EP1951287A2 - Medicament destine a traiter les problemes de fertilite et de grossesse, et les maladies auto-immunes, et a induire une tolerance immunologique chez des patients transplantes, et procede de production du dit medicament - Google Patents

Medicament destine a traiter les problemes de fertilite et de grossesse, et les maladies auto-immunes, et a induire une tolerance immunologique chez des patients transplantes, et procede de production du dit medicament

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Publication number
EP1951287A2
EP1951287A2 EP06805531A EP06805531A EP1951287A2 EP 1951287 A2 EP1951287 A2 EP 1951287A2 EP 06805531 A EP06805531 A EP 06805531A EP 06805531 A EP06805531 A EP 06805531A EP 1951287 A2 EP1951287 A2 EP 1951287A2
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EP
European Patent Office
Prior art keywords
hcg
seq
subunit
mature
precursor
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EP06805531A
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German (de)
English (en)
Inventor
Henry Alexander
Gerolf Zimmermann
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VivoSensMedical GmbH
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Universitaet Leipzig
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders

Definitions

  • the invention relates to agents for the treatment of fertility and pregnancy disorders, autoimmune diseases and induction of immunological tolerance in transplant patients for use in medicine, in particular in gynecology and transplantation medicine, and a method for producing.
  • prematurity is a major medical problem.
  • premature birth the pregnancy ends before the 37th week of pregnancy (normal duration of pregnancy: 40 weeks).
  • the premature birth rate is around 6 to 7%.
  • "Premature babies” have a weight of 500 to 2500 g.
  • the neonatal care of premature births often extends over several months and is a very cost-intensive field of paediatrics Despite intensive medical care of the surviving premature births carry about 7% late damage (neurological damage, physical and mental developmental disorder or retardation, visual or hearing disorders) thereof.
  • Late pregnancy disorders are currently being treated symptomatically with progesterone, magnesium, ß-sympathomimetics or antihypertensives.
  • Fas ligand is also important for immune tolerance (Fandrich F., Lin X., Klöppel G. and Kremer B., 1998, Fandrich F., Lin X, Zhu X, Parwaresch R., Kremer B. and Henne- Bruns D., 1998).
  • the hormones human chorionic gonadotropin (hCG), LH (luteinizing hormone), FSH (follicle stimulating hormone) and TSH (thyroid stimulating hormone) make up a family of glycoprotein hormones. They consist of a non-covalently linked heterodimer of an ⁇ -subunit and a ⁇ -subunit. The ⁇ subunit is identical in all four hormones, the ⁇ subunits are different and define the endocrine functions of the heterodimers (Pierce et al 1981).
  • the ⁇ -subunit of chorongonadotropin differs from the other ⁇ -subunits of glycoprotein hormones mainly in that it is extended at the C-terminus by 23 amino acids - the so-called C-terminal peptide (CTP).
  • CTP C-terminal peptide
  • the ⁇ -subunit of hCG has two asparagine-N-glycosidic side chains, and the C-terminal peptide (CTP) portion (amino acids 122 to 145) has four additional serine-O-glycosidic oligosaccharide side chains.
  • the ⁇ -subunit of LH, FSH, TSH and human chorionic gonadotropin ( ⁇ CG) is encoded by a gene located on chromosome 6 (chromosome 6q21.1-q23), while the ⁇ -subunit of human chorionic gonadotropin ( ⁇ hCG) of the six homologous genes hCG ⁇ 1, ⁇ 2, ⁇ 3, ⁇ 5, ⁇ 7 and ⁇ 8, which are located in a gene cluster on chromosome 19 (chromosome 19q13.3) next to LH ⁇ 4 (Jameson et al., 1993).
  • the ⁇ hCG gene ⁇ 6 is probably an allele of ⁇ 7 with differences in the non-translating nucleotide sequence of the promoter gene (exon 1) and the translational sequence (exon 2) of the ⁇ hCG subunit.
  • hCG heterodimer and free ⁇ CG and ⁇ hCG subunits are formed in the trophoblast of the early embryo (from days 6 to 12 after conception) and later in the syncytiotrophoblast of the placenta and secreted into the blood.
  • This trophoblastic tissue expresses exclusively the ⁇ hCG subunits hCG ⁇ 5, ⁇ 8 and ⁇ 3.
  • ⁇ hCG subunits are therefore called trophoblastic ⁇ hCG (t ⁇ hCG) or type Il - ⁇ hCG.
  • This trophoblastic hCG binds to the corpus luteum, which is thus stimulated to produce and further secrete progesterone needed to maintain pregnancy.
  • the trophoblastic hCG acts on a common membrane-bound G protein-coupled receptor. This is demonstrated in the epithelium, endothelium and stromal cells of the endometrium and other organs, in lymphocytes and macrophages (Reshef et al 1990, Licht et al 1993, Lin et al 1995, Zhang et al 2003, Licht et al ). Next to it works possibly trophoblastic ⁇ hCG also via signaling pathways that are not receptor-mediated (Cruz et al., 1987).
  • hCG heterodimer or free ⁇ CG and ⁇ hCG subunits are also expressed in small amounts in some non-trophoblastic tissues (Rothman et al., 1992; Dirnhofer et al., 1996; Lei et al., 1993; Yokotani et al., 1997; Berger et al., 1994).
  • Non-trophoblastic tissues such as As mamma, lung, prostate, bladder and colon express exclusively the ßhCG subunits hCG ß7 / ß6.
  • Non-trophoblastic hCG is also produced in the endometrial and decidual epithelium of the uterus (Alexander et al 1998b, Wolkersdorfer et al 1998, Zimmermann et al., 2003).
  • the ⁇ hCG subunits ⁇ 7 and the ⁇ 7 allele ⁇ 6 are therefore also designated as non-trophoblastic or epithelial ⁇ hCG or type ⁇ CG (Bellet et al., 1997).
  • the function of the non-trophoblastic hCG has barely been studied.
  • the object of the invention is therefore to provide an agent 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, growth retardation and preeclampsia.
  • the object of the invention is also to specify agents for the treatment of autoimmune diseases or for the induction of immune tolerance in transplant patients.
  • the object is achieved by a medicament, in particular for the treatment of pregnancy disorders, comprising a precursor hCG ⁇ subunit selected from hCG ⁇ 6 according to SEQ ID NO 1 or alternatively SEQ ID NO 2, hCG ⁇ 7 according to SEQ ID NO 5 or a mature hCG ⁇ subunit selected from hCG ⁇ 6 according to SEQ ID NO 3 or alternatively SEQ ID NO 4, from hCG ⁇ 7 according to SEQ ID NO 6 or fragments thereof.
  • a medicament in particular for the treatment of pregnancy disorders, comprising a precursor hCG ⁇ subunit selected from hCG ⁇ 6 according to SEQ ID NO 1 or alternatively SEQ ID NO 2, hCG ⁇ 7 according to SEQ ID NO 5 or a mature hCG ⁇ subunit selected from hCG ⁇ 6 according to SEQ ID NO 3 or alternatively SEQ ID NO 4, from hCG ⁇ 7 according to SEQ ID NO 6 or fragments thereof.
  • Also part of the invention is the use of a precursor hCG ⁇ subunit selected from hCG ⁇ 6 according to SEQ ID NO 1 or SEQ ID NO 2 and hCG ⁇ 7 according to SEQ ID NO 5 or a mature hCG ⁇ subunit selected from hCG ⁇ 6 according to SEQ ID NO 3 or SEQ ID NO 4, hCG ⁇ 7 according to SEQ ID NO 6 or glycan-afflicted oligopeptide fragments thereof for the treatment of pregnancy disorders.
  • the amino acid sequences according to SEQ ID NO 1 or SEQ ID NO 2 are the two possible forms of the amino acid sequences of the precursor of the endometrial or decidual hCG ⁇ 6 subunit.
  • the precursor of the hCG ⁇ 6 subunit consists of 165 Amino acids (numbered 1 to 165 in SEQ ID NO 1 and SEQ ID NO 2, respectively).
  • the amino acid sequence of amino acid 1 to 20 is the signal peptide that is cleaved in the Golgi apparatus.
  • the specific, mature form of the decidual hCG ⁇ 6 subunit corresponds to the amino acid sequence from amino acid 21 to amino acid 165, ie an amino acid sequence according to SEQ ID NO 3 or SEQ ID NO 4.
  • the specific, mature form of the endometrial or decidual hCG ⁇ 6 subunit consists of 145 amino acids (numbered 1 to 145 in SEQ ID NO 3 and SEQ ID NO 4, respectively).
  • the amino acid sequence of the hCG ⁇ 6 subunit contains an alanine instead of an aspartate.
  • At position 2 of the hCG ⁇ 6 subunit is a lysine (SEQ ID NO 3) or an arginine (SEQ ID NO 4).
  • the mature form of the endometria or deciduous hCG ⁇ 6 subunit according to SEQ ID NO 3 or SEQ ID NO 4 and / or the precursor of the decident hCG ⁇ 6 subunit according to SEQ ID NO 1 or SEQ ID NO 2 are preferably contained in the medicament.
  • SEQ ID NO 5 is the precursor of the amino acid sequence of the endometrial or decidual hCG ⁇ 7 subunit.
  • the precursor of the hCG ⁇ 7 subunit consists of 165 amino acids (numbered 1 to 165 in SEQ ID NO 5).
  • the amino acid sequence of amino acid 1 to 20 corresponds to the signal peptide that is cleaved in the Golgi apparatus.
  • the specific, biologically mature form of the endometrial or decidual hCG ⁇ 7 subunit corresponds to the amino acid sequence from amino acid 21 to amino acid 165, i. an amino acid sequence according to SEQ ID NO 6.
  • the mature form of the endometrial or decidual hCG ⁇ 7 subunit consists of 145 amino acids (numbered 1 to 145 in SEQ ID NO 6).
  • the amino acid sequence of the hCG ⁇ 7 subunit contains an alanine instead of an aspartate.
  • amino acid position 2 it contains an arginine instead of a lysine
  • amino acid position 4 it contains a methionine instead of a proline.
  • the mature form of the endometrial or decidual hCG ⁇ 7 subunit according to SEQ ID NO 6 and / or the precursor of the decidual hCG ⁇ 7 subunit according to SEQ ID NO 5 are preferably contained in the medicament according to the invention.
  • this contains, in addition to the endometrial ⁇ 6 and / or ⁇ 7 unit, the trophoblastic subunit hCG ⁇ 5, hCG ⁇ 3 and hCG ⁇ 8 according to SEQ ID NO 7 and / or SEQ ID NO 8
  • SEQ ID NO 7 is the precursor of the amino acid sequence of the trophoblastic ⁇ hCG subunits ⁇ 5, ⁇ 3 and ⁇ 8.
  • the precursors of the trophoblastic ⁇ hCG subunits ⁇ 5, ⁇ 3 and ⁇ 8 each consist of 165 amino acids (numbered 1 to 165 in SEQ ID NO 7).
  • the amino acid sequence of amino acid 1 to 20 corresponds to the signal peptide that is cleaved in the Golgi apparatus.
  • the specific mature forms of trophoblastic ⁇ hCG subunits ⁇ 5, ⁇ 3 and ⁇ 8 correspond to the amino acid sequence from amino acid 21 to amino acid 165, i. an amino acid sequence according to SEQ ID NO 8.
  • the specific mature forms of the trophoblastic ⁇ hCG subunits ⁇ 5, ⁇ 3 and ⁇ 8 consist of 145 amino acids (numbered 1 to 145 in SEQ ID NO 8).
  • amino acid position 2 it contains a lysine
  • amino acid position 4 it contains a proline.
  • the implementation of a causal therapy of pregnancy disorders is made possible for the first time.
  • the loss of decidual hCG, which causes the pregnancy disorders is substituted by the agents according to the invention.
  • the hCG delivered stimulates the formation of hCG in the decidua, which in turn calms the uterine muscles and improves the blood circulation to the placenta.
  • the causal treatment of pregnancy disorders and premature birth is possible in terms of premature birth.
  • the hCG preparations hitherto used in gynecology use purified urinary hCG or a genetically engineered recombinant human choriogonadotropin.
  • This hCG consists of the ßhCG subunit ß5, ß8, ß3 and the ⁇ CG subunit and has its main site of action on the yellow body (corpus luteum) of the ovary. Since the corpus luteum in humans is only essential until the 10th week of pregnancy, according to the state of the art, hCG therapy is also carried out only up to the 10th week of pregnancy.
  • human decidual chorionic gonadotropin which consists of the hCG ⁇ subunit ⁇ 6 or ⁇ 7 and the ⁇ CG subunit, also supports the corpus luteum, it is beyond that mainly necessary for maintaining the immune tolerance of pregnancy.
  • the induction, expression and protein formation of the ⁇ hCG gene ⁇ 7 and / or ⁇ 6 in the endometrial and decidual gland epithelium promotes fertility and pregnancy. The effect takes place via various mechanisms.
  • the pregnancy itself is an immunological paradox.
  • the embryo or fetus is a so-called “semi-allograft", half of which consists of maternal and half of paternal genes and is therefore half "alien".
  • the fruit must be tolerated immunologically until maturity for 38 weeks. This is a complex and multifactorial process whose mechanisms are still poorly understood.
  • the uterus is an immune-privileged place.
  • the endometrial hCG (ß6 / ß7hCG and ⁇ CG) is the main factor for this biological peculiarity that makes a successful pregnancy possible.
  • hCG has an immunosuppressive effect on the uterus.
  • the decidua that surrounds the fruit and also releases the hCG into the amniotic fluid via the membranes, acts as a shield.
  • the hCG of the decidua (ß6 / ß7hCG and ⁇ CG) is also responsible for the chemotactic attraction of mononuclear immune cells, which in turn prevent a rejection reaction.
  • decidual hCG improves blood flow to the uterus and placenta.
  • the medicament according to the invention is used for the treatment of pregnancy disorders.
  • Pregnancy disorders include fertility disorders, implantation disorders, early pregnancy loss, imminent and habitual abortion as well as premature birth, growth retardation and preeclampsia. In particular, such are among them To understand pregnancy disorders caused by a lack of deciduous hCG.
  • a fertility disorder is a disorder characterized by no pregnancy despite regular unprotected intercourse.
  • An imminent abortion is a so-called impending miscarriage, which is usually characterized by bleeding and pelvic pain.
  • a habitual tendency to abortion occurs when a patient has had a miscarriage three or more times in succession.
  • a premature birth occurs when it comes to birth between the 24th and 37th week of pregnancy, or if a living child is born before the 24th week of pregnancy.
  • the fetus In case of an intrauterine growth retardation, the fetus is too small for its age corresponding to the week of pregnancy. 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.
  • Pre-eclampsia is a hypertensive disease in pregnancy (pregnancy hypertension). It also describes the occurrence of edema and protein excretion in the urine. In 20 percent of the cases liver involvement occurs, with increases in transaminases and bilirubin.
  • the agents according to the invention also make it possible to treat autoimmune diseases. Furthermore, the agents according to the invention are also suitable for inducing immune tolerance in transplant patients.
  • 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 overreaction of the immune system against the body's own tissues.
  • the body's own immune system is mistakenly considered to be too Fighting foreign body perceived. This leads to severe systemic or local inflammatory reactions, which lead to damage to the affected organs.
  • hCG is an immunosuppressive substance. Therefore, hCG therapy in the above manner can cause both an immune response of the body to an allograft, i. suppress an organ transplant from another individual, as well as suppress an aberrant immune response against the body's own tissue in the context of an autoimmune disease.
  • the drug additionally contains the precursor of the human chorionic gonadotropin ⁇ CG subunit according to SEQ ID NO 9 or the mature ⁇ CG subunit of the human chorionic gonadotropin according to SEQ ID NO 10 or glycan-linked oligopeptide fragments as parts of these sequences.
  • SEQ ID NO 9 is the amino acid sequence of the hCG ⁇ subunit precursor (J.C. Fiddes and H.M. Goodman 1973).
  • the precursor of the hCG ⁇ subunit consists of 116 amino acids (numbered 1 to 116 here).
  • the amino acid sequence of amino acid 1 to 24 corresponds to the signal peptide that is cleaved in the Golgi apparatus.
  • the specific, mature form of the hCG ⁇ subunit corresponds to the amino acid sequence from amino acid 25 to amino acid 116, i. the amino acid sequence according to SEQ ID NO 10.
  • the specific, mature form of the hCG ⁇ subunit consists of 92 amino acids (numbered 1 to 94 in SEQ ID NO 10).
  • the mature hCG ⁇ subunit according to SEQ ID NO 10 is preferably contained in the medicament according to the invention.
  • hCG ⁇ subunit additionally the ⁇ CG subunit of human chorionic gonadotropin according to SEQ ID NO 9 or SEQ ID NO 10 or glycan-linked oligopeptide Fragments thereof are preferably equimolar amounts of ⁇ hCG subunits and ⁇ CG subunits.
  • the medicament consists, for example, of the hCG ⁇ subunit ⁇ 6 according to SEQ ID NO 1 and the ⁇ subunit of the human chorionic gonadotropin according to SEQ ID NO 9
  • the medicament according to the invention preferably contains equimolar amounts of the hCG ⁇ subunit ⁇ 6 according to SEQ ID NO 1 and the ⁇ subunit of human chorionic gonadotropin according to SEQ ID NO 9.
  • the medicament for the treatment of pregnancy disorders contains various forms of the ⁇ hCG subunit, such as ⁇ hCG ⁇ 6 and / or ⁇ 7
  • the medicament preferably contains an ⁇ CG subunit of the human chorionic gonadotropin according to SEQ ID NO 9 or SEQ ID NO 10 or fragments thereof per in the drug present ⁇ hCG subunit or pro-drug fragments of hCG ⁇ subunits. Contains the drug z. B.
  • the drug also contains as many ⁇ CG subunits according to SEQ ID NO 9 and SEQ ID NO 10 and Glycated oligopeptide fragments of ⁇ CG subunits, that each ßhCG subunit or each fragment of a ⁇ hCG subunit with an ⁇ CG subunit or with the fragment of an ⁇ subunit can form a heterodimer.
  • the subunits of human chorionic gonadotropin used according to the invention include, for example, chorionic gonadotropins isolated from natural sources, recombinantly produced forms, as well as deglycosylated, non-glycosylated, modified glycosylated and other forms.
  • the ßhCG subunit ß6 contained in the medicament of the invention according to SEQ ID 1, SEQ ID NO 2 or SEQ ID NO 3 or SEQ ID NO 4 or the ßhCG subunit ß7 according to SEQ ID NO 5 or SEQ ID NO 6 and the ßhCG Subunits ⁇ 5, ⁇ 3 and ⁇ according to SEQ ID NO 7 or SEQ ID NO 8 of the human chorionic gonadotropin or the glycan-linked oligopeptide fragments thereof are preferably prepared recombinantly. If, in addition, the ⁇ CG subunit of the human chorionic gonadotropin according to SEQ ID NO 9 or SEQ ID NO 10 or fragments thereof are present in the medicament according to the invention, then these are preferably also prepared recombinantly.
  • ⁇ hCG and ⁇ hCG DNA sequences are transfected with cloned ⁇ hCG and ⁇ hCG DNA sequences, and the protein produced by these cells is isolated.
  • suitable cells such as, for example, Chinese hamster ovary cells - CHO cells
  • insect cell lines are preferred for expression of the protein.
  • mammalian epithelial cell lines preferably human epithelial cell lines, more preferably endometrial or decidual, are used for expression. Because of its complex structure, the integrity of the hCG molecule should be ensured in the isolation of the ⁇ CG and ⁇ -hCG DNA fragments. Serine-O- and asparagine-N-linked glycoside side chains (glycans), and optionally also disulfide bonds, guarantee the biological activity of the hCG.
  • glycans asparagine-N-linked glycoside side chains
  • the alpha subunit ( ⁇ -hCG, ⁇ CG) is preferably present on the amino acids Asn-52 and / or Asn-78 of the mature, mature amino acid sequence (SEQ ID NO 10) or Asn-76 and / or Asn-102 of the precursor ( SEQ ID NO 9) N-glycosylated and forms N-glycan chains with specific sugar residue portions.
  • the mature (mature) endometrial or decidual beta subunits hCG ⁇ 6 (SEQ ID NO 3, SEQ ID NO 4) and hCG ⁇ 7 (SEQ ID NO 6) are preferably N-glycosylated on the amino acids Asn-13 and / or Asn-30 and preferably at least one of the CTP positions Ser-121, Ser-127, Ser-132 and SeM 38 O-glycosylated.
  • the precursor hCG ⁇ subunit ⁇ 6 according to SEQ ID NO 1 or SEQ ID NO 2 or ⁇ 7 according to SEQ ID NO 5 is preferably on at least one of the following amino acids Asn-33, Asn-50 n-glycosidic and / or Ser-141, SeM 47, SeM 52, SeM 58 glycosidically glycosidated.
  • the (up to) two Asn-N-glycan chains of the ⁇ hCG and ⁇ 6 or ⁇ 7-hCG subunits are preferably three- to two-membered and tri-, di-, mono- or non-sialylated.
  • the Asn-N-glycan chains each contain preferably 2 to 15, more preferably 4 to 10, sugar residues, preferably with a descending fraction of NAc-glucosamine, sialic acid, galactose, mannose.
  • the (up to) four Ser-O-glycan chains of the CTP region in the ⁇ 6 or ⁇ 7 hCG subunit preferably contain four to two-branched and more strongly sialylated 2 to 10 sugar residues, particularly preferably 4 to 8 sugar residues, preferably with decreasing proportion of sialic acid, NAc-galactosamine, galactose, mannose, fucose.
  • the mature, mature alpha subunit ⁇ hCG particularly preferably contains 3 disulfide bridge bonds between the cysteine pairs AS 10-60, AS 28-82 and AS 59-87 as well as further 2 preferred SH bridges between AS 7-31 and AS 32-84.
  • the mature, mature beta-subunit ß6- or ß7-hCG contains particularly preferably 2 disulfide bridge bonds between the cysteine pairs AS 9-57 and AS 38-90 and further 4 preferred SH bridges between AS 23-72, AS 26-110 , AS 34-88 and AS 93-100.
  • the preferred disulfide bonds in dimeric hCG are responsible for the formation of the typical cysteine knot structure, which is analogous to a number of cysteine knot proteins.
  • altered conditions of SH bridging in hCG show only minor changes in biological activity.
  • epithelial formation of the ⁇ hCG and ⁇ CG subunits is induced by mediators such as estradiol, progesterone, hCG, LPS and Th2 cytokines, and by inhibitors such as Th1 cytokines, cycloheximide and actinomycin D can be reduced. That is, for production of dimeric epithelial hCG having the beta subunit hCG ⁇ 6 or hCG ⁇ 7, epithelial cells of the secretory transformed endometrium are preferably used for this process, or epithelial endometrial cell lines capable of secretory transformation are used. Epithelial endometrial cell lines of carcinogenic origin are usually excluded or must be checked to express no additional beta-subunit hCG ß5, ß and ß3.
  • 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.
  • preference is given to the precursor or mature hCG having the subunit hCG ⁇ 7 according to SEQ ID NO 5 or SEQ ID NO 6 or the precursor or mature hCG ⁇ 6 according to SEQ ID NO 1 to SEQ ID NO 4 or glycan-afflicted oligopeptide fragments thereof dissolved in an injection solution and transferred to a pre-filled syringe.
  • the precursor of the ⁇ CG subunit of the human chorionic gonadotropin according to SEQ ID NO 9 or the mature ⁇ CG subunit of the human chorionic gonadotropin according to SEQ ID NO 10 or glycan-linked oligopeptide fragments thereof are preferably additionally administered.
  • the drug is e.g. administered subcutaneously, intramuscularly, intraamnically, sublingually, intrathecally or intravenously. 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.
  • the dose of endometrial hCG to be administered depends on the condition of the disease and the particular patient to be treated.
  • the drug is adapted so that the amount of human chorionic gonadotropin administered is 1 to 10 ⁇ g, more preferably 3 to 6 ⁇ g, per kg of body weight per day.
  • Preferred unit doses are 50 ⁇ g to 1000 ⁇ g of the described hCG.
  • 250 micrograms of the mature hCG are formed from an endometrial ⁇ subunit ( ⁇ 7 according to SEQ ID NO 6 or the mature hCG ⁇ 6 according to SEQ ID NO 2 or SEQ ID NO 4) and an ⁇ subunit (SEQ ID NO 9 or SEQ ID NO 10) dissolved in 0.5 ml of an injection solution and transferred to a pre-filled syringe.
  • the invention further relates to a method for the treatment of fertility and pregnancy disorders or for the induction of immunological tolerance in patients with autoimmune diseases or transplantation processes in which a precursor hCG ⁇ subunit of the human chorionic gonadotropin selected from hCG ⁇ 6 according to SEQ ID NO 1 or SEQ ID NO 2, hCG ⁇ 7 according to SEQ ID NO 5 or a mature hCG ⁇ subunit selected from hCG ⁇ 6 according to SEQ ID NO 3 or SEQ ID NO 4, hCG ⁇ 7 according to SEQ ID NO 6 or glycan-afflicted oligopeptide fragments of these sequences administered to a patient becomes.
  • the precursor of the ⁇ CG subunit of the human chorionic gonadotropin according to SEQ ID NO 9 or the mature ⁇ CG subunit of the human chorionic gonadotropin according to SEQ ID NO 10 or glycan-afflicted oligopeptide fragments thereof are preferably additionally administered.
  • the amount of human chorionic gonadotropin administered is 1 to 10 ⁇ g, more preferably 3 to 6 ⁇ g, each per kg of body weight per day.
  • Preferred single doses are 50 ⁇ g to 1000 ⁇ g hCG.
  • the injections with the medicament according to the invention take place in the case of imminent premature birth, in preeclampsia or in intrauterine growth retardation, for example daily, in the case of imminent premature birth with the onset of regular labor. After the contractions have subsided, the injection with the medicament according to the invention takes place at intervals of 2 to 4 days.
  • the administration of the medicament according to the invention by intravenous infusion is preferred.
  • the protein dimer - hCG ß7 / ⁇ or hCGß6 / ⁇ (mature hCG ß7 according to SEQ ID NO 6 or mature hCG ß6 according to SEQ ID NO 2 or SEQ ID NO 4 with hCG ⁇ SEQ ID NO 9 or 10) - in one Dissolved solution and administered over a period of preferably four hours.
  • Preferred dose 500 ⁇ g to 1500 ⁇ g, preferably 1000 ⁇ g hCG ⁇ 7 / ⁇ or hCG ⁇ 6 / ⁇ in 500 ml infusion solution.
  • hCG ß7 / ⁇ or hCGß6 / ⁇ is intramnial.
  • Preferred dose 500 ⁇ g to 1500 ⁇ g, preferably 1000 ⁇ g hCG ⁇ 7 / ⁇ or hCG ⁇ 6 / ⁇
  • the patient preferably mononuclear cells are removed, incubated with the above hCG forms in vitro and then returned subcutaneously, intravenously or locally to the respective patient.
  • 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.
  • hCG site of transplantation, joint space, bladder, intestine, skin, cerebrospinal fluid
  • hCG site of transplantation, joint space, bladder, intestine, skin, cerebrospinal fluid
  • Method of Preparation The prior art has produced a recombinant trophoblastic hCG in a culture of Chinese hamster ovary (CHO) cell lines, such as CHO-DUKX fibroblast cells, COS-7 cells, or other CHO cell lines described in the literature (Chappel et al 1992, Matzuk et al., 1989, Garcia-Campayo et al., 2002, Birken et al., 2003).
  • CHO-DUKX fibroblast cells such as CHO-DUKX fibroblast cells, COS-7 cells, or other CHO cell lines described in the literature (Chappel et al 1992, Matzuk et al., 1989, Garcia-Campayo et al., 2002, Birken et al., 2003).
  • glycation of the recombinant hCG produced by the prior art is disadvantageously different from hCG naturally expressed in human endometrium and decidua.
  • Glycolation of hCG has been shown to be astonishingly epithelial-specific. Glycation has strong effects on the specificity and biological activity of hCG.
  • RNA hCG ⁇ 7 or hCG ⁇ 6 or hCG ⁇ 7 + ⁇ 6 expresses in the healthy secretory transformed endometrium and the decidua of humans as RNA hCG ⁇ 7 or hCG ⁇ 6 or hCG ⁇ 7 + ⁇ 6 (see SEQ ID No 11 to 13).
  • the invention therefore further relates to a method for the production of human hCG with the subunits ⁇ -chorionic gonadotropin ( ⁇ CG) and ⁇ -human chorionic gonadotropin ( ⁇ hCG) in isolated human epithelial cells or epithelial cell lines of endometrial or decidual origin.
  • ⁇ CG ⁇ -chorionic gonadotropin
  • ⁇ hCG ⁇ -human chorionic gonadotropin
  • the expressed ⁇ -subunit consists of ⁇ hCG ⁇ 6 and / or ⁇ hCG p7.
  • the production process according to the invention has hitherto had the advantage of producing an epithelium-specific hCG secretion product with epithelium-specific glycosylation, which is more pronounced in the human epithelium. It also avoids the disadvantages which have arisen in the prior art in that the hCG has hitherto been produced in a mammalian cell culture without direct reference to the natural human epithelium-specific glycosylation program.
  • the isolated endometrial and decidual epithelial cells are preferably cell lines of human origin. Derived endometrial epithelial cell lines or epithelial cell lines with additional transfection of ßhCG genes ß6 and ß7 and / or other ßhCG genes (ß5, ß3, ß8, ß1, ß2) and / or of genes for Giykosyl mich of the hormone are hereby expressly included.
  • the epithelial cells are preferably obtained from native endometrial tissue.
  • the hCG expressed in these cells has the above-mentioned preferred glycation pattern and the above-mentioned disulfide bridges.
  • hCG (composed of ⁇ -CG and ⁇ 6 / 7-hCG or the glycosylated oligopeptides) is preferably infused.
  • Preferred dose 500 to 1000 ⁇ g / d hCG.
  • Virus-related carcinomas and sarcomas are treated systemically and locally with doses of preferably 1000 ⁇ g / d.
  • hCG (composed of ⁇ -CG and ⁇ 6 / 7-hCG or the glycosylated oligopeptides) is subcutaneously injected or administered sublingually. Preferred dose: daily 10 ⁇ g hCG.
  • the hCG is subcutaneously implanted into polymer rods or polymer rings are intravaginally appplied.
  • the rods or rings are preferably made of polyethylene-co-vinyl acetate and preferably daily 2, 5 to 20, preferably 4 to 7.5 ug hCG (composed of ⁇ -CG and ß6 / 7-hCG) free
  • hCG-containing gels and creams containing hCG are used in a concentration of preferably 1%.
  • hCG Composed of ⁇ -CG and ⁇ 6 / 7-hCG or the glycosylated oligopeptides
  • Preferred dose daily 500 to 1000 ⁇ g.
  • hCG Composed of ⁇ -CG and ß6 / 7-hCG or the glycosylated oligopeptides
  • Preferred dose daily 50 to 100 ⁇ g.
  • hCG (composed of ⁇ -CG and ⁇ 6 / 7-hCG or the glycosylated oligopeptides) is preferably administered sublingually.
  • Preferred dose 0.5 ⁇ g twice daily
  • Treatment of Autoimmune Disease of the Eye In autoimmune uveitis, a solution composed of ⁇ -CG and ⁇ 6 / 7-hCG or the glycosylated oligopeptides is preferably injected at intervals of 4 to 7 days.
  • Preferred dose 0.5ml with 10 ⁇ g / ml hCG. This therapy can also be prescribed for therapy-resistant glaucoma therapy or at risk of rejection of a cornea transplant.
  • weekly hCG (composed of ⁇ -CG and ß6 / 7-hCG or the glycosylated oligopeptides) is preferably instilled intrathecally.
  • Preferred dose 2 ml at a concentration of 10 ⁇ g / ml
  • hCG composed of ⁇ -CG and ⁇ 6 / 7-hCG or the glycosylated oligopeptides into a biomembrane capsule, which release hCG in the small or large intestine, preferably at a concentration of 5 ⁇ g / ml.
  • autologous and / or xenogeneic islet cells are preferably stimulated for 48 to 48 hours in an hCG emulsion containing 2 ⁇ g / ml hCG prior to transplantation and then injected intravenously.
  • autologous or xenogeneic islet cells are encapsulated in a hCG-releasing biomembrane, preferably composed of a biodegradable polymer, such as poly (s-caprolactone) (PCL), and so implanted.
  • PCL poly (s-caprolactone)
  • a biodegradable implant preferably poly ( ⁇ -caprolactone) (PCL), in the form of a pencil in chronic cystitis or interstitial cystitis, which continuously releases hCG.
  • PCL poly ( ⁇ -caprolactone)
  • hCG Composed of ⁇ -CG and ⁇ 6 / 7-hCG or the glycosylated oligopeptides
  • Administration is preferably daily for 2 weeks.
  • Preferred dose 1000 ⁇ g / ml.
  • FIG. 1 Organization of the ⁇ hCG genes ⁇ 5, ⁇ and ⁇ 3 as well as the ⁇ hCG genes ⁇ 6 and ⁇ 7
  • FIG. 2 Gene expression and nucleotide sequence of the mRNA of ⁇ hCG genes ⁇ 5, ⁇ 6 and ⁇ 7 and of ⁇ 1_H gene ⁇ 4 in conjunction with the encoded amino acid sequence
  • FIG. 3 Localization of different nucleotide sequences of the ⁇ hCG mRNA of gene ⁇ 5, ⁇ 6 and ⁇ 7 in exons 1, 2 and 3
  • Fig. 4 Gene expression of ⁇ hCG and ⁇ CG after RT-PCR in the secretory endometrium
  • Fig. 5 Cycle dependence of the endometrial gene expression of ⁇ hCG
  • Fig. 6 (A) Sequence analysis of the transcript of endometrial gene expression ⁇ hCG ⁇ 6
  • FIG. 7 Determination of the concentration of the endometrial hCG in the endometrium homogenate
  • FIG. 8 Western biot examination for the molecular structure and for the glycolization of the placental and endometrial hCG with.
  • Fig. 9 Western blot studies to distinguish between ⁇ hCG endometrial ( ⁇ 7, ⁇ 6) and trophoblastic ( ⁇ 5) origin
  • the organization of the ⁇ hCG placental genes ⁇ 5, ⁇ and ⁇ 3 as well as the endometrial genes ⁇ 6 and ⁇ 7 is shown in FIG.
  • the genes of the ⁇ hCG subunit consist of three exons and two intervening introns.
  • Exon 1 contains the promoter sequence, the both structural genes exon 2 and exon 3 including the C-terminal peptide (CTP) encode the mature (145h) ⁇ hCG subunit (aa).
  • CTP C-terminal peptide
  • the ⁇ hCG gene is expressed from bp -366 in exon 1 (transcriptional start, ***) via bp +1 in exon 1 (translation start, Tr) to bp +495 in the CTP of exon 3.
  • Exon 1 covers the bp range from -366 to +15, exon 2 from +16 to +183, and exon 3 from +184 to +545.
  • Four intron-spanning ⁇ hCG primer pairs with the resulting amplicons of 548, 423, 378, and 300 bp confirm full-length ⁇ hCG gene expression.
  • the ⁇ hCG genes ⁇ 1 and ⁇ 2 contain a point mutation in the donor splice site of the first intron.
  • An mRNA resulting from alternative splicing of intron 1 encodes proteins of 132 amino acids, but their sequences are not similar to the amino acid sequences of the other ⁇ hCGs (Policastro et al 1983, Talmadge et al 1984, Bo and Boime 1992).
  • the differing nucleotide sequences of the placental ⁇ hCG genes ⁇ 5 are compared with the epithelial ⁇ hCG genes ⁇ 6 and ⁇ 7 (CG6, CG7) and ⁇ 1_H gene ⁇ 4 (LH4) as well as the determined endometrial sequences (endo).
  • the associated different amino acid sequences are listed in the protein molecule.
  • the mRNA of the ⁇ hCG genes covers the nucleotide range from - 366 to +495 bp, the nucleotides from +1 to +496 bp encode the prehormone ( ⁇ hCG precursor) with 165 amino acids, the nucleotides from +60 to +46 to +5 bp encode the mature ( mature) ⁇ -subunit with 145 amino acids.
  • M in the endometrial sequence is C or A, R is G or A and S is G or C.
  • the start of exon 1 is indicated by * ** , exon 2 by ** and exon 3 by * marked.
  • Promoter genes and the structural genes as a whole differ between the genes hCG ß5 and hCG ß7 in 27 nucleotide positions, the hCG gene ß7 differs from hCG ß6 in ten nucleotide positions.
  • the nucleotide sequences of the hCG subunits ⁇ 3, ⁇ 5 and ⁇ in the promoter gene show several differences, but the amino acid sequences of the prehormone and mature hCG subunits ⁇ 3, ⁇ 5 and ⁇ 8 are identical.
  • the count of the bp numbers in Fig. 3 refers to the transcription start or translation start, respectively.
  • the resulting amino acid sequence of the placental hCG does not differ despite its diverse ⁇ hCG ⁇ 5, ⁇ , ⁇ 3 subunit structure.
  • the endometrial hCG with its ßhCG ß7 and ß6- Subunits are in addition to the amino acid +117 in the C-terminal region, however, further amino acids in the N-terminal region to the placental hCG changed.
  • ⁇ CG and ⁇ hCG expressed from different genes combine intracellularly soon after protein synthesis in the endoplasmic reticulum and undergo post translational modifications into the specific, biologically active heterodimeric form (disulfide bonds and glycosylation in the endoplasmic reticulum, heterodimerization, seat belt configuration and Prehormone cleavage in the Golgi apparatus).
  • the resulting amino acid sequences of the mature hCG ⁇ 7 subunit and the hCG ⁇ 3, ⁇ 5, ⁇ 8 subunits differ in the amino acid positions +2 (arginine / lysine), +4 (methionine / proline) and +117 (alanine / aspartate), which are the hCG ⁇ 6- like the hCG ⁇ 7- with the hCG ⁇ 5 subunit also in the amino acid position +117 (alanine / aspartate) and those of the hCG ⁇ 6 and hCG ⁇ 7 subunits still in the amino acid position +2 (lysine / arginine) and +4 (proline / methionine).
  • an arginine with the nucleotide sequence AGG can be displayed in position +2 for hCG ⁇ 6 (b). The differences in the amino acid sequence are summarized in Tab.1.
  • Fig. 4 we can demonstrate for the first time with our laboratory results that in the normal secretory endometrium of healthy women epithelial hCG is expressed.
  • Gene expression of the secretory endometrium includes both the ⁇ CG subunit and the full length RNA of the ⁇ hCG subunit from exon 1 to exon 3, including the CTP moiety.
  • the primer pairs listed in Table 2 were used in an RT-PCR under standard conditions: Table 2: r. Gene primer exon beach primer sequence bp no. Location Amp- Pair- licon * ed
  • GAPDH 335/352 sense 5'-CCATGGAGAAGGCTGGGG-3 1 196 10 0
  • the base pair length of the DNA products amplified by the RT-PCR are indicated in bp.
  • the primers are included with SEQ ID 15 to 24 in the attached Sequence Listing.
  • the specific ⁇ hCG primers did not amplify ⁇ LH mRNA.
  • Fig. 4 the results of the RT-PCR for 4 tissue samples of the endometrium in lanes 3 to 6 ("endometrium") and in lane 8 as control a tissue sample of an "early gestation" placenta ("plac") are shown.
  • a DNA ladder is applied as standard ("stand.") In lane 1. In lane 2, a negative control (without primer, without RNA) was applied.
  • Fig. 4 A the ⁇ hCG specific primers 1 and 4 of Table 2 were used. The comparison with the DNA ladder shows that the amplified DNA has the expected length of 548 bp.
  • Fig. 4B the ⁇ hCG specific primers 2 and 4 of Table 2 were used. The comparison with the DNA ladder shows that the amplified DNA has the expected length of 423 bp.
  • Fig. 4C the ⁇ hCG specific primers 3 and 6 of Table 2 were used. The amplified DNA has the expected length of 378 bp.
  • Fig. 4 D the ⁇ hCG specific primers 3 and 5 of Table 2 were used. The amplified DNA has the expected length of 300 bp.
  • the ⁇ CG-specific primers 7 were obtained in FIGS.
  • the DNA product in Fig. E has the expected length of 231 bp. Without revertase in the cDNA approach (- RTase), the reaction remains, ie, is RNA and not about endogenous DNA.
  • the GAPDH-specific primers 9 and 10 of Table 2 were used as a control and for a semiquantitative determination.
  • the comparison with the DNA ladder shows that the amplified DNA has the expected length of 196 bp.
  • the results show that ⁇ hCG and ⁇ hCG mRNA are expressed in the secretory phase of healthy endometrium at approximately the same concentration as the placenta.
  • FIG. 5 shows that the ⁇ hCG mRNA expression is dependent on the degree of differentiation of the secretory transformation of the endometrium.
  • the endometrial biopsies have always been evaluated by diagnostic pathologists as a cycle fit and normal proliferative phase tissue to the late secretory phase after diagnostic curettage.
  • the endometrial RNA was extracted and semi-quantitatively determined by RT-PCR for the corresponding GAPDH amplification.
  • An optical densitometric evaluation ( ⁇ SEM) was carried out.
  • FIG. 6 shows the results of the sequence analysis for confirming the gene expression of ⁇ hCG mRNA in the secretory endometrium.
  • the cDNA amplicons used were used after RNA extraction of the endometrial tissue and after RT-PCR under standard conditions for sequencing.
  • FIG. 6A after sequencing of the ⁇ hCG amplificate, a nucleotide sequence for ⁇ hCG ⁇ 6, in FIG. 6B a nucleotide sequence for ⁇ hCG ⁇ 7, and in FIG. 6C a nucleotide sequence for ⁇ hCG ⁇ 7 with ⁇ hC ⁇ 6 are shown.
  • sequence sequences confirm in high accuracy the nucleotide sequences assembled in Table 2 for the expression of the endometrial ⁇ hCG ⁇ 7 and ⁇ hCG ⁇ 6 subunits.
  • the determined nucleotide sequence is based on the knowledge of the cDNA amplificate used for the studies with 548 bp on the exon 1 and exon 3. With these results, an mRNA sequence for the expression of an endometrial ⁇ hCG subunit is presented for the first time.
  • the blots are treated with a polyclonal hCG antibody (Dako) and a polyclonal CTP-h CG antibody (Biotrend) as the primary antibody.
  • the blots are treated with a monoclonal ⁇ hCG antibody (INN22) under reducing or non-reducing conditions.
  • the blots are treated with an ⁇ CG monoclonal antibody (INN 132) under reducing or non-reducing conditions.
  • the endometrial tissue samples display predominantly ⁇ hCG subunit bands of about 31 kDa or 29 kDa and ⁇ GK subunit bands of 24 kDa or 21 kDa as well as ⁇ hCG dimer bands of 44 kDa, 38 kDa and 35 kDa or other 24 kDa ⁇ hCG monomer bands. 21 kDa, 17 kDa and 15 kDa, depending on the degree of desialylation or dex- glolyzation,
  • the hCG-, ßhCG- and ⁇ CG-specific antibodies also show the cycle-dependent epithelial hCG secretion in the endometrial glandular and luminal epithelium of the healthy woman on tissue sections, especially the middle and late secretory phase.
  • a TOPO TA cloning kit (Invitrogen or alternatively the pGEM-T vector system Promega) under manufacturer instruction depending for hCG ß6, hCG ß7 and hCG ⁇ (SEQ ID NO 11 to SEQ ID NO 13 and SEQ ID NO 15) and insertion in the expression vector.
  • the introduced DNA is combined with the DNA sequence of dehydrofolic acid reductase (DHFR) required for the synthesis of the nuclear acid precursors in DHFR-deficient mammalian host cells.
  • DHFR dehydrofolic acid reductase
  • the ⁇ CG and ⁇ hCG expression vectors are transfected by Ca coprecipitation into the well-characterized animal CHO (Chinese hamster ovary) cell line, which are DHFR-deficient cells.
  • CHO Choinese hamster ovary
  • the clones ⁇ hCG gene ⁇ 6 or ⁇ hCG gene ⁇ 7 are co-transfected together with ⁇ CG and cultured.
  • the single-cell clones are screened for their ability to produce hCG, their biological activity of hCG, and their genetic stability.
  • E. Establishment of a master cell bank (MCB) from a single cotransfected CHO cell with ⁇ hCG and respective ⁇ hCG expression vectors derived from a clone considered to be optimal.
  • MBB master cell bank
  • Bioreactor attachment and growth of cells, hCG production, collection of hCG culture medium
  • Embodiment 2 is a diagrammatic representation of Embodiment 1:
  • N-glycosidic and O-glycoside glycoprotein side chain production N-glycan and O-glycan
  • Example 1 Continuation of execution selection, establishment of the master cell bank, working cell bank, culture extension, fine cleaning and quality control is carried out as indicated in Example 1.
  • Embodiment 3 Treatment of Fertility Disorders / Treatment of Implantation Disorders / Treatment of Early Pregnancy Losses
  • the patient receives the hCG (composed of ⁇ -CG and ⁇ 6 / 7-hCG) prepared as described in Example 1 or Example 2 on the 21st cycle day and onwards 3 days 250 ⁇ g subcutaneously until the diagnosis of pregnancy.
  • hCG composed of ⁇ -CG and ⁇ 6 / 7-hCG
  • Embodiment 4 Treatment of abortions
  • the patients receive the hCG (composed of ⁇ -CG and ⁇ 6 / 7-hCG) produced as described in Example 1 or Example 2 twice a week 250 ⁇ g ⁇ and ⁇ 6/7 hCG injected subcutaneously. From the 20th week of pregnancy onwards, up to the 28th week of pregnancy, 1000 ⁇ g of the hCG prepared as described in Example 1 (composed of ⁇ -CG and ⁇ 6 / 7 hCG) are additionally instilled into the amniotic fluid.
  • Embodiment 5 Treatment of premature birth
  • the patients are given 1000 ⁇ g of the hCG (composed of ⁇ -CG and ⁇ 6 / 7 hCG) prepared as described in Example 1 or Example 2 instilled into the amniotic fluid. This instillation is performed weekly until the 32nd week of pregnancy. In addition, patients receive 250 ⁇ g ⁇ and ß 6/7 hCG subcutaneously daily.
  • Embodiment 6 Treatment of preeclampsia
  • Embodiment 7 Treatment of Growth Retardation
  • the patient receives subcutaneously every two days until the 34th week of pregnancy, 250 ⁇ g of hCG (composed of ⁇ CG and ⁇ 6 / 7 hCG) prepared in Ex. 1 and Ex.
  • Embodiment 8 Treatment of autoimmune diseases and induction of immunological tolerance in transplant patients
  • 0.5 ⁇ g of the hCG (composed of ⁇ -CG and ⁇ 6 / 7-hCG) produced as in Ex. 1 and Ex. 2, respectively, are administered sublingually three times a day.
  • the patients are subcutaneously administered 50 ⁇ g of the hCG (composed of ⁇ -CG and ⁇ 6 / 7-hCG) prepared as in Example 1 or Example 2.
  • mononuclear blood cells taken in vitro before the transplantation and 12 weeks later with about 3 ⁇ g of the hCG (composed of ⁇ -CG and ⁇ 6 / 7-hCG) prepared as in Ex. 1 and Ex. 2, respectively, are incubated for 8 hours incubated and administered intravenously to the patient.
  • an immune-privileged space In order to enable immune tolerance for transplanted organs, an immune-privileged space must be created for this organ, in which the organs are encased in a tight-fitting biomembrane, from the daily 1 ug hCG produced as in Ex. 1 and Ex (composed of ⁇ -CG and ß6 / 7-hCG) is slowly released.
  • Embodiment 9 Avoidance of a graft-versus-host reaction
  • the preparation should after the donor removal with the hCG (composed of ⁇ -CG and ⁇ 6 / 7-hCG) prepared as in Ex. 1 and Ex. 2 in a dose of 250 ⁇ g 0.5 ml of both the arterial and the venous side.
  • the hCG form is also added to the transport medium in the same concentration.
  • hCG For the treatment of sepsis daily 500 to 1000 ug of hCG as prepared in Ex. 1 and Ex. 2 (composed of ⁇ -CG and ß6 / 7-hCG) are infused. Virus-related carcinomas and sarcomas are treated systemically and locally with doses of 1000 ⁇ g / d.
  • Embodiment 11 Use for Contraception
  • Example 10 ⁇ g of the hCG (composed of ⁇ -CG and ⁇ 6 / 7-hCG) produced as in Example 1 or Example 2 are injected subcutaneously or administered sublingually daily.
  • polyethylene co-vinyl acetate sticks for subcutaneous application or polyethylene co-vinyl acetate rings for intravaginal application may release 5 ⁇ g / day of hCG (composed of ⁇ -CG and ⁇ 6 / 7 hCG) prepared as in Ex. 1 to be used.
  • Exemplary embodiment 12 Use for the prophylaxis of an HIV infection containing hCG-containing gels and creams which contain hCG (composed of ⁇ -CG and ⁇ 6 / 7-hCG) prepared as in Ex. 1 and Ex. 2, respectively, in a concentration of 1%, are used for the prophylaxis of HIV infection.
  • Embodiment 13 Treatment of tissue ischemia and severe necrosis
  • hCG hCG (composed of ⁇ -CG and ⁇ 6 / 7-hCG) prepared as in Example 1 and 2, respectively, are infused daily Burns applied locally in the form of sprays.
  • Embodiment 14 Treatment of allergic-inflammatory reactions
  • hCG human hCG produced as in Ex. 1 and Ex. 2 (composed of ⁇ -CG and ß6 / 7-hCG) Form of a spray (alternatively a cream or gel) used.
  • Embodiment 15 Treatment of Benign Prostate Dysplasia (BPH)
  • Embodiment 16 Treatment of Autoimmune Disease of the Eye
  • a solution containing 10 ⁇ g / ml of the hCG (composed of ⁇ -CG and ⁇ 6 / 7-hCG) prepared as in Ex. 1 or Ex. 2 is injected at intervals of 4 to 7 days.
  • This therapy can also be prescribed for therapy-resistant glaucoma therapy or at risk of rejection of a cornea transplant.
  • Embodiment 17 Treatment of Multiple Sclerosis
  • Embodiment 18 Treatment of Crohn's disease and ulcerative colitis
  • Example 1 Peroral administration of the hCGs prepared as described in Example 1 or Example 2 composed of ⁇ -CG and ⁇ 6 / 7-hCG into a (biomembrane) capsule, which releases hCG in a concentration of 5 ⁇ g / ml only in the small or large intestine ,
  • Exemplary embodiment 19 Transplantation of autologous and xenogeneic islet cells
  • Autologous islet cells are stimulated before transplantation for 48 hours in an emulsion containing 2 ⁇ g / ml of the hCG (composed of ⁇ -CG and ⁇ 6 / 7-hCG) prepared as in Ex. 1 and Ex. 2 and then injected intravenously.
  • encapsulation of autologous and xenogeneic islet cells in hCG (composed of ⁇ -CG and ⁇ 6 / 7-hCG) releasing biomembranes composed of a biodegradable poly ( ⁇ -caprolactone) (PCL).
  • PCL biodegradable poly
  • Embodiment 20 Treatment of interstitial cystitis and chronic cystitis
  • Embodiment 21 Treatment for HIV infection
  • the human epithelial cells are cotransfected with a vector containing proteins important for human N-glycosidic and O-glycosidic glycoprotein side chain production.
  • Embodiment 23 is a diagrammatic representation of Embodiment 23.
  • Example 1 Continuation of execution selection, establishment of the master cell bank, working cell bank, culture extension, fine cleaning and quality control is carried out as indicated in Example 1.
  • Embodiment 24 is a diagrammatic representation of Embodiment 24.
  • Embodiment 25 is a diagrammatic representation of Embodiment 25.

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Abstract

Médicament destiné à traiter en particulier les problèmes de grossesse et à induire une tolérance immunologique chez des patients souffrant d'une maladie auto-immune ou transplantés. Ce médicament contient au moins une sous-unité précurseur ou mature bêta6 ou bêta7 de la choriogonadotropine endométriale ou déciduale humaine (hCG), ainsi qu'une sous-unité précurseur ou mature alpha de la choriogonadotropine humaine ou des fragments glycolysés de ces sous-unités. Au contraire de la hCG trophoblastique avec les sous-unités bêta3, bêta5 et bêta8, la fonction de la hCG bêta6 ou bêta7 non trophoblastique exprimée dans l'endomètre et la caduque n'a pratiquement pas été étudiée.
EP06805531A 2005-11-22 2006-11-21 Medicament destine a traiter les problemes de fertilite et de grossesse, et les maladies auto-immunes, et a induire une tolerance immunologique chez des patients transplantes, et procede de production du dit medicament Withdrawn EP1951287A2 (fr)

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