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EP1605962A1 - Analogues de gnrh non mammiferes et leurs utilisations dans le cadre de la regulation de la fertilite et de la gestation - Google Patents

Analogues de gnrh non mammiferes et leurs utilisations dans le cadre de la regulation de la fertilite et de la gestation

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Publication number
EP1605962A1
EP1605962A1 EP04756015A EP04756015A EP1605962A1 EP 1605962 A1 EP1605962 A1 EP 1605962A1 EP 04756015 A EP04756015 A EP 04756015A EP 04756015 A EP04756015 A EP 04756015A EP 1605962 A1 EP1605962 A1 EP 1605962A1
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Prior art keywords
gnrh
seq
analogs
mammalian
chicken
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German (de)
English (en)
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Theresa M. Siler-Khodr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A61K38/09—Luteinising hormone-releasing hormone [LHRH], i.e. Gonadotropin-releasing hormone [GnRH]; Related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/02—Drugs for disorders of the endocrine system of the hypothalamic hormones, e.g. TRH, GnRH, CRH, GRH, somatostatin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G01N2800/367—Infertility, e.g. sperm disorder, ovulatory dysfunction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G01N2800/368—Pregnancy complicated by disease or abnormalities of pregnancy, e.g. preeclampsia, preterm labour

Definitions

  • the present invention relates generally to the field of regulating reproductive function, fertility and pregnancy. More particularly, it concerns the use of unique non-mammalian peptide hormone analogs of GnRH designed to be useful in male and female fertility regulation, post-coital contraception and as a menses-inducing agent, in the management of ovarian cyst, polycystic ovarian disease, in vitro fertilization protocols, endometriosis, abnormal uterine bleeding, leiomyomas, abnormal pregnancies, ectopic pregnancies, molar pregnancies, and trophoblastic disease, and in the management of disorders of the male reproductive system.
  • LRF hypothalamic luteinizing hormone releasing factor
  • GnRH Gonadotropin-releasing hormone
  • the Placenta Part IV- Function of the Human Placenta.
  • the GnRH receptor in the placenta has not been characterized as fully as the GnRH receptor in the pituitary. It is known that placental GnRH receptors exist, having a Ka of only 10 "6 M . In addition, superagonist or antagonist for the pituitary GnRH receptor shows very different affinity for the placental receptor in primates. GnRH receptor activity, as well as the mRNA for the GnRH receptor, varies throughout gestation in the human placenta.. The receptor is greatest in early gestation and appears to be down regulated by 12-20 weeks.
  • Hum Reprod 6:1063-1069; Szilagyi A, Benz R, Rossmanith WG 1992 The human first- term placenta in vitro: regulation of hCG secretion by GnRH and its antagonist.
  • 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. It acts as a post-proline peptidase, and is inhibited by bacitracin, para-amino-benzamidine, acetopyruvate and certain cations.
  • GnRH is actively degraded by C-ase-1 at neutral pH, having a Km of 10 "8 M.
  • C-ase-1 has been localized by the present inventor in the cytoplasm of the syncytiotrophoblast and syncytial buds. It is secreted into maternal blood, where GnRH is not stable without specific inhibitors of this post- proline peptidase C-ase-1 is present in very high concentrations, and accounts for virtually al GnRH degrading activity in the placenta under physiological conditions.
  • 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. Placenta 10:283-296; Kang IS, Siler-Khodr TM 1992 Chorionic peptidase inactivates GnRH as a post-proline peptidase. Placenta 13:81-87; Kang IS, Gallwitz J, Guzman V, Siler-Khodr TM 1990 Definition of the enzyme kinetics and optimal activity of chorionic peptidase-1.
  • GnRH-like substance which has a specific pulse frequency, amplitude and duration, with increased amplitude during early gestation.
  • Further studies on the action of mammalian GnRH and its analogs in vivo have also demonstrated these paracrine interactions for chorionic GnRH-like activity and numerous other chorionic hormones, and have established the physiologic role of GnRH in the maintenance of normal pregnancy.
  • the secretion of a GnRH-like substance by the peri-implantation rhesus monkey embryo, which precedes the secretion of chorionic gonadotropin has been demonstrated.
  • GnRH gonadotropin-releasing hormone
  • Chicken II GnRH has now been characterized in the guinea pig and in the primate brain. Separate genes for chicken II GnRH and mammalian GnRH have also been described. Other isomers of GnRH, such as salmon GnRH and chicken II GnRH, have a much greater affinity for the placental receptor, yet bind with a lesser affinity to the human pituitary receptor. These data demonstrate the existence of a specific placental receptor for GnRH-like molecules, yet the true ligand for this receptor is not known. In amphibians, a chicken II 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.
  • GnRH human placental gonadotropin-releasing hormone
  • a second gene for gonadotropin-releasing hormone cDNA and expression pattern in the brain.
  • 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, ovarian, fallopian tube, and uterine tissue GnRH receptors as well as human sperm, testicular, scrotal, seminiferous tubule, Leydig cell, Sertoli cell, epididymis, vas deferentia, prostate, seminal vesicle, ejaculatory duct, and urethral GnRH receptors.
  • GnRH gonadotropin releasing hormone
  • Post-proline peptidases have been found to specifically and very actively degrade GnRH in chorionic, ovarian, tubal, and uterine tissues and maternal blood.
  • the non-mammalian GnRH analogs of the present invention may act either as a superagonist at the placental, ovarian, tubal, or uterine GnRH receptor leading to acute stimulation then to its down regulation, or as a pure antagonist at the chorionic, ovarian, tubal, uterine, sperm, testicular, scrotal, seminiferous tubule, Leydig cell, Sertoli cell, epididymis, vas deferentia, prostate, seminal vesicle, ejaculatory duct, and urethral 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. In addition, a direct ovarian luteolytic action may be expected to occur. If trophoblastic and/or ovarian function is jeopardized, premature luteolytic action will occur. If trophoblastic and/or ovarian function is jeopardized, premature luteolysis of the corpus luteum will occur and menses will ensue.
  • the down-regulation or antagonism of endogenous GnRH activity at the testis will provide for a reduction in testosterone production and will affect sperm function.
  • the GnRH analog is also expected to act at the sperm, testicules, scrotum, seminiferous tubule, Leydig cell, Sertoli cell, epididymis, vas deferentia, prostate, seminal vesicle, ejaculatory duct, and urethra to affect these tissues function and thus again affect sperm function.
  • an agent may be used as a post-coital, luteolytic and anti-sperm agent, leading to the induction of menses and sperm inactivation.
  • no such GnRH analog has been found to be active during pregnancy or at the ovary or on the testis or sperm .
  • maturation of the egg and sperm and the process of ovulation, as well as the process of fertilization and maturation of the fertilized egg and sperm, will be affected.
  • Sperm capacitation in the male and female tracts and fertilizing capability will be affected.
  • the activity of the fallopian tube will be affected altering transport and maturation of the morula during transit.
  • uterine hormone and cell functions will be affected both directly and indirectly by non-mammalian GnRH analogs. PGE production is decreased which will lead to decreased vaso-function and vasodilation.
  • the uterine environment will be made hostile to implantation of the blastocyst or the maintenance of pregnancy. The regression of uterine endometrial tissue will result.
  • the inventor has designed non-mammalian GnRH analogs that are active as luteolytic, menses-inducing agents, and anti-sperm agents and/or post-coital contraceptives.
  • the chorionic, ovarian, and uterine and sperm, testicular, scrotal, seminiferous tubule, Leydig cell, Sertoli cell, epididymis, vas deferentia, prostate, seminal vesicle, ejaculatory duct, and urethral receptor binding activity of these particularly designed non-mammalian GnRH analogs has also been characterized in the development of the present analogs.
  • the analogs of the invention may be further defined as resistant to enzymatic degradation by blood, ovarian, uterine, and placental and sperm, testicular, scrotal, seminiferous tubule, Leydig cell, Sertoli cell, epididymis, vas deferentia, prostate, seminal vesicle, ejaculatory duct, and urethral enzymatic activity by specific endopeptidase and post-proline peptidase, such as 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 and ovary, and PGE production from fallopian tubes and uterine tissues and testosterone from the testis and tissue functions of the and testicular, sperm prostate, seminal vesicle, epididymis.
  • the non-mammalian GnRH analogs of the invention may be used to inhibit placental production of hCG and progesterone, and have a direct effect on steroidogenesis at the ovary and prostaglandins in the fallopian tubes and uterus.
  • the effects of the analogs may thus be used to induce luteolysis and menses-induction and anti-implantation, anti-pregnancy activity.
  • the invention provides methods of designing 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 another particular embodiment of the invention.
  • the use of these analogs to directly affect fallopian tube function is still another embodiment of the invention.
  • analogs of this invention may be used in pharmaceutical preparations as a menses-regulating agent, a contraceptive, or as an abortifacient.
  • Non-mammalian GnRH analogs that are superagonists or antagonists at the trophoblastic/placental, ovarian, tubal and/or uterine 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 or at the tubal or uterine level to inhibit PGE production leading to menses induction.
  • the non-mammalian GnRH analogs of the invention thus comprise peptides that are capable of specifically binding the chorionic, ovarian, fallopian tubes and/or uterine GnRH receptors with high affinity, are resistant to degradation by endopeptidase and post-proline peptidase activity and effect either a down-regulation of the GnRH receptor or act as a true antagonist, inhibiting hCG production and ovarian and placental steroidogenesis or directly inhibiting ovarian steroidogenesis and/or inhibiting tubal and/or uterine prostaglandin production, and testicular, sperm prostate, seminal vesicle, epididymis function.
  • the invention comprises a salmon sequence (SEQ ID NO: 4) or chicken II GnRH sequence (SEQ ID NO: 2), which both show greater affinity for the placental, ovarian, uterine, testicular, sperm, prostate, seminal vesicle, and epididymis receptor than mammalian GnRH, and are modified at the C-terminal.
  • An ethylamide or aza-Gly 10 -NH 2 substitution may be used, making the sequence more stable in chorionic, ovarian, tubal, uterine, testicular, sperm, prostate, seminal vesicle, and epididymis tissues and maternal blood.
  • the GnRH analog sequence is substituted at the 6- position with a D-Arg, or other D-amino acid. In yet other embodiments, both of these modifications are made to the GnRH analog peptide sequence.
  • the chicken II or salmon backbone and the substitutions of the molecule are expected to enhance the binding of the molecule, while at the same time the substitutions are designed to inhibit any of the peptidases that are present in blood.
  • the ovarian receptor binding, ovarian metabolic degradation, and the biological activity for progesterone production were studied for each of the specially designed non-mammalian GnRH analogs, and compared to closely related pituitary mammalian GnRH analogs. These studies demonstrated greater stability, binding affinity, and bioactivity of the non-mammalian GnRH analogs compared to the mammalian GnRH analogs examined.
  • the uterine receptor binding and biological activity for the prostaglandin E production were studied for these specially designed non-mammalian GnRH analogs and compared to closely related pituitary mammalian GnRH analogs.
  • the invention provides non-mammalian GnRH analogs with enhanced activity within the uterine, sperm, testicular, scrotal, seminiferous tubule, Leydig cell, Sertoli cell, epididymis, vas deferentia, prostate, seminal vesicle, ejaculatory duct, and urethral tissues, as well as a method for regulating hCG production and thus progesterone production during pregnancy.
  • the activity of these analogs may be useful in the management of threatened abortion or the induction of abortions. Activity of these analogs may also be useful in the management of abnormal pregnancies, ectopic pregnancies, molar pregnancies, or trophoblastic disease and a list of male diseases, impotence, undescented testis, male infertility, azo- or oligospermia and the like.
  • These non-mammalian GnRH analogs also have a direct action on endometrial tissue. This activity may prove beneficial in treatments for endometriosis, abnormal uterine bleeding, and leiomyomas. These non- mammalian GnRH analogs 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. This will be useful to induce menses and as a contraceptive. It is envisioned that these analogs will be administered intra-nasally, orally, intramuscularly, intrauterine, subcutaneously, transdermally 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, but could be continuous.
  • the placebo is envisioned to start on the first day of menses and continue for approximately 13 days, then the analog would be given days 13 through 28, or less to suppress luteal and/or endometrial and anti- sperm function and to induce menses. This could be repeated monthly.
  • Numerous IVF protocols now routinely use mammalian GnRH analogs for ovulation timing and have been shown to be nontoxic, even after weeks of administration.
  • the design of the present non-mammalian analogs considers the specific metabolism of GnRH at extra-pituitary tissues, such as the ovary, fallopian tubes, uterus, placenta, testicle, sperm, prostate, and seminal vesicle, and during pregnancy in maternal blood.
  • Another embodiment of the invention provides non-mammalian GnRH analogs that are resistant to degradation by post-proline peptidases and endopeptidases .
  • This analog will bind the chorionic, ovarian, tubal, and uterine GnRH receptor or non- mammalian GnRH with high affinity so to first stimulate then down-regulate the receptor 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, ovarian, tubal, uterine, sperm, testicular, scrotal, seminiferous tubule, Leydig cell, Sertoli cell, epididymis, vas deferentia, prostate, seminal vesicle, ejaculatory duct, and urethral GnRH receptor.
  • non-mammalian GnRH analogs will be provided that are stable in maternal circulation and in the blood of non-pregnant individuals. It is also anticipated that these non-mammalian GnRH analogs will be biologically active in chorionic tissues, at the ovary, fallopian tube, uterus, sperm, testis, scrotum, seminiferous tubule, Leydig cell, Sertoli cell, epididymis, vas deferentia, prostate, seminal vesicle, ejaculatory duct, and urethra, in the regulation of hormonogenesis that will affect the maintenance of pregnancy and/or the receptivity of the uterus for implantation.
  • the present invention provides non-mammalian GnRH analogs.
  • the human may contain another GnRH defined as salmon GnRH which contains the sequence or a degenerate variant of Salmo salar.
  • Other praline-containing peptides compete for post-proline peptidase activity, such as angiotensin II, and to a lesser extent, thyrotropin releasing hormone and reduced oxytocin.
  • the existing mammalian GnRH analogs are also proline-containing molecules.
  • the present non- mammalian GnRH analogs have been designed to inhibit the former enzymatic activities, and have substitutions in the 5-6 position of the molecule.
  • Some of the present non-mammalian GnRH analogs also have a substitution at the 10 position with an ethylamide which is only a weak inhibitor of the post-proline peptidase.
  • the present non-mammalian GnRH analogs are therefore, resistant to degradation at the pituitary or in the blood of non-pregnant individuals, but not the ovary, fallopian tube, uterus, placenta, sperm, testis, scrotum,, seminiferous tubule, Leydig cell, Sertoli cell, epididymis, vas deferentia, prostate, seminal vesicle, ejaculatory duct, and urethra, or in maternal blood.
  • the existing mammalian GnRH analogs commonly used in medicine can be degraded in the ovarian, endometrial, chorionic, testicular, sperm, prostate, and seminal vesicle tissues, and in maternal blood.
  • the findings of inhibition of placental, ovarian, and uterine function can be explained by recognizing that the decapeptide sequence for mammalian GnRH is not the only active GnRH sequence in ovarian, fallopian tube, uterine, and chorionic GnRH.
  • Substantial data exists that in these tissues there is a receptor and there is a GnRH of which the chemical nature is not identical to mammalian GnRH.
  • Mammalian GnRH acts as a partial agonist of non-mammalian chorionic GnRH. When receptors are available, it acts as an agonist of ovarian, tubal, uterine, sperm, testicular, scrotal, seminiferous tubule, Leydig cell, Sertoli cell, epididymis, vas deferentia, prostate, seminal vesicle, ejaculatory duct, urethral, or chorionic GnRH.
  • GnRH When ovarian, tubal, uterine, sperm, testicular, scrotal, seminiferous tubule, Leydig cell, Sertoli cell, epididymis, vas deferentia, prostate, seminal vesicle, ejaculatory duct, urethral, or placental receptors are low or occupied, mammalian 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.
  • the present inventor has found that certain non-mammalian GnRH analogs can act on the ovarian, uterine, and chorionic GnRH receptor, and with high affinity binding, affect changes in the ovarian and/or intrauterine environment that effect fertility, reproductive function, and the outcome of pregnancy. This finding is the basis of the invention disclosed herein. Thus, the present investigator has developed particular (non-mammalian) GnRH analogs that can be used for regulation of ovarian, tubal, and uterine function, induction of luteolysis and menstruation, and regulation of uterine PGE production.
  • the ability of specific (non-mammalian) GnRH analogs to interact with the physiologic regulation of hCG, progesterone and prostaglandin during luteal phase of the cycle and early pregnancy, may be used to specifically interrupt luteal function and early pregnancy according to the invention as outlined here.
  • the specificity, activity and stability of these non- mammalian GnRH analogs were investigated at the ovary, the endometrium and the pituitary and their acute action was assessed on chorionic tissues. A direct action on ovarian and endometrial tissue was found. A potential direct contraceptive action of these analogs, as well as their placental hCG stimulation followed by inhibition and steroidogenic suppression activity is indicated.
  • the present invention relates to novel preparations of non- mammalian GnRH analogs that can be useful in male fertility regulation essentially acting as a male contraceptive agent.
  • This male contraceptive agent can act within the male reproductive system to reduce or eliminate sperm production or to disable the motility and travel of the sperm through the male reproductive system.
  • the present preparations can provide a reduction in testosterone production.
  • the male contraceptive agent can be introduced into the female along with the semen upon the male's ejaculation during coitus.
  • This non-mammalian GnRH analog may lead to sperm inactivation or inability to capacitate and thus induce infertility.
  • the non-mammalian GnRH analog of the present invention may act either as a superagonist at the placental, ovarian, tubal, or uterine receptor leading to its down regulation, or as a pure antagonist of chorionic, ovarian, tubal, or uterine 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 will occur. If trophoblastic and/or ovarian function is jeopardized, premature luteolysis of the corpus luteum will occur and menses will ensue. Thus, such an agent may be used as a post-coital, luteolytic agent, leading to the induction of menses in the female.
  • maturation of the egg and the process of ovulation, as well as the process of fertilization and maturation of the fertilized egg will be affected. The ability of the sperm to capacitate or to bind or fertilize the egg may be affected.
  • the activity of the fallopian tube will be affected altering transport and maturation of the morula during transit.
  • uterine hormone and cell functions will be affected.
  • PGE production will be decreased which will lead to decreased vaso-function and vasodilation.
  • the uterine environment will be made hostile to implantation of the blastocyst or the maintenance of pregnancy. The regression of uterine endometrial tissue will result.
  • the present invention relates to novel pharmaceutical preparations that include non-mammalian gonadotropin releasing hormone (GnRH) analogs and any biomimetic or chemomimetic agents, i.e., functional mimetics of the present non- mammalian GnRH analogs specifically designed to bind to GnRH receptors in the male reproductive system including human sperm, testicular, scrotal, seminiferous tubule, Leydig cell, Sertoli cell, epididymis, vas deferentia, prostate, seminal vesicle, ejaculatory duct, and urethral GnRH receptors.
  • GnRH gonadotropin releasing hormone
  • any functional mimetics may be used for any purpose as the non-mammalian GnRH analogs of the present invention which can include, among other things, antagonizing the activity of GnRH receptor or as an antigen in a manner described elsewhere herein.
  • Functional mimetics of the non- mammalian GnRH analog of the present invention include but are not limited to truncated polypeptides or synthetic organic or inorganic molecules comprising a comparable GnRH receptor binding site.
  • Polynucleotides encoding each of these functional mimetics may be used as expression cassettes to express each mimetic polypeptide. It is preferred that these cassettes comprise 5' and 3' restriction sites to allow for a convenient means to ligate the cassettes together when desired.
  • cassettes comprise gene expression signals known in the art or described elsewhere herein.
  • These analogs and mimetics are designed to be resistant to degradation by post-proline peptidases and endopeptidases.
  • Post-proline peptidases have been found to specifically and very actively degrade GnRH in male reproductive system tissues.
  • Other proline-containing peptides compete for post-proline peptidase activity, such as angiotensin II, and to a lesser extent, thyrotropin releasing hormone and reduced oxytocin.
  • the existing mammalian GnRH analogs are also proline-containing molecules.
  • the present non- mammalian GnRH analogs have been designed to inhibit the former enzymatic activities, and have substitutions in the 5-6 position of the molecule. Some of the analogs also have a substitution at the 10 position with an ethylamide which is only a weak inhibitor of the post-proline peptidase.
  • the present mammalian analogs are therefore, resistant to degradation at the pituitary or in the blood, seminal fluid, or vaginal fluid of individuals. The even more potent aza-Gly 10 -NH 2 , inhibits degradation by post-proline peptidase.
  • the non-mammalian GnRH analogs of the present invention may act either as a superagonist at the sperm, testicular, scrotal, seminiferous tubule, Leydig cell, Sertoli cell, epididymis, vas deferentia, prostate, seminal vesicle, ejaculatory duct, or urethral receptor leading to its down regulation, or as a pure antagonist of sperm, testicular, scrotal, seminiferous tubule, Leydig cell, Sertoli cell, epididymis, vas deferentia, prostate, seminal vesicle, ejaculatory duct, or urethra GnRH at the GnRH receptor.
  • the invention comprises a salmon sequence (SEQ ID NO: 4) or chicken II GnRH sequence (SEQ ID NO: 2), which both show greater affinity for the sperm, testicular, scrotal, seminiferous tubule, Leydig cell, Sertoli cell, epididymis, vas deferentia, prostate, seminal vesicle, ejaculatory duct, or urethra receptor than mammalian GnRH, that are modified at the C-terminal.
  • SEQ ID NO: 4 the invention comprises a salmon sequence (SEQ ID NO: 4) or chicken II GnRH sequence (SEQ ID NO: 2), which both show greater affinity for the sperm, testicular, scrotal, seminiferous tubule, Leydig cell, Sertoli cell, epididymis, vas deferentia, prostate, seminal vesicle, ejaculatory duct, or urethra receptor than mammalian GnR
  • An ethylamide or aza-Gly 10 -NH 2 substitution may be used, making the sequence more stable in sperm, testicular, scrotal, seminiferous tubule, Leydig cell, Sertoli cell, epididymis, vas deferentia, prostate, seminal vesicle, ejaculatory duct, and urethral tissues.
  • the non-mammalian GnRH analog sequence (SEQ ID NO: 4 and SEQ ID NO: 2) is substituted at the 6-position with a D-Arg, or other D-amino acid.
  • both of these modifications are made to the non- mammalian GnRH analog peptide sequence.
  • the chicken II or salmon backbone and the substitutions of the molecule are expected to enhance the binding of the non- mammalian GnRH analog, while at the same time the substitutions are designed to inhibit any of the peptidases that are present in blood, seminal fluid, or vaginal fluid.
  • These non-mammalian GnRH analogs are expected to have increased binding to the sperm, testicular, scrotal, seminiferous tubule, Leydig cell, Sertoli cell, epididymis, vas deferentia, prostate, seminal vesicle, ejaculatory duct, or urethral receptor and increased metabolic stability.
  • non-mammalian GnRH analogs will be administered intra-nasally, orally, subcutaneously, transdermally or intramuscularly to the male, or intrauterine or intravaginally to the female partner.
  • virtually any mode of administration may be used in the practice of the invention.
  • the non-mammalian GnRH analog can be taken daily.
  • FIG. 4a Inhibition of the Degradation of Mammalian GnRH by Placental Enzyme Chorionic Peptidase-1 by Chicken II GnRH.
  • Figure 4b Inhibition of the Degradation of Mammalian GnRH by Placental Enzyme Chorionic Peptidase-1 by D-Arg-Chicken ll-ethylamide.
  • FIG. 12A and 12B Action of Angiotensin II on Degradation of GnRH.
  • GnRH was actively degraded by C-ase-1. This activity of C-ase-1 was inhibited by, 9 OH-Pro-GnRH, Lamprey, Chicken 1-GnRH, Antide, Chicken 11-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.
  • Both Chicken II GnRH- 10 ethylamide and 6 lm-btl-D-His-GnRH 10 ethylamide were essentially inactive, i.e., ⁇ 0.001 inhibitory activity for GnRH.
  • Figure 14 Effect of des-Gly 10 -lm-Btl-D-His 6 -GnRH-ethylamide on Degradation of GnRH by C-ase-1.
  • GnRH was bound by the placental GnRH receptor with a K d of 10 "6 M.
  • Chicken II GnRH was similar to GnRH.
  • the K d for 6 lm-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.
  • the greatest potency having a K d of 3 non-mammalian, i.e. 33-fold more activity than GnRH .
  • Figure 22 Affinity of Chicken II Analog of Ovarian Receptor.
  • Figure. 23 Degradation of Mammalian GnRH in Baboon Ovary Extract.
  • FIG. 28 Effect of Chicken II GnRH Analog on Baboon Granulosa Cells.
  • Figure 32 Maternal circulating progesterone for each of the five Day 1-6 GnRH II analog-treated animals is compared to the circulating progesterone for saline treated- controls (mean ⁇ sd).
  • Figure 33 Maternal circulating progesterone for each of the five Day 6-11 GnRH II analog-treated animals is compared to the circulating progesterone for saline treated- controls (mean ⁇ sd).
  • FIG 34 Maternal circulating progesterone for each of the five Day 11-17 GnRH II analog-treated animals is compared to the circulating progesterone for saline treated- controls (mean ⁇ sd).
  • FIG. 36 Chicken II GnRH in Human Seminal Vesicle.
  • FIG. 37 Chicken II GnRH in Human Epididymis.
  • “Male fertility” depends on the proper function of a complex system of organs and hormones. The process begins in the area of the brain called the “hypothalamus-pituitary axis” which is a system of glands, hormones, and chemical messengers called “neurotransmitters” critical for reproduction.
  • the first step in fertility is the production of GnRH in the hypothalamus, which prompts the pituitary gland to manufacture follicle-stimulating hormone (FSH) and luteinizing hormone (LH). FSH maintains sperm production while LH stimulates the production of the male hormone testosterone.
  • FSH follicle-stimulating hormone
  • LH luteinizing hormone
  • sperm and testosterone production occur in the two testicles, or "testes", which are contained in the scrotal sac or “scrotum".
  • the sperm are manufactured in several hundred microscopic "seminiferous" tubules which make-up most of the testicles. Surrounding these tubules are “Leydig cells” which manufacture testosterone.
  • the development of sperm begins in "Sertoli cells” located in the lower parts of the seminiferous tubules. As they mature, they are stored in the upper part of the tubules. Young sperm cells are known as "spermatids". When the sperm complete the development of their head and tail, they are released from the cell into the "epididymis”.
  • This C-shaped tube is 1/300 of an inch in diameter and about 20 feet long. It loops back and forth on itself within a space of only about one and a half inches long.
  • the sperm's journey through the epididymis takes about three weeks.
  • the fluid in which the sperm is transported contains fructose sugar, which provides energy as the sperm matures. In the early stages of its passage, the sperm cannot swim in a forward direction and can only vibrate its tail weakly. By the time the sperm reaches the end of the epididymis, however, it is mature.
  • each healthy sperm consists of a head that contains the male DNA and a tail that propels the head forward at about four times its own length every second.
  • vas deferentia A single channel is called a vas deferens.
  • Muscle contractions in the vas deferens from sexual activity propel the sperm past the "seminal vesicles" which contribute “seminal fluid” to the sperm.
  • the vas deferens also collects fluid from the nearby “prostate gland”. This mixture of various fluids and sperm is the “semen”. Semen provides several benefits to the sperm.
  • the semen is forced through the urethra during ejaculation and out of the penis.
  • sperm usually about 100 to 300 million sperm are delivered into the ejaculate at any given time, but, even under normal conditions, only about 15% are healthy enough to fertilize an egg.
  • Sperm that manage to reach the mucous lining in the woman's cervix (the lower part of her uterus) must survive about four more days to reach the woman's fallopian tubes.
  • the egg is positioned for fertilization for only one to two days each month.
  • Capacitation is a one time burst of energy that signals a cascade of events, including speeding up the motion of the sperm and triggering the actions of the "acrosome", a membrane filled with enzymes, which covers the head of the sperm. Dissolving the acrosome is a critical result of the capacitation process.
  • Enzymes in the acrosome are then released that allow the sperm to drill a hole through the tough outer coating of the egg (the corona cells and zona pellucida ). Only one sperm can get through to fertilize the egg.
  • Disorders of the male reproductive system include (a) priapism - a nonsexual, prolonged, painful erection, (b) balanoposthitis (balanitis)- inflammation of the glans penis, (c) cryptorchidism - undescended testes, one or both, (d) epididymitis- inflammation of the epididymus, (e) cancer, (f) prostatitis - acute or chronic inflammation of the prostate gland, (g) benign prostatic hyperplasia, (h) testicular descent, (i) testicular dysfunction, (j) prostate dysfunction, and (k) preservation of testes during chemotherapy.
  • isolated nucleic acid is a nucleic acid the structure of which is not identical to that of any naturally occuring nucleic acid or to that of any fragment of a naturally occuring genomic nucleic acid spanning more than three separate genes.
  • the term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occuring genomic DNA molecule, but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occuring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.
  • the terms "complementary” or “complementarity” refer to the natural binding of polynucleotides by base pairing. For example, the sequence 5'-AGT-3' binds to the complementary sequence 3'-TCA-5'. Complementarity between two single-stranded molecules may be "partial” such that only some of the nucleic acids bind or it may be "complete” such that total complementarity exists between the single stranded molecules.
  • EMF expression modulating fragment
  • a sequence is said to "modulate the expression of an operably linked sequence” when the expression of the sequence is altered by the presence of the EMF.
  • EMFs include, but are not limited to, promoters, and promoter modulating sequences (inducible elements).
  • One class of EMFs are nucleic acid fragments which induce the expression of an operably linked ORF in response to a specific regulatory factor or physiological event.
  • nucleotide sequence or “nucleic acid” or “polynucleotide” or
  • oligonucleotide are used interchangeably and refer to a heteropolymer of nucleotides or the sequence of these nucleotides. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA) or to any DNA-like or RNA-like material.
  • PNA peptide nucleic acid
  • nucleic acid segments provided by this invention may be assembled from fragments of the genome and short oligonucleotide linkers, or from a series of oligonucleotides, or from individual nucleotides, to provide a synthetic nucleic acid which is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon, or a eukaryotic gene.
  • Probes may, for example, be used to determine whether specific mRNA molecules are present in a cell or tissue or to isolate similar nucleic acid sequences from chromosomal DNA as described by Walsh et al. (Walsh, P. S. et al., 1992, PCR Methods Appl 1 :241-250).
  • Probes of the present invention are elaborated in Sambrook, J. et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY; or Ausubel, F. M. et al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons, New York N.Y., both of which are incorporated herein by reference in their entirety.
  • the term "open reading frame,” ORF means a series of nucleotide triplets coding for amino acids without any termination codons and is a sequence translatable into protein.
  • operably linked refers to functionally related nucleic acid sequences.
  • a promoter is operably associated or operably linked with a coding sequence if the promoter controls the transcription of the coding sequence.
  • operably linked nucleic acid sequences can be contiguous and in the same reading frame, certain genetic elements e.g. repressor genes are not contiguously linked to the coding sequence but still control transcription/translation of the coding sequence.
  • transcription/translation of the coding sequence can be contiguous and in the same reading frame, certain genetic elements e.g. repressor genes are not contiguously linked to the coding sequence but still control transcription/translation of the coding sequence.
  • transcription/translation of the coding sequence The term “translated protein coding portion” means a sequence which encodes for the full length protein which may include any leader sequence or any processing sequence.
  • mature protein coding sequence means a sequence which encodes a peptide or protein without a signal or leader sequence.
  • the peptide may have been produced by processing in the cell which removes any leader/signal sequence.
  • the peptide may be produced synthetically or the protein may have been produced using a polynucleotide only encoding for the mature protein coding sequence.
  • derivative refers to polypeptides chemically modified by such techniques as ubiquitination, labeling (e.g., with radionuclides or various enzymes), covalent polymer attachment such as pegylation (derivatization with polyethylene glycol) and insertion or substitution by chemical synthesis of amino acids such as ornithine, which do not normally occur in human proteins.
  • variant refers to any polypeptide differing from naturally occurring polypeptides by amino acid insertions, deletions, and substitutions, created using, e g., recombinant DNA techniques.
  • Guidance in determining which amino acid residues may be replaced, added or deleted without abolishing activities of interest, may be found by comparing the sequence of the particular polypeptide with that of homologous peptides and minimizing the number of amino acid sequence changes made in regions of high homology (conserved regions) or by replacing amino acids with consensus sequence.
  • recombinant variants encoding these same or similar polypeptides may be synthesized or selected by making use of the "redundancy" in the genetic code.
  • codon substitutions such as the silent changes which produce various restriction sites, may be introduced to optimize cloning into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic system.
  • Mutations in the polynucleotide sequence may be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide, to change characteristics such as ligand- binding affinities, interchain affinities, or degradation/turnover rate.
  • amino acid "substitutions" are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e., conservative amino acid replacements.
  • Constant amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
  • nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, praline, phenylalanine, tryptophan, and methionine
  • polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine
  • positively charged (basic) amino acids include arginine, lysine, and histidine
  • negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • “Insertions” or “deletions” are preferably in the range of about 1 to 10 amino acids, more preferably 1 to 5 amino acids. The variation allowed may be experimentally determined by systematically making insertions, deletions, or substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity. Alternatively, where alteration of function is desired, insertions, deletions or non-conservative alterations can be engineered to produce altered polypeptides. Such alterations can, for example, alter one or more of the biological functions or biochemical characteristics of the polypeptides of the invention.
  • such alterations may change the characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate. Further, such alterations can be selected so as to generate peptides that are better suited for expression, scale up and the like in the host cells chosen for expression. For example, cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges.
  • purified or “substantially purified” as used herein denotes that the indicated nucleic acid or polypeptide is present in the substantial absence of other biological macromolecules, e.g., polynucleotides, proteins, and the like.
  • the polynucleotide or polypeptide is purified such that it constitutes at least 95% by weight, more preferably at least 99% by weight, of the indicated biological macromolecules present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000 daltons, can be present).
  • recombinant when used herein to refer to a polypeptide or protein, means that a polypeptide or protein is derived from recombinant (e.g., microbial, insect, or mammalian) expression systems.
  • Microbial refers to recombinant polypeptides or proteins made in bacterial or fungal (e.g., yeast) expression systems.
  • “recombinant microbial” defines a polypeptide or protein essentially free of native endogenous substances and unaccompanied by associated native glycosylation. Polypeptides or proteins expressed in most bacterial cultures, e.g., E. coli, will be free of glycosylation modifications; polypeptides or proteins expressed in yeast will have a glycosylation pattern in general different from those expressed in mammalian cells.
  • the term "recombinant expression vehicle or vector” refers to a plasmid or phage or virus or vector, for expressing a polypeptide from a DNA (RNA) sequence.
  • An expression vehicle can comprise a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription initiation and termination sequences.
  • Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell.
  • recombinant protein is expressed without a leader or transport sequence, it may include an amino terminal methionine residue. This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.
  • recombinant expression system means host cells which have stably integrated a recombinant transcriptional unit into chromosomal DNA or carry the recombinant transcriptional unit extrachromosomally.
  • Recombinant expression systems as defined herein will express heterologous polypeptides or proteins upon induction of the regulatory elements linked to the DNA segment or synthetic gene to be expressed.
  • This term also means host cells which have stably integrated a recombinant genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers.
  • Recombinant expression systems as defined herein will express polypeptides or proteins endogenous to the cell upon induction of the regulatory elements linked to the endogenous DNA segment or gene to be expressed.
  • the cells can be prokaryotic or eukaryotic.
  • secreted includes a protein that is transported across or through a membrane, including transport as a result of signal sequences in its amino acid sequence when it is expressed in a suitable host cell.
  • Stecreted proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially (e.g., receptors) from the cell in which they are expressed.
  • Stecreted proteins also include without limitation proteins that are transported across the membrane of the endoplasmic reticulum.
  • “Secreted” proteins are also intended to include proteins containing non-typical signal sequences (e.g. Interleukin-1 Beta, see Krasney, P. A. and Young, P. R.
  • an expression vector may be designed to contain a "signal or leader sequence" which will direct the polypeptide through the membrane of a cell.
  • sequences of the present invention may be naturally present on the polypeptides of the present invention or provided from heterologous protein sources by recombinant DNA techniques.
  • substantially equivalent can refer both to nucleotide and amino acid sequences, for example a mutant sequence, that varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between the reference and subject sequences.
  • such a substantially equivalent sequence varies from one of those listed herein by no more than about 35% (i.e., the number of individual residue substitutions, additions, and/or deletions in a substantially equivalent sequence, as compared to the corresponding reference sequence, divided by the total number of residues in the substantially equivalent sequence is about 0.35 or less).
  • Such a sequence is said to have 65% sequence identity to the listed sequence.
  • a substantially equivalent, e.g., mutant, sequence of the invention varies from a listed sequence by no more than 30% (70% sequence identity); in a variation of this embodiment, by no more than 25% (75% sequence identity); and in a further variation of this embodiment, by no more than 20% (80% sequence identity) and in a further variation of this embodiment, by no more than 10% (90% sequence identity) and in a further variation of this embodiment, by no more that 5% (95% sequence identity).
  • Substantially equivalent, e.g., mutant, amino acid sequences according to the invention preferably have at least.80% sequence identity with a listed amino acid sequence, more preferably at least 90% sequence identity.
  • nucleotide sequences of the invention can have lower percent sequence identities, taking into account, for example, the redundancy or degeneracy of the genetic code.
  • nucleotide sequence has at least about 65% identity, more preferably at least about 75% identity, and most preferably at least about 95% identity.
  • sequences having substantially equivalent biological activity and substantially equivalent expression characteristics are considered substantially equivalent.
  • sequences having substantially equivalent biological activity and substantially equivalent expression characteristics are considered substantially equivalent.
  • sequence identity may be determined, e.g., using the
  • the term “antibody” includes whole antibodies and fragments thereof, single chain (recombinant) antibodies, "humanized” chimerie antibodies, and immunologically active fragments of antibodies (eg. Fab fragments).
  • the term “degenerate variant” means nucleotide fragments which differ from a nucleic acid fragment of the present invention (e.g., an ORF) by nucleotide sequence but, due to the degeneracy of the genetic code, encode an identical polypeptide sequence.
  • Preferred nucleic acid fragments of the present invention are the ORFs that encode proteins.
  • the amino acids and their corresponding DNA codons can include, but are not limited to, isoleucine (ATT, ATC, ATA), leucine (CTT, CTC, CTA, CTG, TTA, TTG), valine (GTT, GTC, GTA, GTG), phenylalanine (TTT, TTC), methionine (ATG), cysteine (TGT, TGC), alanine (GCT, GCC, GCA, GCG), glycine (GGT, GGC, GGA, GGG), proline (CCT, CCC, CCA, CCG), threonine (ACT, ACC, ACA, ACG), serine (TCT, TCC, TCA, TCG, AGT, AGO, tyrosine (TAT, TAC), tryptophan (TGG), glutamine (CAA, CAG), asparagine (AAT, AAC), hist
  • EXAMPLE I Design of Non-Mammalian GnRH Analogs
  • the present example outlines how analogs of non-mammalian GnRH with increased activity in chorionic, ovarian, tubal and uterine, sperm, testicular, scrotal, seminiferous tubule, Leydig cell, Sertoli cell, epididymis, vas deferentia, prostate, seminal vesicle, ejaculatory duct, and urethral tissues are designed.
  • Existing mammalian GnRH analogs are designed for activity at the pituitary GnRH receptor and with extended stability in the circulation of non-pregnant individuals.
  • the existing data indicate that the ovarian, uterine, and chorionic, sperm, testicular, scrotal, seminiferous tubule, Leydig cell, Sertoli cell, epididymis, vas deferentia, prostate, seminal vesicle, ejaculatory duct, and urethral tissues have a high affinity GnRH receptor which differs from that in the pituitary.
  • the degradation of GnRH is different in the ovary, uterus, and placenta during pregnancy.
  • prior known pituitary mammalian GnRH analogs have not been designed for use at extra-pituitary sites or during pregnancy, and potent non-mammalian GnRH analogs have not previously been designed for use at extra-pituitary sites or during pregnancy.
  • the present invention provides potent non-mammalian GnRH analogs for use at extra pituitary sites.
  • Non-mammalian analogs of GnRH were synthesized by order. They were specifically designed to prevent degradation of the non-mammalian GnRH analog in extra-pituitary tissues, in the maternal circulation as well as within the intrauterine tissues.
  • the chicken II GnRH sequence (SEQ ID NO: 2) and the salmon GnRH sequence (SEQ ID NO: 4) were also modified at the 6 position using D-Arg, making them resistant to degradation by the endopeptidase in blood, and were modified at the 10 position making them stable in maternal blood and the ovarian, tubal, uterine, and, sperm, testicular, scrotal, seminiferous tubule, Leydig cell, Sertoli cell, epididymis, vas deferentia, prostate, seminal vesicle, ejaculatory duct, and urethral tissues, and chorionic tissues.
  • EXAMPLE I I - Placental Receptor Binding Activity Placental Receptor Studies The placental receptor binding activity of the different non-mammalian GnRH analogs of the present invention were compared. There is a human placental GnRH receptor which 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 mammalian GnRH analogs do not demonstrate potent binding activity at the placental receptor as they do at the pituitary receptor.
  • the non-mammalian GnRH analogs of the present invention 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 ovarian, tubal, uterine, and chorionic enzymes, present in maternal circulation as well as the ovary, fallopian tube, uterus, and placenta. Placental binding activity of the newly synthesized non-mammalian GnRH analogs have been compared to that for existing pituitary-active analogs of mammalian GnRH (SEQ ID NO: 5).
  • 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. Addition of enzyme inhibitors for the endogenous C-ase-1 were used as well as agents for receptor stabilization.
  • the receptor binding for D-Arg- chicken II GnRH-aza-Gly-amide (SEQ ID NO: 2) with the Buserelin label exhibited a dissociation constant (Kd) of 175 ⁇ 59 nM (2 fold greater than its natural chicken II GnRH isoform or Buserelin (SEQ ID NO: 10) and 60fold that of mammalian GnRH (SEQ ID NO: 5)).
  • Kd dissociation constant
  • SEQ ID NO: 2 the affinity for the placental GnRH receptor was enhanced 2 fold and that for mammalian GnRH (SEQ ID NO: 5) was decreased 1.5 times.
  • Figure 1 compares the average Kd observed for the three different placental membrane preparations for mammalian GnRH (SEQ ID NO: 5), Buserilin (SEQ ID NO: 10), D-Arg(6) chicken II GnRH-aza-Gly (l ⁇ )-amide (SEQ ID NO: 2) using the D- Arg(6)-chicken II GnRH-aza-Gly-amide (SEQ ID NO: 2) radiolabeled analog.
  • the most potent affinity constant was observed for the D-Arg(6)-chicken II GnRH -aza- Gly(lO) amide analog (SEQ ID NO: 2), having a Ks of 68 nM when using the placenta 2 membrane preparation as illustrated in Figure 2.
  • the average binding affinity for this analog was 93 ⁇ 23 nM (25 fold that observed for mammalian GnRH(SEQ ID NO: 5)).
  • Example III Placental Stability Studies of GnRH Analogs
  • the present example demonstrates 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 SEQ ID NO: 5
  • the present non-mammalian GnRH analogs had not been examined for placental receptor binding.
  • the added stability of these non-mammalian GnRH analogs would effect a substantial increase in bioactivity alone. Thus, both stability and binding studies were performed.
  • C-ase-1 A chorionic peptidase activity 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 maternal 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 of the present invention were designed with these specific criteria in mind.
  • the stability of these non-mammalian GnRH analogs to the enzymatic activity of C- ase-1 and in placental homogenate was examined.
  • the ability of the analogs to competitively inhibit the degradation of mammalian GnRH (SEQ ID NO: 5) by C-ase-1 was studied.
  • the stability of most potent receptor-active non-mammalian GnRH analogs in the presence of C-ase-1 and placental homogenate was identified. Using the incubation system developed for the C-ase-1 activity, the degradation of each analog was tested.
  • the product formed was calculated by subtraction, and its inverse plotted against the inverse of the original substrate concentrations to determine the nature of the competition.
  • the K was to be determined by plotting the inverse of the product that formed verses the inhibitor used.
  • 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 1 1. Placenta 10:283-296. Studies using whole placental homogenate were also performed.
  • the enzymatic degradation of mammalian GnRH (SEQ ID NO: 5) was studied as described above, replacing C-ase-1 with placental homogenate.
  • the remaining GnRH was measured using a radioimmunoassay specific for mammalian GnRH (SEQ ID NO: 5) having less than 0.1% cross-reactivity for any of the analogs or isoforms tested.
  • the concentration of the product of the degraded GnRH was quantified by subtracting the remaining mammalian GnRH from the starting concentrations of mammalian GnRH.
  • Analogs and isoforms of GnRH studied were Buserelin (SEQ ID NO: 10), Leuprolide (SEQ ID NO: 11), chicken I I GnRH (SEQ ID NO: 6), and its D-Arg (6), Des-Gly (10) GnRH -ethylamide, and D-Arg (6), aza-Gly (l ⁇ )-amide (SEQ ID NO: 2) analogs.
  • the Ks for the degradation of mammalian GnRH was calculated from the x axis intercept using Lineweaver-Burke double reciprocal plot of the concentration of the product formed versus the concentration of the substrate used.
  • the inhibitor constant Ki was also calculated from the point of converging lines formed from the plot of the concentration of the product formed using a given concentration of mammalian GnRH in the presence of different concentrations of competing analogs or isoform.
  • the Ks for mammalian GnRH (SEQ ID NO: 5) degradation by C-ase-1 was —30 nM.
  • Buserelin SEQ ID NO: 10
  • Buserelin SEQ ID NO: 10
  • Chicken II GnRH (SEQ ID NO: 6) competed for the degradation of mammalian GnRH (SEQ ID NO: 5) with a Ki of 200 nM (one-sixth that of the mammalian GnRH (SEQ ID NO: 5)).
  • the D-Arg-chicken II GnRH-ethylamide (SEQ ID NO: 2) had a Ki of more than 200 nM and D-Arg (6)-aza- Gly(lO) amide analog (SEQ ID NO: 2) of chicken II GnRH was essentially not degraded (Ki of >400 nM).
  • 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 non-mammalian 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 non-mammalian GnRH analogs.
  • 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. Replicate cultures were studied, thus allowing for comparison of different doses of each non-mammalian GnRH analog to mammalian GnRH (SEQ ID NO: 5), as well as direct competition assays.
  • Biol Reprod 35:312-319 Using an in vitro system to define bio-potency is expected to be predictive of in vivo activity. In addition to placental action, these newly synthesized non- mammalian GnRH analogs are also expected to act directly at the corpus luteum to inhibit steroidogenesis. These analogs are also expected to be active at the ovarian level.
  • an explant culture system was used to determine the effect of mammalian GnRH (SEQ ID NO: 5), chicken II GnRH (SEQ ID NO: 6), or the D-Arg(6)-chicken II GnRH-aza-Gly(10)-amide analog (SEQ ID NO: 2) on the release of the hCG, progesterone, and prostaglandin E 2 .
  • Human term placentas were dissected free of membranes, minced into fragments of 5 mm, rinsed in medium, and a total weight of —100 mg (20 explants) was placed on a sterile filter paper resting on an organ culture grid such that they touched the surface of the culture medium, but were not immersed in it.
  • the medium contained penicillin, streptomycin, and fungizone at 100 U/mL, 100 ug/mL, and 2.5 ug/mL respectively with and without varying doses of GnRH isoforms or analogs was added to each Petri dish.
  • Triplicate chambers for each media were made and incubated at 37 C in a humidified chamber with an atmosphere of 5% C0 2 and 95% air. Spent media were collected and replaced after 2 hours, 24 hours, and 48 hour of culture and stored frozen at -20° C until assayed for hormones.
  • HCG, progesterone, and PGE 2 were measured using specific double antibody procedures as described previously.
  • the chicken II GnRH analog (SEQ ID NO: 2) was studied using four different human term placentas, and the native chicken II GnRH isoform was also studied using one human term placenta. Gibbons JM, Mitnick M, Chieffo V 1975 In vitro biosynthesis, of TSH- and LH-releasing factors by the human placenta. Am J Obstet Gynecol 121 :127- 131.
  • the biopotency of the D-Arg(6) chicken II GnRH-aza-Gly(lO) amide analog (SEQ ID NO: 2) was compared with that of mammalian GnRH (SEQ ID NO: 5).
  • D-Arg- chicken II GnRH -aza-Gly-amide (SEQ ID NO: 2) (0.25 -1.00 uM) resulted in as much as a three fold stimulation of hCG during the first two hours of exposure using the lowest concentration of analog tested (250 nM) as illustrated in Figures 5a and 5b.
  • D-Arg-chicken II GnRH-aza-Gly-amide (SEQ ID NO: 2) was biphasic i.e. an inhibition of hCG was observed using the higher concentrations (1-9 uM) of the chicken II GnRH analog (SEQ ID NO: 2).
  • D-Arg(6)-chicken II GnRH-NH 2 analog (SEQ ID NO: 2) has bioactivity in the regulation of hCG and progesterone in the human term placenta. These studies demonstrate specific binding of non-mammalian GnRH analogs to the human GnRH placental receptor, which is unique from the pituitary receptor.
  • the most potent analogs were chicken II GnRH derivatives, particularly the D-Arg(6)- chicken II GnRH-aza-Gly 10 NH 2 (SEQ ID NO: 2). This analog may be used in the regulation of chorionic GnRH activity.
  • EXAMPLE V Inhibition Of Chorionic peptidase-1 (C-ase-1) Activity by Analogues of GnRH
  • C-ase-1 C-ase-1
  • 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), thyrotropin releasing hormone (TRH), reduced oxytocin, and Angiotensin II (Ang-ll). See Figures 10, 1 1 , 12A, and 12B.
  • GnRH gonadotropin releasing hormone
  • TRH thyrotropin releasing hormone
  • Ang-ll Angiotensin II
  • peptides contain a proline residue where the chorionic peptidase-1 (C-ase-1) is to cleave its C-terminal peptide bond.
  • C-ase-1 chorionic peptidase-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 s of the reaction.
  • the inhibitory activity of Antide (SEQ ID NO: 12), 6 lm-btl-D-His-GnRH- 10 ethylamide, 9 OH-Prl-GnRH, chicken II GnRH- 10 ethylamide, chicken II GnRH (SEQ ID NO: 6), chicken I GnRH (SEQ ID NO: 13), salmon GnRH (SEQ ID NO: 7) and lamprey GnRH (SEQ ID NO: 14) 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 (SEQ ID NO: 14), chicken 1-GnRH (SEQ ID NO: 13), Antide (SEQ ID NO: 12), chicken 11-GnRH (SEQ ID NO: 6) and salmon GnRH (SEQ ID NO: 7) with a relative potency of 1.5, 1.5, 0.6, 0.6, 0.2 and 0.2, respectively, compared to that for GnRH.
  • 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 (SEQ ID NO: 12) (see Figure 13) are degraded with reduced activity, while other analogs such as chicken II GnRH- 10 aza- Gly-amide and 6 lm-btl-D-His-GnRH 10 ethylamide are resistant to degradation by this endogenous chorionic enzyme. See Figure 14. These analogs will be useful in the regulation of chorionic GnRH activity.
  • GnRH compared to that of the pituitary receptor.
  • GnRH was bound by the placental GnRH receptor with a K d of 10 "6 M.
  • Chicken II GnRH was similar to GnRH.
  • the K d for - 6 lm-btl-D-His- GnRH 10 ethylamide was half the potency of GnRH, while Buserelin (SEQ ID NO: 10) and 6 D-Trp-GnRH- 10 ethylamide were twice as active as GnRH.
  • the greatest potency was for chicken II GnRH ethylamide, having a K d of 30 non-mammalian, i.e. 33-fold more activity than GnRH. See Figure 15.
  • the stability of the D-Arg-(6)-chicken II GnRH aza-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.
  • EXAMPLE VIII Non-Mammalian GnRH and Methods for Maintaining Pregnancy
  • 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.
  • As 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 II GnRH analog (SEQ ID NO: 2) or salmon GnRH analog (SEQ ID NO: 4). 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 incorporated herein by reference for this purpose.
  • the present example demonstrates the utility of the present invention for use as a post-coital contraceptive preparation.
  • the non-mammalian GnRH 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 may be prepared as part of a suitable vector, such as in an adenovirus or retroviral vector, and administered to the animal.
  • a suitable vector such as in an adenovirus or retroviral vector
  • 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.
  • the non-mammalian GnRH 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.
  • the activity of the present non-mammalian GnRH analogs would prove useful in the treatment of ovarian conditions, such as polycystic ovarian disease and ovarian cysts.
  • EXAMPLE X - Antibodies Specific for Non-Mammalian GnRH and GnRH Receptor The present example demonstrates the utility for using the present 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, or the receptors therefor. 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, or the GnRH receptor, 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-ll GnRH analog (SEQ ID NO: 2) or the salmon GnRH analog (SEQ ID NO: 4) 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.
  • an acceptable antibody level for the preparation of monoclonal antibodies, one would follow standard techniques for the immunization of an animal, again using the decapeptide non- mammalian GnRH peptide. Once sufficiently high acceptable antibodies are reached (titer) in the animal, 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.
  • 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 or GnRH receptor would then be collected from the media of the cell culture using techniques well know to those of skill in the art.
  • the textbook Sambrook et al (1989) Molecular Cloning, A Laboratory Manual, 2 nd Ed., Cold Springs Harbor Laboratory, Cold Springs Harbor, N.Y. is specifically incorporated herein by reference. All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure.
  • EXAMPLE XI - HCG Stimulating and Inhibiting Activity of the Non-Mammalian GnRH Analogs The acute activity of the chicken II GnRH analogs (SEQ ID NO: 2) on hCG release was studied using a human placental explant perifusion system. Prior studies have demonstrated the long term effect of these newly synthesized non-mammalian GnRH analogs and their biological action using a static culture system. A dose- related biphasic response was noted over time. In the perifusion studies, the dynamic of the response to continuous exposure of the chorionic GnRH agonist can be better defined.
  • the system better emulates the in vivo situation and provides a better understanding of the effect of the analogs on chorionic hormonogenesis and the applicable dose appropriate for future studies.
  • Chicken II GnRH analog with D-Arg at position 6 and aza-Gly-amide at position 10 (SEQ ID NO: 2) and commercially available mammalian GnRH agonist, Buserelin (SEQ ID NO: 10), were used in four different placental perfusion studies. Placental tissues that are normally discarded were obtained from unidentified patients following first trimester pregnancy termination (early human placental explants). Tissue fragments, dissected of vessels and membranes, were placed in a perfusion system for study of 20 replicate chambers of the same tissue.
  • hCG released into the effluent media, as well as the production of GnRH were measured using sensitive and specific radioimmunoassays.
  • the release of hCG at each time point was normalized to its zero treatment release and the release throughout the treatment period and its average release was compared for various treatment regimens. These studies were repeated using tissues from three different first trimester placentas. The results of this experiment are illustrated in Figure 20.
  • hCG release from control chambers decreased over the five hours of treatment to approximately 60% of its initial release.
  • the addition of varying concentrations of chicken II GnRH (SEQ ID NO: 2) analog to the perifusing media resulted in a biphasic stimulation of hCG from these early placental tissues.
  • non-mammalian GnRH analogs are capable of inhibiting hCG and progesterone production in placental tissues after extended exposure.
  • a direct action on ovarian and/or endometrial tissue was demonstrated.
  • a potential direct contraceptive action of these non-mammalian GnRH analogs, as well as their placental hCG and steroidogenic suppression activity is indicated.
  • Such non-mammalian GnRH analogs could be used as a menses regulator, contraceptive, abortifacient or in any other manner to function in regulating reproductive function and disorder and would be valuable agents in population control.
  • the non-mammalian GnRH analogs of the present invention have high affinity for the ovarian, tubal, uterine, and placental receptor and limited degradation by the chorionic enzyme C-ase-1.
  • a similar receptor and enzyme appears to be acting in the ovary and uterus, but not the pituitary.
  • the present study was designed to define the receptor binding of newly synthesized non- mammalian GnRH analogs in the ovary, uterus, and the pituitary and to compare them to the receptor binding of known mammalian GnRH analogs.
  • the receptor binding activity of newly synthesized non-mammalian GnRH analogs was studied in ovarian and uterine tissues.
  • Mammalian GnRH (SEQ I D NO: 5) was rapidly degraded by baboon ovarian cytosol fractions, yet the chicken II GnRH analog (SEQ ID NO: 2) was resistant to such degradation.
  • One study using human granulosa cells was performed and receptor binding for the GnRH analog was observed. Due to the limited number of cells it was not possible to precisely define the affinity of this receptor.
  • the chicken II GnRH analog (SEQ ID NO: 2) receptor affinity was found to be approximately 60 nM. Baboon uterine tissue was also demonstrated to have a GnRH receptor with high binding affinity for the chicken II GnRH analog (SEQ ID NO: 2).
  • chicken II GnRH analogs may have particular applicability for regulation of implantation and in uterine tissue conditions, such as endometriosis, abnormal uterine bleeding, and leiomyomas.
  • high affinity receptors for chicken II GnRH analogs have been defined in baboon ovary and uterus tissues.
  • EXAMPLE XIII - Stability of GnRH Analogs in Ovarian Homogenates The stability of newly synthesized chicken II GnRH analogs (SEQ ID NO: 2) in ovarian, endometrial, and pituitary homogenates was determined using enzyme activity assays. Chorionic peptidase which actively degrades GnRH in the placenta will be called chorionic peptidase 1 (C-ase-1 ).
  • the enzyme acts as a post-proline peptidase and is present in maternal circulation. In non-pregnant individuals very little post- proline peptidase activity is present in the circulation. Thus, GnRH analogs in the prior art have not been designed to be resistant to this activity.
  • the endogenous peptidase specific activity in the degradation of GnRH was tenfold that of placental cytosolic fractions.
  • an incubation system was used similar to that developed to study GnRH and its isoforms and analogs in the presence of chorionic tissues except baboon ovarian homogenates were substituted for C-ase-1.
  • the reaction was stopped by heating and the remaining GnRH substrate was quantified by radioimmunoassay.
  • Figure 24 illustrates the ability of D-Arg-6-chicken II GnRH-aza-Gly-amide (SEQ ID NO: 2) to act as a competitive inhibitor of GnRH for the baboon ovarian enzymatic activity. Since this analog did not significantly compete with the degradation of mammalian GnRH (SEQ ID NO: 5) in the baboon ovary, it is therefore, for all essential purposes, stable in the baboon ovary. Three different ovaries were tested and similar results were obtained as illustrated in Figure 24.
  • Endometrial Prostaglandin E2 PGE 2
  • Pituitary Luteinizing Hormone (LH) Release The bioactivity of the newly synthesized non-mammalian GnRH analogs on ovarian estradiol and progesterone production, endometrial stromal prolactin and endothelial prostaglandin production and pituitary luteinizing hormone release was determined using ovarian, endometrial, and pituitary cell cultures. It was anticipated that certain non-mammalian GnRH analogs tested in the stability and receptor assays would be active in culture.
  • the dose-related action of a stable analog of chicken II GnRH (SEQ ID NO: 2) on progesterone production was defined using baboon granulosa cell cultures. After a two hour basal study period, the medium was supplemented with either Buserilin (SEQ ID NO: 10) (10 7 ) or a chicken II GnRH analog (SEQ ID NO: 2] dO '9 , 10 '8 , 10 '7 , 10 "6 M) leaving four control wells. The average progesterone releases normalized to each well's basal release after 22 and 46 hours was compared among groups as seen in Figures 28 and 29.
  • Buserilin SEQ ID NO: 10 7
  • a chicken II GnRH analog SEQ ID NO: 2] dO '9 , 10 '8 , 10 '7 , 10 "6 M
  • non-mammalian GnRH analogs particularly chicken II GnRH (SEQ ID NO: 2) and salmon analogs (SEQ ID NO: 4)
  • non-mammalian GnRH analogs can be used in the treatment of conditions of or regulation of the reproductive system and the tissues therein including
  • Such treatment or regulation may be for endometriosis, polycystic ovarian disease, ovarian cysts, tubals, abnormal uterine bleeding, leiomyomas, endometrial polyps, fallopian tube mobility, function or obstruction, ectopic pregnancy, molar pregnancy, trophoblastic disease, abnormal placentation, such as pre-eclampsia, intrauterine growth retardation, preterm labor, preservation of the ovary during chemotherapy, in vitro fertilization, and ovarian atresia.
  • Conventional methods known to those of ordinary skill in the art of medicine, can be used to administer the pharmaceutical formulation(s) to the patient.
  • the pharmaceutical formulation will be administered to the patient by intramuscular injection, subdermal pellet, or nasal spray.
  • the pharmaceutical formulation(s) can also be administered via other conventional routes (e.g., oral, subcutaneous, intrapulmonary, transmucosal, intraperitoneal, intrauterine, vaginal, sublingual, or intrathecal routes) by using standard methods.
  • the pharmaceutical formulations can be administered to the patient via injection depot routes of administration such as by using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods.
  • the therapeutical agent typically is administered at a daily dosage of 0.00 lug to 30 mg/kg of body weight of the patient.
  • the pharmaceutical formulation can be administered in multiple doses per day, if desired, to achieve the total desired daily dose or as a long acting depot.
  • the effectiveness of the method of treatment can be assessed by monitoring the patient for known signs or symptoms of the disorder.
  • Common symptoms of endometriosis include onset of increasing painful periods, steady dull to severe lower abdominal pain, pelvic or low back pain that may occur at any time during the menstrual cycle, severe pelvic cramps or abdominal pain that may start 1 to 2 weeks before the menstrual cycle, more frequent or totally irregular periods, premenstrual spotting, pain during or following sexual intercourse, pain with bowel movements, and infertility.
  • a laparoscopy is typically performed to make the determination.
  • the symptoms include abnormal uterine bleeding (lengthened, shortened, absent, or irregular menstrual cycle), constant dull aching pelvic pain, pain with intercourse or pelvic pain during movement, pelvic pain shortly after onset or cessation of menses, nausea/vomiting or breast tenderness similar to that associated with pregnancy.
  • Prolonged symptoms that may be associated with polycystic ovarian disease include abnormally light or lack of menstrual periods, infertility, obesity, swollen abdomen, abdominal mass, and hirsutism.
  • Hormonal level tests are typically ordered including FSH, LH, estrogen, and pregnanediol.
  • a serum hCG test may be done to rule out pregnancy.
  • the symptoms of abnormal uterine bleeding, uterine fibroids, or leiomyomas may include menorrhagia, menometrorrhagia, severe pressure or pain, urinary or bowel complaints, recurrent abortions, and infertility. Some patients may however be asymptomatic. Diagnosis is made by pelvic examination and can be confirmed by ultrasonography, CT or MRI. While discussion has been made concerning specifically the female reproductive system, this invention have great applicability in the male reproductive system and conditions of the male reproductive system as the developmental reproductive biology of males and females is known by those skilled in the art to have a common origin.
  • the present invention in the treatment of conditions of or regulation of male reproductive tissues has particular applicability in testicular descent, testicular function, prostate function and preservation of testis during chemotherapy.
  • the non-mammalian GnRH analogs of the present invention can be used in the regulation of the immune system in pregnant and non-pregnant individuals and in systemic lupus erythematosus.
  • EXAMPLE XVI-Design of Non-Mammalian GnRH Analogs for Use in the Male Reproductive System The present example outlines how analogs of non-mammalian GnRH with increased activity in male reproductive system tissues including human sperm, testicular, scrotal, seminiferous tubule, Leydig cell, Sertoli cell, epididymis, vas deferentia, prostate, seminal vesicle, ejaculatory duct, and urethral tissues are designed.
  • Existing mammalian GnRH analogs are designed for activity at the pituitary GnRH receptor and with extended stability in the circulation of individuals.
  • the existing data indicate that the human sperm, testicular, scrotal, seminiferous tubule, Leydig cell, Sertoli cell, epididymis, vas deferentia, prostate, seminal vesicle, ejaculatory duct, and urethral tissues have a high affinity GnRH receptor which differs from that in the pituitary.
  • the degradation of GnRH is different in the human sperm, testicules, scrotum, seminiferous tubule, Leydig cell, Sertoli cell, epididymis, vas deferentia, prostate, seminal vesicle, ejaculatory duct, and urethra.
  • Non-mammalian analogs of GnRH were synthesized by order. They were specifically designed to prevent degradation of the analog in extra-pituitary tissues as well as in the male and female reproductive system tissue. This allows for the maintenance of sufficient concentrations cf analog to remain active when administered via the individual and to reach the extra-pituitary and male and female reproductive system tissue.
  • Analogs of the salmon (SEQ ID NO: 4) and chicken II GnRH (SEQ ID NO: 2) sequences that both show greater affinity for the human sperm, testicular, scrotal, seminiferous tubule, Leydig cell, Sertoli cell, epididymis, vas deferentia, prostate, seminal vesicle, ejaculatory duct, and urethral receptor than for the pituitary receptor, were modified to the tenth amino acid to ethylamide or aza-Gly 10 -NH 2 analog to make them resistant to degradation in the circulation and by post-proline peptidases.
  • the chicken II GnRH sequence (SEQ ID NO: 6) and the salmon GnRH sequence (SEQ ID NO: 7) were also modified at the 6 position using D-Arg, making them resistant to degradation by the endopeptidase in blood, and were modified at the 10 position making them stable in blood, seminal fluid, vaginal fluid and the human sperm, testicular, scrotal, seminiferous tubule, Leydig cell, Sertoli cell, epididymis, vas deferentia, prostate, seminal vesicle, ejaculatory duct, and urethral tissues.
  • GnRH receptor There is a human sperm, testicular, scrotal, seminiferous tubule, Leydig cell, Sertoli cell, epididymis, vas deferentia, prostate, seminal vesicle, ejaculatory duct, and urethral GnRH receptor which is distinct from that at the pituitary.
  • Prior mammalian GnRH analogs have been designed to increase activity at the pituitary GnRH receptor and stab ;l ity in the circulation of individuals.
  • the non-mammalian GnRH analogs of the present invention have been designed to interact with preference at the human sperm, testicular, scrotal, seminiferous tubule, Leydig cell, Sert ⁇ 'i cell, epididymis, vas deferentia, prostate, seminal vesicle, ejaculatory duct, and urethral receptor and not the pituitary receptor.
  • Example XVIII - Stability Studies of GnRH Analogs in the Male Reproductive System The present example demonstrated the utility of using the present invention in controlling and modulating the activity of the human sperm, testicles, scrotum, seminiferous tubule, Leydig cells, Sertoli cells, epididymis, vas deferentia, prostate gland, seminal vesicle, ejaculatory duct, and urethra of the mammal.
  • the present non-mammalian GnRH analogs had not previously been examined for receptor binding.
  • the human male reproductive system GnRH receptor shows different kinetic constants for GnRH compared to that of the pituitary receptor.
  • EXAMPLE XXI - Non-mammalian GnRH Analogs and Male Contraception The present example demonstrates the utility of the present invention for use as a contraceptive preparation.
  • the non-mammalian GnRH analogs defined here, and conservative variants thereof may be formulated into a pharmaceutically acceptable preparation, and then administered to a male mammal during the 24 hours prior to coitus. Relatively high doses of about 0.1 gram to about 10 grams of the non- mammalian GnRH analog could be given daily.
  • the cDNA sequence for the non-mammalian GnRH of SEQ ID NO: 1 may be prepared as part of a suitable vector, such as in an adenovirus or retroviral vector, and administered to the animal.
  • a suitable vector such as in an adenovirus or retroviral vector
  • 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 male fertility is a novel clinical regimen.
  • EXAMPLE XXII - Antibodies Specific for Non-Mammalian GnRH in the Male Reproductive System 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 human sperm, testicular, scrotal, seminiferous tubule, Leydig cell, Sertoli cell, epididymis, vas deferentia, prostate, seminal vesicle, ejaculatory duct, or urethral GnRH receptor or any other non-pituitary GnRH peptide or protein, or the receptors therefor.
  • 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, or the GnRH receptor, present in a sample as an indicator molecule.
  • 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-ll GnRH analog (SEQ ID NO: 2) or the salmon GnRH analog (SEQ I D NO: 4) 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.
  • an acceptable antibody level for the preparation of monoclonal antibodies, one would follow standard techniques for the immunization of an animal, again using the decapeptide non- mammalian GnRH peptide. Once sufficiently high acceptable antibodies are reached (titer) in the animal, 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.
  • 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.
  • the textbook Sambrook et al (1989) Molecular Cloning, A Laboratory Manual, 2 nd Ed., Cold Springs Harbor Laboratory, Cold Springs Harbor, N.Y. is specifically incorporated herein by reference.
  • the pharmaceutical formulation(s) can be administered to the patient by intramuscular injection, subdermal pellet, or nasal spray.
  • the pharmaceutical formulation(s) can also be administered via other conventional routes (e.g., oral, subcutaneous, intrapulmonary, transmucosal, intraperitoneal, sublingual, or intrathecal routes) by using standard methods.
  • the pharmaceutical formulations can be administered to the patient via injection depot routes of administration such as by using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods.
  • the therapeutical agent typically is administered at a daily dosage of 0.001 ug to 30 mg/kg of body weight of the patient.
  • the pharmaceutical formulation can be administered in multiple doses per day, if desired, to achieve the total desired daily dose or as a long acting depot.
  • the effectiveness of the method of treatment can be assessed by monitoring the patient for known signs or symptoms of the disorder.
  • EXAMPLE XXIV - Localization of Non-Mammalian GnRH in Tissues of the Reproductive System Tissue of the male and female reproductive system were examined for the presence of non-mammalian GnRH in their cells.
  • the tissues were then washed with phosphate buffered saline and anti-rabbit gamma globulin conjugated with biotin and incubated for 4 minutes at 55 C.
  • the slides were rinsed in buffer followed by blocking of the endogenous peroxidase activity.
  • streptavidin horse radish peroxidase was added and incubated for 4 minutes at 55 C.
  • Stable diaminobenzidine (5 minutes at 55 C) was used to generate the signal.
  • the slides were rinsed, mounted and read. The presence of non- mammalian GnRH was localized via the DAB (diaminobenzidine) using microscopy.
  • testis In each of these reproductive tissues examined, the testis, seminal vesicle (See Figure 36), epididymis (See Figure 37), ovarian, uterus and placental tissues, non- mammalian GnRH was visualized. Tissues such as atrium and liver were negative.
  • EXAMPLE XXV - Contraceptive Activity of Non-Mammalian GnRH Analog in Vivo Mammalian GnRH (SEQ ID NO: 5) was previously thought to be the only isoform of GnRH expressed in mammals, but chicken II GnRH (SEQ ID NO: 6) has now been identified in numerous mammalian tissues, including the ovary, uterus, placenta and brain. Specific, high affinity receptors, which bind chicken II GnRH (SEQ ID NO: 6) and its analogs, have been identified throughout the reproductive tract. Studies using ovarian tissues or placental explants in vitro have shown that chicken II GnRH (SEQ ID NO: 6) can regulate progesterone and hCG production.
  • chicken II GnRH acts as a paracrine factor to regulate extra-hypothalamic tissue functions, and when delivered chronically chicken II GnRH (SEQ ID NO: 6) would be an effective contraceptive agent.
  • chicken II GnRH analog SEQ ID NO: 2
  • saline via osmotic mini-pumps from Day 1-6, Day 6-11 or Day 1 1-17 to regularly cycling monkeys mated with fertile males around the time of ovulation.
  • Circulating estradiol (E 2 ) and progesterone (P) in the luteal phase was observed as well as cycle length in this and subsequent cycle. Implantation and pregnancy were confirmed by circulating CG and continued progesterone production.
  • This chicken II GnRH analog (SEQ ID NO: 2) had no significant effect on progesterone production or cycle length when administered on day 1 — 6. See Figure 32. In these animals no pregnancies resulted, but in the saline treated controls five of eight animals (62.5%) became pregnant. In animals treated on day 6 — 1 1 or days 11 — 17, no effect on luteinization or cycle length was noted; and two of five (40.0%), and one of three animals (33.3%), respectively, implanted with normal pregnancies resulting.
  • EXAMPLE XXVI - Sperm Motility Studies of GnRH Analogs in vitro The present example demonstrates the utility of using the present non- mammalian GnrH analog decapeptides to effect the motility of sperm.
  • Human sperm motility is an important factor for normal sperm function.
  • Sperm motility is graded as a percent of motile sperm. The grade of sperm motility ranges from 0-4 with 4 being the most active in forward movement. It is anticipated that the present chicken II GnRH analogs (SEQ ID NO: 2) will effect sperm motility.
  • chicken II GnRH (SEQ ID NO: 6) is produced in the male reproductive tract and sperm have a receptor for chicken I I GnRH (SEQ ID NO: 6).
  • Normal human sperm (20,000 million/ml) were obtained with seminal plasma and the motility of the sperm was assessed microscopically. Both the fraction of motile sperm and the forward sperm motion were assessed. These parameters were measured just prior to a 1 :5 dilution with phosphate buffered saline containing 0 to 200 nM chicken II GnRH analog (SEQ ID NO: 2). The motility and motion of the sperm were assessed at 2, 30, 60, 90, 120 and 180 minutes after incubation at 37 C.
  • the motility was initially inhibited with 20 to 200 nM chicken II GnRH analog (SEQ ID NO: 2); inhibition took 30 minutes using 2 nM of this analog. By 180 minutes the motility of the control had fallen to that of all the treated sperm.

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Abstract

La présente invention concerne des décapeptides analogues de la GnRH de saumon et poulet II, qui sont résistants à la dégradation par la peptidase comprenant D-arginine, D-leucine, D-tBu-sérine, D-Trp ou autre D-acide aminé actif, en position 6 et éthylamide, aza-Gly-amide au autre Gly-amide en position 10. Les analogues montrent une liaison préférentielle avec les récepteurs de la GnRH du système reproducteur mâle et femelle. L'efficacité biologique est plus importante dans le système reproducteur que l'hypophyse. Ces analogues de GnRH non mammifères peuvent être utilisés dans des préparations pharmaceutiques, et plus spécifiquement dans différents procédés de traitement, en tant qu'agents contraceptifs ou agents contraceptifs post-coïtaux. Ces analogues de GnRH non mammifères peuvent également être intégrés à des préparations pharmaceutiques qui peuvent être utilisées cliniquement pour préserver la gestation lorsqu'elles sont utilisées à très faibles doses et administrées de façon pulsatoire, ainsi que dans des préparations pour traiter des troubles du système reproducteur mâle et femelle. Des analogues non mammifères d'amide aza-Gly (10) peuvent selon l'invention être utilisés en tant qu'analogues de GnRH non mammifères.
EP04756015A 2003-08-12 2004-06-24 Analogues de gnrh non mammiferes et leurs utilisations dans le cadre de la regulation de la fertilite et de la gestation Withdrawn EP1605962A1 (fr)

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US63974903A 2003-08-12 2003-08-12
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US20050043245A1 (en) * 2003-08-11 2005-02-24 Siler -Khodr Theresa M. Non-mammalian GnRH analogs and uses thereof in the immune system
US20050054576A1 (en) * 2003-08-12 2005-03-10 Siler-Khodr Theresa M. Non-mammalian GnRH analogs and uses thereof in regulation of fertility and pregnancy

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US6323179B1 (en) * 1999-10-15 2001-11-27 Theresa Siler-Khodr Chicken GNRH analogs and uses thereof in regulation of fertility and pregnancy
US6635739B2 (en) * 1999-10-15 2003-10-21 Theresa Siler-Khodr Non-mammalian GnRH analogs and uses thereof in regulation of fertility and pregnancy
US7834141B1 (en) * 2000-03-31 2010-11-16 Theresa Siler-Khodr Non-mammalian GnRH analogs and uses thereof in tumor cell growth regulation and cancer therapy
AU2002355954A1 (en) * 2001-08-17 2003-03-03 Theresa M. Siler-Khodr Non-mammalian gnrh analogs and uses thereof in regulation of fertility and pregnancy
US20040152639A1 (en) * 2001-08-28 2004-08-05 Siler-Khodr Theresa M. Non-mammalian GnRH analogs and uses thereof in regulation of fertility and pregnancy

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