EP2148694A2 - Pharmaceutical agent for the treatment of fertility and gestation disorders, immunological diseases, and transplantation for use in veterinary medicine, particularly in horses, and methods for the production and treatment monitoring thereof - Google Patents

Pharmaceutical agent for the treatment of fertility and gestation disorders, immunological diseases, and transplantation for use in veterinary medicine, particularly in horses, and methods for the production and treatment monitoring thereof

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Publication number
EP2148694A2
EP2148694A2 EP08759974A EP08759974A EP2148694A2 EP 2148694 A2 EP2148694 A2 EP 2148694A2 EP 08759974 A EP08759974 A EP 08759974A EP 08759974 A EP08759974 A EP 08759974A EP 2148694 A2 EP2148694 A2 EP 2148694A2
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EP
European Patent Office
Prior art keywords
ecg
seq
treatment
disorders
pro
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EP08759974A
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German (de)
French (fr)
Inventor
Henry Alexander
Gerolf Zimmermann
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Universitaet Leipzig
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Universitaet Leipzig
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Publication of EP2148694A2 publication Critical patent/EP2148694A2/en
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    • 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
    • A61K38/22—Hormones
    • A61K38/24—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors

Definitions

  • the invention relates to agents for the treatment of fertility and pregnancy disorders (pregnancy disorders) and immunologically-related diseases (autoimmune diseases) and transplants, and to processes for their preparation and therapy control for use in veterinary medicine, in particular in horses.
  • the object of the invention is therefore to provide an agent for the treatment of pregnancy disorders (pregnancy disorders), in particular for the treatment of fertility disorders, implantation disorders, early pregnancy losses, imminent pregnancy losses and habitual abortions as well as premature birth and growth retardation, for use in veterinary medicine, in particular in horses.
  • pregnancy disorders pregnancy disorders
  • fertility disorders in particular for the treatment of fertility disorders, implantation disorders, early pregnancy losses, imminent pregnancy losses and habitual abortions as well as premature birth and growth retardation
  • the object of the invention is further to provide agents for the treatment of infectological and immunological diseases of the intestine and the lungs and the joints or for transplantation (cornea, skin) for use in veterinary medicine, in particular in horses.
  • this object is achieved by a medicament containing at least one protein component of equine chorionic gonadotropin (eCG) or a nucleic acid sequence (eCG) coding for a protein component of equine chorionic gonadotropin.
  • eCG equine chorionic gonadotropin
  • eCG nucleic acid sequence
  • the protein component of equine chorionic gonadotropin is a prehormonal subunit (pre- ⁇ -eCG or pre- ⁇ -eCG), an alpha or beta subunit of equine chorionic gonadotropin ( ⁇ -eCG or ⁇ -eCG) or a Fragment of the alpha or beta subunit.
  • the fragments of the alpha unit preferably have a length of 10 to 95 amino acids.
  • Fragments of the beta unit preferably have a length of 20 to 148 Amino acids. Particular preference is given in each case to fragments having a length of from 25 to 50 amino acids.
  • the medicament is advantageously suitable for the treatment of pregnancy disorders, in particular for the treatment of fertility disorders, implantation disorders, early pregnancy loss, imminent and habitual abortion and premature birth, and growth retardation in veterinary medicine, especially in Perissodactyla and Equinae, such as horses.
  • the equine chorionic gonadotropin preferably consists of a non-covalently linked heterodimer of an ⁇ -subunit ( ⁇ -eCG) and a ⁇ -subunit ( ⁇ -eCG).
  • ⁇ -eCG ⁇ -subunit
  • ⁇ -eCG ⁇ -subunit
  • ⁇ -eCG ⁇ -subunit
  • the ⁇ -subunits including the CTP sections ("C-terminal repeats") of the human ( ⁇ -hCG amino acids 121-145) and the equine ⁇ subunit ( ⁇ -eCG amino acids 121-149) differ in about 50% of the amino acids and in the number of N- and O-glycosidic side chains (1 N- and 12-O-glycosidic side chains in ⁇ -eCG and 2 N and 4 O-glycosidic side chains in ⁇ -hCG) .beta.-eCG In contrast to ⁇ -hCG, only one gene is read in.
  • eCG expressed in the placenta (ie in the Girdle cells of the chorio-villous cup structure of the conceptus (embryo)) and the hypophysis-expressed el_H (luteinizing hormone
  • the el_H and the eCG differ in the glycan side chain and thus by their biological activity at the receptor and in the half-life.
  • the equine chorionic gonadotropin (eCG) is formed by a further glycosylation of equine LH with a carbohydrate moiety of From 40% to 45%, eCG is the most abundant glycosylated mammalian hormone in the placenta.
  • the placental eCG is more glycosylated than eLH.
  • the eCG also has a lower sulphin group content and a higher sialic acid content than the eLH.
  • eCG is not only expressed in embryonic (or fetal) tissue, but also in the endometrium and the decidua of the non-pregnant or pregnant mare.
  • the endometrial (maternal) eCG of the non-pregnant horse in the normal luteal phase as detected in the invention differs from the pituitary el_H, fetal (placental, chorio-villous) eCG and deciduous eCG of the pregnant horse in terms of the structure of the glycan side chains and may hCG and chorio-villous eCG can be used as drugs.
  • the equine chorionic gonadotropin has a very extensive and diverse spectrum of activity.
  • eCG supports the corpus luteum function to generate progesterone, which in turn stimulates eCG secretion in the endometrium.
  • eCG is a growth and differentiation hormone and promotes blood flow to the uterus. It also has immunosuppressant, antiviral and antibacterial effects. In addition, eCG keeps the smooth muscles of the uterus quiet.
  • Equine chorionic gonadotropin is an immune protection hormone as well as a barrier protection and anti-infective hormone that acts especially during the second half of the cycle and throughout the gestation period.
  • Chorionic gonadotropin plays an essential role in reproductive physiology. Although the amino acid sequence of equine chorionic gonadotropin is significantly different from human chorionic gonadotropin, surprisingly similar effects are obtained. Disturbances of the endometrial eCG production lead to infections of the uterus and the fruit, which not infrequently induces fetal abortion and death. Lack of eCG formation, especially in early pregnancy, causes growth retardation and, in later pregnancy, placental insufficiency.
  • equine chorionic gonadotropin binds as a drug at equine eLH / eCG receptors in the pituitary gland (a), in the Girdle cells of the chorio-villous cup structure of the equine concept (b) and probably already in the normal luteal endometrium non-pregnant mare (c).
  • el_H and eCG bind with different affinity to the receptors.
  • This eCG antibody allows post-ovulatory monitoring and pregnancy monitoring by ELISA testing.
  • the equine chorionic gonadotropin secreted with the pregnancy of the mare in the Girdle cells of the chorio-villous cup structure is preferably determined in the peripheral blood with a peak from the 39th to the 130th but also to the 200th day of gestation.
  • the equine chorionic gonadotropin has a paracrine meaning in addition to the endocrine.
  • This effect of the endometrial eCG can already be used in the normal luteal phase of the non-pregnant uterus of the mare and continues to take further protective effect on the course of gestation with the chorio-villous eCG of the conceptus implantation.
  • the equine chorionic gonadotropin formed in the maternal endometrium of the uterus and the well-known chorio-villous eCG of Girdle cells of the conceptus are of importance throughout pregnancy (pregnancy). During the first 150 days of pregnancy, an abortion often goes unnoticed. Early pregnancy losses are associated with a lack of early eCG secretion in both the maternal endometrium and fetal chorio-villous Girdle cells of the mare. This lack of eCG is often not recognized or recognized too late.
  • the eCG's barrier protection and infection protection function can also be used outside the pregnancy in horses for the treatment of diseases of the bronchial tract (chronic obstructive inflammation), the gastrointestinal tract (colic) and the joints (osteoarthritides).
  • ECG administered daily at low doses of 3 to 6 ⁇ g / kg body weight may advantageously stimulate endogenous eCG production.
  • the equine chorionic gonadotropin used according to the invention is preferably produced recombinantly.
  • the recombinant production is preferably carried out in a eukaryotic expression system.
  • the synthesis preferably takes place as prehormonal subunits pre- ⁇ -eCG and pre- ⁇ -eCG.
  • the cleavage of the N-termini and thus the generation of the mature subunits ⁇ -eCG and ⁇ -eCG preferably takes place by proteases or during the synthesis of the proteins.
  • the equine chorionic gonadotropin preferably contains both the alpha ( ⁇ -eCG) and beta subunits ( ⁇ -eCG) of the eCG.
  • the alpha subunit has SEQ ID no. 3 ( ⁇ -eCG) or SEQ ID NO. 4.
  • the beta subunit ( ⁇ -eCG) has SEQ ID no. 1 or SEQ ID NO. Second
  • the sequence SEQ ID no. 1 and SEQ ID NO. 3 are each the amino acid sequences of the pre-hormonal subunits (pre- ⁇ -eCG or pre- ⁇ -eCG).
  • the mature ⁇ -eCG subunit (SEQ ID No 4) is formed by cleavage of the first 24 amino acids from pre- ⁇ -eCG (SEQ ID No. 3).
  • the mature ⁇ -eCG subunit (SEQ ID No 2) is formed by cleavage of the first 20 amino acids from pre- ⁇ -eCG (SEQ ID No. 1). The cleavage of the amino acid sequences in the prehormonal subunits to obtain the mature subunits occurs either before administration as a drug or after administration in the organism to be treated.
  • An alternative pre- ⁇ -eCG or ⁇ -eCG sequence is described in SEQ ID no. 10 or SEQ ID no. 11 indicated. Included here are in each case sequences which belong to one of these sequences SEQ ID no. 1 to 4 and 10 or 11 have a homology of at least 90%, preferably of at least 95%.
  • the alpha subunit ( ⁇ -eCG) is preferably at the amino acids selected from Asp-56, Asp-82 glycanome the specified amino acid positions refer to the sequence of the mature ⁇ -eCG, for the pre- ⁇ -eCG shift the positions corresponding to +24 amino acids (Asp-80, Asp-106).
  • the beta subunit ( ⁇ -eCG) is preferably selected from the amino acids selected from Asp-13 Ser-123, Thr-127, Ser-128, Thr-129, Ser-130, Thr-131, Thr-133, Ser-137 , Ser-140, Ser-141, Thr-148, Ser-149 glycated (the indicated amino acid positions refer to the sequence of the mature ß-eCG, for the pre-ß-eCG positions shift accordingly by +20 amino acids, Asp-33, Ser-143, .).
  • the glycan chains each contain preferably 2 to 15, more preferably 2 to 7 sugar residues.
  • Preferred sugar residues are N-acetylglucosamine (GlcNac), galactose, manose, fucose, sialic acid (N-acetylneuraminic acid).
  • the glycan side chains of the eCG beta subunit of the luteal eCG in the normal endometrium of the non-pregnant mare may differ slightly from the above-described glycan chains of the chorio-villous eCG.
  • the alpha subunit contains 2 to 5 disulfide bridges between the cysteine pairs selected from Cys-11 with Cys 36, Cys 14 with Cys-35, Cys-63 or Cys-64 with Cys-91, Cys-86 Cys-88 and Cys-32 with Cys-63 or Cys-64 (the indicated amino acid positions refer to the sequence of the mature ⁇ -eCG, whereas for the pre- ⁇ -eCG the positions shift accordingly by +24 amino acids).
  • the beta subunit contains 2 to 6 disulfide bridges between the cysteine pairs selected from.
  • the indicated amino acid positions refer to the sequence of the mature ß-eCG, for the pre-ß-eCG the positions shift accordingly by +20 amino acids).
  • Preferred nucleic acid sequences coding for the alpha subunit ( ⁇ -eCG) are SEQ ID NO. 7 and SEQ ID NO. 8, wherein SEQ ID no. 7 encoded for the prehomonal alpha subunit (pre- ⁇ -eCG).
  • Preferred nucleic acid sequences coding for the beta subunit ( ⁇ -eCG) are SEQ ID NO. 5 and SEQ ID NO. 6, wherein SEQ ID no. 5 encoded for the prehomonal beta subunit (pre- ⁇ -eCG). Included are also sequences which belong to one of these sequences SEQ ID no. 5 to 8 have a homology of at least 90%.
  • SEQ ID no. 1 to 11 or fragments thereof for the treatment of pregnancy disorders, in particular for the treatment of fertility disorders, implantation disorders, early pregnancy loss, early and habitual abortion and premature birth and growth retardation, in veterinary medicine, especially in Perissodactyla, especially in Equinae, such as horses.
  • the invention also encompasses the use of SEQ ID NO. 1 to 11 or fragments thereof for the treatment of infectious and immunological diseases, in particular of the intestine and the lungs and the joints, in veterinary medicine, especially in Perissodactyla, in particular in Equinae, such as horses.
  • Another object of the invention is the use of SEQ ID NO. 1 to 11 or fragments thereof to aid in transplantation, d. H. for the prevention of rejection reactions, in particular in the transplantation of the cornea and the skin, in veterinary medicine, in particular in perissodactyla, in particular in Equinae, such as horses.
  • the invention also provides the use of SEQ ID no. 1 to 1 1 or fragments thereof as a contraceptive in veterinary medicine, especially in Perissodactyla, especially in Equinae, such as horses.
  • the fragments of the alpha unit preferably have a length of 10 to 95 amino acids. Fragments of the beta unit preferably have a length of 20 to 148 amino acids. Particular preference is given in each case to fragments having a length of from 25 to 50 amino acids.
  • the eCG has hitherto only been produced in insect cells. In the method according to the invention, however, the production takes place in a culture of mammalian cells, such as.
  • CHO Chinese hamster ovary
  • the equine chorionic gonadotropin is administered intramuscularly, intravenously, intraamnically, orally, intra-articularly or in the form of aerosols, preferably 3-6 ⁇ g / kg body weight daily, preferably distributed over several single doses.
  • the serum chorionic gonadotropin levels are determined during pregnancy. In case of suspected fetomaternal functional unit therapy should be started. In case of impaired early pregnancy injections should i. m. be applied. After the serum eCG levels have dropped, diagnosis can be made by determining the chorionic gonadotropin in the amniotic fluid and substituted accordingly. Depending on the level of the amnial eCG, treatment should be limited to the completion of the 10. Pregnancy month.
  • nucleic acid sequence coding for the equine chorionic gonadotropin takes place according to the known methods of gene therapy, e.g. B. by means of adenoviral vectors.
  • the invention also provides a method for the treatment of pregnancy disorders, in particular for the treatment of fertility disorders, implantation disorders, early pregnancy losses, imminent and habitual abortions, as well as premature birth and growth retardation, treatment of infectious and immunological-related diseases, in particular of the intestine and the lungs and the Joints, arthrides and ischaemias and to assist transplantation, in particular of the cornea and the skin, in veterinary medicine, in particular in horses by administering equine chorionic gonadotropin or an equine chorionic gonadotropin encoding nucleic acid sequence (ecg) or fragments of the protein eCG or fragments of the for the equine chorionic gonadotropin-encoding nucleic acid sequence.
  • ecg equine chorionic gonadotropin encoding nucleic acid sequence
  • the agents according to the invention for the first time the implementation of a causal therapy of pregnancy disorders in Perissodactyla, especially in Equinae, such as horses allows.
  • the loss of decidual eCG which causes the pregnancy disorders is substituted by the agents according to the invention.
  • the delivered eCG stimulates the formation of eCG in the endometrium, which in turn calms the uterine musculature and improves circulation to the placenta.
  • the causal treatment of pregnancy disorders and premature birth in terms of premature birth are possible.
  • the medicament according to the invention is used in particular for the treatment of pregnancy disorders.
  • Pregnancy disorders include fertility disorders, implantation disorders, early pregnancy loss, abortion and habitual abortion, as well as premature birth and growth retardation.
  • a fertility disorder is a disorder characterized by the absence of pregnancy despite regular insemination or treatment by in vitro fertilization.
  • An abortion is a so-called miscarriage, which is characterized by an early death or resorption of a fruit.
  • a habitual abortion is present when a mare had a miscarriage three or more times in succession.
  • An intrauterine growth retardation of a fetus occurs when the fetus is too small and in growth for its gestation period.
  • the deviation of the estimated weight by two standard deviations is below the normal value. This variation in size growth is determined by measuring the fetus with ultrasound and then comparing it to growth curves.
  • the agents according to the invention also make it possible to treat infectious and immunologically-related diseases, in particular of the intestine, the lungs and the joints. Furthermore, the agents according to the invention are also suitable for inducing immune tolerance in transplants, in particular the cornea and the skin.
  • Immune tolerance refers to the absence of an immune response after administration of a particular antigen.
  • the autoimmune disease is an umbrella term for diseases whose cause is an excessive reaction of the immune system against the body's own tissue. The body's own tissue is mistakenly perceived as a foreign body to be controlled by the immune system. This leads to severe systemic or local inflammatory reactions, which lead to damage to the affected organs.
  • the medicament of the invention is administered by injection, for example.
  • a particularly preferred embodiment of the medicament is adapted to allow parenteral administration of the drug.
  • parenteral administration of the medicament according to the invention for example, 250 micrograms of the mature eCG are dissolved in 0.5 ml of an injection solution and transferred to a pre-filled syringe.
  • the drug is e.g. administered intramuscularly, intra-amnially, intravenously, orally, intra-articularly or in the form of aerosols. Under emergency conditions, intravenous administration is preferred.
  • administration of the drug is preferably by subcutaneous injection.
  • the drug is adapted so that the amount of equine chorionic gonadotropin administered is 3 to 6 ⁇ g per kg of body weight per day.
  • Injections are prescribed at the onset of regular labor in the event of premature birth. After the contractions have subsided, the injection with the medicament according to the invention takes place at intervals of 2 to 4 days.
  • intravenous infusion e.g. 1000 ⁇ g of the mature eCG dissolved in 500 ml of an infusion solution and administered over a period of four hours.
  • the injection is intraamnial.
  • mononuclear cells are removed from the animal, incubated with the abovementioned eCG forms in vitro and then again intramuscularly, intravenously, orally or in the form of aerosols to be treated Animal back given.
  • the mononuclear cells especially monocytes, NK cells and T cells
  • the mononuclear cells are changed in their properties so that they cause an immune tolerance.
  • the invention also includes a method for cycle synchronization of mares but also for the prevention of pregnancy (contraception) by administration of at least one of SEQ ID NO. 1 to 11 or fragments thereof as, especially in Perissodactyla, especially in Equinae, such as horses.
  • the eCG is preferably injected intramuscularly in the form of depot sticks or inserted intravaginally monthly in ring form.
  • the invention further relates to a method for the production of equine eCG with the subunits ⁇ -chorionic gonadotropin ( ⁇ -eCG) and ⁇ -chorionic gonadotropin ( ⁇ -eCG) in isolated endometrial epithelial cells, decidual cells or cells of the membranes (chorion or amnion).
  • ⁇ -eCG ⁇ -chorionic gonadotropin
  • ⁇ -eCG ⁇ -chorionic gonadotropin
  • the isolated endometrial epithelial cells are preferably cell lines of equine origin.
  • the epithelial cells are preferably obtained from native endometrial tissue.
  • the eCG expressed in these cells has the above-mentioned preferred glycation pattern and the above-mentioned disulfide bridges.
  • the invention also relates to the use of eCG or its fragments in medical diagnostics in particular for the diagnosis of said pregnancy disorders or implantation disorders or infectious and immunological-related diseases, arthrides and ischaemias.
  • the invention further relates to the differentiated diagnosis of the el_H, as well as the eCG of the endometrium and eCG of the placenta, preferably from the serum. Endometrial eCG and placental eCG make a distinction through the sugar side chains.
  • the invention further relates to the use of eCG or its fragments as a contraceptive. Preference is given thereto that eCG or its fragments are administered intramuscularly or intraperitoneally in implants.
  • the subject matter of the invention is also an antibody which specifically recognizes eCG.
  • This is preferably not cross-reactive with human CG (hCG) and preferably recognizes an epitope on the peptide according to SEQ ID no. 9th
  • FIG. 1 shows that eCG is also formed in the endometrium in the sexually mature mare in the second half of the cycle.
  • the eCG was detected by classical immunohistochemistry staining with rabbit anti-eCG (10 ⁇ resolution).
  • FIG. 2 shows that maternal eCG is already formed in the sexually mature mare from the early proliferative (estrus) phase until the second half of the cycle (secretory, diestrus phase) in the gland epithelium of the endometrium.
  • A inactive endometrium, an oestrus or early oestrus phase (proliferative phase);
  • B periovulatory oestrus phase;
  • C oestrus phase, beginning diestrus phase (secretory phase);
  • Fig. 3 shows that fetal eCG is released in pregnant mares in the Girdle cells of the chorio-villous cup structure (placenta, upper arrow) of the conceptus and, in addition, maternal eCG in the gland epithelium of the endometrium (decidua, lower arrows) at gestation (magnification 1: 100).
  • A human placenta as negative or positive control.
  • B to (D) different sections of equine chorio-villous cup structure and endometrium.
  • Left column polyclonal antibody to equine CG (anti-eCG).
  • Right column polyclonal antibody against humans CG (anti-hCG).
  • Embodiment 1 Genetic engineering of recombinant hCG (based on Loumaye et al., 1995)
  • the cDNA sequences coding for the mature ⁇ -eCG and ⁇ -eCG are respectively cloned into the expression vector with the pGEM-T vector system (Promega) according to the manufacturer's instructions.
  • the dehydrofolic acid reductase (DHFR) DNA sequence is cloned into the expression vector.
  • the thus-obtained ⁇ -eCG and the ⁇ -eCG expression vectors are cotransfected into the well-characterized DHFR-deficient CHO (Chinese hamster ovary) cell line, cultured and selected individual clones.
  • the single cell clones are screened for their ability to produce eCG, their eCG biological activity and their genetic stability.
  • the best clones are then cultured in a bioreactor for the production of the recombinant eCG.
  • the secreted into the culture medium eCG is collected and purified by ultrafiltration and conventional chromatography methods and sterile filtered.
  • Embodiment 2 is a diagrammatic representation of Embodiment 1:
  • the CNBr-activated Sepharose 4B column is coupled with the eCG peptide ligand (SEQ ID No. 9) and buffer washed.
  • eCG peptide ligand SEQ ID No. 9
  • buffer washed After capacity calculation of the AK binding, up to 50 ml of eCG antibody serum are applied, prepared and washed after buffer washing with an eluate buffer from the column.
  • the affinity-purified monospecific IgG buffer solutions of the eCG antibody are used for WB and ICH in the dilution 1: 200 to 1: 5000.
  • Example 4 Detection and Diagnostic Recording of the eCG Expression: Detection and Diagnostic Recording of the eCG Release in the Equine Endometrium / Decidua of the Non-Pregnant and Pregnant Mare Using Western Blot and Immunohistochemistry (ICH) for Therapy Control of the eCG Application with the eCG Described -specific antibodies according to SEQ ID No 9 (FIG. 1, FIG. 2, FIG. 3 and FIG. 4)
  • the recombinant eCG produced as in Example 1 or 2 is used for therapy in the following examples:
  • Embodiment 5 Sterility If sterility is suspected, eCG is given intramuscularly every 4 days to induce ovulation and to support the luteal phase at a dose of 3 - 6 ⁇ g / kg body weight until a sufficient increase in eCG is achieved.
  • Embodiment 6 Osteoarthritides
  • 300 ⁇ g / ml eCG is injected into the joint space of the affected joints every 3 to 4 days for a period of 4 weeks.
  • Embodiment 7 Treatment of Growth Retardation
  • Exemplary embodiment 8 Treatment of imminent abortion
  • Embodiment 9 Treatment of fertility disorders, implantation disorders and early pregnancy loss
  • the animals are injected with 500-1000 ⁇ g eCG intramuscularly on the 24th cycle day and further every 4 days.
  • Embodiment 10 Treatment of habitual abortion
  • the animals are given intramuscular injection of 1000 ⁇ g eCG once a week after the diagnosis of pregnancy until the 36th week of pregnancy.
  • Embodiment 11 Treatment for Induction of Contraception
  • 500-1000 ⁇ g eCG is intramuscularly injected in the incipient proliferative (oestrus) cycle phase of the endometrium.
  • administration is by an intraperitoneal eCG implant.
  • Embodiment 12 Treatment of immunological diseases and induction of immunological tolerance in transplantations
  • the eCG is prescribed for the treatment of immunological diseases of the lungs, intestines and joints.
  • the eCG in a dosage of 3 to 5 ug / ml in aerosol form is prescribed to the animals.
  • the eCG in capsule form is administered at a concentration of 3000 ⁇ g per capsule daily for a period of 4 weeks.
  • the animals are injected intraarticularly with 1000 ⁇ g for 3 weeks.
  • the grafts are transported in a solution of 3 ⁇ g / ml eCG to avoid a graft-versus-host reaction.
  • an eCG medium is created at the recipient sites by eCG rinsing in a concentration of 3-6 ⁇ g / ml, which is continued for 3 weeks by injecting the transplant in the same concentration.
  • biomembranes are applied for 4 to 6 weeks, which release eCG.
  • Embodiment 13 Ovulation timing and contraception of the mare
  • eCG depot sticks of 5000 ⁇ g are injected intramuscularly every quarter or intravaginally administered monthly in ring form.
  • Embodiment 14 Treatment of Acute Viral and Bacterial Inflammations
  • eCG is injected intravenously at a daily dose of three times 500 to 1000 ⁇ g.
  • Embodiment 15 Treatment of severe tissue ischemia
  • SEQ ID No. 1 to 4 the positions to which the respective protein carries N-glycosidic side chains are emphasized by simple underlining, positions with O-glycoside side chains are underlined twice.
  • SEQ ID no. 5 to 8 are corresponding nucleic acid sequences.

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Abstract

The invention relates to pharmaceutical agents for the treatment of fertility and pregnancy disorders, and immunological diseases (autoimmune diseases) and transplantation, and a method for the production thereof for use in veterinary medicine, particularly in horses. According to the invention, a protein constituent of equine chorionic gonadotropin (eCG) or a nucleic acid sequence (ecg) coding for a protein constituent of equine chorionic gonadotropin is utilized for the treatment or diagnosis of pregnancy disorders, particularly of fertility disorders, implantation disorders, premature loss of pregnancies, imminent and habitual abortion, premature delivery, and growth retardation, and for the treatment and diagnostics of infectological and immunological diseases, arthritis and ischemia, for facilitating transplantation, for cycle synchronization, and for contraception in veterinary medicine, particularly for perissodactyla, particularly in equines.

Description

Arzneimittel zur Behandlung von Fertilitäts- und Trächtigkeitsstörungen, immunologischbedingten Erkrankungen und Transplantation für die Anwendung in der Tiermedizin, insbesondere bei Pferden sowie Verfahren zur Herstellung und Therapiekontrolle Medicines for the treatment of fertility and pregnancy disorders, immunological disorders and transplantation for use in veterinary medicine, in particular in horses, as well as methods of production and therapy control
Die Erfindung betrifft Mittel zur Behandlung von Fertilitäts- und Schwangerschaftsstörungen (Trächtigkeitsstörungen) und immunologisch-bedingten Erkrankungen (Autoimmunerkrankungen) und Transplantationen sowie Verfahren zur deren Herstellung und Therapiekontrolle für die Anwendung in der Tiermedizin, insbesondere bei Pferden.The invention relates to agents for the treatment of fertility and pregnancy disorders (pregnancy disorders) and immunologically-related diseases (autoimmune diseases) and transplants, and to processes for their preparation and therapy control for use in veterinary medicine, in particular in horses.
Eine gestörte Reproduktion durch Aborte (Verfehlen) und Resorption in der Frühträchtigkeit stellt in der Pferdezucht eine große wirtschaftliche Belastung für die Besitzer dar. Jedes verlorene Fohlen bedeutet einen finanziellen Verlust. Dies spielt vor allem bei der Zucht von wertvollen Pferden eine bedeutend Rolle. Deshalb wird in der Reproduktionsmedizin des Pferdes viel Augenmerk auf die frühzeitige Erkennung von drohenden Aborten bzw. frühen Trächtigkeits- verlusten durch Resorption (Mare Reproductive Loss Syndrome, MRLS: early pregnancy loss and term/close-to term abortations) gelegt. Als Ursache für die Aborte werden gehäuft virale und bakterielle Entzündung im Bereich der Gebärmutter und der Frucht beschrieben. Bei einer Trächtigkeitsdauer von 335 bis 342 Tagen treten die Aborte gehäuft im 7. bis 10. Monat auf.Disturbed reproduction through miscarriage (missing) and absorption in the early pregnancy is a great economic burden for the owners in the horse breeding. Each lost foal means a financial loss. This plays a significant role especially in the breeding of valuable horses. Therefore, in the reproductive medicine of the horse much attention is paid to the early detection of threatening abortions or early loss of pregnancy due to absorption (Mare Reproductive Loss Syndrome, MRLS: early pregnancy loss and term / close-to term abortations). The cause of the abortions is described as frequent viral and bacterial inflammation in the area of the uterus and the fruit. With a gestation period of 335 to 342 days, the abortions occur frequently in the 7th to 10th month.
Aufgabe der Erfindung ist es daher, ein Mittel zur Behandlung von Schwangerschaftsstörungen (Trächtigkeitsstörungen), insbesondere zur Behandlung von Fertilitätsstörungen, Implantationsstörungen, frühen Trächtigkeitsverlusten, imminenten Schwangerschaftsverlusten und habituellen Aborten sowie Frühgeburt und Wachstumsretardierung, zur Anwendung in der Tiermedizin, insbesondere bei Pferden bereitzustellen.The object of the invention is therefore to provide an agent for the treatment of pregnancy disorders (pregnancy disorders), in particular for the treatment of fertility disorders, implantation disorders, early pregnancy losses, imminent pregnancy losses and habitual abortions as well as premature birth and growth retardation, for use in veterinary medicine, in particular in horses.
Aufgabe der Erfindung ist es weiter, Mittel zur Behandlung von infektologischen und immunologisch-bedingten Erkrankungen des Darmes und der Lunge und der Gelenke bzw. zur Transplantation (Cornea, Haut) zur Anwendung in der Tiermedizin, insbesondere bei Pferden anzugeben.The object of the invention is further to provide agents for the treatment of infectological and immunological diseases of the intestine and the lungs and the joints or for transplantation (cornea, skin) for use in veterinary medicine, in particular in horses.
Erfindungsgemäß wird die Aufgabe gelöst durch ein Arzneimittel, das mindestens einen Proteinbestandteil des equinen Choriongonadotropins (eCG) oder eine für einen Proteinbestandteil des equinen Choriongonadotropin codierende Nukleinsäuresequenz (eCG) enthält.According to the invention, this object is achieved by a medicament containing at least one protein component of equine chorionic gonadotropin (eCG) or a nucleic acid sequence (eCG) coding for a protein component of equine chorionic gonadotropin.
Der Proteinbestandteil des equinen Choriongonadotropins (eCG) ist dabei eine prehormonelle Untereinheit (pre-α-eCG bzw. pre-ß-eCG), eine alpha- oder beta-Untereinheit des equinen Choriongonadotropins (α-eCG bzw. ß-eCG) oder ein Fragment der alpha- oder beta- Untereinheit. Die Fragmente der alpha-Einheit haben bevorzugt eine Länge von 10 bis 95 Aminosäuren. Fragmente der beta-Einheit haben bevorzugt eine Länge von 20 bis 148 Aminosäuren. Besonders bevorzugt sind jeweils Fragmente mit einer Länge von 25 bis 50 Aminosäuren.The protein component of equine chorionic gonadotropin (eCG) is a prehormonal subunit (pre-α-eCG or pre-β-eCG), an alpha or beta subunit of equine chorionic gonadotropin (α-eCG or β-eCG) or a Fragment of the alpha or beta subunit. The fragments of the alpha unit preferably have a length of 10 to 95 amino acids. Fragments of the beta unit preferably have a length of 20 to 148 Amino acids. Particular preference is given in each case to fragments having a length of from 25 to 50 amino acids.
Überraschend wurde festgestellt, dass die Administration einer therapeutisch wirksamen Menge von equinen Choriongonadotropin bei Stuten während der Schwangerschaft die Abortrate deutlich reduziert.Surprisingly, it was found that the administration of a therapeutically effective amount of equine chorionic gonadotropin in mares during pregnancy significantly reduces the abortion rate.
Das Arzneimittel eignet sich vorteilhaft zur Behandlung von Schwangerschaftsstörungen, insbesondere zur Behandlung von Fertilitätsstörungen, Implantationsstörungen, frühen Schwangerschaftsverlusten, drohenden und habituellen Abort sowie Frühgeburt, und Wachstumsretardierung in der Tiermedizin, vor allem bei Perissodactyla und Equinae, wie Pferden.The medicament is advantageously suitable for the treatment of pregnancy disorders, in particular for the treatment of fertility disorders, implantation disorders, early pregnancy loss, imminent and habitual abortion and premature birth, and growth retardation in veterinary medicine, especially in Perissodactyla and Equinae, such as horses.
Das equine Choriongonadotropin (eCG) besteht bevorzugt aus einem nicht-kovalent verknüpften Heterodimer aus einer α-Untereinheit (α-eCG) und einer ß-Untereinheit (ß-eCG). Die Sequenzen der α-eCG und der ß-eCG unterscheiden sich beträchtlich von den korrespondierenden humanen hCG-Sequenzen. So unterscheiden sich die ß-Untereinheiten einschließlich der CTP-Abschnitte („C-terminal repeats") der humanen (ß-hCG Aminosäuren 121-145) und der equinen ß-Untereinheit (ß-eCG Aminosäuren 121-149) in etwa 50 % der Aminosäuren und in der Anzahl der Anzahl der N- und O-glykosidischen Seitenketten (1 N- und 12-O-glykosidische Seitenketten in ß-eCG und 2 N- und 4 O-glykosidische Seitenketten in ß-hCG). ß-eCG wird im Unterschied zu ß-hCG nur von einem Gen abgelesen. Bei Pferden hat das in der Plazenta (d. h. in den Girdle-Zellen der chorio-villösen Cupstruktur des Konzeptus (Embryo)) exprimierte eCG und das in der Hypophyse exprimierte el_H (luteinisierendes Hormon) die gleiche Aminosäuresequenz. Das el_H und das eCG unterscheiden sich jedoch in der Glycanseitenketten und damit durch ihre biologische Aktivität am Rezeptor und in der Halbwertzeit. Das equine Choriongonadotropin (eCG) entsteht durch eine weitere Glykosilierung des equinen LH. Mit einem Karbohydratanteil von 40 bis 45% ist das eCG das am ausgeprägteste glykosilierte Säugetierhormon der Plazenta. Das plazentare eCG ist dabei stärker glykosiliert als eLH. Das eCG weist zudem einen niedrigeren Sulfangruppen-Anteil und einen höheren Sialinsäureanteil auf als das eLH.The equine chorionic gonadotropin (eCG) preferably consists of a non-covalently linked heterodimer of an α-subunit (α-eCG) and a β-subunit (β-eCG). The sequences of the α-eCG and the β-eCG differ considerably from the corresponding human hCG sequences. Thus, the β-subunits, including the CTP sections ("C-terminal repeats") of the human (β-hCG amino acids 121-145) and the equine β subunit (β-eCG amino acids 121-149) differ in about 50% of the amino acids and in the number of N- and O-glycosidic side chains (1 N- and 12-O-glycosidic side chains in β-eCG and 2 N and 4 O-glycosidic side chains in β-hCG) .beta.-eCG In contrast to β-hCG, only one gene is read in. In horses, eCG expressed in the placenta (ie in the Girdle cells of the chorio-villous cup structure of the conceptus (embryo)) and the hypophysis-expressed el_H (luteinizing hormone However, the el_H and the eCG differ in the glycan side chain and thus by their biological activity at the receptor and in the half-life.The equine chorionic gonadotropin (eCG) is formed by a further glycosylation of equine LH with a carbohydrate moiety of From 40% to 45%, eCG is the most abundant glycosylated mammalian hormone in the placenta. The placental eCG is more glycosylated than eLH. The eCG also has a lower sulphin group content and a higher sialic acid content than the eLH.
Neuere Erkenntnisse zeigen, dass eCG nicht nur in embryonalem (bzw. fetalem) Gewebe, sondern auch im Endometrium und der Dezidua der nicht-trächtigen bzw. trächtigen Stute exprimiert wird. Das in der Erfindung nachgewiesene endometriale (maternale) eCG des nicht-trächtigen Pferdes in der normalen Lutealphase unterscheidet sich vom hypophysären el_H, vom fetalen (plazentaren, chorio-villösen) eCG und vom dezidualen eCG des trächtigen Pferdes hinsichtlich der Struktur der Glycanseitenketten und kann wie hCG und chorio-villöses eCG als Arzneimittel eingesetzt werden.Recent findings show that eCG is not only expressed in embryonic (or fetal) tissue, but also in the endometrium and the decidua of the non-pregnant or pregnant mare. The endometrial (maternal) eCG of the non-pregnant horse in the normal luteal phase as detected in the invention differs from the pituitary el_H, fetal (placental, chorio-villous) eCG and deciduous eCG of the pregnant horse in terms of the structure of the glycan side chains and may hCG and chorio-villous eCG can be used as drugs.
Das equine Choriongonadotropin weist ein sehr umfangreiches und vielfältiges Wirkungsspektrum auf. eCG unterstützt die Corpus-luteum-Funktion zur Bildung von Progesteron, welches wiederum die eCG Sekretion im Endometrium stimuliert. eCG ist ein Wachstums- und Differenzierungshormon und fördert die Durchblutung der Gebärmutter. Es besitzt auch immunsuppressive, antivirale und antibakterielle Wirkungen. Außerdem stellt eCG die glatte Muskulatur der Gebärmutter ruhig.The equine chorionic gonadotropin has a very extensive and diverse spectrum of activity. eCG supports the corpus luteum function to generate progesterone, which in turn stimulates eCG secretion in the endometrium. eCG is a growth and differentiation hormone and promotes blood flow to the uterus. It also has immunosuppressant, antiviral and antibacterial effects. In addition, eCG keeps the smooth muscles of the uterus quiet.
Das equine Choriongonadotropin ist ein Immunschutzhormon sowie ein Barriereschutz- und Infektschutzhormon, dass besonders in der zweiten Zyklushälfte und während der gesamten Trächtigkeit seine Wirkung entfaltet. Dem Choriongonadotropin kommt in der Reproduktionsphysiologie eine essentielle Bedeutung zu. Obwohl sich die Aminosäuresequenz des equinen Choriongonadotropin deutlich vom humanen Choriongonadotropin unterscheiden, werden überraschenderweise gleichartige Effekte erzielt. Störungen der endometrialen eCG Produktion führt zu Infekten der Gebärmutter sowie der Frucht, was nicht selten den Abort und den Tod des Feten induziert. Eine mangelnde eCG Bildung vor allem in der Frühträchtigkeit verursacht Wachstumsretardierungen sowie in der späteren Trächtigkeit eine Plazentainsuffizienz.Equine chorionic gonadotropin is an immune protection hormone as well as a barrier protection and anti-infective hormone that acts especially during the second half of the cycle and throughout the gestation period. Chorionic gonadotropin plays an essential role in reproductive physiology. Although the amino acid sequence of equine chorionic gonadotropin is significantly different from human chorionic gonadotropin, surprisingly similar effects are obtained. Disturbances of the endometrial eCG production lead to infections of the uterus and the fruit, which not infrequently induces fetal abortion and death. Lack of eCG formation, especially in early pregnancy, causes growth retardation and, in later pregnancy, placental insufficiency.
Das Choriongonadotropin ist wichtig für die Implantation und normale Entwicklung des Embryo. Das equine Choriongonadotropin (eCG) bindet als Arzneimittel an den equinen eLH/eCG- Rezeptoren des Pferdes in der Hypophyse (a), in den Girdle-Zellen der chorio-villösen Cupstruktur des equinen Konzeptus (b) und wahrscheinlich bereits im normalen lutealen Endometrium der nichtträchtigen Stute (c). el_H und eCG binden jedoch mit unterschiedlicher Affinität an die Rezeptoren.Chorionic gonadotropin is important for implantation and normal development of the embryo. The equine chorionic gonadotropin (eCG) binds as a drug at equine eLH / eCG receptors in the pituitary gland (a), in the Girdle cells of the chorio-villous cup structure of the equine concept (b) and probably already in the normal luteal endometrium non-pregnant mare (c). However, el_H and eCG bind with different affinity to the receptors.
Eigene Versuche (s. insbesondere Fig. 2 und Fig. 3) zeigen, dass ein eCG bei der geschlechtsreifen Stute bereits postovulatorisch und auch bei ausbleibender Implantation während der zweiten Zyklushälfte im Endometriums und anderen Epithelien der inneren Oberfläche gebildet wird. Dieses endometriale equine Choriogonadotropin schafft damit im nicht-trächtigen Uterus einen immun-privilegierten Raum, in dem der semiallogene Embryo bzw. Fetus toleriert wird und sich entwickeln kann. Die Freisetzung des endometrialen eCG im normalen lutealen Drüsenepithels kann mit dem entwickelten pferdespezifischen und bevorzugt nicht hCG-kreuzreaktivem eCG-Anitkörper immunhistochemisch und durch Western Blot bewiesen werden (s. Fig. 2, Fig. 3 und Fig. 4). Dieser eCG-Antikörper gestattet ein post- ovulatorisches Monitoring und die Überwachung der Trächtigkeit durch ELISA-Testung. Das zudem mit der Trächtigkeit der Stute in den Girdle-Zellen der chorio-villösen Cupstruktur sezernierte equine Choriongonadotropin wird im peripheren Blut bevorzugt mit einem Peak vom 39. bis zum 130. aber auch bis zum 200. Tag der Trächtigkeit bestimmt. Das equine Choriongonadotropin hat neben der endokrinen auch eine parakrine Bedeutung. Diese Wirkung des endometrialen eCG kann bereits in der normalen Lutealphase des nichtträchtigen Uterus der Stute einsetzen und nimmt weiter mit dem chorio-villösem eCG des Konzeptus ab Implantation weiteren protektiven Einfluß auf den Verlauf der Trächtigkeit ein. Das im maternalen Endometrium des Uterus gebildete equine Choriongonadotropin und das bekannte chorio-villöse eCG der Girdle-Zellen des Konzeptus ist während der gesamten Zeit der Trächtigkeit (Schwangerschaft) von Bedeutung. Während der ersten 150 Tage der Trächtigkeit verläuft ein Abort nicht selten unbemerkt ab. Frühe Trächtigkeitsverluste sind mit einer mangelnden frühen eCG-Sekretion sowohl im maternalen Endometriums und in der fetalen chorio-villösen Girdle-Zellen der Stute verbunden. Dieser Mangel an eCG wird häufig nicht oder erst zu spät erkannt.Our own experiments (see in particular Fig. 2 and Fig. 3) show that an eCG is formed in the sexually mature mare postovulatory and even in the absence of implantation during the second half of the cycle in the endometrium and other epithelia of the inner surface. This endometrial equine choriogonadotropin thus creates an immune-privileged space in the non-pregnant uterus, in which the semiallogenic embryo or fetus is tolerated and can develop. The release of endometrial eCG in the normal luteal gland epithelium can be immunohistochemically and by western blotting with the developed horse-specific and preferably non-hCG cross-reactive eCG antibody are proved (see Fig. 2, Fig. 3 and Fig. 4). This eCG antibody allows post-ovulatory monitoring and pregnancy monitoring by ELISA testing. The equine chorionic gonadotropin secreted with the pregnancy of the mare in the Girdle cells of the chorio-villous cup structure is preferably determined in the peripheral blood with a peak from the 39th to the 130th but also to the 200th day of gestation. The equine chorionic gonadotropin has a paracrine meaning in addition to the endocrine. This effect of the endometrial eCG can already be used in the normal luteal phase of the non-pregnant uterus of the mare and continues to take further protective effect on the course of gestation with the chorio-villous eCG of the conceptus implantation. The equine chorionic gonadotropin formed in the maternal endometrium of the uterus and the well-known chorio-villous eCG of Girdle cells of the conceptus are of importance throughout pregnancy (pregnancy). During the first 150 days of pregnancy, an abortion often goes unnoticed. Early pregnancy losses are associated with a lack of early eCG secretion in both the maternal endometrium and fetal chorio-villous Girdle cells of the mare. This lack of eCG is often not recognized or recognized too late.
Die Barriereschutz- und Infektschutzfunktion des eCG kann auch außerhalb der Schwangerschaft beim Pferd für die Therapie von Erkrankungen des Bronchialtraktes (chronisch-obstruktive Entzündung), des Gastrointestinaltraktes (Koliken) und der Gelenke (Osteoarthritiden) genutzt werden.The eCG's barrier protection and infection protection function can also be used outside the pregnancy in horses for the treatment of diseases of the bronchial tract (chronic obstructive inflammation), the gastrointestinal tract (colic) and the joints (osteoarthritides).
In niedrigen Dosen von 3 bis 6 μg/kg Körpergewicht täglich verabreichtes eCG kann vorteilhaft die endogene eCG-Bildung stimulieren.ECG administered daily at low doses of 3 to 6 μg / kg body weight may advantageously stimulate endogenous eCG production.
Das erfindungsgemäß eingesetzte equine Choriongonadotropin wird bevorzugt rekombinant hergestellt. Die rekombinante Herstellung erfolgt bevorzugt in einem eukaryontischen Expressionsystem. Die Synthese erfolgt bevorzugt als prehormonelle Untereinheiten pre-α-eCG und pre-ß-eCG. Die Abspaltung der N-Termini und damit die Generierung der maturen Untereinheiten α-eCG und ß-eCG erfolgt bevorzugt durch Proteasen oder während der Synthese der Proteine.The equine chorionic gonadotropin used according to the invention is preferably produced recombinantly. The recombinant production is preferably carried out in a eukaryotic expression system. The synthesis preferably takes place as prehormonal subunits pre-α-eCG and pre-β-eCG. The cleavage of the N-termini and thus the generation of the mature subunits α-eCG and β-eCG preferably takes place by proteases or during the synthesis of the proteins.
Das equine Choriongonadotropin enthält bevorzugt sowohl die alpha (α-eCG) als auch die beta- Untereinheit (ß-eCG) des eCG. Bevorzugt hat die alpha-Untereinheit die SEQ ID No. 3 (α-eCG) oder SEQ ID No. 4. Bevorzugt hat die beta-Untereinheit (ß-eCG) die SEQ ID No. 1 oder SEQ ID No. 2. Die Sequenz SEQ ID No. 1 und SEQ ID No. 3 sind jeweils die Aminosäuresequenzen der prehormonellen Untereinheiten (pre-ß-eCG bzw. pre-α-eCG). Die reife α-eCG Untereinheit (SEQ ID No 4) wird durch Abspaltung der ersten 24 Aminosäuren aus pre-α-eCG (SEQ ID No. 3) gebildet. Die reife ß-eCG Untereinheit (SEQ ID No 2) wird durch Abspaltung der ersten 20 Aminosäuren aus pre-ß-eCG (SEQ ID No. 1 ) gebildet. Die Abspaltung der Aminosäuresequenzen in den prehormonellen Untereinheiten, um die reifen Untereinheiten zu erhalten erfolgt entweder vor der Verabreichung als Arzneimittel oder nach der Verabreichung im zu behandelnden Organismus. Eine alternative pre-ß-eCG bzw. ß-eCG Sequenz wird in SEQ ID No. 10 bzw. SEQ ID No. 11 angegeben. Mit umfasst sind dabei jeweils Sequenzen, die zu einer dieser Sequenzen SEQ ID No. 1 bis 4 und 10 oder 11 eine Homologie von mindestens 90 %, bevorzugt von mindestens 95 %, aufweisen.The equine chorionic gonadotropin preferably contains both the alpha (α-eCG) and beta subunits (β-eCG) of the eCG. Preferably, the alpha subunit has SEQ ID no. 3 (α-eCG) or SEQ ID NO. 4. Preferably, the beta subunit (β-eCG) has SEQ ID no. 1 or SEQ ID NO. Second The sequence SEQ ID no. 1 and SEQ ID NO. 3 are each the amino acid sequences of the pre-hormonal subunits (pre-β-eCG or pre-α-eCG). The mature α-eCG subunit (SEQ ID No 4) is formed by cleavage of the first 24 amino acids from pre-α-eCG (SEQ ID No. 3). The mature β-eCG subunit (SEQ ID No 2) is formed by cleavage of the first 20 amino acids from pre-β-eCG (SEQ ID No. 1). The cleavage of the amino acid sequences in the prehormonal subunits to obtain the mature subunits occurs either before administration as a drug or after administration in the organism to be treated. An alternative pre-β-eCG or β-eCG sequence is described in SEQ ID no. 10 or SEQ ID no. 11 indicated. Included here are in each case sequences which belong to one of these sequences SEQ ID no. 1 to 4 and 10 or 11 have a homology of at least 90%, preferably of at least 95%.
Wegen der komplexen Struktur des eCG muss bei der Zubereitung des Arzneimittels darauf geachtet werden, dass die Integrität des e-CG-Moleküls erhalten bleibt. Disulfidbrücken- bindungen sowie Threonin-O-, Serin-O- und Asparagin-N-gebundene Glycosaccharid- Seitenketten garantieren die biologische Aktivität des eCG.Because of the complex structure of the eCG, care must be taken in the preparation of the drug to maintain the integrity of the e-CG molecule. Disulfide bridge bonds as well as threonine O, serine O and asparagine N-linked glycosaccharide side chains guarantee the biological activity of the eCG.
Die alpha-Untereinheit (α-eCG) ist bevorzugt an den Aminosäuren ausgewählt aus Asp-56, Asp- 82 glycanisiert die angegebenen Aminosäurepositionen beziehen sich dabei auf die Sequenz des reifen α-eCG, für das pre-α-eCG verschieben sich die Positionen entsprechend um +24 Aminosäuren (Asp-80, Asp-106).The alpha subunit (α-eCG) is preferably at the amino acids selected from Asp-56, Asp-82 glycanisiert the specified amino acid positions refer to the sequence of the mature α-eCG, for the pre-α-eCG shift the positions corresponding to +24 amino acids (Asp-80, Asp-106).
Die beta-Untereinheit (ß-eCG) ist bevorzugt an den Aminosäuren ausgewählt aus Asp-13 Ser- 123, Thr-127, Ser-128, Thr-129, Ser-130, Thr-131 , Thr-133, Ser-137, Ser-140, Ser-141 , Thr- 148, Ser-149 glycanisiert (die angegebenen Aminosäurepositionen beziehen sich dabei auf die Sequenz des reifen ß-eCG, für das pre-ß-eCG verschieben sich die Positionen entsprechend um +20 Aminosäuren, Asp-33, Ser-143, ....).The beta subunit (β-eCG) is preferably selected from the amino acids selected from Asp-13 Ser-123, Thr-127, Ser-128, Thr-129, Ser-130, Thr-131, Thr-133, Ser-137 , Ser-140, Ser-141, Thr-148, Ser-149 glycated (the indicated amino acid positions refer to the sequence of the mature ß-eCG, for the pre-ß-eCG positions shift accordingly by +20 amino acids, Asp-33, Ser-143, ....).
Die Glycanketten enthalten jeweils bevorzugt 2 bis 15, besonders bevorzugt 2 bis 7 Zuckerreste. Bevorzugte Zuckerreste sind N-Acetylglucosamin (GlcNac), Galactose, Manose, Fucose, Sialinsäure (N-Acetylneuraminsäure).The glycan chains each contain preferably 2 to 15, more preferably 2 to 7 sugar residues. Preferred sugar residues are N-acetylglucosamine (GlcNac), galactose, manose, fucose, sialic acid (N-acetylneuraminic acid).
Die Glycanseitenketten der eCG-beta-Untereinheit des lutealen eCG im normalen Endometrium der nicht-trächtigen Stute können sich in geringem Maße von den oben-beschriebenen Glycanketten des chorio-villösen eCG unterscheiden.The glycan side chains of the eCG beta subunit of the luteal eCG in the normal endometrium of the non-pregnant mare may differ slightly from the above-described glycan chains of the chorio-villous eCG.
Bevorzugt enthält die alpha-Untereinheit (α-eCG) 2 bis 5 Disulfidbrücken zwischen den Cysteinpaaren ausgewählt aus Cys-11 mit Cys 36, Cys 14 mit Cys-35, Cys-63 oder Cys-64 mit Cys-91 , Cys-86 mit Cys-88 und Cys-32 mit Cys-63 oder Cys-64 (die angegebenen Amino- säurepositionen beziehen sich dabei auf die Sequenz des reifen α-eCG, für das pre-α-eCG verschieben sich die Positionen entsprechend um +24 Aminosäuren).Preferably, the alpha subunit (α-eCG) contains 2 to 5 disulfide bridges between the cysteine pairs selected from Cys-11 with Cys 36, Cys 14 with Cys-35, Cys-63 or Cys-64 with Cys-91, Cys-86 Cys-88 and Cys-32 with Cys-63 or Cys-64 (the indicated amino acid positions refer to the sequence of the mature α-eCG, whereas for the pre-α-eCG the positions shift accordingly by +24 amino acids).
Bevorzugt enthält die beta-Untereinheit (ß-eCG) 2 bis 6 Disulfidbrücken zwischen den Cysteinpaaren ausgewählt aus. (die angegebenen Aminosäurepositionen beziehen sich dabei auf die Sequenz des reifen ß-eCG, für das pre-ß-eCG verschieben sich die Positionen entsprechend um +20 Aminosäuren).Preferably, the beta subunit (β-eCG) contains 2 to 6 disulfide bridges between the cysteine pairs selected from. (The indicated amino acid positions refer to the sequence of the mature ß-eCG, for the pre-ß-eCG the positions shift accordingly by +20 amino acids).
Bevorzugte für die alpha-Untereinheit (α-eCG) codierende Nukleinsäuresequenzen sind SEQ ID No. 7 und SEQ ID No. 8, wobei die SEQ ID No. 7 für die prehomonelle alpha-Untereinheit (pre- α-eCG) codiert. Bevorzugte für die beta-Untereinheit (ß-eCG) codierende Nukleinsäuresequenzen sind SEQ ID No. 5 und SEQ ID No. 6, wobei die SEQ ID No. 5 für die prehomonelle beta- Untereinheit (pre-ß-eCG) codiert. Mit umfasst sind Sequenzen, die zu einer dieser Sequenzen SEQ ID No. 5 bis 8 eine Homologie von mindestens 90 % aufweisen.Preferred nucleic acid sequences coding for the alpha subunit (α-eCG) are SEQ ID NO. 7 and SEQ ID NO. 8, wherein SEQ ID no. 7 encoded for the prehomonal alpha subunit (pre-α-eCG). Preferred nucleic acid sequences coding for the beta subunit (β-eCG) are SEQ ID NO. 5 and SEQ ID NO. 6, wherein SEQ ID no. 5 encoded for the prehomonal beta subunit (pre-β-eCG). Included are also sequences which belong to one of these sequences SEQ ID no. 5 to 8 have a homology of at least 90%.
Bestandteil der Erfindung ist auch die Verwendung der SEQ ID No. 1 bis 11 oder Fragmenten daraus zur Behandlung von Schwangerschaftsstörungen, insbesondere zur Behandlung von Fertilitätsstörungen, Implantationsstörungen, frühen Schwangerschaftsverlusten, frühem und habituellen Abort sowie Frühgeburt und Wachstumsretardierung, in der Tiermedizin, vor allem bei Perissodactyla, insbesondere bei Equinae, wie Pferden.Also part of the invention is the use of SEQ ID no. 1 to 11 or fragments thereof for the treatment of pregnancy disorders, in particular for the treatment of fertility disorders, implantation disorders, early pregnancy loss, early and habitual abortion and premature birth and growth retardation, in veterinary medicine, especially in Perissodactyla, especially in Equinae, such as horses.
Die Erfindung umfasst auch die Verwendung der SEQ ID No. 1 bis 11 oder Fragmenten daraus zur Behandlung von infektologischen und immunologisch-bedingten Erkrankungen, insbesondere des Darmes und der Lunge und der Gelenke, in der Tiermedizin, vor allem bei Perissodactyla, insbesondere bei Equinae, wie Pferden.The invention also encompasses the use of SEQ ID NO. 1 to 11 or fragments thereof for the treatment of infectious and immunological diseases, in particular of the intestine and the lungs and the joints, in veterinary medicine, especially in Perissodactyla, in particular in Equinae, such as horses.
Ein weiterer Gegenstand der Erfindung ist die Verwendung der SEQ ID No. 1 bis 1 1 oder Fragmenten daraus zur Unterstützung der Transplantation, d. h. zur Verhinderung von Abstoßungsreaktionen, insbesondere bei der Transplation von Cornea und der Haut, in der Tiermedizin, vor allem bei Perissodactyla, insbesondere bei Equinae, wie Pferden.Another object of the invention is the use of SEQ ID NO. 1 to 11 or fragments thereof to aid in transplantation, d. H. for the prevention of rejection reactions, in particular in the transplantation of the cornea and the skin, in veterinary medicine, in particular in perissodactyla, in particular in Equinae, such as horses.
Gegenstand der Erfindung ist auch die Verwendung der SEQ ID No. 1 bis 1 1 oder Fragmenten daraus als Kontrazeptivum in der Tiermedizin, vor allem bei Perissodactyla, insbesondere bei Equinae, wie Pferden.The invention also provides the use of SEQ ID no. 1 to 1 1 or fragments thereof as a contraceptive in veterinary medicine, especially in Perissodactyla, especially in Equinae, such as horses.
Die Fragmente der alpha-Einheit haben bevorzugt eine Länge von 10 bis 95 Aminosäuren. Fragmente der beta-Einheit haben bevorzugt eine Länge von 20 bis 148 Aminosäuren. Besonders bevorzugt sind jeweils Fragmente mit einer Länge von 25 bis 50 Aminosäuren. Im Stand der Technik wurde das eCG bisher nur in Insektenzellen hergestellt. Bei dem erfindungsgemäßen Verfahren erfolgt die Herstellung jedoch in einer Kultur von Säugetierzellen, wie z. B. in einer Kultur von Ovarienzelle des Chinesischen Hamsters (CHO-Zellen), bevorzugt jedoch in einer Kultur von Zellen des equinen (bevorzugt zyklischen) Endometriums der nichtträchtigen Stute, der Dezidua (maternal), der Plazenta bzw. Girdle-Zellen der chorio-villösen Cupstruktur, oder des Epithels der Eihäute (Chorion bzw. Amnion).The fragments of the alpha unit preferably have a length of 10 to 95 amino acids. Fragments of the beta unit preferably have a length of 20 to 148 amino acids. Particular preference is given in each case to fragments having a length of from 25 to 50 amino acids. In the prior art, the eCG has hitherto only been produced in insect cells. In the method according to the invention, however, the production takes place in a culture of mammalian cells, such as. In a culture of Chinese hamster ovary (CHO) cells, but preferably in a culture of cells of the equine (preferably cyclic) endometrium of the non-pregnant mare, the decidua (maternal), the placenta or girdle cells of the chorionic villous cup structure, or the epithelium of the membranes (chorion or amnion).
Das equine Choriongonadotropin wird intramuskulär, intravenös, intraamnial, oral, intraartikulär bzw. in Form von Aerosolen verabreicht, bevorzugt werden 3 - 6 μg/kg Körpergewicht täglich, bevorzugt verteilt auf mehrere Einzelgaben gegeben.The equine chorionic gonadotropin is administered intramuscularly, intravenously, intraamnically, orally, intra-articularly or in the form of aerosols, preferably 3-6 μg / kg body weight daily, preferably distributed over several single doses.
Zur Orientierung eines eCG-Mangels werden in der Schwangerschaft die Serum- Choriongonadotropin-Werte bestimmt. Bei Verdacht auf eine gestörte fetomaternale Funktionseinheit sollte die Therapie begonnen werden. Bei gestörter Frühträchtigkeit sollten die Injektionen i. m. appliziert werden. Nach dem Abfall der Serum-eCG-Werte kann eine Diagnostik über die Bestimmung des Choriongonadotropin in der Amnionflüssigkeit vorgenommen werden und entsprechend substituiert werden. Eine Behandlung sollte je nach Höhe des amnialen eCG bis maximal zum Abschluss des10. Trächtigkeitsmonats erfolgen.For the orientation of an eCG deficiency, the serum chorionic gonadotropin levels are determined during pregnancy. In case of suspected fetomaternal functional unit therapy should be started. In case of impaired early pregnancy injections should i. m. be applied. After the serum eCG levels have dropped, diagnosis can be made by determining the chorionic gonadotropin in the amniotic fluid and substituted accordingly. Depending on the level of the amnial eCG, treatment should be limited to the completion of the 10. Pregnancy month.
Die Verabreichung einer für das equine Choriongonadotropin codierenden Nukleinsäuresequenz erfolgt nach den bekannten Methoden der Gentherapie, z. B. mittels adenoviraler Vektoren.The administration of a nucleic acid sequence coding for the equine chorionic gonadotropin takes place according to the known methods of gene therapy, e.g. B. by means of adenoviral vectors.
Gegenstand der Erfindung ist auch eine Methode zur Behandlung von Schwangerschaftsstörungen, insbesondere zur Behandlung von Fertilitätsstörungen, Implantationsstörungen, frühen Schwangerschaftsverlusten, imminentem und habituellen Aborten, sowie Frühgeburt und Wachstumsretardierung, Behandlung von infektologischen und immunologisch-bedingten Erkrankungen, insbesondere des Darmes und der Lunge und der Gelenke, Arthriden sowie Ischämien und zur Unterstützung der Transplantation, insbesondere von Cornea und der Haut, in der Tiermedizin, insbesondere bei Pferden durch Verabreichung von equinem Choriongonadotropin bzw. einer für das equine Choriongonadotropin codierenden Nukleinsäuresequenz (ecg) oder Fragmente des Proteins eCG oder Fragmente der für das equine Choriongonadotropin codierenden Nukleinsäuresequenz.The invention also provides a method for the treatment of pregnancy disorders, in particular for the treatment of fertility disorders, implantation disorders, early pregnancy losses, imminent and habitual abortions, as well as premature birth and growth retardation, treatment of infectious and immunological-related diseases, in particular of the intestine and the lungs and the Joints, arthrides and ischaemias and to assist transplantation, in particular of the cornea and the skin, in veterinary medicine, in particular in horses by administering equine chorionic gonadotropin or an equine chorionic gonadotropin encoding nucleic acid sequence (ecg) or fragments of the protein eCG or fragments of the for the equine chorionic gonadotropin-encoding nucleic acid sequence.
Mit den erfindungsgemäßen Mitteln wird erstmals die Durchführung einer kausalen Therapie von Schwangerschaftsstörungen bei Perissodactyla, insbesondere bei Equinae, wie Pferden ermöglicht. Der für die Schwangerschaftsstörungen ursächliche Verlust an dezidualem eCG wird durch die erfindungsgemäßen Mittel substituiert. Gleichzeitig stimuliert das zugeführte eCG die Bildung von eCG im Endometrium, was wiederum die Gebärmuttermuskulatur ruhig stellt und die Durchblutung für die Plazenta verbessert. Somit sind die ursächliche Behandlung von Schwangerschaftsstörungen und der vorzeitige Geburtsbeginn im Sinne einer Frühgeburt möglich.With the agents according to the invention for the first time the implementation of a causal therapy of pregnancy disorders in Perissodactyla, especially in Equinae, such as horses allows. The loss of decidual eCG which causes the pregnancy disorders is substituted by the agents according to the invention. At the same time, the delivered eCG stimulates the formation of eCG in the endometrium, which in turn calms the uterine musculature and improves circulation to the placenta. Thus, the causal treatment of pregnancy disorders and premature birth in terms of premature birth are possible.
Somit ist bei einem Mangel an endometrialem eCG dessen Substitution während der gesamten Schwangerschaft bis zum 10. Monat notwendig.Thus, in the absence of endometrial eCG its substitution during the entire pregnancy up to the 10th month is necessary.
Das erfindungsgemäße Arzneimittel dient insbesondere der Behandlung von Schwangerschaftsstörungen. Unter Schwangerschaftsstörungen sind dabei Fertilitätsstörungen, Implantationsstörungen, frühe Schwangerschaftsverluste, Aborte und habitueller Abort sowie Frühgeburt und Wachstumsretardierung zu verstehen.The medicament according to the invention is used in particular for the treatment of pregnancy disorders. Pregnancy disorders include fertility disorders, implantation disorders, early pregnancy loss, abortion and habitual abortion, as well as premature birth and growth retardation.
Bei einer Fertilitätsstörung handelt es sich um eine Störung, die dadurch gekennzeichnet ist, das trotz regelmäßiger Besamung bzw. Behandlungen mittels In-vitro Fertilisation keine Schwangerschaft eintritt.A fertility disorder is a disorder characterized by the absence of pregnancy despite regular insemination or treatment by in vitro fertilization.
Eine Implantationsstörung liegt vor, wenn zwar eine Befruchtung der Eizelle erfolgt bzw. ein Embryotransfer vorgenommen wurde, diese sich jedoch nicht in die Gebärmutterschleimhaut implantieren.There is an implantation disorder if fertilization of the egg or embryo transfer has taken place, but these do not implant in the uterine lining.
Frühe Schwangerschaftsverluste (early pregnancy loss) sind dadurch gekennzeichnet, dass sich ein Embryo zwar in die Gebärmutterschleimhaut einnistet, der Embryo jedoch kurz nach der Implantation abstirbt und resorbiert wird.Early pregnancy loss is characterized by an embryo implanting in the uterine lining, but the embryo dies shortly after implantation and is resorbed.
Ein Abort ist eine sogenannte Fehlgeburt, die durch ein frühzeitiges Absterben bzw. Resorption einer Frucht gekennzeichnet ist. Während hingegen eine habituelle Abort dann vorliegt, wenn eine Stute drei- und mehrfach hintereinander eine Fehlgeburt hatte.An abortion is a so-called miscarriage, which is characterized by an early death or resorption of a fruit. In contrast, a habitual abortion is present when a mare had a miscarriage three or more times in succession.
Eine intrauterine Wachstumsretardierung eines Feten besteht dann, wenn der Fetus für seine Trächtigkeitsdauer zu klein und im Wachstum zurückgeblieben ist. Dabei liegt die Abweichung des geschätzten Gewichtes um zwei Standardabweichungen unter dem Normalwert. Diese Abweichung des Größenwachstums wird nach Abmessen des Fetus mit dem Ultraschall und dem anschließenden Vergleich mit Wachstumskurven festgestellt.An intrauterine growth retardation of a fetus occurs when the fetus is too small and in growth for its gestation period. The deviation of the estimated weight by two standard deviations is below the normal value. This variation in size growth is determined by measuring the fetus with ultrasound and then comparing it to growth curves.
Mit den erfindungsgemäßen Mitteln wird auch die Behandlung von infektologischen und immunologisch-bedingten Erkrankungen, insbesondere des Darmes, der Lunge und der Gelenke ermöglicht. Weiterhin sind die erfindungsgemäßen Mittel auch zur Induktion einer Immuntoleranz bei Transplantationen, insbesondere der Cornea und der Haut, geeignet. Unter der Immuntoleranz wird das Ausbleiben einer Immunreaktion nach Gabe eines bestimmten Antigens verstanden. Die Autoimmunerkrankung ist ein Überbegriff für Krankheiten, deren Ursache in einer überschießenden Reaktion des Immunsystems gegen körpereigenes Gewebe besteht. Dabei wird vom Immunsystem körpereigenes Gewebe irrtümlicherweise als ein zu bekämpfender Fremdkörper wahrgenommen. Dadurch kommt es zu schweren systemischen oder lokalen Entzündungsreaktionen, die zu Schäden an den betroffenen Organen führen.The agents according to the invention also make it possible to treat infectious and immunologically-related diseases, in particular of the intestine, the lungs and the joints. Furthermore, the agents according to the invention are also suitable for inducing immune tolerance in transplants, in particular the cornea and the skin. Immune tolerance refers to the absence of an immune response after administration of a particular antigen. The autoimmune disease is an umbrella term for diseases whose cause is an excessive reaction of the immune system against the body's own tissue. The body's own tissue is mistakenly perceived as a foreign body to be controlled by the immune system. This leads to severe systemic or local inflammatory reactions, which lead to damage to the affected organs.
Das erfindungsgemäße Arzneimittel wird beispielsweise durch Injektion verabreicht. Eine besonders bevorzugte Ausführungsform des Arzneimittels ist so angepasst, dass die parenterale Verabreichung des Arzneimittels ermöglicht wird. Zur parenteralen Verabreichung des erfindungsgemäßen Arzneimittels werden beispielsweise 250 Mikrogramm des maturen eCG in 0,5 ml einer Injektionslösung gelöst und in eine Fertigspritze überführt. Das Arzneimittel wird z.B. intramuskulär, intraamnial, intravenös, oral, intraartikulär oder bzw. in Form von Aerosolen verabreicht. Unter Notfallbedingungen ist dabei die intravenöse Verabreichung bevorzugt. Im Fall einer Störung der frühen Schwangerschaft wie z.B. bei Implantationsstörungen, frühen Schwangerschaftsverlusten, imminentem oder habituellem Abort erfolgt die Verabreichung des Arzneimittels bevorzugt durch subkutane Injektion.The medicament of the invention is administered by injection, for example. A particularly preferred embodiment of the medicament is adapted to allow parenteral administration of the drug. For parenteral administration of the medicament according to the invention, for example, 250 micrograms of the mature eCG are dissolved in 0.5 ml of an injection solution and transferred to a pre-filled syringe. The drug is e.g. administered intramuscularly, intra-amnially, intravenously, orally, intra-articularly or in the form of aerosols. Under emergency conditions, intravenous administration is preferred. In the case of a disorder of early pregnancy, e.g. In case of implantation disorders, early pregnancy loss, or imminent or habitual abortion, administration of the drug is preferably by subcutaneous injection.
Bevorzugt ist das Arzneimittel so angepasst, dass die Menge an verabreichtem equinen Choriongonadotropin 3 bis 6 μg pro kg Körpergewicht pro Tag beträgt.Preferably, the drug is adapted so that the amount of equine chorionic gonadotropin administered is 3 to 6 μg per kg of body weight per day.
Die Injektionen werden mit dem Beginn regelmäßiger Wehen bei einer drohenden Frühgeburt verordnet. Nach Abklingen der Wehen erfolgt die Injektion mit dem erfindungsgemäßen Arzneimittel in Abständen von 2 bis 4 Tagen.Injections are prescribed at the onset of regular labor in the event of premature birth. After the contractions have subsided, the injection with the medicament according to the invention takes place at intervals of 2 to 4 days.
Im Falle eines akuten Wehenbeginns bei fortgeschrittener Eröffnung des Muttermundes erfolgt die Verabreichung des erfindungsgemäßen Arzneimittels, ebenso bei einer drohenden Frühgeburt oder bei intrauteriner Wachstumsretardierung beispielsweise täglich, bevorzugt im Falle einer drohenden Frühgeburt durch intravenöse Infusion. Hierbei werden z.B. 1000 μg des maturen eCG in 500 ml einer Infusionslösung gelöst und über einen Zeitraum von vier Stunden verabreicht. Alternativ erfolgt die Injektion intraamnial.In the case of an acute onset of labor in advanced opening of the cervix, the administration of the medicament according to the invention, as well as in imminent premature birth or in intrauterine growth retardation, for example daily, preferably in the case of imminent premature birth by intravenous infusion. Here, e.g. 1000 μg of the mature eCG dissolved in 500 ml of an infusion solution and administered over a period of four hours. Alternatively, the injection is intraamnial.
Zur Behandlung von immunologisch-bedingten Erkrankungen und zur Induktion einer immunologischen Toleranz bei Transplantationen werden dem Tier bevorzugt mononukleäre Zellen entnommen, mit den oben genannten eCG Formen in vitro inkubiert und danach wieder intramuskulär, intravenös, oral bzw. in Form von Aerosolen an das zu behandelnde Tier zurück gegeben. Bei diesem Schritt werden die mononukleären Zellen (vor allem Monozyten, NK-Zellen und T-Zellen) in ihren Eigenschaften so verändert, dass sie eine Immuntoleranz bewirken.For the treatment of immunologically-related diseases and for the induction of immunological tolerance in transplants, preferably mononuclear cells are removed from the animal, incubated with the abovementioned eCG forms in vitro and then again intramuscularly, intravenously, orally or in the form of aerosols to be treated Animal back given. In this step, the mononuclear cells (especially monocytes, NK cells and T cells) are changed in their properties so that they cause an immune tolerance.
Dabei induziert die Inkubation von mononukleären Zellen mit eCG in vitro die Bildung und Sekretion von eCG dieser Zellen. Dieser Effekt lässt sich hauptsächlich bei Monozyten und T- Zellen nachweisen. Eine systemische eCG Gabe wirkt ebenfalls über diesen Effekt.The incubation of mononuclear cells with eCG in vitro induces the formation and secretion of eCG of these cells. This effect is mainly found in monocytes and T cells. Systemic eCG administration also has this effect.
Eine Aufrechterhaltung dieser Immunität kann durch intravenöse und lokale Applikation der oben genannten eCG Formen oder deren Fragmente erzielt werden. Durch die lokale eCG Applikation (Ort der Transplantation, Gelenkspalt, Blase, Darm, Haut, Liquor) in Form von Injektion, Instillation, Cremes, Sprays, Kapseln wird der chemotaktische Effekt des eCG auf die mononukleären Zellen, die eine Immuntoleranz induzieren, ausgenutzt.Maintenance of this immunity can be achieved by intravenous and local administration of the above eCG forms or fragments thereof. By local eCG application (site of transplantation, joint space, bladder, intestine, skin, cerebrospinal fluid) in the form of injection, instillation, creams, sprays, capsules, the chemotactic effect of the eCG on the mononuclear cells, which induce immune tolerance, is exploited.
Die Erfindung umfasst auch eine Methode zur Zyklussynchronisation von Stuten aber auch zur Verhütung einer Schwangerschaft (Kontrazeption) durch Verabreichung mindestens einer der SEQ ID No. 1 bis 11 oder Fragmenten daraus als, vor allem bei Perissodactyla, insbesondere bei Equinae, wie Pferden. Dazu wird das eCG vorzugsweise in Form von Depotstäbchen intramuskulär injiziert oder monatlich in Ringform intravaginal eingelegt.The invention also includes a method for cycle synchronization of mares but also for the prevention of pregnancy (contraception) by administration of at least one of SEQ ID NO. 1 to 11 or fragments thereof as, especially in Perissodactyla, especially in Equinae, such as horses. For this purpose, the eCG is preferably injected intramuscularly in the form of depot sticks or inserted intravaginally monthly in ring form.
Die Erfindung betrifft weiter ein Verfahren zur Herstellung von equinem eCG mit den Untereinheiten α-Choriongonadotropin (α-eCG) und ß-Choriongonadotropin (ß-eCG) in isolierten endometrialen Epithelzellen, Dezidualzellen oder Zellen der Eihäute (Chorion bzw. Amnion).The invention further relates to a method for the production of equine eCG with the subunits α-chorionic gonadotropin (α-eCG) and β-chorionic gonadotropin (β-eCG) in isolated endometrial epithelial cells, decidual cells or cells of the membranes (chorion or amnion).
Die isolierten endometrialen Epithelzellen sind bevorzugt Zelllinien equinen Ursprungs. Die Epithelzellen werden bevorzugt aus nativem endometrialen Gewebe gewonnen.The isolated endometrial epithelial cells are preferably cell lines of equine origin. The epithelial cells are preferably obtained from native endometrial tissue.
Vorteilhaft weist das in diesen Zellen exprimierte eCG, das oben erwähnte bevorzugte Glykolisierungsmuster und die oben erwähnten Disulfidbrücken auf.Advantageously, the eCG expressed in these cells has the above-mentioned preferred glycation pattern and the above-mentioned disulfide bridges.
Mit dem erfindungsgemäßen Verfahren wird es daher möglich ein eCG bereitzustellen, dessen Glykolisierung, Faltung und Disulfidbrücken den natürlichen Gegebenheiten, u. a. epithelspezifische Glykosylierung, entsprechen.With the method according to the invention it is therefore possible to provide an eCG whose glycosylation, folding and disulfide bridges the natural conditions, u. a. epithelium-specific glycosylation.
Die Erfindung betrifft auch die Verwendung von eCG oder dessen Fragmenten in der medizinischen Diagnostik insbesondere zur Diagnostik der genannten Schwangerschaftstörungen bzw. Implantationstörungen bzw. infektologischen und immunologisch-bedingten Erkrankungen, Arthriden und Ischämien. Die Erfindung betrifft weiter die differenzierte Diagnostik des el_H, sowie des eCG vom Endometrium und von eCG der Plazenta, bevorzugt aus dem Serum. Endometriales eCG und plazentares eCG lassen durch die Zuckerseiten ketten unterscheiden.The invention also relates to the use of eCG or its fragments in medical diagnostics in particular for the diagnosis of said pregnancy disorders or implantation disorders or infectious and immunological-related diseases, arthrides and ischaemias. The invention further relates to the differentiated diagnosis of the el_H, as well as the eCG of the endometrium and eCG of the placenta, preferably from the serum. Endometrial eCG and placental eCG make a distinction through the sugar side chains.
Die Erfindung betrifft weiter die Verwendung von eCG oder dessen Fragmenten als Kontrazeptivum. Bevorzugt wird dazu dass eCG oder dessen Fragmente dazu intramuskulär oder in Implantaten intraperitoneal verabreicht.The invention further relates to the use of eCG or its fragments as a contraceptive. Preference is given thereto that eCG or its fragments are administered intramuscularly or intraperitoneally in implants.
Gegenstand der Erfindung ist auch ein Antikörper, der spezifisch eCG erkennt. Dieser ist bevorzugt nicht kreuzreaktiv mit humanem CG (hCG) und erkennt bevorzugt ein Epitop auf dem Peptid gemäß SEQ ID No. 9.The subject matter of the invention is also an antibody which specifically recognizes eCG. This is preferably not cross-reactive with human CG (hCG) and preferably recognizes an epitope on the peptide according to SEQ ID no. 9th
Anhand folgender Ausführungsbeispiele wird die Erfindung näher erläutert ohne diese zu beschränken.Reference to the following embodiments, the invention is explained in more detail without limiting this.
Fig. 1 zeigt, dass eCG bei der geschlechtsreifen Stute in der zweiten Zyklushälfte auch im Endometriums gebildet wird. Der Nachweis des eCG erfolgte durch klassische Immunohistochemiefärbung mit Kaninchen-anti-eCG (Auflösung 10 x).FIG. 1 shows that eCG is also formed in the endometrium in the sexually mature mare in the second half of the cycle. The eCG was detected by classical immunohistochemistry staining with rabbit anti-eCG (10 × resolution).
Fig. 2 zeigt, dass maternales eCG bei der geschlechtsreifen Stute bereits ab der frühen proliferativen (Östrus-) Phase bis in die zweite Zyklushälfte (sekretorische, Diöstrusphase) im Drüsenepithel des Endometriums gebildet wird. (A): inaktives Endometrium, AnÖstrus- oder früher Östrusphase (proliferative Phase); (B) periovulatorischer Östrusphase; (C) Östrusphase, beginnende Diöstrusphase (sekretorische Phase); (D) und (E) seketorische Diöstrusphase des Zyklus.FIG. 2 shows that maternal eCG is already formed in the sexually mature mare from the early proliferative (estrus) phase until the second half of the cycle (secretory, diestrus phase) in the gland epithelium of the endometrium. (A): inactive endometrium, an oestrus or early oestrus phase (proliferative phase); (B) periovulatory oestrus phase; (C) oestrus phase, beginning diestrus phase (secretory phase); (D) and (E) seketoric diestrus phase of the cycle.
Linke Spalte: Der Nachweis des eCG erfolgte mit dem hergestellten Kaninchen-anti-eCG- Antikörper (Verdünnung 1 :3000) und immunohistochemischer Färbung mit DAB unter Verwendung eines Visualisierungskits (Vergrößerung A bis D 1 :200, E 1 :100). Rechte Spalte: Das Endometrium der Stute zeigt keine Färbung oder Kreuzreaktion mit dem polyklonalen anti- hCG-Antikörper (Verdünnung 1 :1000).Left column: The detection of the eCG was carried out with the prepared rabbit anti-eCG antibody (dilution 1: 3000) and immunohistochemical staining with DAB using a visualization kit (magnification A to D 1: 200, E 1: 100). Right column: The endometrium of the mare shows no staining or cross-reaction with the polyclonal anti-hCG antibody (dilution 1: 1000).
Fig. 3 zeigt, dass fetales eCG bei trächtigen Stuten in den Girdle-Zellen der chorio-villösen Cupstruktur (Plazenta, oberer Pfeil) des Konzeptus und weiterhin zusätzlich maternales eCG im Drüsenepithel des Endometriums (Dezidua; untere Pfeile) bei Trächtigkeit freigesetzt wird (Vergrößerung 1 :100). (A) humane Plazenta als negativ bzw. positiv Kontrolle. (B) bis (D) verschiedene Abschnitte der equinen chorio-villösen Cupstruktur und des Endometriums. Linke Spalte: polyklonaler Antikörper gegen equines CG (anti-eCG). Rechte Spalte: polyklonaler Antikörper gegen humans CG (anti-hCG). Fig. 4 zeigt im Western Blot, daß die Reinsubstanz des hCG (Sigma) in den Konzentrationen 200, 100 und 20 mM vom polyklonalen hCG-Antikörper (Dako, B), aber nicht vom polyklonalen eCG-Antikörper, hergestellt nach SEQ ID No. 9, erkannt und spezifisch nachweisen kann (A). Linke Spalte: polyklonaler Antikörper gegen equines CG (anti-eCG). Rechte Spalte: poly- klonaler Antikörper gegen humanes CG (anti-hCG).Fig. 3 shows that fetal eCG is released in pregnant mares in the Girdle cells of the chorio-villous cup structure (placenta, upper arrow) of the conceptus and, in addition, maternal eCG in the gland epithelium of the endometrium (decidua, lower arrows) at gestation (magnification 1: 100). (A) human placenta as negative or positive control. (B) to (D) different sections of equine chorio-villous cup structure and endometrium. Left column: polyclonal antibody to equine CG (anti-eCG). Right column: polyclonal antibody against humans CG (anti-hCG). Fig. 4 shows in the Western blot that the pure substance of hCG (Sigma) in the concentrations 200, 100 and 20 mM of the polyclonal hCG antibody (Dako, B), but not the polyclonal eCG antibody prepared according to SEQ ID NO. 9, recognized and specifically detectable (A). Left column: polyclonal antibody to equine CG (anti-eCG). Right column: polyclonal antibody to human CG (anti-hCG).
Ausführungsbeispiel 1 : Gentechnische Herstellung von rekombinantem hCG (angelehnt an Loumaye et al. 1995)Embodiment 1: Genetic engineering of recombinant hCG (based on Loumaye et al., 1995)
Die für die reifen α-eCG und ß-eCG codierenden cDNA-Sequenzen (SEQ ID No. 5 und 7) werden mit dem pGEM-T Vektorsystem (Promega) gemäß Hersteller-Anweisung jeweils in den Expressionsvektor cloniert. In den Expressionsvektor wird zusätzlich die Dehydrofolsäurereduktase (DHFR)-DNA-Sequenz kloniert.The cDNA sequences coding for the mature α-eCG and β-eCG (SEQ ID Nos. 5 and 7) are respectively cloned into the expression vector with the pGEM-T vector system (Promega) according to the manufacturer's instructions. In addition, the dehydrofolic acid reductase (DHFR) DNA sequence is cloned into the expression vector.
Die so erhaltenen α-eCG und der ß-eCG-Expressionsvektoren werden in die gut charakterisierte DHFR-defiziente CHO-Zelllinie (Chinese hamster ovary) zusammen kotransfiziert, kultiviert und einzelne Klone selektiert. Die aus je einer Zelle stammenden Klone werden überprüft hinsichtlich ihrer Fähigkeit zur Bildung von eCG, ihrer biologischen Aktivität des eCG und ihrer genetischen Stabilität. Die besten Klone werden anschließend für die Produktion des rekombinanten eCG in einem Bioreaktor kultiviert. Das in das Kulturmedium sekretierte eCG wird gesammelt und mittels Ultrafiltration und gängigen Chromatographiemethoden gereinigt und sterilfiltriert.The thus-obtained α-eCG and the β-eCG expression vectors are cotransfected into the well-characterized DHFR-deficient CHO (Chinese hamster ovary) cell line, cultured and selected individual clones. The single cell clones are screened for their ability to produce eCG, their eCG biological activity and their genetic stability. The best clones are then cultured in a bioreactor for the production of the recombinant eCG. The secreted into the culture medium eCG is collected and purified by ultrafiltration and conventional chromatography methods and sterile filtered.
Ausführungsbeispiel 2:Embodiment 2:
Gentechnische Herstellung von rekombinantem eCG in equinen Epithelzellen des sekretorisches Endometrium oder der Dezidua:Genetic engineering of recombinant eCG in equine epithelial cells of the secretory endometrium or decidua:
- Verwendung eines TOPO TA Cloning Kit (Invitrogen) unter Hersteller-Anweisung je für α-eCG und ß-eCG (SEQ ID No. 5 und 7) und Insertion in den Expressionsvektor entsprechend Ausführungsbeispiel 1.Use of a TOPO TA cloning kit (Invitrogen) under manufacturer instruction for each of α-eCG and β-eCG (SEQ ID NOS: 5 and 7) and insertion into the expression vector according to Example 1.
- Zellseparation und Kultivation von menschlichen Epithelzellen des sekretorischen Endometriums oder der Dezidua.- Cell separation and cultivation of human epithelial cells of the secretory endometrium or decidua.
- Inkorporation (Transfektion) der Vektoren entsprechend Ausführungsbeispiel 1.Incorporation (transfection) of the vectors according to embodiment 1.
- Nutzung der nativen Syntheseleistung der menschlichen Epithelzelle des Endometriums oder der Dezidua zur N-glykosidischen und O-glykosidischen Glykoproteinseitenketten- Produktion (N-Glycan und O-Glycan) von aCG und bhCG.Use of the native synthesis output of the human epithelial cell of the endometrium or the decidua to the N-glycosidic and O-glycosidic glycoprotein side chains Production (N-Glycan and O-Glycan) of aCG and bhCG.
- Ansonsten wird, wie in Ausführungsbeispiel 1 angegeben verfahren. Ausführungsbeispiel 3: Herstellung eines eCG-spezifischen polyklonalen Antikörpers:- Otherwise, as stated in Example 1 procedure. Exemplary Embodiment 3 Production of an eCG-Specific Polyclonal Antibody
Herstellung eines eCG-spezifischen Antikörpers unter Verwendung des Peptid-Antigens nach SEQ ID No 9 für den diagnostischen Nachweisdes eCG mit ELISA, Western Blot und IHC im Blut, Gewebeflüssigkeiten und -homogenaten und Endometriumbiopsien zur Diagnostik und Therapiekontrolle der eCG-Applikation in Körperflüssigkeiten und Geweben: a. Auswahl der Peptidsequenz von 15 AS (SEQ ID No. 9) aus der eCG-beta-Untereinheit nach den Kriterien optimaler Epitop-Analyse und maximaler AS-Differenz zum humanen CG. Synthese des Peptides im Maßstab 15 mg und einer Reinheit von 80-90%. Kopplung des Peptides an einem Carrier (BSA). b. Herstellung der Peptidantiseren nach dem üblichen Standardprotokoll für die Gewinnung polyklonaler Antikörper im Kaninchen (Immunisierung von 2-3 Tieren): I .Tag Erstimmunisiering und 1 ,5 ml Präimmunserumabnahme, 7. Tag 2.lmmunisiering, 14. Tag 3. Immunisierung, 28. Tag 4. Immunisierung, 35. Tag Blutabnahme (20 ml). Erneute Boosterung und Blutabnahme (20ml) nach 4 Wochen für mehrere Monate. Elisa-Titer-Testung im Immunserum mit Titer jeweils über 1 :50.000, Einsatz dieser Antiseren im WB und ICH mit Verdünnung 1 :2.000 bis 1 :8.000. c. Affinitätssäulen-Reinigung zur Isolierung der monospezifischen IgG aus bis zu 50 ml gepoolten Antiserums eines Tieres nach dem Standardprotokoll: Die CNBr-aktivierte Sepharose 4B-Säule wird mit dem eCG-Peptidliganden (SEQ ID No. 9) gekoppelt und puffer-gewaschen. Nach Kapazitätsberechnung der AK-Bindung werden bis zu 50 ml eCG-Antikörperserum aufgetragen, gebuden und nach Pufferwaschung mit einem Eluatpuffer von der Säule gewaschen. Die affinitätsgereinigten monospezifischen IgG-Pufferlösungen des eCG- Antikörpers wird für WB und ICH in der Verdünnung 1 :200 bis 1 :5000 eingesetzt.Preparation of an eCG-specific antibody using the peptide antigen according to SEQ ID No 9 for the diagnostic detection of eCG in the blood with ELISA, Western blot and IHC, tissue fluids and homogenates and endometrial biopsies for the diagnosis and therapy control of eCG application in body fluids and tissues : a. Selection of the peptide sequence of 15 AS (SEQ ID No. 9) from the eCG beta subunit according to the criteria of optimal epitope analysis and maximal AS difference to the human CG. Synthesis of the peptide on a scale of 15 mg and a purity of 80-90%. Coupling of the peptide to a carrier (BSA). b. Preparation of the peptide antisera according to the usual standard protocol for the production of polyclonal antibodies in rabbits (immunization of 2-3 animals): 1st day initial immunization and 1.5 ml pre-immune serum intake, 7th day 2nd immunization, 14th day 3rd immunization, 28. Day 4. Immunization, 35th day Blood sample (20 ml). Renewed Boosterung and blood collection (20ml) after 4 weeks for several months. Elisa titer testing in immune serum with titers above 1: 50,000, use of these antisera in WB and ICH with dilution 1: 2,000 to 1: 8,000. c. Affinity column purification to isolate monospecific IgG from up to 50 ml of pooled antiserum of an animal according to the standard protocol: The CNBr-activated Sepharose 4B column is coupled with the eCG peptide ligand (SEQ ID No. 9) and buffer washed. After capacity calculation of the AK binding, up to 50 ml of eCG antibody serum are applied, prepared and washed after buffer washing with an eluate buffer from the column. The affinity-purified monospecific IgG buffer solutions of the eCG antibody are used for WB and ICH in the dilution 1: 200 to 1: 5000.
Ausführungsbeispiel 4: Nachweis und diagnostische Erfassung der eCG-Expression: Nachweis und diagnostische Erfassung der eCG-Freisetzung im equinen Endometrium/Dezidua der nicht-trächtigen und trächtigen Stute mit Western Blot und Immunhistochemie (ICH) zur Therapiekontrolle der eCG-Applikation mit dem beschriebenen eCG-spezifischen Antikörper nach SEQ ID No 9 (Fig.1 , Fig.2, Fig.3 und Fig.4)Example 4 Detection and Diagnostic Recording of the eCG Expression: Detection and Diagnostic Recording of the eCG Release in the Equine Endometrium / Decidua of the Non-Pregnant and Pregnant Mare Using Western Blot and Immunohistochemistry (ICH) for Therapy Control of the eCG Application with the eCG Described -specific antibodies according to SEQ ID No 9 (FIG. 1, FIG. 2, FIG. 3 and FIG. 4)
Das wie in Ausführungsbeispiel 1 oder 2 hergestellte rekombinante eCG wird in den nachfolgenden Beispielen zur Therapie eingesetzt:The recombinant eCG produced as in Example 1 or 2 is used for therapy in the following examples:
Ausführungsbeispiel 5: Sterilität Bei Verdacht auf Sterilität wird zur Ovulationsauslösung und zur Stützung der Lutealphase eCG des in einer Dosierung von 3 - 6 μg/kg Körpergewicht alle 4 Tage intramuskulär gegeben bis zu einem suffizienten Anstieg des eCG. Ausführungsbeispiel 6: OsteoarthritidenEmbodiment 5: Sterility If sterility is suspected, eCG is given intramuscularly every 4 days to induce ovulation and to support the luteal phase at a dose of 3 - 6 μg / kg body weight until a sufficient increase in eCG is achieved. Embodiment 6: Osteoarthritides
Bei chronischen Arthritiden werden 300 μg/ml eCG alle 3 bis 4 Tage über einen Zeitraum von 4 Wochen in den Gelenkspalt der betroffenen Gelenke injiziert.For chronic arthritis, 300 μg / ml eCG is injected into the joint space of the affected joints every 3 to 4 days for a period of 4 weeks.
Ausführungsbeispiel 7: Behandlung der WachstumsretardierungEmbodiment 7: Treatment of Growth Retardation
Zur Behandlung der Wachstumsretardierung erhalten die Tiere alle zwei bis drei Tage 1000 bisFor the treatment of the growth retardation the animals receive 1000 bis every two to three days
3000 μg eCG intramuskulär injiziert..3000 μg eCG injected intramuscularly.
Ausführungsbeispiel 8: Behandlung des drohenden AbortesExemplary embodiment 8: Treatment of imminent abortion
Zur Behandlung des drohenden Abortes bzw. zur Abortprophylaxe erhalten die Tiere alle 4 TageFor the treatment of threatened abortion or abort prophylaxis, the animals receive every 4 days
250 μg eCG intramuskulär injiziert.250 μg eCG injected intramuscularly.
Ausführungsbeispiel 9: Behandlung von Fertilitätsstörungen, Implantationsstörungen und frühen SchwangerschaftsverlustenEmbodiment 9: Treatment of fertility disorders, implantation disorders and early pregnancy loss
Zur Behandlung von Fertilitäts- und Implantationsstörungen sowie zur Behandlung von frühen Schangerschaftsverlusten erhalten die Tiere am 24. Zyklustag und weiter aller 4 Tage 500 - 1000 μg eCG intramuskulär injiziert.For the treatment of fertility and implantation disorders as well as for the treatment of early pregnancy losses, the animals are injected with 500-1000 μg eCG intramuscularly on the 24th cycle day and further every 4 days.
Ausführungsbeispiel 10: Behandlung von habituellem AbortEmbodiment 10: Treatment of habitual abortion
Zur Behandlung des habituellen Abortes erhalten die Tiere 1000μg eCG einmal wöchentlich nach der Diagnostik der Schwangerschaft bis zur 36. Schwangerschaftswoche intramuskulär injiziert.For the treatment of habitual abortion, the animals are given intramuscular injection of 1000 μg eCG once a week after the diagnosis of pregnancy until the 36th week of pregnancy.
Ausführungsbeispiel 11 : Behandlung zur Induktion der Kontrazeption:Embodiment 11: Treatment for Induction of Contraception
Für die Behandlung zur Auslösung der Kontrazeption bei der Stute werden in der beginnenden proliferativen (Östrus) Zyklusphase des Endometriums 500-1000 μg eCG intramuskulär injiziert.For the treatment to induce contraception in the mare, 500-1000 μg eCG is intramuscularly injected in the incipient proliferative (oestrus) cycle phase of the endometrium.
Alternativ erfolgt die Administration durch ein intraperitonales eCG-lmplantat.Alternatively, administration is by an intraperitoneal eCG implant.
Ausführungsbeispiel 12: Behandlung von immunologisch-bedingten Erkrankungen und zur Induktion einer immunologischen Toleranz bei Transplantationen Das eCG wird zur Behandlung von immunologisch-bedingten Erkrankungen der Lungen, des Darmes und der Gelenke verordnet. Bei chronisch-entzündlichen bzw. allergischen Erkrankungen der Lunge wird das eCG in einer Dosierung von 3 bis 5 μg/ml in Aerosolform den Tieren verordnet.Embodiment 12: Treatment of immunological diseases and induction of immunological tolerance in transplantations The eCG is prescribed for the treatment of immunological diseases of the lungs, intestines and joints. In chronic inflammatory or allergic diseases of the lung, the eCG in a dosage of 3 to 5 ug / ml in aerosol form is prescribed to the animals.
Bei immunologisch-bedingten Darmerkrankungen wird das eCG in Kapselform mit einer Konzentration von 3000 μg pro Kapsel täglich dreimal über einen Zeitraum von 4 Wochen verabreicht.In immunological bowel disease, the eCG in capsule form is administered at a concentration of 3000 μg per capsule daily for a period of 4 weeks.
Bei immunologisch-bedingten Gelenkerkrankungen (Osteoarthritiden) werden den Tieren über 3 Wochen 1000 μg intraartikülär injiziert.In the case of immunologically-related joint diseases (osteoarthritides), the animals are injected intraarticularly with 1000 μg for 3 weeks.
Im Zusammenhang mit Hornhaut- bzw. Hauttransplantaten werden die Grafts zur Vermeidung einer Graft-versus-Host Reaktion in einer Lösung von 3 μg/ml eCG transportiert. Vor der Transplantation wird an den Empfängerarealen durch eine eCG Spülung in einer Konzentration von 3-6 μg /ml ein eCG Milieu geschaffen, dass über 3 Wochen durch Unterspritzung des Transplantates in gleicher Konzentration fortgesetzt wird. Zur Immuntoleranz der Grafts werden Biomembrane über 4 bis 6 Wochen aufgelegt, die eCG freisetzen.In connection with corneal or skin grafts, the grafts are transported in a solution of 3 μg / ml eCG to avoid a graft-versus-host reaction. Before the transplantation, an eCG medium is created at the recipient sites by eCG rinsing in a concentration of 3-6 μg / ml, which is continued for 3 weeks by injecting the transplant in the same concentration. For immune tolerance of the grafts, biomembranes are applied for 4 to 6 weeks, which release eCG.
Ausführungsbeispiel 13: Ovulationstiming und Kontrazeption der StuteEmbodiment 13: Ovulation timing and contraception of the mare
Zur Zyklussynchronisation von Stuten und zur Verhütung von Schwangerschaften werden vierteljährlich eCG-Depotstäbchen von 5000 μg intramuskulär injiziert bzw. monatlich in Ringform intravaginal appliziert.For cycle synchronization of mares and for the prevention of pregnancies, eCG depot sticks of 5000 μg are injected intramuscularly every quarter or intravaginally administered monthly in ring form.
Ausführungsbeispiel 14: Behandlung von akuten viralen und bakteriellen Entzündungen:Embodiment 14: Treatment of Acute Viral and Bacterial Inflammations
Zur Behandlung von schweren viralen und bakteriellen Entzündungen wird eCG in einer täglichen Dosis von drei mal 500 bis 1000 μg intravenös injiziert.For the treatment of severe viral and bacterial infections, eCG is injected intravenously at a daily dose of three times 500 to 1000 μg.
Ausführungsbeispiel 15: Behandlung schwerer Gewebsischämien:Embodiment 15: Treatment of severe tissue ischemia
Zur Behandlung schwerer postpartaler cerebraler Ischämie werden täglich 500 bis 1000 μg eCG intravenös injiziert.For the treatment of severe postpartum cerebral ischemia, 500 to 1000 μg eCG are injected intravenously daily.
In den nachfolgenden Proteinsequenzen (SEQ ID No. 1 bis 4) sind die Positionen, an den das jeweilige Protein N-glykosidische Seitenketten trägt durch einfache Unterstreichung hervorgehoben, Postionen mit O-glykosdische Seitenketten sind doppelt unterstrichen. SEQ ID No. 5 bis 8 sind korrespondierende Nukleinsäuresequenzen. SEQ ID No. 1In the following protein sequences (SEQ ID No. 1 to 4), the positions to which the respective protein carries N-glycosidic side chains are emphasized by simple underlining, positions with O-glycoside side chains are underlined twice. SEQ ID no. 5 to 8 are corresponding nucleic acid sequences. SEQ ID no. 1
<210> 1<210> 1
<211> 169<211> 169
<212> PRT<212> PRT
<213> equus<213> equus
<223> pre-beta-eCG<223> pre-beta eCG
<400> 1<400> 1
Met GIu Thr Leu GIn GIy Leu Leu Leu Trp Met Leu Leu Ser VaI GIy 1 5 10 15Met GIu Thr Leu GIn Gily Leu Leu Leu Trp Met Leu Leu Ser Vai GIy 1 5 10 15
GIy VaI Trp AIa Ser Arg GIy Pro Leu Arg Pro Leu Cys Arg Pro He 20 25 30GIy VaI Trp AIa Ser Arg GIy Pro Leu Arg Pro Leu Cys Arg Pro He 20 25 30
Asn AIa Thr Leu AIa AIa GIu Lys GIu AIa Cys Pro He Cys He Thr 35 40 45Asn AIa Thr Leu AIa AIa GIu Lys GIu AIa Cys Pro He Cys He Thr 35 40 45
Phe Thr Thr Ser He Cys AIa GIy Tyr Cys Pro Ser Met VaI Arg VaI 50 55 60Phe Thr Thr Ser He Cys Ala GIy Tyr Cys Pro Ser Met VaI Arg VaI 50 55 60
Met Pro AIa AIa Leu Pro AIa He Pro GIn Pro VaI Cys Thr Tyr Arg 65 70 75 80Met Pro AIa AIa Leu Pro AIa He Pro GIn Pro VaI Cys Thr Tyr Arg 65 70 75 80
GIu Leu Arg Phe AIa Ser He Arg Leu Pro GIy Cys Pro Pro GIy VaI 85 90 95Glu Leu Arg Phe Ala Ser He Arg Leu Pro Gly Cys Pro Pro Gly VaI 85 90 95
Asp Pro Met VaI Ser Phe Pro VaI AIa Leu Ser Cys His Cys GIy Pro 100 105 HOAsp Pro Met VaI Ser Phe Pro VaI Ala Leu Ser Cys His Cys Gly Per 100 105 HO
Cys GIn He Lys Thr Thr Asp Cys GIy VaI Phe Arg Asp GIn Pro Leu 115 120 125Cys GIn He Lys Thr Thr Asp Cys GIy VaI Phe Arg Asp GIn Pro Leu 115 120 125
AIa Cys AIa Pro GIn AIa Ser Ser Ser Ser Lys Asp Pro Pro Ser GIn 130 135 140 AIa Cys AIa Pro GIn AI Ser Ser Ser Lys Asp Pro Pro Ser GIn 130 135 140
Ser His Pro Leu Pro He Lys Thr Ser 165 Ser His Pro Leu Pro He Lys Thr Ser 165
SEQ ID No. 2SEQ ID no. 2
<160><160>
<210> 2<210> 2
<211> 149<211> 149
<212> PRT<212> PRT
<213> equus caballus<213> equus caballus
<223> beta-eCG<223> beta-eCG
<400> 2<400> 2
Ser Arg GIy Pro Leu Arg Pro Leu Cys Arg Pro He Asn AIa Thr Leu 1 5 10 15Ser Arg Gly Pro Leu Arg Pro Leu Cys Arg Pro He Asn Ala Thr Leu 1 5 10 15
AIa AIa GIu Lys GIu AIa Cys Pro He Cys He Thr Phe Thr Thr Ser 20 25 30Ala Ala GIu Lys GIu Ala Cys Pro He Cys He Thr Phe Thr Thr Ser 20 25 30
He Cys AIa GIy Tyr Cys Pro Ser Met VaI Arg VaI Met Pro AIa AIa 35 40 45He Cys AIa GIy Tyr Cys Pro Ser Met VaI Arg VaI Met Pro AIa AIa 35 40 45
Leu Pro AIa He Pro GIn Pro VaI Cys Thr Tyr Arg GIu Leu Arg Phe 50 55 60Leu Pro AIa He Pro GIn Pro VaI Cys Thr Tyr Arg Giu Leu Arg Phe 50 55 60
AIa Ser He Arg Leu Pro GIy Cys Pro Pro GIy VaI Asp Pro Met VaI 65 70 75 80AIa Ser He Arg Leu Pro Gly Cys Pro Pro Gly VaI Asp Pro Met VaI 65 70 75 80
Ser Phe Pro VaI AIa Leu Ser Cys His Cys GIy Pro Cys GIn He Lys 85 90 95Ser Phe Pro VaI Ala Leu Ser Cys His Cys Gly Pro Cys GIn He Lys 85 90 95
Thr Thr Asp Cys GIy VaI Phe Arg Asp GIn Pro Leu AIa Cys AIa Pro 100 105 HOThr Thr Asp Cys GIy VaI Phe Arg Asp GIn Pro Leu AIa Cys AIa Pro 100 105 HO
GIn AIa Ser Ser Ser Ser Lys Asp Pro Pro Ser GIn Pro Leu Thr Ser 115 120 125GIn Al Ser Ser Ser Lys Asp Pro Pro Ser GIn Pro Leu Thr Ser 115 120 125
Thr Ser Thr Pro Thr Pro GIy AIa Ser Arg Arg Ser Ser His Pro Leu Ϊ3C) 135 TΛÖThr Ser Thr Pro Thr Pro GIy AIa Ser Arg Arg Ser Ser Pro Leu Ϊ3C) 135 TΛÖ
Pro He Lys Thr Ser 145 Pro He Lys Thr Ser 145
SEQ ID No. 3SEQ ID no. 3
<210> 3<210> 3
<211> 120<211> 120
<212> PRT<212> PRT
<213> equus caballus<213> equus caballus
<223> pre-alpha-eCG<223> pre-alpha eCG
<400> 3<400> 3
Met Asp Tyr Tyr Arg Lys His AIa AIa VaI He Leu AIa Thr Leu Ser 1 5 10 15Met Asp Tyr Tyr Arg Lys His Ala Ala VaI He Leu Ala Thr Leu Ser 1 5 10 15
VaI Phe Leu His He Leu His Ser Phe Pro Asp GIy GIu Phe Thr Thr 20 25 30VaI Phe Leu His He Leu His Ser Phe Pro Asp GIy GIu Phe Thr Thr 20 25 30
GIn Asp Cys Pro GIu Cys Lys Leu Arg GIu Asn Lys Tyr Phe Phe Lys 35 40 45GIn Asp Cys Pro Glu Cys Lys Leu Arg Glu Asn Lys Tyr Phe Phe Lys 35 40 45
Leu GIy VaI Pro He Tyr GIn Cys Lys GIy Cys Cys Phe Ser Arg AIa 50 55 60Leu Giy VaI Pro He Tyr GIn Cys Lys Gly Cys Cys Phe Ser Arg AIa 50 55 60
Tyr Pro Thr Pro AIa Arg Ser Arg Lys Thr Met Leu VaI Pro Lys Asn 65 70 75 80Tyr Pro Thr Pro AIa Arg Ser Arg Lys Thr Met Leu VaI Pro Lys Asn 65 70 75 80
He Thr Ser GIu Ser Thr Cys Cys VaI AIa Lys AIa Phe He Arg VaI 85 90 95He Thr Ser GIu Ser Thr Cys Cys VaI Ala Lys Ala Phe He Arg VaI 85 90 95
Thr VaI Met GIy Asn He Lys Leu GIn Asn His Thr GIn Cys Tyr Cys 100 105 HOThr VaI Met GIy Asn He Lys Leu GIn Asn His Thr GIn Cys Tyr Cys 100 105 HO
Ser Thr Cys Tyr His His Lys He 115 120Ser Thr Cys Tyr His His Lys He 115 120
SEQ ID No. 4SEQ ID no. 4
<210> 4<210> 4
<211> 96<211> 96
<212> PRT<212> PRT
<213> equus caballus<213> equus caballus
<223> alpha-eCG<223> alpha eCG
<400> 4<400> 4
Phe Pro Asp GIy GIu Phe Thr Thr GIn Asp Cys Pro GIu Cys Lys Leu 1 5 10 15Phe Pro Asp GIy GIu Phe Thr Thr GIn Asp Cys Pro Giu Cys Lys Leu 1 5 10 15
Arg GIu Asn Lys Tyr Phe Phe Lys Leu GIy VaI Pro He Tyr GIn Cys 20 25 30Arg Giu Asn Lys Tyr Phe Phe Lys Leu Giy VaI Pro He Tyr GIn Cys 20 25 30
Lys GIy Cys Cys Phe Ser Arg AIa Tyr Pro Thr Pro AIa Arg Ser Arg 35 40 45Lys Gly Cys Cys Phe Ser Arg AIa Tyr Pro Thr Pro AIa Arg Ser Arg 35 40 45
Lys Thr Met Leu VaI Pro Lys Asn He Thr Ser GIu Ser Thr Cys Cys 50 55 60Lys Thr Met Leu VaI Pro Lys Asn He Thr Ser Glu Ser Thr Cys Cys 50 55 60
VaI AIa Lys AIa Phe He Arg VaI Thr VaI Met GIy Asn He Lys Leu 65 70 75 80VaI Ala Lys Ala Phe He Arg VaI Thr VaI Met GIy Asn He Lys Leu 65 70 75 80
GIn Asn His Thr GIn Cys Tyr Cys Ser Thr Cys Tyr His His Lys He 85 90 95 SEQ ID No. 5GIn Asn His Thr Gin Cys Tyr Cys Ser Thr Cys Tyr His His Lys He 85 90 95 SEQ ID no. 5
<160><160>
<210> 5<210> 5
<211> 507<211> 507
<212> cDNA resp. RNA<212> cDNA resp. RNA
<213> equus caballus<213> equus caballus
<223> pre-beta-eCG<223> pre-beta eCG
<400> 5<400> 5
1 atggagacgc tccaggggct gctgctgtgg atgctgctga gtgttggcgg ggtctgggca1 atggagacgc tccaggggct gctgctgtgg atgctgctga gtgttgggggg ggtctgggca
61 tccagggggc cactgcggcc actgtgccgg cccatcaacg ccactctggc tgctgagaag61 tccagggggc cactgcggcc actgtgccgg cccatcaacg ccactctggc tgctgagaag
121 gaggcctgcc ccatctgcat caccttcacc accagcatct gtgccggcta ctgccccagc121 gaggcctgcc ccatctgcat caccttcacc accagcatct gtgccggcta ctgccccagc
181 atggtgcggg tgatgccagc tgccctgccg gccattcccc agccagtgtg cacctacctg181 atggtgcggg tgatgccagc tgccctgccg gccattcccc agccagtgtg cacctacctg
241 gagctgcgct ttgcttccat ccggctcccc ggctgcccgc ctggtgtgga ccccatggtc241 gagctgcgct ttgcttccat ccggctcccc ggctgcccgc ctggtgtgga ccccatggtc
301 tccttccccg tggccctcag ttgtcactgc gggccctgcc agatcaagac cactgactgc301 tccttccccg tggccctcag ttgtcactgc gggccctgcc agatcaagac cactgactgc
361 ggggttttca gagaccagcc cttggcctgt gccccccagg cctcctcttc ctctaaggatGccccccagg cctctcttc ctctaaggat
421 cccccatccc aacctctcac atccacatcc accccaactc ctggggccag cagacgttcc421 cccccatccc aacctctcac atccacatcc accccaactc ctggggccag cagacgttcc
481 tctcatcccc tcccaataaa gacttct481 tctcatcccc tcccaataaa gacttct
SEQ ID No. 6SEQ ID no. 6
<160><160>
<210> 6<210> 6
<211> 447<211> 447
<212> cDNA resp. RNA<212> cDNA resp. RNA
<213> equus caballus<213> equus caballus
<223> beta-eCG<223> beta-eCG
<400> 6<400> 6
1 tccagggggc cactgcggcc actgtgccgg cccatcaacg ccactctggc tgctgagaag1 tccagggggc cactgcggcc actgtgccgg cccatcaacg ccactctggc tgctgagaag
61 gaggcctgcc ccatctgcat caccttcacc accagcatct gtgccggcta ctgccccagc61 gaggcctgcc ccatctgcat caccttcacc accagcatct gtgccggcta ctgccccagc
121 atggtgcggg tgatgccagc tgccctgccg gccattcccc agccagtgtg cacctacctg121 atggtgcggg tgatgccagc tgccctgccg gccattcccc agccagtgtg cacctacctg
181 gagctgcgct ttgcttccat ccggctcccc ggctgcccgc ctggtgtgga ccccatggtc181 gagctgcgct ttgcttccat ccggctcccc ggctgcccgc ctggtgtgga ccccatggtc
241 tccttccccg tggccctcag ttgtcactgc gggccctgcc agatcaagac cactgactgc241 tccttccccg tggccctcag ttgtcactgc gggccctgcc agatcaagac cactgactgc
301 ggggttttca gagaccagcc cttggcctgt gccccccagg cctcctcttc ctctaaggat301 ggggttttca gagaccagcc cttggcctgt gccccccagg cctctcttc ctctaaggat
361 cccccatccc aacctctcac atccacatcc accccaactc ctggggccag cagacgttcc361 cccccatccc aacctctcac atccacatcc accccaactc ctggggccag cagacgttcc
421 tctcatcccc tcccaataaa gacttct421 tctcatcccc tcccaataaa gacttct
SEQ ID No. 7SEQ ID no. 7
<160><160>
<210> 7<210> 7
<211> 361<211> 361
<212> cDNA resp. RNA<212> cDNA resp. RNA
<213> equus caballus<213> equus caballus
<223> pre-alpha-eCG<223> pre-alpha eCG
<400> 7<400> 7
1 aggagagcta tggattacta cagaaaacat gcagctgtca tcctggccac attgtccgtg1 aggagagcta tggattacta cagaaaacat gcagctgtca tcctggccac attgtccgtg
61 tttctgcata ttctccattc ctttcctgat ggagagttta caacgcagga ttgcccagaa61 tttctgcata ttctccattc ctttcctgat ggagagttta caacgcagga ttgcccagaa
121 tgcaagctaa gggaaaacaa gtacttcttc aaactgggcg tcccgattta ccagtgtaag121 tgcaagctaa gggaaaacaa gtacttcttc aaactgggcg tcccgattta ccagtgtaag
181 ggctgctgct tctccagagc gtaccccact ccagcaaggt ccaggaagac aatgttggtc181 ggctgctgct tctccagagc gtaccccact ccagcaaggt ccaggaagac aatgttggtc
241 ccaaagaaca tcacctcaga atccacatgc tgtgtggcca aagcatttat cagggtcaca241 ccaaagaaca tcacctcaga atccacatgc tgtgtggcca aagcatttat cagggtcaca
301 gtgatgggaa acatcaagtt ggagaaccac acccagtgct attgcagcac ttgctatcac301 gtgatgggaa acatcaagtt ggagaaccac acccagtgct attgcagcac ttgctatcac
361 cacaagattt aaaggtttca ccaagtg SEQ ID No. 8361 cacaagattt aaaggtttca ccaagtg SEQ ID no. 8th
<160><160>
<210> 8<210> 8
<211> 288<211> 288
<212> cDNA resp. RNA<212> cDNA resp. RNA
<213> equus caballus<213> equus caballus
<223> alpha-eCG<223> alpha eCG
<400> 8<400> 8
1 ttcccggatg gagagtttac aacgcaggat tgcccagaat gcaagctaag ggaaaacaag1 ttcccggatg gagagtttac aacgcaggat tgcccagaat gcaagctaag ggaaaacaag
61 tacttcttca aactgggcgt cccgatttac cagtgtaagg gctgctgctt ctccagagcg61 tacttcttca aactgggcgt cccgatttac cagtgtaagg gctgctgctt ctccagagcg
121 taccccactc cagcaaggtc caggaagaca atgttggtcc caaagaacat cacctcagaa121 taccccactc cagcaaggtc caggaagaca atgttggtcc caaagaacat cacctcagaa
181 tccacatgct gtgtggccaa agcatttatc agggtcacag tgatgggaaa catcaagttg181 tccacatgct gtgtggccaa agcatttatc agggtcacag tgatgggaaa catcaagttg
241 gagaaccaca cccagtgcta ttgcagcact tgctatcacc acaagatt241 gagaaccaca cccagtgcta ttgcagcact tgctatcacc acaagatt
SEQ ID No. 9SEQ ID no. 9
<160><160>
<210>9<210> 9
<211>15<211> 15
<212>PRT<212> PRT
<213>equus caballus<213> equus caballus
<223>beta-eCG<223> beta-eCG
<400>9<400> 9
AIa GIu Lys GIu AIa Cys Pro He Cys He Thr Phe Thr Thr Ser 1 5 10 15AIa GIu Lys GIu AIa Cys Pro He Cys He Thr Phe Thr Thr Ser 1 5 10 15
SEQ ID No. 10SEQ ID no. 10
<210> 10<210> 10
<211> 169<211> 169
<212> PRT<212> PRT
<213> equus<213> equus
<223> pre-beta-eCG<223> pre-beta eCG
<400> 10<400> 10
Met GIu Thr Leu GIn GIy Leu Leu Leu Trp Met Leu Leu Ser VaI GIy 1 5 10 15Met GIu Thr Leu GIn Gily Leu Leu Leu Trp Met Leu Leu Ser Vai GIy 1 5 10 15
GIy VaI Trp AIa Ser Arg GIy Pro Leu Arg Pro Leu Cys Arg Pro He 20 25 30GIy VaI Trp AIa Ser Arg GIy Pro Leu Arg Pro Leu Cys Arg Pro He 20 25 30
Asn AIa Thr Leu AIa AIa GIu Lys GIu AIa Cys Pro He Cys He Thr 35 40 45Asn AIa Thr Leu AIa AIa GIu Lys GIu AIa Cys Pro He Cys He Thr 35 40 45
Phe Thr Thr Ser He Cys AIa GIy Tyr Cys Pro Ser Met VaI Arg VaI 50 55 60Phe Thr Thr Ser He Cys Ala GIy Tyr Cys Pro Ser Met VaI Arg VaI 50 55 60
Met Pro AIa AIa Leu Pro AIa He Pro GIu Pro VaI Cys Thr Tyr Arg 65 70 75 80Met Pro AIa AIa Leu Pro AIa He Pro GIu Pro VaI Cys Thr Tyr Arg 65 70 75 80
GIu Leu Arg Phe AIa Ser He Arg Leu Pro GIy Cys Pro Pro GIy VaI 85 90 95Glu Leu Arg Phe Ala Ser He Arg Leu Pro Gly Cys Pro Pro Gly VaI 85 90 95
Asp Pro Met VaI Ser Phe Pro VaI AIa Leu Ser Cys His Cys GIy Pro 100 105 HO Cys GIn He Lys Thr Thr Asp Cys GIy VaI Phe Arg Asp GIn Pro Leu 115 120 125Asp Pro Met VaI Ser Phe Pro VaI Ala Leu Ser Cys His Cys Gly Per 100 105 HO Cys GIn He Lys Thr Thr Asp Cys GIy VaI Phe Arg Asp GIn Pro Leu 115 120 125
AIa Cys AIa Pro GIn AIa Ser Ser Ser Ser Lys Asp Pro Pro Ser GIn 130 135 140AIa Cys AIa Pro GIn AI Ser Ser Ser Lys Asp Pro Pro Ser GIn 130 135 140
Pro Leu Thr Ser Thr Ser Thr Pro Thr Pro GIy AIa Ser Arg Arg Ser 145 150 155 160Pro Leu Thr Ser Thr Ser Thr Pro Thr Pro GIy AIa Ser Arg Arg Ser 145 150 155 160
Ser His Pro Leu Pro He Lys Thr Ser 165 Ser His Pro Leu Pro He Lys Thr Ser 165
SEQ ID No. 11SEQ ID no. 11
<160><160>
<210> 11<210> 11
<211> 149<211> 149
<212> PRT<212> PRT
<213> equus caballus<213> equus caballus
<223> beta-eCG<223> beta-eCG
<400> 11<400> 11
Ser Arg GIy Pro Leu Arg Pro Leu Cys Arg Pro He Asn AIa Thr Leu 1 5 10 15Ser Arg Gly Pro Leu Arg Pro Leu Cys Arg Pro He Asn Ala Thr Leu 1 5 10 15
AIa AIa GIu Lys GIu AIa Cys Pro He Cys He Thr Phe Thr Thr Ser 20 25 30Ala Ala GIu Lys GIu Ala Cys Pro He Cys He Thr Phe Thr Thr Ser 20 25 30
He Cys AIa GIy Tyr Cys Pro Ser Met VaI Arg VaI Met Pro AIa AIa 35 40 45He Cys AIa GIy Tyr Cys Pro Ser Met VaI Arg VaI Met Pro AIa AIa 35 40 45
Leu Pro AIa He Pro GIu Pro VaI Cys Thr Tyr Arg GIu Leu Arg Phe 50 55 60Leu Pro AIa He Pro GIu Pro VaI Cys Thr Tyr Arg Giu Leu Arg Phe 50 55 60
AIa Ser He Arg Leu Pro GIy Cys Pro Pro GIy VaI Asp Pro Met VaI 65 70 75 80AIa Ser He Arg Leu Pro Gly Cys Pro Pro Gly VaI Asp Pro Met VaI 65 70 75 80
Ser Phe Pro VaI AIa Leu Ser Cys His Cys GIy Pro Cys GIn He Lys 85 90 95Ser Phe Pro VaI Ala Leu Ser Cys His Cys Gly Pro Cys GIn He Lys 85 90 95
Thr Thr Asp Cys GIy VaI Phe Arg Asp GIn Pro Leu AIa Cys AIa Pro 100 105 HOThr Thr Asp Cys GIy VaI Phe Arg Asp GIn Pro Leu AIa Cys AIa Pro 100 105 HO
GIn AIa Ser Ser Ser Ser Lys Asp Pro Pro Ser GIn Pro Leu Thr Ser 115 120 125GIn Al Ser Ser Ser Lys Asp Pro Pro Ser GIn Pro Leu Thr Ser 115 120 125
Thr Ser Thr Pro Thr Pro GIy AIa Ser Arg Arg Ser Ser His Pro Leu 130 135 140Thr Ser Thr Pro Thr Pro GIy AIa Ser Arg Arg Ser Ser Pro Leu 130 135 140
Pro He Lys Thr Ser 145 Pro He Lys Thr Ser 145
Literaturverzeichnis:Bibliography:
Birken, S., O. Yershova, R. V. Myers, M. P. Bernard, and W. Moyle. 2003. Analysis of human choriogonadotropin core 2 o-glycan isoforms. Mol. Cell Endocrinol. 204:21-30.Birken, S., O. Yershova, R.V. Myers, M.P. Bernard, and W. Moyle. 2003. Analysis of human choriogonadotropin core 2 o-glycan isoforms. Mol. Cell Endocrinol. 204: 21-30.
Garcia-Campayo, V., T. Sugahara, and I. Boime. 2002. Unmasking a new recognition signal for O-linked glycosylation in the chorionic gonadotropin beta subunit. Mol. Cell Endocrinol. 194:63- 70.Garcia-Campayo, V., T. Sugahara, and I. Boime. 2002. Unmasking a new recognition signal for O-linked glycosylation in the chorionic gonadotropin beta subunit. Mol. Cell Endocrinol. 194: 63-70.
Howles, C. M. 1996. Genetic engineering of human FSH (Gonal-F). Hum.Reprod. Update. 2:172- 191.Howles, C.M. 1996. Genetic engineering of human FSH (Gonal-F). Hum.Reprod. Update. 2: 172-191.
Loumaye, E., R. Campbell, and J. Salat-Baroux. 1995. Human follicle-stimulating hormone produced by recombinant DNA technology: a review for clinicians. Hum.Reprod. Update. 1 :188- 199.Loumaye, E., R. Campbell, and J. Salat-Baroux. 1995. Human follicle-stimulating hormone produced by recombinant DNA technology: a review for clinicians. Hum.Reprod. Update. 1: 188-199.
Matzuk, M. M., A. J. W. Hsueh, P. Lapolt, A. Tsafriri, J. L. Keene, and I. Boime. 1993. The biological role of carboxyl-terminal extension of human chorionic gonadotropin beta-subunit. Endocrinology 126:376-383.Matzuk, M.M., A.J.W. Hsueh, P. Lapolt, A. Tsafriri, J.L. Keene, and I. Boime. 1993. The biological role of carboxyl terminal extension of human chorionic gonadotrophin beta subunit. Endocrinology 126: 376-383.
Matzuk, M. M., J. L. Keene, and I. Boime. 1989. Site specificity of the chorionic gonadotropin N- linked Oligosaccharides in signal transduction. J.Biol.Chem. 264:2409-2414.Matzuk, M.M., J.L. Keene, and I. Boime. 1989. Site specificity of the chorionic gonadotropin N-linked oligosaccharides in signal transduction. J. Biol. 264: 2409-2414.
Murphy B. D. and S.D. Martinuk. 1991. Equine Chorionic Gonadotropin. Endocrine Rev. 12:27- 44.Murphy B.D. and S.D. Martinuk. 1991. Equine Chorionic Gonadotropin. Endocrine Rev. 12: 27-44.
W. R. (Twink) Allen. 2001. Fetomaternal interactions and influences during equine pregnancy. Reproduction. 121 :513-527.W.R. (Twink) Allen. 2001. Fetomaternal interactions and influenza during equine pregnancy. Reproduction. 121: 513-527.
Bousfield R. G, VI. Y. Butnev and Vi. Y. Butnev. 2001. Identfication of twelf O-glycosylation sites in equine chorionic gonadotropin ß and equine luteinizing hormone ß by solid-phase edman degradation. Biol. Reprod. 64:136-147.Bousfield R. G, VI. Y. Butnev and Vi. Y. Butnev. 2001. Identification of twelf O-glycosylation sites in equine chorionic gonadotropin ß and equine luteinizing hormone ß by solid-phase edman degradation. Biol. Reprod. 64: 136-147.
Legardinier S., M. Duonor-Cerutti, G. Devauchelle, Y. Combarnous and C. Cahoreau. 2005. Biological activities of recombinant equine luteinizing hormone/chorionic gonadotropin (eLH/CG) expressed in Sf9 and mimic insect cell lines. J. Mol. Endocrinol. 34:47-60.Legardinier S., M. Duonor-Cerutti, G. Devauchelle, Y. Combarnous and C. Cahoreau. 2005. Biological activities of recombinant equine luteinizing hormone / chorionic gonadotropin (eLH / CG) expressed in Sf9 and mimic insect cell lines. J. Mol. Endocrinol. 34: 47-60.
Legardinier S., D. Klett, J. -C. Poirier, Y. Combarnous and C. Cahoreau. 2005. Mammalian-like nonsialyl complex-type N-glycosylation of equine gonadotropins in mimic insect cells. Glycobiology. 15:776-790. Legardinier S., D. Klett, J. -C. Poirier, Y. Combarnous and C. Cahoreau. 2005. Mammalian-like nonsialyl complex-type N-glycosylation of equine gonadotrophins in mimic insect cells. Glycobiology. 15: 776-790.

Claims

Patentansprüche claims
1. Arzneimittel enthaltend mindestens einen Proteinbestandteil des equinen Choriongona- dotropins (eCG) oder eine für einen Proteinbestandteil des equinen Chorion- gonadotropin codierende Nukleinsäuresequenz {ecg).1. A medicament containing at least one protein component of equine chorionic gonadotropin (eCG) or a nucleic acid sequence coding for a protein component of the equine chorionic gonadotropin {ecg).
2. Arzneimittel nach Anspruch 1 , dadurch gekennzeichnet, dass es mindestens eine der Proteinsequenzen ausgewählt aus den Sequenzen SEQ ID 1 bis 4 oder SEQ ID 9 bis 11 oder mindestens eine Nukleinsäuresequenz ausgewählt aus den Sequenzen SEQ ID 5 bis 8 enthält oder eine Sequenz, die zu einer dieser Sequenzen eine Homologie von mindestens 90 % aufweist.2. Medicament according to claim 1, characterized in that it contains at least one of the protein sequences selected from the sequences SEQ ID 1 to 4 or SEQ ID 9 to 11 or at least one nucleic acid sequence selected from the sequences SEQ ID 5 to 8 or a sequence containing to one of these sequences has a homology of at least 90%.
3. Verwendung eines Arzneimittel nach Anspruch 1 oder 2 zur Behandlung von Schwangerschaftsstörungen, insbesondere zur Behandlung von Fertilitätsstörungen, Implantationsstörungen, frühen Schwangerschaftsverlusten, imminentem und habituellen Abort, Frühgeburt und Wachstumsretardierung, sowie zur Behandlung von infektologischen und immunologisch-bedingten Erkrankungen und zur Transplantation in der Tiermedizin, vor allem bei Perissodactyla, insbesondere bei Equinae3. Use of a medicament according to claim 1 or 2 for the treatment of pregnancy disorders, in particular for the treatment of fertility disorders, implantation disorders, early pregnancy loss, imminent and habitual abortion, premature birth and growth retardation, and for the treatment of infectious and immunological-related diseases and transplantation in the Veterinary medicine, especially in Perissodactyla, especially in Equinae
4. Verwendung eines Proteinbestandteil des equinen Choriongonadotropins (eCG) oder eine für einen Proteinbestandteil des equinen Choriongonadotropin codierende Nukleinsäuresequenz (ecg) zur Behandlung oder Diagnostik von Schwangerschaftsstörungen, insbesondere von Fertilitätsstörungen, Implantationsstörungen, frühen Schwangerschaftsverlusten, imminentem und habituellen Abort, Frühgeburt und Wachstumsretardierung, sowie zur Behandlung oder Diagnostik von infektologischen und immunologisch-bedingten Erkrankungen, Arthriden und Ischämien, zur Unterstützung der Transplantation, zur Zyklussynchronisation und zur Kontrazeption in der Tiermedizin, vor allem bei Perissodactyla, insbesondere bei Equinae.4. Use of a protein component of equine chorionic gonadotropin (eCG) or an equine chorionic gonadotropin-encoding nucleic acid sequence (ecg) for the treatment or diagnosis of pregnancy disorders, in particular fertility disorders, implantation disorders, early pregnancy loss, imminent and habitual abortion, premature birth and growth retardation, and for the treatment or diagnosis of infectious and immunological diseases, arthrides and ischaemias, for the support of transplantation, for cycle synchronization and for contraception in veterinary medicine, especially for perissodactyla, in particular in Equinae.
5. Verwendung nach Anspruch 4 dadurch gekennzeichnet, dass der Proteinbestandteil mindestens eine der Proteinsequenzen ausgewählt aus den Sequenzen SEQ ID 1 bis SEQ ID No. 4 oder SEQ ID No. 9 bis 11 oder die für den Proteinbestandteil codierende Nukleinsäuresequenz (ecg) mindestens eine Nukleinsäuresequenz ausgewählt aus den Sequenzen SEQ ID 5 bis SEQ ID No. 8 enthält oder eine Sequenz, die zu einer dieser Sequenzen gemäß SEQ ID 1 bis SEQ ID No. 8 eine Homologie von mindestens 90 % aufweist. 5. Use according to claim 4, characterized in that the protein constituent at least one of the protein sequences selected from the sequences SEQ ID 1 to SEQ ID NO. 4 or SEQ ID NO. 9 to 11 or the nucleic acid sequence coding for the protein constituent (ecg) at least one nucleic acid sequence selected from the sequences SEQ ID 5 to SEQ ID NO. 8 contains or a sequence corresponding to one of these sequences according to SEQ ID 1 to SEQ ID NO. 8 has a homology of at least 90%.
6. Verwendung des eCG-Peptides nach SEQ ID No 9 zur Herstellung eines eCG- spezifischen Antikörpers oder für die Diagnostik und Therapiekontrolle der eCG- Applikation bei Tieren, insbesondere bei Pferden.6. Use of the eCG peptide according to SEQ ID No 9 for the production of an eCG-specific antibody or for the diagnosis and therapy control of the eCG application in animals, in particular in horses.
7. Antikörper, der spezifisch eCG erkennt.7. Antibody that specifically recognizes eCG.
8. Verwendung des Antikörpers nach Anspruch 7 für die Diagnostik und Therapiekontrolle der eCG-Applikation bei Tieren, insbesondere bei Pferden. 8. Use of the antibody according to claim 7 for the diagnosis and therapy control of eCG application in animals, in particular in horses.
EP08759974A 2007-05-23 2008-05-23 Pharmaceutical agent for the treatment of fertility and gestation disorders, immunological diseases, and transplantation for use in veterinary medicine, particularly in horses, and methods for the production and treatment monitoring thereof Withdrawn EP2148694A2 (en)

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PCT/EP2008/056374 WO2008142162A2 (en) 2007-05-23 2008-05-23 Pharmaceutical agent for the treatment of fertility and gestation disorders, immunological diseases, and transplantation for use in veterinary medicine, particularly in horses, and methods for the production and treatment monitoring thereof

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BR102015032660B1 (en) 2015-12-28 2019-05-28 Ouro Fino Saúde Animal Participações S.A. PROCESS OF PRODUCTION OF A RECOMBINANT EQUINE CHORIONIC GONADOTROFIN (RECG): VETERINARY COMPOSITION AND USE

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WO1990002757A1 (en) * 1988-09-02 1990-03-22 Integrated Genetics, Inc. Heteropolymeric protein
AU4964197A (en) * 1996-11-12 1998-06-03 Teikoku Hormone Mfg. Co., Ltd. Recombinant single-stranded equine chorionic gonadotropin
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Title
MAUREL M-C ET AL: "Immunochemical study of equine chorionic gonadotropin (eCG/PMSG): Antigenic determinants on alpha- and beta-subunits", BIOCHIMICA ET BIOPHYSICA ACTA, ELSEVIER, NL, vol. 1159, no. 1, 1 January 1992 (1992-01-01), pages 74 - 80, XP009030538, ISSN: 0006-3002 *

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