novel genes relating to pain and use of the genes for pharmaceuticals
US20030124558A1 (en) Cadherin-11 as an indicator of viable pregnancy

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20021120

AK Designated contracting states

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

AX Request for extension of the european patent

Extension state: AL LT LV MK RO SI

RIN1 Information on inventor provided before grant (corrected)

Inventor name: CHWALISZ, KRISTOF

Inventor name: LESSEY, BRUCE

Inventor name: HAENDLER, BERNARD

Inventor name: HESS-STUMP, HOLGER

17Q First examination report despatched

Effective date: 20040720

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20041201