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WO2003016331A2 - Analogues de gnrh non-mammaliens et leurs utilisations dans la regulation de la fertilite et de la gestation - Google Patents

Analogues de gnrh non-mammaliens et leurs utilisations dans la regulation de la fertilite et de la gestation Download PDF

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WO2003016331A2
WO2003016331A2 PCT/US2002/025619 US0225619W WO03016331A2 WO 2003016331 A2 WO2003016331 A2 WO 2003016331A2 US 0225619 W US0225619 W US 0225619W WO 03016331 A2 WO03016331 A2 WO 03016331A2
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gnrh
analogs
mammalian
placental
analog
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WO2003016331A3 (fr
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Theresa M. Siler-Khodr
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Siler-Khodr Theresa M
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/23—Luteinising hormone-releasing hormone [LHRH]; Related peptides
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates generally to the field of regulating fertility and parturition. More particularly, it concerns the use of unique non-mammalian peptide hormone analogs of GnRH designed to be useful in fertility regulation, post-coital contraception and as a menses-inducing agent.
  • GnRH mammalian GnRH
  • the human placenta and the chononic membranes have also been observed to contain a GnRH-like substance.
  • the present investigator has recently localized, quantified and demonstrated the synthesis of a GnRH-like substance by the human placenta.
  • the concentration of immunoreactive GnRH-like material in the placenta and maternal blood has been found to vary with gestational age, following a pattern similar to that of hCG Siler-Khodr TM, Khodr GS, Valenzuela G 1984 Immunoreactive gonadotropin-releasing hormone level in maternal circulation throughout pregnancy.
  • chorionic GnRH axis has also been identified as having an observed feedback interaction for activin, inhibin, follistatin, neurotransmitter, prostaglandin and steriods, Shi LY, Zhang ZW, Li WX 1994 Regulation of human chorionic gonadotropin secretion and messenger ribonucleic acid levels by follistatin in the NUCC-3 choriocarcinoma cell line.
  • GnRH Gonadotropin-releasing hormone
  • Mol Cell Endocrinol 117 121-130; Dong KW, Yu KL, Chen ZG, Chen YD, Roberts JL 1997 Characterization of multiple promoters directing tissue-specific expression of the human gonadotropin-releasing hormone gene.
  • Endocrinology 138:2754-2762 Steroid regulatory sites on the promoter have also been identified. Chandran UR, Attardi B, Friedman R, Dong KW, Roberts JL, DeFranco DB 1994 Glucocorticoid receptor-mediated repression of gonadotropin-releasing hormone promoter activity in GTI hypothalamic cell lines. Endocrinology 134:1467-1474; Dong KW, Chen ZG, Cheng KW, Yu KL 1996 Evidence for estrogen receptor-mediated regulation of human gonadotropin-releasing hormone promoter activity in human placental cells. Mol Cell Endocrinol 117:241-246. The functionality of this promoter is supported by showing that GnRH mRNA can be regulated by steroids.
  • the GnRH in the placenta has not been characterized as fully as the GnRH receptor in the pituitary.
  • Sealfon SC Weinstein H, Millar RP 1997 Molecular mechanism of ligand interaction with the gonadotropin-releasing hormone receptor.
  • Endocr Rev 7:44-66 It is known that two populations of placental GnRH receptors exist, one having a Ka of 10 "9 M and the other with a significantly lower affinity of 10 "7 M.
  • GnRH GnRH
  • Other isomers of GnRH such as salmon GnRH and Chicken U GnRH, have a much greater affinity for the placental receptor, yet bind with a lesser affinity to the human pituitary receptor.
  • GnRH receptor In amphibians, a chicken ⁇ GnRH receptor as well as a mammalian GnRH receptor have been shown. The specificity and evolutionary aspects of the GnRH receptor have been studied in many species. Mammalian GnRH has been reported to be active in many vertebrate classes. Other GnRHs, such as chicken U GnRH and salmon GnRH, have reduced affinity for the mammalian pituitary receptor.
  • GnRH receptor activity as well as the mRNA for the GnRH receptor, varies throughout gestation in the human placenta.
  • Bramley TA McPhie CA, Menzies GS 1994 Human placental gonadotropin-releasing hormone (GnRH) binding sites: 111. Changes in GnRH binding levels with stage of gestation. Placenta 15:733-745; Lin LS, Roberts VJ, Yen SS 1997 Expression of human gonadotropin-releasing hormone receptor gene in the placenta and its functional relationship to human chorionic gonadotropin secretion. J Clin Endocrinol Metab 80:580-585. The receptor is greatest in early gestation and appears to be down regulated by 12-20 weeks.
  • GnRH gonadotropin releasing hormone
  • Hum Reprod 6:1063-1069 Other studies of Szilagyi et al. and Currie et al. indicate that pituitary GnRH agonist can down-regulate the placental GnRH receptor.
  • the enzyme that degrades GnRH differs during pregnancy from the enzyme that degrades GnRH in the pituitary or the blood of non-pregnant individuals.
  • the primary enzymatic activity for the degradation of GnRH is chorionic peptidase- 1 (C-ase-1), a post-proline peptidase.
  • C-ase-1 is a glycoprotein with a molecular weight of 60,000.
  • Siler-Khodr TM Kang IS, Jones MA, Harper MJK, Khodr GS, Rhode J 1989 Characterization and purification of a placental protein that inactivates GnRH, TRH and Angiotensin 11.
  • GnRH is actively degraded by C-ase-1 at neutral pH, having a Km of 10 "8 M.
  • Vancouver Abstract #311: 144(Abstr.).
  • C-ase-1 has been localized by the present inventor in the cytoplasm of the syncytiotrophoblast and syncytial buds.
  • GnRH is not stable without specific inhibitors of this post-proline peptidase. Benuck M, Marka N 1976 Differences in the degradation of hypothalamic releasing factors by rat and human serum. Life Sci 19:1271-1276. C-ase-1 is present in very high concentrations, and accounts for virtually all GnRH degrading activity in the placenta under physiological conditions.
  • Gallinelli A de Vita D, Caruso A, Genazzani AR 1994 Pulsatile fluctuations of plasma- gonadotropin-releasing hormone and corticotropin-releasing factor levels in healthy pregnant women. Acta Obstet Gynecol Scand 73:284-289.
  • Other investigators using rhesus monkey embryos have demonstrated the secretion of a GnRH-like substance by the peri-implantation embryo, which precedes the secretion of chorionic gonadotropin. Seshagiri PB, Terasawa E, Hearn JP 1994 The secretion of gonadotropin-releasing hormone by peri-implantation embryos of the rhesus monkey: comparison with the secretion of chorionic gonadotropin. Hum Reprod 9:1300-1307.
  • Gupta SK Singh M 1985 Characteristics and bioefficacy of monoclonal antigonadotropin releasing hormone antibody.
  • the present invention in a general and overall sense, relates to novel pharmaceutical preparations that include non-mammalian gonadotropin releasing hormone (GnRH) analogs specifically designed to bind human chorionic GnRH receptor and ovarian GnRH receptors. These analogs are designed to be resistant to degradation by chorionic peptidase 1 (C-ase-1). C-ase-1 has been found to specifically and very actively degrade GnRH in chorionic tissues and maternal blood.
  • GnRH gonadotropin releasing hormone
  • the non-mammalian GnRH analogs of the present invention may act either as a superagonist at the placental receptor leading to its down regulation, or as a pure antagonist of chorionic GnRH at the GnRH receptor.
  • the down-regulation or antagonism of endogenous chorionic GnRH will provide for a reduction in human chorionic gonadotropin (hCG) production. This will also provide a reduction in ovarian and placental steroidogenesis.
  • hCG human chorionic gonadotropin
  • a direct ovarian luteolytic action may be expected to occur. If trophoblastic and/or ovarian function is jeopardized, premature luteolytic action might occur.
  • the analogs of the invention may be further defined as resistant to enzymatic degradation by C-ase-1.
  • the agonist and antagonists with the greatest receptor affinity and tissue stability are expected to effectively inhibit hCG and progesterone release from human placenta.
  • the non-mammalian GnRH analogs of the invention may be used to inhibit placental production of hCG, and have a direct effect on steroidogenesis at the ovary. This physiological effect of the analogs may thus be used to induce luteolysis and menses-induction.
  • the invention provides methods of synthesizing analogs of non- mammalian GnRH having increased activity in the chorionic tissues.
  • Methods to inhibit hCG production by placental tissues, that in turn provide a reduction of ovarian and placental steroidogenesis, i.e., luteolysis and menses-induction, are provided in another aspect of the present invention.
  • the use of these analogs directly on the ovary is yet another particular embodiment of the invention.
  • the analogs of the invention may be used in pharmaceutical preparations as a menses-regulating agent, a contraceptive, or as an abortifacient.
  • Non-mammalian chorionic GnRH analogs that are superagonist or antagonists at the trophoblastic/placental level constitute yet other embodiments of the invention. Such a non- mammalian analog would provide for the inhibition of steroidogenesis during pregnancy, acting both as an anti-chorionic and anti-luteal agent by inhibiting steroidogenesis leading to menses induction.
  • the chorionic GnRH analogs of the invention thus comprise peptides that are capable of specifically binding the chorionic and/or ovarian GnRH receptors with high affinity, are resistant to degradation by the C-ase-1 and effect either a down-regulation of the chorionic GnRH receptor or act as a true antagonist, inhibiting hCG production and ovarian and placental steroidogenesis or directly inhibiting ovarian steroidogenesis.
  • the invention comprises a salmon or chicken II GnRH sequence, which both show greater affinity for the placental receptor than mammalian GnRH, that are modified at the C-terminal.
  • GnRH analog sequence is substituted at the 6-position with a D-Arg, or other D-amino acid.
  • both of these modifications are made to the GnRH analog peptide sequence. These modifications are expected to enhance the binding of the molecule, while at the same time inhibit any of the endopeptidases that are present in blood. These analogs are expected to have increased binding to the placental or ovarian receptor and increased metabolic stability.
  • the invention provides non-mammalian GnRH analogs with enhanced activity within the intrauterine tissues, as well as a method for regulating hCG production and thus progesterone production during pregnancy.
  • These non-mammalian GnRH analogs may also have a direct action at the ovary. Luteolysis may be affected by a dual mechanism i.e., through inhibition of hCG and thus reduction of ovarian steroidogenesis and/or direct inhibition of ovarian steroidogenesis.
  • these analogs will be administered intra-nasally, orally, intramuscularly or vaginally. However, virtually any mode of administration may be used in the practice of the invention. Treatment with these analogs may require one to three days of active non-mammalian GnRH analog when used as a post coital contraceptive. As a monthly contraceptive, the placebo is envisioned to start on the first day of menses and continue for approximately 13 days, then the analog would be given 13 through 28, or less when menses is induced. This could be repeated monthly.
  • Another embodiment of the invention provides non-mammalian GnRH analogs that are resistant to degradation by C-ase-1. This analog will bind the chorionic GnRH receptor or non-mammalian GnRH with high affinity so to displace the endogenous GnRH-like activity and block its action.
  • the invention provides more potent non-mammalian GnRH analogs that will specifically bind to the placental and the ovarian GnRH receptor.
  • analogs will be provided that are stable in maternal circulation and in the blood of non-pregnant individuals. It is also anticipated that these analogs will be biologically active in chorionic tissues and at the ovary in the regulation of hormonogenesis that will affect the maintenance of pregnancy and/or the receptivity of the uterus for implantation. Due to the specificity of these analogs and their relatively short half- life, the present invention provides non-mammalian GnRH analogs.
  • proline-containing peptides compete for C-ase-1 activity, such as angiotensin TJ, and to a lesser extent, thyrotrophin releasing hormone and reduced oxytocin.
  • angiotensin TJ angiotensin TJ
  • thyrotrophin releasing hormone and reduced oxytocin Siler- Khodr TM, Kang IS, Jones MA, Harper MJK, Khodr GS, Rhode J 1989 Characterization and purification of a placental protein that inactivates GnRH, TRH and Angiotensin ⁇ .
  • the initial findings of inhibition can be explained by recognizing that the decapeptide sequences for mammalian GnRH and chorionic GnRH are not identical. Substantial data exists that the receptor and the chemical nature of chorionic GnRH are not identical to GnRH. Postulating that chorionic GnRH differs from the decapeptide, GnRH, and that there is a placental receptor specific for chorionic GnRH, explains the biphasic response of placental hormones. Mammalian GnRH acts as a partial agonist of chorionic GnRH. When receptors are available, it acts as an agonist of chorionic GnRH. When placental receptors are low or occupied, GnRH competes with the more potent chorionic GnRH resulting in an antagonistic action.
  • GnRH-like substances have been found by the present inventor to be decreased at mid-pregnancy in women who later have pre-term labor, and increased in those with post term deliveries. In more recent studies, a GnRH binding substance has been demonstrated in their circulation and in these cases hCG was abnormally reduced and pregnancy loss was observed. Thus, the current studies of GnRH-like substance production during pregnancy indicate that chorionic GnRH is of significance to the maintenance of normal pregnancy.
  • GnRH was actively degraded by C-ase-1. This activity of C-ase-1 was inhibited by, 9 OH-Pro-GnRH, Lamprey, Chicken I-GnRH, Antide, Chicken H-GnRH and Salmon
  • GnRH with a relative potency of 1.5, 1.5, 0.6, 0.6, and 0.2 and 0.2, respectively to that for
  • GnRH GnRH.
  • Both Chicken ⁇ GnRH- 10 ethylamide and 6 Im-btl-D-His-GnRH 10 ethylamide were essentially inactive, i.e., O.001 inhibitory activity for GnRH.
  • GnRH was bound by the placental GnRH receptor with a Kj of 10 " ° M. Chicken JJ GnRH was similar to GnRH.
  • the I j for 6 Im-btl-D-His-GnRH '10 ethylamide was half the potency of GnRH , while Buserilin and 6 D-T ⁇ -GnRH "10 ethylamide were twice as active as GnRH.
  • the greatest potency having a K d of 3 non-mammalian, i.e. 33-fold more activity than GnRH.
  • FIG. 5A and 5B Action of Angiotensin U on Degradation of GnRH.
  • the chorion is described as the highly vascularized outer embryonic membrane that is associated with the allantois in the formation of the placenta.
  • the present example outlines how analogs of non-mammalian GnRH with increased activity in chorionic and ovarian tissues are synthesized.
  • Non-mammalian analogs of GnRH were synthesized. They were specifically designed to prevent degradation of the analog both in the matemal circulation as well as within the intrauterine tissues. This allows for the maintenance of sufficient concentrations of analog to remain active when administered via the maternal system and to reach the intrauterine tissue. Due to the particular specificity of the placental receptor and specific peptidase in matemal blood and placental tissue, the particular analogs of the invention were designed.
  • Analogs of the salmon and chicken II GnRH sequences that both show greater affinity for the placental receptor than for the pituitary receptor, were modified to the ⁇ -aza-Gly 10 -NH 2 analog to make them resistant to degradation in the circulation and by C-ase-1 (chorionic GnRH analogs 1 and 2).
  • the chicken II GnRH sequence and the salmon GnRH sequence were also modified at the 6 position using D- Arg, making it resistant to degradation by the endopeptidase in blood, and was modified at the 10 position making it stable in maternal blood and the chorionic tissues (chorionic GnRH analogs 2 and 4). These analogs are expected to have increased binding to the placental receptor and increased metabolic stability.
  • the placental receptor binding activity of the different non-mammalian GnRH analogs of the present invention were compared.
  • the human placental GnRH receptor is distinct from that at the pituitary.
  • Prior mammalian GnRH analogs have been designed to increase activity at the pituitary GnRH receptor and stability in the circulation of non- pregnant individuals. These analogs do not demonstrate potent binding activity at the placental receptor as they do at the pituitary receptor.
  • the non-mammalian GnRHs have been designed to interact with preference at the placental receptor and not the pituitary receptor. They have also been designed to limit degradation by the chorionic enzyme, C- ase-1, present in maternal circulation as well as the placenta.
  • Placental binding activity of the newly synthesized chorionic GnRH analogs has been compared to that for existing pituitary-active analogs of mammalian GnRH.
  • Method and Analysis The newly synthesized non-mammalian GnRH analogs and other commercially available analogs were used in placental receptors binding and enzyme stability studies described here. On the basis of these studies, the most receptor potent and most enzyme-stable analogs were chosen for further biopotency studies.
  • GnRH receptors were purified from the membrane fractions from placentas. The purification procedure for the placental GnRH receptor was performed using a modification of the method described by Bramley et al., which reference is specifically incorporated herein by reference for the purpose.
  • GnRH binding sites 111. Changes in GnRH binding levels with stage of gestation. Placenta 15:733-745. Addition of enzyme inhibitors for the endogenous C-ase- 1 were used as well as agents for receptor stabilization. Initially, receptor-binding assays using 125 1-Buserilin as label were performed. The competitive binding of each of the analogs was studied over a dose range of 10 "11 to 10 "6 M. Incubation was at room temperature and receptor bound label was precipitated with polyethyleneglycol. Specific and nonspecific binding was determined. The data was subjected to Scatchard analysis.
  • the non- mammalian analogs' ability to bind to the placental GnRH receptor was compared to that for synthetic mammalian GnRH, Buserilin and the newly synthesized non-mammalian GnRH analogs.
  • the more potent analogs were then studied in homologous receptor assays using newly synthesized non-mammalian GnRH analog as the radioiodinated label. This way, the receptor affinity for that analog could be precisely determined.
  • Receptors from three different term placentas were used to study each of these analogs. The most potent analogs were used for the C-ase-1 stability studies.
  • the present example demonstrated the utility of using the present invention in controlling and modulating the activity of the placenta, such as in a placenta of a pregnant mammal.
  • Mammalian GnRH and its analogs bind to placental receptors.
  • the present non-mammalian analogs had not been examined for placental receptor binding.
  • the added stability of these non-mammalian analogs would effect a substantial increase in bioactivity alone.
  • both stability and binding studies were performed.
  • Chorionic Peptidase- 1 Stability Studies The enzymatic degradation of the non- mammalian GnRH analogs were studied using the C-ase-1 enzyme activity assay as well as whole placental homogenate assays.
  • chorionic peptidase- 1 A chorionic peptidase that actively degrades GnRH in the placenta, named chorionic peptidase- 1 (C-ase-1), was used.
  • This enzyme acts as a post-proline peptidase, and is present in the placenta and in matemal circulation. In a non-pregnant individual very little post-proline peptidase activity is present in blood.
  • mammalian GnRH analogs have not been designed to be resistant to degradation by this activity.
  • Non- mammalian GnRH analogs were designed with these specific criteria in mind.
  • the hCG inhibiting activity of the chorionic GnRH analogs was studied using an in vitro human placental explant system.
  • the present example demonstrates the utility of using the present non-mammalian analogs to regulate hCG levels in a mammal and in the regulation of pregnancy.
  • the newly synthesized non-mammalian GnRH analogs are resistant to enzyme degradation and are potent binders of the placental GnRH receptor. Bio-potency was studied using a placental explant system, and by determining the release of hCG, progesterone and prostanoids.
  • hCG is the luteotropin of pregnancy, and known to be critical to the maintenance of the corpus luteum during pregnancy. Thus, it is a primary parameter of interest.
  • the production of progesterone by the placenta and the ovary is affected by hCG, as well as being independently regulated by a GnRH-like substance.
  • Progesterone is primary to the maintenance of uterine quiescence and thus the maintenance of pregnancy, and therefore is of primary interest to these studies. Also, of interest is the effect of these GnRH analogs on prostaglandin production. Prostaglandins are required for abortifacient activity, and thus, the maintenance or increase in their production may be necessary for the proposed action of the analogs.
  • Method and analysis The biological activity of the newly synthesized non- mammalian GnRH analogs was studied using a static implant culture system. This system allows for inexpensive extended activity studies. Mammalian GnRH action on the human placenta release of hCG, progesterone and prostaglandins were defined using this system.
  • the present example demonstrates the isolation of an enzyme from human placentas, and the action of the enzyme as a post-proline peptidase. It actively degrades peptides, such as gonadotropin releasing hormone (GnRH), thyrotrophin releasing hormone (TRH), oxytocin, and Angiotensin II (Ang-H). See Figures 3, 4, 5A, and 5B. These peptides contain a proline residue enzyme, chorionic peptidase- 1 (C-ase-1). The present example also defines enzyme inhibitors of C-ase-1 action on GnRH, such that it might regulate GnRH concentrations within the intrauterine tissues.
  • C-ase-1 enzyme activity studies were done by incubating GnRH with C-ase-1 in the presence of varying concentrations of the non-mammalian GnRH analogs. The reaction was stopped by heating at 85°C for 10 minutes. The remaining GnRH was determined using a specific radioimmunoassay. The formation of product, i.e., the N-terminal nonapeptide of GnRH, was calculated by subtraction and its inverse was plotted versus the inverse of the initial substrate to determine the K ⁇ of the reaction.
  • the inhibitory activity of Antide, °Im-btl-D-His-GnRH- 10 ethylamide, 9 OH-Pro-GnRH, chicken II GnRH- 10 ethylamide, chicken ⁇ GnRH, chicken I GnRH, salmon GnRH and lamprey GnRH was studied. The relative potency of each analog was compared.
  • GnRH was actively degraded by C-ase-1. This activity of C-ase-1 was inhibited by, 9 OH-Pro-GnRH, lamprey, chicken I-GnRH, Antide, chicken U-GnRH and salmon GnRH with a relative potency of 1.5, 1.5, 0.6, 0.6, 0.2 and 0.2, respectively, compared to that for GnRH. Both chicken ⁇ GnRH- 10 ethylamide and 6 Im-btl-D-His-GnRH 10 ethylamide were essentially inactive, i.e., ⁇ 0.001 inhibitory activity for GnRH. See Figure 1.
  • Chorionic peptidase- 1 which is a post-proline peptidase with high specificity for the degradation of GnRH, can also degrade other GnRH species.
  • the synthetic mammalian GnRH analogs such as antide are degraded with reduced activity, while other analogs such as chicken ⁇ GnRH- 10 ethylamide and 0 Im-btl-D-His-GnRH 10 ethylamide are resistant to degradation by this endogenous chorionic enzyme. See Figure 7. These analogs will be useful in the regulation of chorionicGnRH activity.
  • EXAMPLE VI Comparison Of GnRH And Its Synthetic And Naturally Occurring Analogs For Binding Degradation Action in The Human Placental Receptor
  • the human placental GnRH receptor shows different kinetic constants for GnRH compared to that of the pituitary receptor.
  • Receptor assays were performed by incubating human term placental GnRH receptors with varying concentrations of GnRH or its analogs in the presence of 125 I-
  • GnRH was bound by the placental GnRH receptor with a Kj of 10 "6 M.
  • Chicken II GnRH was similar to GnRH.
  • the K_ for - 6 Im-btl-D-His-GnRH 10 ethylamide was half the potency of GnRH, while buserilin and 6 D-Trp-GnRH- 10 ethylamide were twice as active as GnRH.
  • GnRH analogs were examined for their stability in the presence of C-ase-1 and placental homogenate. Using the incubation system developed for the C-ase-1 activity, the degradation of each analog was studied. Previously, this method was used to determine the degradation of GnRH by C-ase-1. Each of these analogs was studied for their ability to act as competitive inhibitors of GnRH for C-ase-1 activity (Table 1). The inverse of the product was plotted against the inverse of the original substrate concentrations to determine K s of the competition. The Kj was determined by plotting the inverse of the product formed verses the inhibitor used.
  • GnRH for C-ase-1 degradation but not as markedly as did the Im-btl-D-His(6) or chicken II GnRH-ethylamides.
  • Ethylamides of the latter two GnRHs were greater than 200-fold less active in the inhibition of GnRH degradation by C-ase-1. Thus, these ethylamides appear to be very stable in the presence of the C-ase-1 enzyme.
  • the Im-btl-D-His(6) analog has reduced receptor potency.
  • the stability of the D-Arg-(6)-Chicken U GnRHaza-Gly-amide was found to be at least 200-fold that of GnRH.
  • the stability of these analogs in the present of whole placental homogenates was examined.
  • the ethylamide derivative has a slowed degradation rate as compared to GnRH, but can be degraded.
  • Chicken II and its ethylamide analog are more stable than the mammalian GnRH analogs analyzed to date.
  • the hCG inhibiting activity of the GnRH analogs was studied using an in vitro human placental explant system.
  • the newly synthesized GnRH analogs are resistant to enzyme degradation and one potent binders of the placental receptor.
  • the bio-potency was done with a placental explant system, and the release of hCG, progesterone and prostaglandin E 2 was assessed.
  • hCG is the luteotropin of pregnancy and known to be important in the maintenance of the co ⁇ us luteum during pregnancy.
  • the production of progesterone by the placenta is affected by hCG, and may be independently regulated by GnRH as well.
  • Progesterone is primary to the maintenance of uterine quiescence and thus the maintenance of pregnancy. Of interest was the effect of these GnRH analogs on prostaglandin production. Prostaglandins are required for abortifacient activity.
  • D-Arg(6)-chicken TJ GnRH analog-NH 2 has bioactivity in the regulation of hCG and progesterone in the human term placenta.
  • the present example defines a method by which the present invention may be used to maintain pregnancy in a pregnant mammal.
  • the mammal in some embodiments is a pregnant human.
  • a proposed dose regimen it is anticipated that a pregnant female between 100 lbs and 150 lbs would be administered about 10 nanogram to 1.0 gram of chicken U GnRH Analog or salmon GnRH analog. This would be expected to be effective for promoting the maintenance of pregnancy in the mammal when administered.
  • the dosing regimen will comprise a pulsatile administration of the chicken II GnRH over a 24-hour period, wherein the daily dosage is administered in relatively equal l/24 th fractions.
  • the daily dosage is about 2.4 micrograms
  • the patient would be administered about 0.1 micrograms per hour over a 24- hour period.
  • Such a daily pulsatile administration would create a hormonal environment in the patient sufficient to maintain pregnancy.
  • the particular pharmaceutical preparations may be created by one of skill in the pharmaceutical arts. Remington's Pharmaceutical Sciences Remington: The Science and Practice of Pharmacy, 19 th edition, Vol. 102, A.R. Gennaro, ed., Mack Publishing co. Easton, PA (1995), is specifically inco ⁇ orated herein by reference for this pu ⁇ ose.
  • the present example demonstrates the utility of the present invention for use as a post-coital contraceptive preparation.
  • analogs defined here, and conservative variants thereof may be formulated into a pharmaceutically acceptable preparation, and then administered to a female mammal having been inseminated during the prior 24 to 72 hours (prior 1 to 3 days). Relatively high doses of about 0.1 gram to about 10 grams of the non-mammalian GnRH analog would be given daily for 2 to 5 days, on the average about 3 days.
  • the cDNA sequence for the non- mammalian GnRH of SEQ ID NO: 1 (Chicken ⁇ GnRH) or SEQ ID NO:3 (Salmon GnRH) may be prepared as part of a suitable vector, such as in an adenovirus or retroviral vector, and administered to the animal. Once the sequence is inco ⁇ orated into the cell, the peptide product will be translated and peptide supplied. Because this method of treatment would not require that the peptide travel in the blood circulation in order to reach the site of action, there would be no requirement that the analog possess enzyme degradation resistance. This mode of treatment has not thus far been proposed, and hence the use of such a method in the regulation of female fertility is a novel clinical regimen.
  • non-mammalian analogs are also contemplated to be useful to directly affect the ovary.
  • this technique renders the system useful as a contraceptive.
  • the non-mammalian GnRH analog would be given daily from the start of ovulation and continue 8 days to two weeks, stopping with onset of menses.
  • NON-MAMMALIAN GNRH The present example demonstrates the utility for using the present invention non- mammalian GnRH analog decapeptides to prepare antibodies that preferentially bind the GnRH peptide sequences, or that bind the ovarian, placental or any other non-pituitary GnRH peptide or protein. It is anticipated that these non-mammalian GnRH analog antibodies may be used in a variety of screening assays. For example, these antibodies may be used to determine levels of GnRH present in a sample as an indicator molecule. The levels of such GnRH may be used to monitor and follow a patient's pregnancy, as well as an indicator of the length of gestation.
  • the antibodies to non-mammalian GnRH may be monoclonal or polyclonal antibodies.
  • Polyclonal antibodies may be created by standard immunization techniques, wherein the immunogen used will be the non-mammalian chicken-11 GnRH analog or the salmon GnRH analog decapeptide described herein. These peptides may be used either alone or together in a pharmaceutically acceptable adjuvant.
  • the animal, such as a rabbit, would be administered several doses of the decapeptide preparation, and the levels of the animal's antibody blood levels monitored until an acceptable antibody level (titer) had been reached.
  • the spleen of the animal would be harvested, and then fused with an immortalized cell line, such as a cancer cell line, to produce a population of hybridoma cells.
  • an immortalized cell line such as a cancer cell line
  • This hybridoma population of cells would then be screened for those that produce the highest amount of antibody that specifically bind the non-mammalian GnRH analog decapeptide.
  • Such hybridoma cells would be selected, and then cultured.
  • the antibody to non- mammalian GnRH would then be collected from the media of the cell culture using techniques well know to those of skill in the art.

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  • Veterinary Medicine (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Reproductive Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • Public Health (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne des décapeptides analogues de GnRH non mammaliens résistant à la dégradation par l'enzyme placentaire C-ase-1, ou par une peptidase post-proline. Les analogues de GnRH sont également définis comme des analogues de GnRH de poulet II ou de GnRH de saumon. Ces analogues non mammaliens comprennent de l'arginine-D, de la leucine-D, de la Serine tBu-D ou Trp-D en position 6 et de l'éthylamide ou de l'amide Gly-azaat en position 10. L'invention concerne également les analogues de l'éthylamide-GnRH de poulet II D-Arg (6), l'amide aza-Gly (10)- GnRH de poulet II D-Arg (6), l' éthylamide GnRH de saumon D-Arg(6), et l'amide- aza-Gly (10)- GnRH de saumon D-Arg(6). Ces analogues mettent en évidence une fixation de préférence au récepteur de GnRH chorionique qui est supérieur par rapport à la fixation de ces analogues au récepteur de GnRH de l'hypophyse. Ces analogues de GnRH non mammaliens peuvent être utilisés dans des compositions pharmaceutiques, et spécifiquement dans plusieurs procédés de traitement en tant qu'agents contraceptifs ou contraceptifs post-coïtal. Les analogues de GnRH non mammaliens sont aussi produits dans des compositions pharmaceutiques qui peuvent être utilisées cliniquement pour le maintien de la gestation lorsqu'il est utilisé en faibles doses et est administré de façon pulsative. Dans un autre mode de réalisation, les analogues de GnRH non mammaliens sont utilisés en tant qu'agents lutéolytiques. Les analogues non mammaliens de l'amide aza-Gly (10) sont encore d'autres modes de réalisation des analogues de GnRH non mammaliens décrits dans l'invention.
PCT/US2002/025619 2001-08-17 2002-08-13 Analogues de gnrh non-mammaliens et leurs utilisations dans la regulation de la fertilite et de la gestation WO2003016331A2 (fr)

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US93214901A 2001-08-17 2001-08-17
US09/932,149 2001-08-17

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005018660A1 (fr) * 2003-08-12 2005-03-03 Siler-Khodr Theresa M Analogues de gnrh non mammiferes et leurs utilisations dans le cadre de la regulation de la fertilite et de la gestation
EP1586581A3 (fr) * 2004-04-08 2005-12-21 Theresa M. Siler-Khodr Analogues de GnRH non mammifère et leurs utilisations pour la regulation de la fertilité et de la gestastion
EP1626055A3 (fr) * 2004-08-10 2006-03-08 Theresa M. Siler-Khodr Analogues de GnRH non mammifère et leur utilisation pour la régulation du système immunitaire

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4410514A (en) * 1982-12-06 1983-10-18 The Salk Institute For Biological Studies GnRH Agonists
IT1178775B (it) * 1983-12-23 1987-09-16 Konzponti Valto Es Hitelbank R Derivati della gonadoliberina e procedimento per la loro preparazione
CA2029018A1 (fr) * 1989-11-01 1991-05-02 Robert P. Millar Analogues de l'hormone liberant de la gonadotrophine

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005018660A1 (fr) * 2003-08-12 2005-03-03 Siler-Khodr Theresa M Analogues de gnrh non mammiferes et leurs utilisations dans le cadre de la regulation de la fertilite et de la gestation
EP1586581A3 (fr) * 2004-04-08 2005-12-21 Theresa M. Siler-Khodr Analogues de GnRH non mammifère et leurs utilisations pour la regulation de la fertilité et de la gestastion
EP1626055A3 (fr) * 2004-08-10 2006-03-08 Theresa M. Siler-Khodr Analogues de GnRH non mammifère et leur utilisation pour la régulation du système immunitaire

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WO2003016331A3 (fr) 2003-11-20

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