EP1157099A2 - Substances destinees au controle de fertilite - Google Patents

Substances destinees au controle de fertilite

Info

Publication number
EP1157099A2
EP1157099A2 EP00910742A EP00910742A EP1157099A2 EP 1157099 A2 EP1157099 A2 EP 1157099A2 EP 00910742 A EP00910742 A EP 00910742A EP 00910742 A EP00910742 A EP 00910742A EP 1157099 A2 EP1157099 A2 EP 1157099A2
Authority
EP
European Patent Office
Prior art keywords
lhop
fertility
substances
administered
substance
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Withdrawn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
EP00910742A
Other languages
German (de)
English (en)
Inventor
Monika Lessl
Ekkehard Brockstedt
Michaele Peters-Kottig
Christa Hegele-Hartung
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
Original Assignee
Schering AG
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 filed Critical Schering AG
Publication of EP1157099A2 publication Critical patent/EP1157099A2/fr
Withdrawn legal-status Critical Current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
    • C07K14/575—Hormones
    • C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
    • 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
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/10—Drugs for disorders of the endocrine system of the posterior pituitary hormones, e.g. oxytocin, ADH
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to a substance and to methods for obtaining fertility control agents.
  • the female contraceptive that is used most often is the classic pill. It consists of an estrogen and a progestogen component. Regular intake leads to inhibition of ovulation in women.
  • the principle of this method is that by taking the pill it suppresses the endogenous
  • Steroid hormone production in the ovary leads to an inhibition of ovulation.
  • One disadvantage is that the woman no longer has a natural cycle.
  • side effects such as breast tensions, weight gain, etc. can occur in connection with taking the pill in patients with risk potential.
  • the present invention relates to methods of providing fertility control agents.
  • the methods are characterized in that a potentially active substance is tested for its ability to induce, stimulate or inhibit the LH-dependent ovary protein (LHOP). This is preferably done in test methods suitable for this purpose, in which the LHOP activity is induced, stimulated or inhibited. As well as the use of these substances for the production of fertility control agents.
  • LHOP LH-dependent ovary protein
  • the invention relates to fertility promotion agents which contain at least a portion of the amino acid primary structure of the LHOP.
  • Fertility-controlling agents are understood to mean all agents which are suitable both for promoting fertility and for inhibiting fertility, both for women and for men.
  • Stimulating substances according to the present invention are understood to mean those which increase the LHOP enzyme activity.
  • Inducing substances according to the present invention are understood to mean those which increase the LHOP mRNA or amount of protein.
  • Inhibitory substances according to the present invention are understood to mean those which reduce and / or inhibit the LHOP enzyme activity and / or reduce the LHOP mRNA or amount of protein.
  • the combination of 2-dimensional gel electrophoresis and subsequent mass spectrometry was used to identify a protein in mouse ovaries that was induced by the luteinizing hormone (LH).
  • LH luteinizing hormone
  • a database search showed that this is a protein which has a high protein sequence homology to mouse vas deferens protein (MVDP) and to human ARL-1 (Cao et al. 1998, J. Biol. Chem. 273: 11429 - 11435) shows.
  • the MVDP was first described by Taragnat et al, 1988 (J. Reprod. Fert. 83: 835-842) in the vas deferens of male mice.
  • LHOP LH-dependent ovary protein
  • LHOP protein belongs to the family of Aldo keto reductases. This group of enzymes reduces aldehydes or ketones from various substrates. Steroids with an aldehyde or keto group also belong to the substrates of the family of aldo keto reductases. Inhibition of LHOP (at the mRNA, protein and / or enzyme level) therefore leads to an inhibition of egg maturation, follicle maturation, steroidogenesis and / or ovulation. Stimulation of the protein (at the mRNA, protein and / or enzyme level) or administration of the recombinant protein has the opposite effect and promotes fertility (for example in the case of ovarian failure or if polycystic ovaries occur).
  • the MVD protein which is very similar to the LHO protein, occurs in large quantities in the vas deferens. Inhibition of the LHOP and / or the MVDP will thus have a negative influence on the maturation and / or the transport of the sperm by the vas deferens and lead to an inhibition of fertility. In contrast, stimulation of the LHOP and / or the MVDP or administration of recombinant protein has the opposite effect and contributes to the promotion of male fertility.
  • a first aspect of the invention relates to fertility enhancers, e.g. in in vitro or in vivo fertilization, which comprise at least a portion of the amino acid primary structure or the entire protein of the MVDP or ARL-1.
  • Another aspect of the invention relates to fertility enhancers, e.g. in in vitro or in vivo fertilization, which comprise at least a section of the amino acid primary structure or the entire protein of the LHOP.
  • Another aspect of the invention relates to methods for providing fertility control agents.
  • the methods are characterized in that a potentially active substance is tested for its ability to specifically induce or inhibit the LHO protein. This is preferably done in test methods suitable for this purpose, in which the LHOP activity is stimulated or inhibited.
  • the test procedure can be automated, for example, as a high-pressure throughput screening procedure. Substances that at this high pressure throughput
  • the substances found in this way can be small molecules, antisense RNA, ribozymes, GeneBloc or other substances which influence the induction of the mRNA, the protein or the enzymatic activity of the LHOP.
  • An enzyme assay is particularly suitable for finding substances which stimulate or inhibit LHOP enzyme activity (reduction of aldehydes and ketones).
  • the LHO protein is purified.
  • the coding region of the Ihop gene can be inserted into an expression vector and the enzyme overexpressed in pro- or eukaryotic cells (Ausubel er al, Current protocols in molecular biology, 1997, Vol. 2; p. 16.0.1).
  • the recombinant protein obtained in this way can then be purified using known chromatographic methods, for example by size exclusion and / or cation and / or anion exchange chromatography, and after incubation in phosphate buffer with NADPH can be used in the enzyme test.
  • substances can be tested that stimulate or inhibit the reduction of substrates of LHOP Aldo ketoreductase.
  • the stimulating substances thus found are then used to produce fertility enhancers in women and men.
  • the inhibiting substances are used to produce agents that inhibit fertility in women and / or in men.
  • AS antisense
  • RB ribozymes
  • GB Genebioc reagents
  • the model of the perfused ovary is suitable, for example, as a test method for finding molecules which inhibit or induce the LHOP at the mRNA, protein and / or enzyme level.
  • the ovary is prepared in rats or mice, with infusion catheters placed in the aorta and vena cava.
  • the model is exactly from Brännström et al. 1987 (Acta Physiol. Scand. 130: 107-14). With the help of this model, substances can be tested that inhibit or induce LHOP expression at the mRNA protein and / or enzyme level.
  • the inhibitory substances tested in this way are then used for the production of fertility inhibitors, the inducing substances are used for the production of fertility promoters, both in men and women.
  • pharmacologically acceptable substances are tested. As already stated, these include low molecular weight pharmacological active ingredients, but in particular small molecules as well as AS / RB or GB molecules.
  • Suitable pharmaceutical dosage forms contain the active ingredient in an amount that either prevents fertility or promotes it.
  • the active substance can be administered alone or in combination with other substances either simultaneously or sequentially.
  • the pharmaceutical composition can contain other pharmaceutically customary substances.
  • the substance can be administered either orally, nasally, transdermally, pulmonally or parenterally, for example by injection. Implants and vaginal rings or intrauterine systems are also conceivable.
  • Orally administrable compositions may be in the form of tablets, capsules, powders or liquids. Compositions which can be administered by injection are usually in the form of a parenterally acceptable aqueous solution or suspension.
  • the invention also relates to a method for promoting fertility, wherein an effective amount of a substance which stimulates or induces the LHO protein is administered to a patient, in particular a human patient.
  • the invention further relates to a method for inhibiting fertility, wherein an effective amount of a substance which inhibits the LHO protein is administered to a patient, in particular a human patient.
  • the invention further relates to a method for inhibiting fertility, wherein an effective amount of at least a portion of the amino acid primary structure of the LHOP, the MVDP or the ARL-1 is administered to a patient, in particular a human patient.
  • the invention relates to antibodies which are directed against or recognize at least a section of the amino acid primary structure or the entire protein of the LHOP of the MVDP or the ARL-1 (Sambrook et al., 1989, Production of Antibodies 18.3; Plückthun, Bio / Technology 9 (1991), 545-551 and references cited therein). As well as the use of these antibodies or agents and kits containing them for the diagnosis of fertility in men or women.
  • the invention further relates to antisense molecules which are directed against or recognize at least a portion of the nucleic acid sequence or the entire sequence of the Ihop, mvdp or arl-1 (Schaeren-Wiemers and Gerfin-Moser (Histochemistry 1993 Vol. 100, 431-440 )). As well as the use of these antisense molecules or agents and kits containing them for the diagnosis of fertility in men or women.
  • Figure 1 Identification of the LHO protein in the ovary.
  • mice Ovaries from immature, FSH-primed mice (age: 19-23 days) were removed before or 14 h after LH application and prepared for further analysis. With the help of the high-resolution two-dimensional gel electrophoresis, a protein spot was detected that is expressed significantly variant under the influence of LH.
  • Figure 2 In situ hybridization to detect the Ihop mRNA in the mouse ovary.
  • FSH-primed mice Ovaries from immature, FSH-primed mice (ages 19-25 days) were removed before (A) or after LH application (B; 6h after LH treatment) and carried out in tissue teck to perform an in situ
  • the Ihop mRNA could be detected specifically in ovaries (in theca and stromal cells) after LH treatment (B).
  • Figure 3 Northem blot of mouse ovary mRNA. After LH application, a 300-fold increase in the Ihop mRNA was measured.
  • Figure 4 Comparison between the MVDP and ARL-1 protein.
  • FIG. 5 SDS-PAGE shows protein fractions before / after overexpression of LHOP in E. coli and before / after purification of the overexpressed LHOP using a NiNTA affinity column. Purified LHOP protein fraction (lane 5) is used in the enzyme assay.
  • the coding region of the LHOP cDNA was cloned into the expression vector pQE30 (Qiagen company), taking into account the reading frame, between an IPTG-inducible promoter and an N-terminal 6xHistidine signal.
  • the vector was transformed into the E. coli strain DH5 ⁇ .
  • the native protein purification was carried out on a nickel affinity column (NiNTA agarose, Qiagen company) according to the manufacturer (The QIAexpressionist, Qiagen).
  • the eluted LHOP protein fraction was dialyzed against 10 mM phosphate buffer and used in the enzyme assay.
  • Standard enzyme reaction approach contained 135 mM sodium phosphate buffer (pH 6.3), 0.2 mM NADPH and adequate substrate and enzyme concentrations. The enzyme reaction took place at 28 ° C and the substrate conversion was determined spectrometrically via the acceptance of the A 340 from NADPH. The reaction was started by adding enzyme. It was by default
  • Controls measured without substrate and without enzyme One of the standard substrates for Aldo ketoreductases is DL-glyceraldehyde. With the help of the enzyme assay described, substances can be tested that stimulate the reduction of substrates of LHOP Aldo ketoreductase. 2) Enzyme assay for testing substances that inhibit LHOP enzyme activity (reduction of aldehydes and ketones). The enzyme assay described above should be used in a manner analogous to that described under 1) to find substances which inhibit LHOP activity.
  • the ovary is prepared by rats or mice, with infusion catheters placed in the aorta and vena cava.
  • the model is exactly from Brännström et al. 1987 (Acta Physiol. Scand. 130: 107-14).
  • substances can be found which inhibit or induce LHOP expression at the mRNA, protein and / or enzyme level.
  • the inhibiting substances found in this way are suitable for inhibiting fertility, the inducing substances are suitable for promoting fertility, both in men and women.
  • hypophysectomized rat model has been described in detail by JS Richard, 1980 (Physiol. Rev. 60: 51-89). In this model, follicular maturation and ovulation are induced by a precisely defined hormone regime (see literature reference above). The injection of 17 ⁇ -estradiol followed by
  • FoHicle stimulating hormone stimulates follicular maturation, the subsequent injection of LH triggers egg maturation, steroidogenesis and ovulation.
  • FSH FoHicle stimulating hormone
  • substances can be found which inhibit or induce LHOP expression at the mRNA, protein and / or enzyme level. The inhibitory substances found in this way are for
  • Fertility inhibition the inducible for fertility promotion, suitable for both men and women.
  • Follicular maturation induced synchronously in many ovary follicles. After 48 h with 10 IU LH the egg maturation, steroidogenesis and ovulation triggered. With the help of this model, substances can also be found that inhibit or induce LHOP expression at the mRNA, protein and / or enzyme level. The substances found in this way can be used for fertility control in men and women.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Endocrinology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Reproductive Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • General Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Public Health (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Diabetes (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Toxicology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gynecology & Obstetrics (AREA)
  • Pregnancy & Childbirth (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Plant Substances (AREA)
  • Peptides Or Proteins (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne une substance et des procédés de production de produits destinés au contrôle de la fertilité.
EP00910742A 1999-03-03 2000-03-02 Substances destinees au controle de fertilite Withdrawn EP1157099A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19910394 1999-03-03
DE19910394A DE19910394A1 (de) 1999-03-03 1999-03-03 Substanzen für die Fertilitätskontrolle
PCT/EP2000/001733 WO2000052148A2 (fr) 1999-03-03 2000-03-02 Substances destinees au controle de fertilite

Publications (1)

Publication Number Publication Date
EP1157099A2 true EP1157099A2 (fr) 2001-11-28

Family

ID=7900280

Family Applications (1)

Application Number Title Priority Date Filing Date
EP00910742A Withdrawn EP1157099A2 (fr) 1999-03-03 2000-03-02 Substances destinees au controle de fertilite

Country Status (5)

Country Link
EP (1) EP1157099A2 (fr)
JP (1) JP2002538180A (fr)
AU (1) AU3284500A (fr)
DE (1) DE19910394A1 (fr)
WO (1) WO2000052148A2 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10152598A1 (de) * 2001-10-19 2003-05-22 Schering Ag ARL-2-für die Fertilitätskontrolle
WO2003046165A1 (fr) * 2001-11-26 2003-06-05 Bayer Healthcare Ag Régulation de la protéine humaine de type aldose réductase

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2680229B2 (ja) * 1992-02-12 1997-11-19 三菱瓦斯化学 株式会社 人アルドース還元酵素の測定方法
US5563059A (en) * 1993-02-23 1996-10-08 Genentech, Inc. Use of human inhibin and human activin to increase the number of mature primate oocytes
EP0839831B1 (fr) * 1994-02-18 2005-12-28 Washington University Gonadotropin monochaine

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0052148A2 *

Also Published As

Publication number Publication date
WO2000052148A3 (fr) 2001-03-29
WO2000052148A9 (fr) 2001-09-13
WO2000052148A2 (fr) 2000-09-08
AU3284500A (en) 2000-09-21
DE19910394A1 (de) 2000-09-07
JP2002538180A (ja) 2002-11-12

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