JP2007533748A - Use of GPCR54 ligand for the treatment of infertility - Google Patents

Abstract

  The present invention is directed to methods and compositions for the treatment of infertility.

Description

  The present invention is generally directed to treating reproductive diseases. More specifically, the present invention relates to the treatment of such diseases by administering a composition comprising a Kiss-1 derived peptide.

  In 1998, the American Reproductive Medicine Society predicted that there would be 6.1 million couples with infertility problems in the United States. Much progress has been made in the area of assisted reproductive technology (ART) by efforts to overcome these individual infertility problems by using sophisticated technical means to combine sperm and eggs. Most couples experience many infertility assessments and treatments before considering ART therapy. ART typically includes only in vitro fertilization techniques combined with various hormones and / or surgical interventions.

  Treatment of infertility often requires ovulation induction (OI) and / or superovulation induction (COH). OI is involved in passing a single follicle through the cycle to ovulation as a single oocyte, while COH is for use in various in vitro ART tools (eg, in vitro Targeted when collecting a large number of oocytes (for fertilization). Both of these measures rely on the use of two major hormones (named follicle stimulating hormone (FSH) and luteinizing hormone (LH)) that control ovarian follicular development and ovulation in women.

  Women with normal ovarian function ovulate at some mid-period of each menstrual cycle. During each menstrual cycle, many follicles are recruited for oocyte maturation. At the beginning of the menstrual cycle of approximately 28 days, follicles exist in the primordial form, i.e. an oocyte surrounded by a monolayer of cells. When follicular growth and maturation is activated by FSH, a multilayer of granulosa cells forms around the initial monolayer of cells (the process that continues to mid-menstrual phase). These granulosa cells nourish the oocytes and are involved in the production and release of estrogens. FSH produced by the pituitary gland induces aromatase activity in granulosa cells, thereby increasing estrogen production. Thus, there is an increase in estrogen production, early in the 28-day menstrual cycle, simultaneously with follicular maturation. The follicle also contains a receptor for LH, the second pituitary gonadotropin. As the follicle grows and matures to mid-menstrual period (approximately 14 days), a space (cavity) develops in the granulosa cells. In mid-menstrual period, increased LH production acts on LH receptors, rupturing the follicle and releasing oocytes down the fallopian tube. The oocyte can later be fertilized.

  Normal ovulating women mobilize approximately 300 immature oocytes for each menstrual cycle. During a normal cycle, all but one follicle are degenerated (closed), a single dominant follicle appears and releases the oocyte. In vitro fertilization of human oocytes (IVF), a commonly used treatment for the decline in fertility in women and men, is mature human oocytes followed by fertilization of mature oocytes with sperm. Based on cell recovery. The recruitment of human mature oocytes is achieved by hormonal therapy. For example, standard IVF treatment protocols include long-term hormonal stimulation (eg, 30 days) in female patients. This protocol begins by suppressing the patient's own FSH and LH with the gonadotropin-releasing hormone GnRH or an analog of GnRH, and then injecting exogenous gonadotropins, such as FSH and / or LH, to develop a number of preovulatory follicles. to be certain. At the appropriate stage of follicular growth, a large number of oocytes are collected by aspiration immediately before ovulation. The aspirated oocyte is then fertilized in vitro and is typically cultured for 3 days before transferring the resulting embryo to the uterus at the 4-8 cell stage.

  In women who do not naturally ovulate, the initial goal of the intervention is to induce ovulation of at least one oocyte. Several drugs are available and are used to artificially induce and sustain follicular development. These agents include clomiphene citrate (Clomid®, Serophene®) or letrozole (Femera®), or injectable hormones such as humans if the patient does not respond to clomiphene Menopausal urinary gonadotropin (hMG) (Repronex, Progonal®, Humegon®), FSH (Gonal-F®, Follistim®, Bravelle®), alone or Human chorionic gonadotropin (hCG; Profasi®) causing release of eggs administered in combination with GnRH antagonists such as Lupron®, Synarel®, Antigon® Mark), Pregnyl (registered trademark), Novarel (registered trademark), Ovudrel (registered trademark)), or Cetrotide (R) and the like to prevent the occurrence of increase in the natural LH.

  Clomiphene citrate is the first drug often used in infertility treatment because it is relatively inexpensive and available in tablet form, and 60-80% of women treated with this drug will ovulate Let's go. Clomiphene acts as an anti-estrogen in the central nervous system, causing an increase in pulse frequency and concentration of FSH released by the pituitary gland, resulting in stimulation of LH rupture, thereby providing gentle gonadotropin stimulation to the ovary. Clomiphene is administered in doses that can typically start at 50 mg / day and increase to 200 mg in later cycles over the first 5 days of the cycle.

  However, clomiphene administration has significant side effects. For example, there is the possibility of a well-characterized multiple pregnancy. Furthermore, it has been suggested that long-term use of clomiphene can increase the risk of ovarian cancer. Furthermore, clomiphene leads to an increase in ovulation rate, while the pregnancy rate of women stimulated with clomiphene is surprisingly reduced. Various reasons for this difference between ovulation rate and pregnancy rate have been suggested. Clomiphene appears to have an anti-estrogenic effect on the endometrium, causing a decrease in the endometrium. Secondly, clomiphene's cervical antiestrogenic action reduces the amount of mucus produced from the cervix. Moreover, clomiphene administration appears to cause a decrease in uterine blood flow, which can reduce the production of placental protein 14 and have deleterious effects on the oocyte. (Tarlatiz & Grimbais, Hum. Reprod., 13: 9 2356-2358, 1998; Out et al., Hum. Reprod., 13: 9 2358-2361, 1998; Dickey et al. Hum. See). Finally, clomiphene is an artificial chemical rather than a “natural” ovarian stimulant derived from a natural protein or other molecule, so such chemical intervention is considered to be a safe stimulant. There are concerns that the side effects (hot flashes, mood swings, insomnia, dizziness, visual impairment) are too broad and diverse for this intervention. In fact, it is suggested that "if you ask for registration today, it is very unlikely that clomiphene citrate will allow you to enter the market." (See Out et al., Hum. Reprod., 13: 9 2358-2361, 1998).

  Thus, there are obvious problems with the use of clomiphene-induced ovarian stimulation. Hormonal stimulation also has drawbacks including risk of ovarian hyperstimulation syndrome (OHSS), weight gain, bloating, nausea, vomiting, time associated with the surveillance process, and risk of unknown long-term cancer. Thus, there remains a need for new and effective methods for OI and / or COH.

SUMMARY OF THE INVENTION The present invention is directed to methods and compositions for replacing or augmenting existing methods for ovarian stimulation and for infertility treatment. In certain embodiments, the present invention contemplates a method for stimulating ovulation in a mammal comprising administering to the mammal a first composition comprising a Kiss-1 derived protein. In a more particular embodiment, the Kiss-1 derived protein is a mature Kiss-1 protein comprising the sequence of SEQ ID NO: 2. In another aspect, the method relates to the use of a Kiss-1 derived protein that is a peptide derived from a mature Kiss-1 protein. Preferably, the Kiss-1 derived protein is a Kiss-1 derived peptide capable of activating the GPCR54 receptor. In a specific embodiment, the Kiss-1 derived protein is a peptide comprising the amino acid sequence of SEQ ID NO: 7.

  As described throughout this specification, many Kiss-1 peptides are encompassed herein. In a specific preferred embodiment, the Kiss-1 derived protein is a peptide having the sequence of SEQ ID NO: 3, an analog of the peptide of SEQ ID NO: 3, a fragment of SEQ ID NO: 3, or an analog of the fragment of SEQ ID NO: 3. It is. Consider variants, mutants, homologs and analogs. In a specific embodiment, the analog of SEQ ID NO: 3 is a peptide that is a conservative variant of SEQ ID NO: 3.

  The peptide composition related to Kiss-1 of the present invention can be used in combination with other compositions. For example, the method of stimulating ovulation can further comprise administering a second composition that stimulates ovulation. Compositions that stimulate ovulation are generally known to those skilled in the art and include ovulation hormones and chemical stimulants. In an exemplary embodiment, the chemical stimulant of ovulation is clomiphene citrate or letrazol. Other agents that are analogs or variants of clomiphene citrate or letrazole, or other agents that act with a similar mechanism of action, can also be used in the methods described herein. The ovulatory hormone stimulant can be any hormone used for such purposes. Exemplary such hormones include gonadotropins selected from the group consisting of human menopausal urinary gonadotropin (hMG), follicle stimulating hormone (FSH), luteinizing hormone (LH), and human chorionic gonadotropin (hCG). Hormones. In a further embodiment, the gonadotropin hormone can be combined with the administration of a composition comprising a GnRH antagonist. In a particularly preferred embodiment, the gonadotropin is FSH.

  The methods of the invention using administration of FSH can be further combined with administration of non-FSH gonadotropin hormones. In certain embodiments, the non-FSH hormone is LH. FSH and LH can be administered in equal amounts or can be administered in unequal amounts. FSH can be administered simultaneously or separately.

  In certain exemplary embodiments, FSH is administered at a dose in the range of about 5-450 IU / day. In other embodiments, FSH is administered at a dose in the range of about 5-75 IU / day. In a specific aspect, the hormone can be administered by any route of administration used for hormone treatment. More particularly, gonadotropin hormone is administered by infusion. The methods of the invention are preferably related to human treatment.

  A further aspect of the invention includes a method of stimulating FSH production in a mammal comprising administering to the mammal a composition comprising a Kiss-1 derived protein. The Kiss-1 derived protein can be any protein derived from the mature Kiss-1 protein comprising the sequence of SEQ ID NO: 2. The protein can be a peptide derived from a mature Kiss-1 protein. Preferably, the method relates to the use of a Kiss-1 derived peptide capable of activating G-protein coupled receptor 54. More specifically, the Kiss-1 derived protein is a peptide comprising the amino acid sequence of SEQ ID NO: 7. The Kiss-1 derived protein is preferably a peptide having the sequence of SEQ ID NO: 3, an analog of the peptide of SEQ ID NO: 3, a fragment of SEQ ID NO: 3, or an analog of the fragment of SEQ ID NO: 3. In a specific embodiment, the analog of SEQ ID NO: 3 is a peptide that is a conservative variant of SEQ ID NO: 3. The method can further comprise administering a second composition that stimulates FSH production in the mammal. A second composition that stimulates FSH production comprises an anti-estrogen compound. For example, the anti-estrogen compound is selected from the group consisting of clomiphene and letrazole.

  Also described herein is a method of stimulating LH production in a mammal comprising administering to the mammal a composition comprising a Kiss-1 derived protein.

  The method of the invention comprises inhibiting the production of endogenous gonadotropin in a mammal, administering to the mammal an amount of a composition of Kiss-1 related peptides effective to stimulate ovarian follicular growth in the mammal; And a method for therapeutic intervention of infertility in a mammal comprising inducing ovulation and producing an egg in the mammal. The fertility treatment method can further comprise administering a gonadotropin preparation to stimulate ovarian follicle growth. Such gonadotropin preparations can comprise urinary or recombinant FSH or HMG, with or without recombinant LH. In certain embodiments, the gonadotropin preparation comprises recombinant FSH. In a specific embodiment, the induction of ovulation comprises administering to the mammal a composition comprising HCG, natural LHRH, LHRH agonist or recombinant LH. In a more specific embodiment, the inhibition of endogenous gonadotropin product in the mammal comprises administering a GnRH antagonist.

  In a preferred embodiment, the Kiss-1 derived peptide is administered in combination with a complex amount of an anti-estrogen agent effective to stimulate FSH production in a mammal. In other aspects of the invention, the methods described herein can be used in mammals that do not respond to standard gonadotropin stimulation of normal ovulation. In a specific embodiment, the infertility disorder is hypogonadotropic hypogonadism. Other methods are associated with the treatment of polycystic ovary syndrome, an infertility disease.

  The method of treating infertility described herein further comprises intrauterine insemination of an egg by sperm injection. In another embodiment, the method further comprises collecting oocytes 12 days after the initial stimulation of ovarian follicle production. In a more specific embodiment, the method further comprises fertilizing the collected oocytes in vitro and culturing the collected and fertilized oocytes to the 4-8 cell stage. In a preferred embodiment, the method further comprises transferring a fertilized oocyte at the 4-8 cell stage to the mammalian uterus. In a specific embodiment, the fertilized oocyte is transferred to the same mammalian uterus from which the oocyte was collected. However, in other embodiments, the fertilized oocyte is transferred to a different mammalian uterus from which the oocyte was collected.

  The methods of the present invention also reduce one or more symptoms of hypogonadotropic hypogonadism in a subject by individually administering a therapeutically effective amount of a Kiss-1 derived peptide. In such an embodiment, the subject is a male subject. A subject suffering from hypogonadotropic hypogonadism can be a female subject.

  The present invention further contemplates a composition comprising a Kiss-1 derived protein for use in the treatment of infertility. In a further embodiment, the present invention provides a kit for the treatment of infertility. Here, the kit comprises a first composition comprising a Kiss-1 derived protein in a pharmaceutically acceptable formulation and a second composition for the treatment of infertility. Preferably, the second composition for the treatment of infertility comprises the FSH preparation in a pharmaceutically acceptable formulation. In a specific embodiment, the FSH preparation is selected from the group consisting of a urinary FSH preparation and a recombinant FSH. Preferably the FSH is human FSH. In a specific embodiment, FSH is present in a unit dose of about 5 IU FSH to about 75 IU FSH. The kit can further comprise a third composition comprising LH in a pharmaceutically acceptable formulation. More particularly, LH is present in a unit dose of about 75 IU LH to about 150 IU LH.

  Other features and advantages of the present invention will become apparent from the following detailed description. However, since various changes and modifications within the spirit and scope of the invention will be apparent to those skilled in the art from this detailed description, the detailed description and specific examples, while indicating preferred embodiments of the invention, It should be understood that this is given by way of example only.

DESCRIPTION OF PREFERRED EMBODIMENTS Infertility has been treated through a variety of mechanisms. Most of them rely on stimulating or enhancing the effects of FSH and / or LH. Prior to the present invention, the first course of intervention against infertility used anti-estrogenic compounds such as clomiphene or letrazole. However, it has been discovered that these drugs have unwanted side effects. Therefore, there is still a need for effective additional treatments for infertility. The present invention provides novel methods and compositions for stimulating endogenous gonadotropin production. More particularly, the methods and compositions of the present invention cause stimulation of FSH and / or LH production and / or release. This stimulus is beneficial in that it can be used to treat various infertility diseases.

  The present invention describes a method for treating infertility by administering a composition comprising a Kiss-1 peptide-related protein. Methods and compositions for achieving this beneficial result are described in further detail herein below.

The present invention is directed to a method of treating various infertility diseases by administering a Kiss-1 related protein. In a preferred aspect of the present invention, Kiss-1 and peptides derived therefrom are the modulators of the hypothalamic pituitary gland (HPG) axis and various hormones such as luteinizing release hormone (LHRH) (GnRH). It has been found that it can be used to regulate the production and blood levels of FSH, LH, and gonadal steroids such as estrogen and testosterone. In a specific example, it was demonstrated that agonists of GPCR54 derived from Kiss-1 protein stimulate the production and release of FSH and / or LH. For example, the inventors have shown that a single administration of the 10 amino acid RFamide of SEQ ID NO: 3 is sufficient to produce LH and FSH as well as GnRH. Importantly, this finding reveals that the level of induction of FSH production is within the appropriate range for therapy. Since the HPG axis is related in various aspects of the reproductive system, including but not limited to the timing of puberty, duration of fertilization, and the beginning of menopause, the present invention These stimulants can be used to treat various reproductive diseases. The following part is aimed at explaining the Kiss-1 protein and the compounds obtainable therefrom that may be useful in the treatment of infertility.

  G protein-coupled receptors (GPCRs) are a large family of membrane receptors. Many genes belonging to this family have been identified, but in many cases their ligands and functions are still unclear. GPCR54 is an orphan GPCR that was first cloned from rat brain and found to be widely expressed in the rat central nervous system, such as the hypothalamus, midbrain, bridge, bone marrow, hippocampus, and spasms. One. The gene encoding human GPCR54 is present in a bacterial artificial chromosome (GenBank accession number AC023583) that maps to the second chromosome. Recently, placental tissue extracts have been shown to contain active peptides derived from the metastasis suppressor gene KiSS-1 (SEQ ID NO: 1; see GenBank accession number NM_002256 and SwissProt accession number Q15726), which is an agonist of GPCR54 (Kotani et al., J. Biol. Chem., 276 (37): 34631-34636, 2001). The peptide described in Kotani (referred to as kisspeptin) is 54-, 14-, and 13-amino acids long (SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively) and C -It contained RF-amide at the end. These peptides are obtained from the full length Kiss-1 protein (SEQ ID NO: 2). The present invention first describes a composition comprising Kiss-1 and kisspeptin may be used to treat infertility and to enhance production and / or release of gonadotropins (especially FSH and / or LH). it can.

  As used herein, the term “Kiss-1 derived protein” is intended to encompass any protein derived from the sequence of SEQ ID NO: 2, a fragment of SEQ ID NO: 2, or a GPCR54 receptor Alternatively, it is an analog or conservative variant of the protein of SEQ ID NO: 2 having any agonistic effect on a variant of the GPCR54 receptor. In the present invention, a peptide having the sequence of YNWNSFGLRF (SEQ ID NO: 3) derived from Kiss-1 is a particularly preferred kisspeptin that can be used in the treatment of infertility. However, it is to be understood that any variant, analog or fragment of SEQ ID NO: 2 can be used in the methods of the invention. In fact, it was found that an RF amide peptide derived from Kiss-1 protein having a length of 5 amino acids has an activity as an agonist of GPCR54. Thus, sequences such as FGLRF (SEQ ID NO: 7); SFGLRF (SEQ ID NO: 8); NSFGLRF (SEQ ID NO: 9); WNSFGLRF (SEQ ID NO: 10); NWNSFGLRF (SEQ ID NO: 11) It is contemplated that those variants or analogs having are useful in the present invention.

  Other specific kisspeptins that may be useful herein include a fragment of Kiss-1 having amino acids 93-121 of SEQ ID NO: 1 (where the C-terminus of the fragment is 120- A fragment of Kiss-1 having amino acids 106-121 of SEQ ID NO: 1 (where the C-terminus of the fragment is positions 120-121 of SEQ ID NO: 1). A fragment of Kiss-1 having amino acids 108-121 of SEQ ID NO: 1 (where the C-terminus of the fragment is from RF at positions 120-121 of SEQ ID NO: 1). The resulting RF amide), a fragment of Kiss-1 having amino acids 68-121 of SEQ ID NO: 1 (where The C-terminus of the fragment is an RF amide obtained from RF at positions 120-121 of SEQ ID NO: 1; a fragment of Kiss-1 having amino acids 94-121 of SEQ ID NO: 1 (where C The end is an RF amide obtained from RF at positions 120-121 of SEQ ID NO: 1; a fragment of Kiss-1 having amino acids 107-121 of SEQ ID NO: 1, where the C-terminus of the fragment is A fragment of Kiss-1 having amino acids 109-121 of SEQ ID NO: 1, wherein the C-terminus of the fragment is SEQ ID NO: An RF amide obtained from RF at positions 120-121 of 1); having amino acids 112-121 of SEQ ID NO: 1 A fragment of Kiss-1 (where the C-terminus of the fragment is an RF amide obtained from RF at positions 120-121 of SEQ ID NO: 1); and Kiss- having amino acids 114-121 of SEQ ID NO: 1 1 fragment (wherein the C-terminus of the fragment is an RF amide obtained from RF at positions 120-121 of SEQ ID NO: 1), but is not limited thereto.

  In a preferred embodiment, the peptide comprises amino acids 110-121 of SEQ ID NO: 1; amino acids 109-121 of SEQ ID NO: 1, 108-121 of SEQ ID NO: 1; 107-121 of SEQ ID NO: 1; SEQ ID NO: 1 106-121; SEQ ID NO: 1: 105-121; SEQ ID NO: 1: 104-121; SEQ ID NO: 1: 103-121; SEQ ID NO: 1: 102-121; SEQ ID NO: 1: 101-121; SEQ ID NO: 1: 100-121; SEQ ID NO: 1: 99-121; SEQ ID NO: 1: 98-121; SEQ ID NO: 1: 97-121; SEQ ID NO: 1: 96-121; SEQ ID NO: 1 95-121; 94-121 of SEQ ID NO: 1; 93-121 of SEQ ID NO: 1; 92-121 of SEQ ID NO: 1; 91-121 of SEQ ID NO: 1; 90-121 of SEQ ID NO: 1; SEQ ID NO: 1: 89-121; SEQ ID NO: 1: 87-121; SEQ ID NO: 1: 86-121; SEQ ID NO: 1: 85-121; SEQ ID NO: 1: 84 -121; SEQ ID NO: 1: 83-121; SEQ ID NO: 1: 82-121; SEQ ID NO: 1: 81-121; SEQ ID NO: 1: 80-121; SEQ ID NO: 1: 79-121; SEQ ID NO: 1: 78-121; SEQ ID NO: 1: 77-121; SEQ ID NO: 1: 76-121; SEQ ID NO: 1: 75-121; SEQ ID NO: 1: 74-121; SEQ ID NO: 1: 73- 121; SEQ ID NO: 1: 72-121; SEQ ID NO: 1: 71-121; SEQ ID NO: 1: 70-121; SEQ ID NO: 1: 69-121; SEQ ID NO: 1: 68-121; SEQ ID NO: 1 67-121; SEQ ID NO: 1 66-12 SEQ ID NO: 1: 65-121; SEQ ID NO: 1: 64-121; SEQ ID NO: 1: 63-121; SEQ ID NO: 1: 62-121; SEQ ID NO: 1: 61-121; SEQ ID NO: 1 60-121; SEQ ID NO: 1: 59-121; SEQ ID NO: 1: 58-121; SEQ ID NO: 1: 57-121; SEQ ID NO: 1: 56-121; SEQ ID NO: 1: 55-121; SEQ ID NO: 1: 54-121; SEQ ID NO: 1: 53-121; SEQ ID NO: 1: 52-121; SEQ ID NO: 1: 51-121; SEQ ID NO: 1: 50-121; SEQ ID NO: 1 49-121; SEQ ID NO: 1: 48-121; SEQ ID NO: 1: 47-121; SEQ ID NO: 1: 46-121; SEQ ID NO: 1: 45-121; SEQ ID NO: 1: 44-121; No. 1 43-121; SEQ ID No. 1 42- 121; SEQ ID NO: 1: 41-121; SEQ ID NO: 1: 40-121; SEQ ID NO: 1: 39-121; SEQ ID NO: 1: 38-121; SEQ ID NO: 1: 37-121; SEQ ID NO: 36-121 of SEQ ID NO: 1; 35-121 of SEQ ID NO: 1; 34-121 of SEQ ID NO: 1; 33-121 of SEQ ID NO: 1; 32-121 of SEQ ID NO: 1; 31-121 of SEQ ID NO: 1 SEQ ID NO: 1: 30-121; SEQ ID NO: 1: 29-121; SEQ ID NO: 1: 28-121; SEQ ID NO: 1: 27-121; SEQ ID NO: 1: 26-121; SEQ ID NO: 1 25-121; SEQ ID NO: 1: 24-121; SEQ ID NO: 1: 23-121; SEQ ID NO: 1: 22-121; SEQ ID NO: 1: 21-121; SEQ ID NO: 1: 20-121; SEQ ID NO: 1: 19-121; SEQ ID NO: 1 8-121; SEQ ID NO: 1: 17-121; SEQ ID NO: 1: 16-121; SEQ ID NO: 1: 15-121; SEQ ID NO: 1: 14-121; SEQ ID NO: 1: 13-121; SEQ ID NO: 1: 11-121; SEQ ID NO: 1: 10-121; SEQ ID NO: 1: 9-121; SEQ ID NO: 1: 8-121; SEQ ID NO: 1: 7 -121; 6-121 of SEQ ID NO: 1; 5-121 of SEQ ID NO: 1; 4-121 of SEQ ID NO: 1; 3-121 of SEQ ID NO: 1; 2-121 of SEQ ID NO: 1; SEQ ID NO: : 1-121.

  A particularly preferred peptide of the present invention is a peptide having the sequence of SEQ ID NO: 3. However, it still retains the three-dimensional conformational structure of the peptide of SEQ ID NO: 3 and / or the function of the peptide of SEQ ID NO: 3 as an agonist of GPCR54 receptor and / or a stimulator of FSH and / or LH production It is also contemplated that conservative substitutions of amino acid residues of this peptide can be made that retain activity. As used herein, the term “conservative substitution” refers to another residue that is biologically similar in terms of hydrophobicity, hydrophilicity, cationic charge, anionic charge, shape, polarity, conformational tendency, etc. Means the substitution of an amino acid residue by Examples of conservative substitutions include substitution of one hydrophobic residue such as isoleucine, valine, leucine, alanine, cysteine, glycine, phenylalanine, proline, tryptophan, tyrosine, norleucine or methionine, or one Substitution of another polar residue, for example, substitution of arginine with lysine, substitution of glutamic acid with aspartic acid, or substitution of glutamine with asparagine, etc. Neutral hydrophilic amino acids that can be substituted for each other include asparagine, glutamine, serine and threonine. The term “conservative substitution” also includes the use of a substituted or modified amino acid in place of the unsubstituted parent amino acid if the raised antibody against the substituted polypeptide also immunoreacts with the unsubstituted polypeptide. By “substitution” or “modification”, the present invention includes those amino acids that have been altered or modified in nature.

  Thus, in the context of the present invention, it should be understood that a conservative substitution is recognized in the art as a substitution of one amino acid for another amino acid having similar properties. Typical conservative substitutions have been described. For example, conservative amino acids can be grouped as described in Lehninger [Biochemistry, Second Edition; Worth Publishers, Inc. NY: NY (1975), pp. 71-77]. One skilled in the art knows many tables showing specific conservative substitutions. One typical such table is provided below:

  Any conservative variant of YNWNSFGLRF (SEQ ID NO: 3) may be used as long as such variant retains the properties of an agonist of GPCR54 and / or the ability to stimulate the production of FSH and / or LH. It is considered a useful peptide. Such activity can be readily assessed as described herein below.

  Other novel peptides include mutants of any of the sequences discussed herein above that have similar conformational tendencies. Similarity of amino acid conformational tendencies can be obtained by comparing their Ramachandran plots, as shown in the following table:

  In addition to the basic amino acid structure of peptides, Kiss-1-derived proteins / peptides may be modified to enhance their absorption, circulation, and / or other modifications that make the peptides more therapeutically effective. Consider what you can do. For example, it has now been discovered that kisspeptin and Kiss-1 derived proteins have a beneficial effect on stimulation of fertilization-related hormones by acting on the central nervous system and possibly sites within the blood brain barrier. Thus, any modification that allows targeting / uptake to a site within the HPG axis of the peptide / protein is useful.

For example, it is desirable to prevent its degradation in order to prolong the effect of the peptide. This can be accomplished by using non-hydrolyzable peptide bonds (known in the art) in conjunction with procedures for synthesizing peptides containing such bonds. Non-hydrolyzable bonds include:-[CH ₂ NH]-reduced amide peptide bond,-[COCH ₂ ]-ketomethylene peptide bond,-[CH (CN) NH]-(cyanomethylene) Amino peptide bonds,-[CH ₂ CH (OH)]-hydroxyethylene peptide bonds,-[CH ₂ O]-peptide bonds, and-[CH ₂ S]-thiomethylene peptide bonds (eg, US Pat. No. 6,172,043), and peptoids. Peptoids are N-substituted glycines that can be used for optimal absorption of the drug of interest. Peptoids are used to make cationic lipid-like compounds (Murphy et al., Proc, Natl. Acad. Sci. 95: 1517, 1998) and can be synthesized using standard methods (eg, Zuckermann). J. Am. Chem. Soc. 114: 10646, 1992; Zuckermann et al. Int. J. Peptide Protein Res. 40: 497, 1992). Combinations of cationic lipids and peptoids, liptoids can also be used for optimal absorption of the subject compositions (Hunag et al. Chemistry and Biology. 5: 345, 1998). Liptoids can be synthesized by synthesizing peptoids and attaching amino-terminal submonomers to lipids through their amino groups (Hunag et al. Chemistry and Biology. 5: 345, 1998). One skilled in the art may refer, for example, to US Patent Publication No. 20030187188, which is incorporated herein by reference. This describes the synthesis of peptoid substituted compounds. Methods for making peptoids containing serine protease inhibitors are described in US Pat. No. 6,548,638; US Patent Publication No. 20030176642, which is incorporated herein by reference.

  Kiss-1 derived proteins useful in the present invention can be linear or cyclic, or can be cyclized by natural or synthetic means. For example, disulfide bonds between cysteine residues can cyclize the peptide sequence. Bifunctional reagents can provide a bond between two or more amino acids of a peptide. Other methods for cyclizing peptides such as Anwer et al. (Int. J Pep. Protein Res. 36: 392-399, 1990) and Rivera--Baeza et al. (Neopeptides 30: 327-333, 1996). What is also known in the industry.

  Logical drug design can be used to produce structural analogs of currently known biologically active Kiss-1 derived polypeptides. Making such analogs can affect the function of Kiss-1 derived proteins that are more active or stable than natural molecules, Kiss-1 derived proteins with different susceptibility to modification, or various other molecules. Kiss-1 derived proteins can be created. In one approach, a three-dimensional structure for the noted Kiss-1 derived protein or fragment thereof is generated. For example, this can be achieved by x-ray crystallography, computer modeling or a combination of both approaches. An alternative method, “Alanine Scan”, involves the random substitution of residues in the molecule with alanine to determine the effect on the resulting function.

  Multiple sequence alignments of Kiss-1 and its sequence analogs, such as from different species or related ligand families, provide further guidance on logical substitution. Residues in the Kiss-1 sequence can be replaced by corresponding residues from related species from different species or related ligand families.

  It is also possible to isolate a Kiss-1 specific antibody selected by functional analysis and analyze its crystal structure. In principle, this approach creates a pharma core that can be the basis for subsequent drug design. By creating anti-idiotype antibodies against functional pharmacologically active antibodies, it is possible to bypass protein crystallography as a whole. As a mirror image of the mirror image, the anti-idiotype binding site is predicted to be an analog of the original antigen. The anti-idiotype can then identify and isolate the peptide from a bank of chemically or biologically generated peptides. The selected peptide then serves as the pharmacore. Anti-idiotypes can be generated by producing antibodies specific for Kiss-1 and then using such antibodies as antigens.

  Therefore, it is possible to design an agent having improved Kiss-1 derived protein activity or acting as a stimulant or agonist of Kiss-1 protein or GPCR54 receptor. Due to the availability of cloned Kiss-1 sequences, sufficient quantities of various Kiss-1 derived proteins can be produced to perform crystallographic studies. In addition, knowledge of polypeptide sequences allows prediction of structure-function relationships using computers.

  In addition, non-peptide analogs of Kiss-1 derived proteins that provide stabilized structures or reduced biodegradation are also contemplated. Based on Kiss-1 derived proteins, peptidomimetic analogs can be prepared by replacing one or more amino acid residues of a protein of interest with a non-peptide moiety. Preferably, the non-peptide moiety allows the peptide to retain its native conformation or to stabilize a preferred (eg, biologically active) conformation. One example of a method for preparing non-peptidomimetic analogs from peptides is described by Nachman et al., Regul. Pept. 57: 359-370 (1995). The peptides used herein include all of the above.

  The Kiss-1 derived proteins used in the therapeutic methods of the present invention can be modified to improve their therapeutic effectiveness. Such modifications of therapeutic compounds can be used to reduce toxicity, increase circulation time, or alter biodistribution. For example, the toxicity of potentially important therapeutic compounds can be significantly reduced by combination with various drug carrier excipients that alter biodistribution. A great example is Mylotarg®, a recently approved Wyeth-Ayerst chemotherapeutic drug. It is composed of calicheamicin, a potent cytotoxic agent conjugated to an antibody that targets CD33 (found on the surface of certain leukemia cells). This antibody-drug conjugate had a dramatically improved therapeutic index relative to the free drug. Kiss-1 derived proteins have a therapeutic effect on the HPG axis by acting on the pituitary gland and stimulating the production and / or release of FSH. Thus, any modification that allows the peptide to be incorporated and has an effect along the HPG axis is useful.

  A strategy for improving drug survival is the use of water-soluble polymers. A variety of water-soluble polymers have been shown to alter biodistribution, improve the mode of cell absorption, change the permeability of physiological barriers, and change the degree of clearance from the body. (Greenwald et al., Crit Rev Therap Drug Carrier System. 2000; 17: 101-161; Kopecek et al., J Controlled Release., 74: 147-158, 2001). In order to achieve either targeting or sustained release effects, water-soluble polymers have been synthesized that contain drug moieties as end groups, as part of the backbone, or as pendant groups on the polymer chain.

  Polyethylene glycol (PEG) is widely used as a drug carrier and provides a high degree of biocompatibility and ease of modification. Harris et al., Clin Pharmacokinet. 2001; 40 (7): 539-51. Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and reduce toxicity. (Greenwald et al., CritRev Drug Drug Carrier Sys. 2000; 17: 101-161; Zalipsky et al., Bioconjug Chem. 1997; 8: 111-118). PEG can be attached to the active agent by a hydroxyl group at the end of the chain and by other chemical methods; however, PEG itself is limited to at most two active agents per molecule. In a different approach, it retains the biocompatible properties of PEG, but has the added advantage of many attachment points per molecule (providing greater drug loading) and is synthesized to suit a variety of applications Copolymers of PEG and amino acids have been explored as new biomaterials that can be engineered (Nathan et al., Macromolecules. 1992; 25: 4476-4484; Nathan et al., Bioconj Chem. 1993; 4: 54- 62).

  One skilled in the art knows PEGylation techniques for effective modification of drugs. For example, drug delivery polymers consisting of alternating polymers of PEG and trifunctional monomers such as lysine and cysteine have been used by VectraMed (Plainsboro, NJ) or Shearwater (NJ). The PEG chain (typically 2000 daltons or less) is linked to the lysine a- and e-amino groups by a stable urethane linkage. Such copolymers retain the desired properties of PEG while at the same time providing reactive pendant groups (lysine carboxylic acid groups) at tightly controlled intervals along the polymer chain. Reactive pendant groups can be used for derivatization, crosslinking, or conjugation with other molecules. These polymers are useful for producing stable, long-circulating prodrugs by altering the molecular weight of the polymer, the molecular weight of the PEG moiety, and the cleavable linkage between the drug and the polymer. The molecular weight of the PEG moiety affects the spacing of the drug / binding group complex, and the amount of drug per molecular weight of the complex (less PEG moieties provide more drug loading). In general, increasing the overall molecular weight of the block copolymer complex will increase the circulation half-life of the complex. Nevertheless, the complex must have a molecular weight that is easily degraded or less than the glomerular filtration with a limited threshold (eg, less than 45 kDa). Accordingly, the PEGylated Kiss-1 derived peptides of the present invention should preferably be in the molecular weight range of 20 kDa to 35 kDa.

  Effective modification of drugs is not limited to PEGylation techniques. Glycosylation of a molecule, fusion to another molecule such as an antibody or human serum albumin or combinations thereof are examples of other modifications that can be used to facilitate more effective or efficient drug delivery.

  Furthermore, for drug backbones that are important in maintaining circulation half-life and biodistribution, the therapeutic agent is a prodrug until it is released from the backbone polymer by a specific trigger (typically enzymatic activity in the target tissue). A linker can be used to hold in the form of For example, this type of tissue activated drug delivery is particularly useful when delivery to a specific site of biodistribution is required and the therapeutic agent is released at or near the site of the lesion. Binding group libraries for use in activated drug delivery are known to those of skill in the art and can be based on enzyme kinetics, prevalence of active enzymes, and openness specificity of selected disease-specific enzymes (See, for example, the technology established by VectraMed (Plainsboro, NJ)). Such linkers can be used in modifying the Kiss-1 derived proteins described herein for therapeutic delivery.

Methods for Making Kiss-1 Derived Peptides The present invention provides proteins and peptides for use in drugs for the treatment of infertility. Such proteins or peptides can be produced by conventional automated peptide synthesis methods or recombinant expression. The general principles for designing and making proteins are well known to those skilled in the art.

A. Automated Solid Phase Peptide Synthesis Even the peptides of the present invention or indeed the full length Kiss-1 can be synthesized in solution or on a solid support according to the prior art. Various automatic synthesizers are commercially available and can be utilized according to known protocols. For example, Stewart and Young, Solid Phase Peptide Synthesis, 2d. ed. , Pierce Chemical Co. (1984); Tam et al., J. MoI. Am. Chem. Soc, 105: 6442, (1983); Merrifield, Science, 232: 341-347, (1986); and Barany and Merrifield, The Peptides, Gross and Meienhofer, eds, Academic 79, Academic 79 ) (Each incorporated herein by reference). The novel Kiss-1-derived proteins of the present invention can be readily synthesized and screened in a GPCR54 receptor binding / activity assay.

For example, the peptides can be synthesized by solid phase techniques using a typical peptide synthesizer, such as Applied Biosystems Inc. Model 433A. The purity of any given peptide substrate created by automated peptide synthesis or recombinant methods can be determined using reverse phase HPLC analysis. The chemical reliability of each peptide can be established by any method well known to those skilled in the art. In a preferred embodiment, the reliability can be established by mass spectrometry. In addition, peptides can be quantified using amino acid analysis, which performs microwave hydrolysis. Such analysis can use a microwave oven, such as CEM Corporation's MDS 2000 microwave oven. The peptide (approximately 2 μg protein) is contacted with 6N HCl (Pierce Constant Boiling eg about 4 ml) containing, for example, approximately 0.5% (volume to volume) phenol (Mallinckrodt). Prior to hydrolysis, flow discharged alternately sample with N _2. Protein hydrolysis is performed using a two-step process. During the first stage, the peptide is subjected to a reaction temperature of about 100 ° C. and held at that temperature for 1 minute. Immediately after this stage, the temperature is increased to 150 ° C. and held at that temperature for about 25 minutes. After cooling, the sample is dried and the amino acids from the hydrolyzed peptide sample are derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate to obtain a stable urea that fluoresces at 395 nm (Waters AccQ Tag Chemistry Package ). Samples can be analyzed by reverse phase HPLC and quantification can be achieved using an enhanced integrator.

B. Production of recombinant proteins As an alternative to automated peptide synthesis, recombinant DNA techniques can be used. Here, as described herein below, a nucleotide sequence encoding a peptide of the invention is inserted into an expression vector, transformed or transfected into a suitable host cell, and under conditions suitable for expression. Incubate. Recombinant methods are particularly preferred for producing longer polypeptides comprising the peptide sequences of the invention.

  A variety of expression vector / host systems can be utilized to contain and express sequences encoding a peptide or protein. These include microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell lines infected with viral expression vectors (eg, baculovirus); viruses Plant cell lines transfected with expression vectors (eg cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (eg Ti or pBR322 plasmid); or animal cell lines. , But not limited to them. Mammalian cells useful for the production of recombinant proteins include VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (eg COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and 293 cells are included, but are not limited to these. Exemplary protocols for the recombinant expression of peptide substrates or fusion polypeptides in bacteria, yeast and other invertebrates are known to those skilled in the art and are briefly described herein below.

  Expression vectors for use in prokaryotic hosts generally comprise a selectable marker gene of one or more phenotypes. Such genes generally encode proteins that confer, for example, antibiotic resistance or provide auxotrophy. A variety of such vectors are readily available from commercial sources. Examples include pSPORT vector, pGEM vector (Promega), pPROEX vector (LTI, Bethesda, MD), Bluescript vector (Stratagene), pET vector (Novagen) and pQE vector (Qiagen). DNA sequences encoding a given peptide substrate or fusion polypeptide are amplified by PCR, such vectors, such as fusion proteins comprising glutathione-S-transferase (GST) encoded by the vector, and vector cloning sites Cloning into p-GEX-3X (Pharmacia, Piscataway, NJ) designed to produce the protein encoded by the DNA fragment inserted into. Primers for PCR can be created to include appropriate open sites, for example. Treatment of a recombinant fusion protein with thrombin or factor Xa (Pharmacia, Psicaway, NJ) is expected to open the fusion protein and release the substrate containing the substrate or polypeptide from the GST moiety. pGEX-3X / Kiss-1 peptide construct E. coli XL-1 Blue cells (Stratagene, La Jolla CA) were transformed and individual transformants were isolated and grown. Plasmid DNA from individual transformants is purified and partially sequenced using an automated sequencer to confirm the presence of an appropriately oriented nucleic acid insert encoding the desired peptide or polypeptide. If a GST / Kiss-1 derived protein fusion protein is produced in bacteria as a water soluble protein, it can be purified using the GST purification module (Pharmacia Biotech).

Alternatively, the DNA sequence encoding the predicted substrate containing the fusion polypeptide can be cloned into a plasmid containing the desired promoter and optionally a leader sequence (eg, Better et al., Science, 240: 1041-43). 1988). The sequence of this construct can be confirmed by automatic sequencing. The plasmid is then transformed into E. coli using standard procedures using bacterial CaCl ₂ incubation and heat shock treatment. Transform into E. coli (Sambrook et al., Supra). The transformed bacteria are grown in LB medium supplemented with carbenicillin, and production of the expressed protein is induced by growth in an appropriate medium. If present, the leader sequence results in secretion of the mature Kiss-1 peptide or fusion protein and is cleaved during secretion.

  Secreted recombinant protein is purified from bacterial media by the methods described throughout this specification. Similar systems for recombinant proteins in yeast host cells are readily commercially available, such as the Pichia expression system (Invitrogen, San Diego, CA) (according to manufacturer's instructions). Another alternative recombinant production can be achieved using an insect system. Insect systems for protein expression are well known to those skilled in the art. In one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) expresses foreign genes as vectors in Spodoptera frugiperda cells or Trichoplusia larvae. The sequence encoding Kiss-1 is cloned into a nonessential region of the virus, such as the polyhedrin gene, and placed under the control of the polyhedrin promoter. Successful insertion of Kiss-1 inactivates the polyhedrin gene and produces a recombinant virus that lacks the coat protein. Recombinant virus is used to infect Kiss-1 expressing yogurt cells or Trichopulcia larvae (Smith et al., J Virol 46: 584, 1983; Engelhard EK et al., Proc Nat Acad Sci 91: 3224-7, 1994). ).

  Mammalian host systems for the expression of recombinant proteins are also well known to those skilled in the art. Host cell lines can be selected for their particular ability to produce specific post-translational modifications useful in processing the expressed protein or providing protein activity. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing that cleaves the “prepro” form of the protein may also be important for correct insertion, folding and / or function. Different host cells, such as CHO, HeLa, MDCK, 293, WI38, etc. have specific cellular and characteristic mechanisms for such post-translational activity, and the precise modification of introduced foreign proteins and A choice can be made to ensure processing.

  Transformed cells are preferably used for long-term, high-yield protein production, and such stable expression is desirable. Once such cells have been transformed with a vector containing a selectable marker along with the desired expression cassette, they can be grown in a rich medium for 1-2 days before being transferred to a selective medium. Selectable markers are designed to confer resistance to selection, and their presence allows the growth and recovery of cells that successfully express the introduced sequences. Resistant groups of stably transformed cells can be propagated using tissue culture techniques suitable for the cells.

  A number of selection systems can be used to recover transformed cells for recombinant protein production. Such selection systems include, but are not limited to, HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase, and adenine phosphoribosyltransferase genes (in each of tk-, hgprt-, or aprt-cells). Also, anti-metabolite resistance is dhfr (which provides resistance to methotrexate); gpt (which provides resistance to mycophenolic acid); neo (which provides resistance to aminoglycoside G418); als (which is chlor Confer resistance to sulphurone); and hygro (which confer resistance to hygromycin). Additional selectable genes that may be useful include trpB (which allows cells to use indole instead of tryptophan), or hisD (which utilizes histinol instead of histidine) Is possible). Markers that give visual indications for identification of transformants include anthocyanins, b-glucuronidase and its substrate (GUS), and luciferase and its substrate (luciferin).

C. Expression constructs for recombinant protein production In the recombinant production of the Kiss-1 derived protein of the present invention, it may be necessary to use a vector comprising a polynucleotide molecule encoding the Kiss-1 derived protein. Methods for preparing such vectors and methods for producing host cells transformed with such vectors are well known to those skilled in the art. The polynucleotide molecules used in such attempts can be conjugated to a vector generally containing a selectable marker and origin of replication for propagation in the host. These elements of the expression construct are well known to those skilled in the art. In general, an expression vector comprises DNA encoding a given protein operably linked to appropriate transcriptional or translational control sequences, such as those obtained from mammalian, microbial, viral, or insect genes. Examples of control sequences include transcriptional promoters, operators or enhancers, mRNA ribosome binding sites, and appropriate sequences that control transcription and translation.

  The terms “expression vector”, “expression construct” or “expression cassette” are used interchangeably throughout this specification and a nucleic acid encoding a gene product into which some or all of the nucleic acid coding sequence can be transcribed. It is intended to include any type of genetic construct comprising

  The selection of an appropriate expression vector for expression of the peptides or polypeptides of the invention will, of course, depend on the particular host cell used and is within the skill of those in the art. Methods for construction of mammalian expression vectors include, for example, Okayama and Berg (MoI. Cell. Biol. 3: 280 (1983)); Cosman et al. (MoI. Immunol. 23: 935 (1986)); Cosman et al. ( Nature 312: 768 (1984)); EP-A-0367566; and WO 91/18982.

  The expression construct can further comprise a selectable marker that allows detection of expression of the peptide or polypeptide. In general, inclusion of drug selectable markers aids in cloning and selection of transformants, such as neomycin, puromycin, hygromycin, DHFR, zeocin and histidinol. Alternatively, enzymes such as herpes simplex virus thymidine kinase (tk) (eukaryotic), b-galactosidase, luciferase, or chloramphenicol acetyltransferase (CAT) (prokaryotic) can be used. Immunological markers can also be used. For example, epitope tags, such as FLAG system (IBI, New Haven, CT), HA and 6xHis system (Qiagen, Chatsworth, CA) can be used. Furthermore, a glutathione S-transferase (GST) system (Pharmacia, Piscataway, NJ) or a maltose binding protein system (NEB, Beverley, MA) can also be used. The selectable marker used is not believed to be important if it can be expressed simultaneously with the nucleic acid encoding the gene product. Further examples of selectable markers are well known to those skilled in the art.

  Expression requires that an appropriate signal be provided in the vector, for example an enhancer / promoter from both viral and mammalian sources that can be used to express the nucleic acid of interest in a host cell, for example. It is. Usually, the nucleic acid to be expressed is under the transcriptional control of a promoter. A “promoter” refers to a DNA sequence that is recognized by, or introduced with, a cellular synthetic machinery necessary to initiate specific transcription of a gene. A nucleotide sequence is operably linked when the control sequence is functionally associated with the DNA encoding the peptide substrate or fusion polypeptide. Thus, when a promoter nucleotide sequence is intended for transcription of a predetermined DNA sequence, the promoter nucleotide sequence is operably linked to the DNA sequence. Similarly, the phrase “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid that controls RNA polymerase initiation and gene expression. Any promoter that expresses the nucleic acid can be used. The particular promoter used to control the expression of the nucleic acid sequence of interest is not believed to be important if it can direct the expression of the nucleic acid in the target cell. Therefore, when a human cell is targeted, it is preferred that the nucleic acid coding region be located near or under the control of a promoter that can be expressed in the human cell. In general, such promoters can include either human or viral promoters. Common promoters include, for example, human cytomegalovirus (CMV) immediate early gene promoter, SV40 early promoter, Rous sarcoma virus long terminal repeat, β-actin, rat insulin promoter, phosphoglycerol kinase promoter and glyceraldehyde 3- A phosphate dehydrogenase promoter is mentioned. All of them are promoters well known to those skilled in the art and are readily available and can be used to obtain high levels of expression of the coding sequence of interest. If the level of expression is sufficient for a given purpose, also consider the use of other viral, mammalian or bacterial phage promoters well known in the art to obtain expression of the coding sequence of interest. . By using promoters with well-known properties, the level and pattern of expression of the protein of interest can be optimized after transfection or transformation. Inducible promoters can also be used.

  Another regulatory element used in protein expression is an enhancer. There are genetic elements that increase transcription from a promoter located at a distance on the same molecule of DNA. Where the expression construct uses a cDNA insert, it is typically desirable to include a polyadenylation signal sequence that results in proper polyadenylation of gene transcription. Any polyadenylation signal sequence recognized by cells of the selected transgenic animal species is suitable for the practice of the present invention. For example, human or bovine growth hormone and SV40 polyadenylation signal.

  A terminator is also considered as an element of the expression cassette. These elements can help to increase message levels and minimize read-through from the cassette to other sequences. Since termination areas for applications (eg, those contemplated by the present invention) appear to be relatively interchangeable, the termination area used primarily will be chosen for convenience. The termination region can be native with transcription initiation, can be native to the DNA sequence of interest, or can be obtained from another source.

D. Site-directed mutagenesis Site-directed mutagenesis is another technique useful in the preparation of individual Kiss-1-derived proteins for use in the methods of the invention. This technique uses specific mutagenesis of the underlying DNA, which encodes the amino acid sequence that is the target of the modification. This technique further provides a rapid ability to prepare and test sequence variants, incorporating one or more of the above considerations, by introducing mutations of one or more nucleotide sequences into the DNA. Site-directed mutagenesis allows the production of variants through the use of a specific oligonucleotide sequence encoding the DNA sequence of the desired mutation, as well as a sufficient number of contiguous nucleotides, of traversed deletion junctions. Provide primer sequences that are large enough and of sufficient sequence complexity to form a stable duplex on both sides. Typically, a primer about 17-25 nucleotides in length is preferred, with about 5-10 residues being altered on either side of the junction of the sequence.

  This technique typically uses a bacteriophage vector that exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as M13 phage. These phage vectors are commercially available and their use is generally well known to those skilled in the art. Double-stranded plasmids are also routinely used in site-directed mutagenesis, which eliminates the step of transferring the gene of interest from the phage to the plasmid.

  In general, site-directed mutagenesis techniques first obtain a single-stranded vector or two strands of a double-stranded vector that contain within its sequence a DNA sequence encoding the desired protein. Perform by thawing. Oligonucleotide primers having the desired mutated sequence are prepared synthetically. Next, when selecting hybridization (annealing) conditions, the primer is annealed with the single-stranded DNA preparation, taking into account the degree of mismatch, and DNA polymerase, e.g. Apply to the E. coli polymerase IKlenow fragment to complete the synthesis of the chain with the mutation In this way, the first strand encodes the original unmutated sequence and the second strand forms a heteroduplex with the desired mutation. The heteroduplex vector is then transformed into a suitable cell, such as A clone is selected that contains a recombinant vector that is used to transform E. coli cells and has a mutated sequence arrangement.

  Of course, the above approach for site-directed mutagenesis is not the only way to generate potentially useful mutant peptide species and is not intended to be limiting. The present invention also contemplates other methods of obtaining mutagenesis. For example, a recombinant vector having a gene of interest is treated with a mutagen, such as hydroxylamine, to obtain a sequence variant. Other examples that produce potentially useful mutant peptides include yeast or phage display methods.

E. Protein Purification It may be desirable to purify the peptides of the present invention. Protein purification techniques are well known to those skilled in the art. These techniques involve, at a certain level, a crude fraction of the cellular environment into polypeptide and non-polypeptide fractions. Once the peptides or polypeptides of the invention are separated from other proteins, the polypeptide or peptide of interest is further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or homogeneity). Purification) can be achieved. Analytical methods particularly suitable for the preparation of pure peptides are ion exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. A particularly efficient method for purifying peptides is high performance protein liquid chromatography (FRLC) or high performance liquid chromatography (HPLC).

  One aspect of the present invention relates to purification, and in certain embodiments, substantial purification of the encoded polypeptide, protein or peptide. As used herein, the term “purified polypeptide, protein or peptide” is intended to refer to a composition isolated from other components. Here, the polypeptide, protein or peptide is purified to any degree as compared to the state obtained in nature. Thus, a purified polypeptide, protein or peptide also refers to a polypeptide, protein or peptide that has been released from the environment in which it can naturally occur.

  In general, “purified” refers to a polypeptide, protein or peptide composition that has been fractionated to remove various other components, which composition substantially retains the expressed biological activity. To do. When the term “substantially purified” is used, this indication refers to a composition in which the polypeptide, protein or peptide forms the major component of the composition, eg, about 50%, about 60% in the composition. About 70%, about 80%, about 90%, about 95% or more.

  Various techniques suitable for use in protein purification will be well known to those skilled in the art. These include, for example, precipitation with ammonium sulfate, PEG, antibodies, etc. or by thermal denaturation after centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; Gel electrophoresis; and combinations of such or other techniques. As is generally known in the art, the order in which the various purification steps are performed can be varied, or certain steps can be omitted to prepare a more substantially purified polypeptide, protein or peptide. It is believed that it can bring the right way to do it.

Method for Determining GPCR54 Receptor Agonist Activity of Kispeptin As indicated herein above, the Kiss-1 derived protein used herein is an agonist of the GPCR receptor. This receptor is known by those skilled in the art and is encoded, for example, by the sequence described in GenBank Accession No. AY0229541 (incorporated herein by reference). Methods for transfecting cells, eg, mammalian cells, with expression constructs encoding such receptors that are expressed on the surface of the cell are well known in the art. (For example, U.S. Pat. Nos. 5,053,337; 5,155,218; 5,360,735; 5,472,866; 5,476,782; No. 5,516,653; No. 5,545,549; No. 5,556,753; No. 5,595,880; No. 5,602,024; No. 5,639, No. 652, No. 5,652,113; No. 5,661,024; No. 5,766,879; No. 5,786,155; and No. 5,786,157 The disclosures of all of which are incorporated herein by reference.)

  Cells produced by such transfection are readily tested for the binding activity of the Kiss-1 derived peptides described herein to the orphan GPCR54 receptor and serve as agonists of this receptor. Can be used. (For example, U.S. Pat. Nos. 5,053,337; 5,155,218; 5,360,735; 5,472,866; 5,476,782; No. 5,516,653; No. 5,545,549; No. 5,556,753; No. 5,595,880; No. 5,602,024; No. 5,639, No. 652, No. 5,652,113; No. 5,661,024; No. 5,766,879; No. 5,786,155; and No. 5,786,157 These disclosures are all incorporated herein by reference).

  The term “agonist” is used throughout this application to refer to any Kiss-1 derived protein, peptide or peptidomimetic of a Kiss-1 derived protein compound that increases the activity of any GPCR54 receptor of the subject invention. . Preferably, such substances also stimulate production and / or release of FSH and / or LH. Preferably, such stimulation of the release of one or other of these hormones is comparable to the GnRH stimulated release of one or other of these hormones. These peptides may also be useful for stimulating GnRH release.

  Typically, cells or membrane preparations of cells or cells that are transfected and express GPCR54 receptor are prepared, or cells or membrane preparations are obtained from cells known to express the receptor. Thus, receptor binding activity is determined by testing with Kiss-1 derived proteins under conditions that allow receptor-ligand binding. Exemplary such conditions are provided in Kotani et al. (J. Biol. Chem. 276 (37): 34661-34636, 2001). Following a test, such as the receptor binding test described in Kotani et al., Will readily allow the determination of the receptor binding activity of the Kiss-1 derived protein to be tested. Typically, such activity may be comparable to the binding of kisspeptin known to bind to the receptor.

Ligand Binding Test The binding properties of Kiss-1 derived proteins can be tested using a labeled ligand binding test. For example, cells expressing the orphan receptor of the present invention can be used to test the activity of Kiss-1 protein, eg, by a labeled ligand binding test. For example, a membrane preparation that expresses the receptor in a multi-well microtiter plate with a labeled kisspeptin, eg, SEQ ID NO: 4, 5, or 6, along with an unlabeled test Kiss-1 protein to be tested and a binding buffer. Or can be contacted with either intact cells. The binding reaction mixture is incubated for times and the temperature is determined to be optimal in a separate equilibrium binding test. Typically such tests are given in Kotani et al. The reaction is stopped by filtration through GF / B filters, use of cell harvesters, or direct measurement of bound ligand. If the ligand is labeled with a radioisotope, such as ³ H, ¹⁴ C, ¹²⁵ I, ³⁵ S, ³² P, ³³ P, etc., the bound ligand can be liquid scintillation counting, scintillation proximity, or any radioisotope detecting radioisotope It can be detected by using other methods. When the ligand is labeled with a fluorescent compound, the bound labeled ligand can be measured by methods such as, but not limited to, fluorescence intensity, time-resolved fluorescence, fluorescence polarization, fluorescence transfer, or fluorescence correlation spectroscopy. In this way, agonist Kiss-1 derived proteins that bind to orphan receptors can be identified. This is because they inhibit the binding of labeled ligands to membrane proteins or intact cells expressing the receptor. Non-specific binding, high concentrations (e.g., 100~1000xK _D) is defined as the amount of labeled ligand remaining after incubation of membrane protein in the presence of unlabeled ligand.

In the equilibrium saturation binding test, orphan receptor transfected membrane preparations or intact cells are incubated in the presence of increasing concentrations of labeled compound to determine the binding affinity of the labeled ligand. The binding affinity of the unlabeled compound can be determined in an equilibrium competitive binding test using a fixed concentration of labeled compound in the presence of varying concentrations of the displacement ligand. For example, the Kiss-1 derived protein of the invention may also be a competitive inhibitor of the natural ligand of SEQ ID NO: 4, 5, or 6 or other GPCR54 receptor for GPCR54 receptor binding. An ELISA-type approach can be used to determine the relative binding affinity of selected Kiss-1 derived proteins. For example, to examine the binding affinity for the GPCR54 receptor, serial dilutions of competing kisspeptins of SEQ ID NO: 4, 5 or 6 and candidate Kiss-1 derived peptides labeled with a subsaturating concentration of myc epitopes were obtained from GPCR54. And incubate until equilibrium is established. The plate is then washed to remove unbound protein. Peptides bound to the receptor-coated plate are detected using an easily detectable label, such as an anti-myc antibody conjugated with horseradish peroxidase. The binding affinity (EC ₅₀ ) can be calculated as the concentration of competing Kiss-1 derived protein that results in half-maximal binding. These values can be compared to those obtained from tests of kisspeptin of SEQ ID NO: 4, 5 or 6 to determine the relative binding affinity for the receptor of the tested Kiss-1 derived protein.

Functional testing Kiss-1 derived proteins can be tested to determine if the test protein increases any signaling activity associated with the GPCR54 receptor. Thereby, it is determined whether the Kiss-1 derived protein is a GPCR54 receptor agonist. This signal response can be a second messenger response of GPCR54 such as chloride channel activation, changes in intracellular calcium levels, release of inositol phosphates, activation of MAP kinase, changes in cAMP levels, release of arachidonic acid, Or other second messenger responses typically associated with GPCR 54.

  As an alternative to ligand binding studies, cells expressing GPCR54 receptor DNA can be used to screen for ligands for receptors by functional testing. It is well known to those skilled in the art that overexpression of G protein coupled receptors can lead to constitutive activation of intracellular signaling pathways. In a similar manner, overexpression of orphan receptors in any of the cell lines described above can result in the activation of the functional response described below, and any test described herein can be performed using GPCR54 orphans. It can be used to screen the activity of Kiss-1 derived proteins as receptor agonist ligands. Agonists thus identified will be useful in the therapeutic methods of the invention.

  As indicated above, a wide range of tests can be used to screen for the presence of GPCR receptor ligands. These tests are based on traditional measurements of total inositol phosphate accumulation, cAMP levels, intracellular calcium mobilization, and potassium currents, for example; systems that measure these similar second messengers, but at higher throughput Systems that are modified and adapted to be more general and more sensitive; cell-based, reporting more general cellular events resulting from receptor activation, eg metabolic changes, differentiation, cell division / proliferation Extend to the trials. A typical such test is described in detail in US Patent Application Publication No. 2003 / 0022839A1, specifically in paragraphs 0100-0135. This document is specifically incorporated in its entirety as a teaching method for determining receptor binding and functional activity of the Kiss-1 derived protein of the present invention. Any protein or peptide produced as described herein is tested in a test method such as that described in US Patent Application Publication No. 2003/002839, and such protein or peptide is a receptor agonist. It can be determined whether the activity is retained. If the protein or peptide produced retains activity in any one or more of such typical tests (eg, increased cytosolic calcium or ERK phosphorylation in transfected CHO cells) Oxidation), which can be used in the present invention.

Methods of Treating Infertility As described throughout this specification, Kiss-1 derived proteins can be used to enhance, stimulate, promote or otherwise increase the release of FSH and / or LH. Was discovered. Thus, any Kiss-1 peptide / protein having activity similar to that of SEQ ID NO: 3 can be used for the treatment of infertility diseases. Infertility diseases that can be treated herein include any disease that can benefit from increased amounts of FSH signal, LH signal, or both. Preferably, the increase in FSH production provided by the treatment method of the present invention results in a significant increase in the blood concentration of FSH. In a preferred embodiment, the method results in an increase in blood concentration of FSH that is more than 2-fold, but any increase in FSH appears to be beneficial. It is contemplated that the peptide / protein based compositions of the present invention can be used in any and all protocols where agents such as clomiphene are currently used in the treatment of infertility diseases. Thus, the protein / peptide-based therapy of the present invention includes treatments in the induction of ovulation in anovulatory women, and anovulatory infertility patients whose cause of infertility is functional and does not depend on primary ovarian disorders Can be used in the treatment of pregnancy, or in individual treatments suffering from hypogonadotropic hypogonadism. In men, these peptides may be useful for inducing spermatogenesis in men with primary and secondary hypogonadotropic hypogonadism. These peptides may also be useful for the treatment of endometriosis.

  The protocol for administration of Kiss-1 derived protein can be similar to the protocol for administration of other stimulants of gonadotropin release. As a general guide, the protocol developed for the administration of clomiphene is a starting point for the administration of the peptides of the invention, as both clomiphene and Kiss-1 related peptides are used to stimulate gonadotropin production. Dots can be formed. Thus, for example to stimulate ovulation, a composition based on Kiss-1 derived protein (eg, a peptide of the amino acid sequence of SEQ ID NO: 3) is typically in each cycle of 5 days, typically each specific day. Can be prescribed as a once-daily administration. However, the dose can be increased or decreased by the physician based on the individual response of the patient. In typical treatments (such as clomiphene) it may be necessary to perform a Kiss-1 protein loading test to determine if the patient responds to Kiss-1 derived protein. The clomiphene loading test is well known to those skilled in the art (see, eg, US Pat. No. 5,091,170). Such a test can be altered by replacing clomiphene with a Kiss-1 derived protein used as a stimulant.

  In a typical protocol for ovulation stimulation, Kiss-1 derived protein can be administered first starting on cycle 3 and taken daily until cycle 7 days. At the 9th or 10th day of the cycle, the patient's LH and FSH levels are measured. If the LH level is 2-3 times higher than the FSH level, the Kiss-1 derived protein may not be effective. At cycle 12 the patient's urine is tested daily for elevated LH (ie, a signal that ovulation occurs within 24-36 hours). For this test, an ovulation prediction kit (LH prediction) can be used. If no rise occurs by the 16th day of the cycle, an ultrasonography can be performed to confirm follicular development and to measure the uterine lining thickness. After LH rise, ovulation should occur in 2 days.

  If pregnancy does not occur, a further cycle of Kiss-1 derived protein therapy is initiated. In such subsequent cycles, pelvic examination and / or ultrasonography can be performed prior to the third day at the onset of menstrual bleeding.

  In an alternative embodiment, if the above protocol fails to conceive, intrauterine insemination can be used to improve the possibility of fertilization. Such intrauterine insemination can be combined with one or more of clomiphene, letrozole, HMG / insemination or Gonal-F / foristim infusion and intrauterine insemination. In intrauterine insemination, baseline ultrasound is performed on cycle 3 or prior to cycle 3. Start Kiss-1 derived protein therapy on day 3 and continue until day 7 of the cycle. In combination therapy, patients can be treated with Kiss-1 derived protein in combination with clomiphene (or letrozole) / FSH or HMG / and intrauterine insemination. In such a protocol, an infusion of 150 units of FSH or HMG (e.g. Brabele / Gonal-F or Lepronex) can be performed on the 8th or 9th day. LH and FSH are measured on the 9th or 10th day.

  If the patient does not have an increase in LH by day 16 of the cycle, an ultrasonography should be performed to confirm follicular development and to measure the thickness of the uterine lining. If it is determined that the follicle size is greater than 19 mm and the endometrial thickness is greater than 7 mm, an hCG injection can be performed to cause the release of the ovum.

Additional Compounds / Compositions to be Administered with Kiss-1 Protein As discussed above, protocols for administration of Kiss-1 derived proteins can be combined with other substances that provide, for example, ovulation stimulation. Thus, Kiss-1 derived proteins are considered sufficient to provide increased FSH release, increased LH, or both of these effects (and thus can be used to improve ovulation stimulation and overcome infertility). However, it is contemplated that the Kiss-1 derived protein can also be administered in therapy in combination with other treatments for infertility. Such additional treatments include administration of other stimulants of gonadotropin release, such as clomiphene and letrozole, and various gonadotropins that enhance ovulation induction and / or follicular maturation.

  In order to achieve an appropriate therapeutic outcome in the combination therapy considered herein, i.e., to achieve increased ovulation induction, resulting in increased FSH production, resulting in elevated LH, and ovulation in the mammal being treated In general, the subject is administered an amount of a Kiss-1-derived protein composition effective to provide the desired therapeutic outcome, such as to cause an increase in the number of healthy oocytes. In addition, additional exogenous FSH can be provided in the course of daily administration lasting from 7 to 12 days. Details of the FSH composition will be described below.

  FSH is a pituitary glycoprotein hormone composed of two subunits. The α-subunit is common to FSH and other glycoproteins (LH, hCG and TSH), and the β-subunit confers FSH specificity. The field of infertility treatment has progressed and there are many FSH preparations that are currently commercially available and can be used in the methods of the present invention. Such commercial preparations include urine-derived FSH compositions and recombinant FSH compositions. These compositions include, for example, typical administrations described in Pergonal® (Serono Laboratories Inc., Randolph, MA; eg, PDR®, 52nd Edn. 1998, pages 2773-2775. Protocols and compositions), Fertinex® (Serono Laboratories Inc., Randolph, MA; for example, typical administrations described in PDR®, 52nd Edn. 1998, pages 2771-2773. Protocols and compositions), Repronex® (Ferring Pharmaceutical Inc., Tarrytown, NJ; PDR®, 57th Edn. 2003, Protocols and compositions for administration as described in US Pat. No. 1325-1327) (Ferring Pharmaceutical Inc., Tarrytown, NJ; see, eg, PDR®, 57th Edn. 2003, pages 135-1327 Humegon (R) (Organon, West Orange, NJ; for example, PDR (R), 52nd Edn. 1998, pages 1949-951, and protocols for typical administration and Composition), Gonal-F ((PDR (R), 57th Edn. 2003, pages 3124-3128), Follistim (R), which are merely exemplary commercial FSH preparations, The trader It will be appreciated that it may be possible to produce other such FSH preparations for use in the methods and compositions of the present invention, said compositions comprising FSH formulations that can be used and They are discussed in more detail below as long as they provide exemplary guidance on doses, but such doses and formulations can be readily varied and should not be used for such administration. Compared to the number of oocytes produced, when administered in combination with a Kiss-1 derived protein, the FSH dose and formulation may increase the therapeutically effective LH and / or in vivo ovulatory eggs It should be understood that it can still be useful in the context of the present invention as long as it results in an increase in the number of mother cells.

  In addition to these commercially available compositions, those skilled in the art will choose to purify FSH from natural sources, such as postmenopausal female urine, using techniques well known to those skilled in the art. (See, eg, US Pat. No. 5,767,067).

  Alternatively, one skilled in the art can select for production of recombinant FSH using techniques well known to those skilled in the art. Special consideration is given to long-lasting FSH agonists will be useful in the methods of the invention. For example, hCG is known to have a longer half-life than FSH. Both of these gonadotropins share a common α-subunit with specific activity conferred by the β-subunit. It has previously been demonstrated that the α-subunit of one gonadotropin can even be used with another β-subunit to obtain a physiologically active chimeric gonadotropin. Furthermore, the increased biopotency of hCG compared to LH has been demonstrated to be due to the carboxy-terminal peptide of the β-subunit of hCG (Matzuk et al., Endocrinology 126: 376-383, 1990). Long-lasting agonists of FSH can be produced that include a carboxy-terminal peptide extension of the hCGβ-subunit at the carboxy terminus of the FSHb subunit. (LaPoIt et al., Endocrinology, 131: 6, 2514-2520, 1992). Such chimeric molecules have been shown to have a significantly increased circulating half-life and efficacy compared to wild type FSH (Fares et al., Proc. Nat'l Acad. Sci, 89: 4304-4308. 1992). As described elsewhere in this application, longer half-life can also be achieved by additional glycosylation or PEGylation.

  FSH can be administered by any route commonly used for administration of gonadotropin hormones. Most preferably, this administration is by either intramuscular or subcutaneous injection. Through the treatment protocol, the patient is observed for signs of rejection, such as signs of OHSS.

  In addition to FSH, other gonadotropin hormones are used in the methods of the invention and packaged in the kits described herein. Such a hormone includes hCG. It is commercially available as Novalel® (Ferring Pharmaceutical Inc., Tarrytown, NJ) and is available from Physician's Desk Reference (eg, PDR®, 57th Edn. 2003, page 1324- 1325) and is gonadotropin produced by human placenta or obtained from the urine of a pregnant woman. Another commercial preparation of hCG is Pregnyl® (Organon, West Orange, NJ). Properties, suggestions and protocols regarding the use of this hormone are discussed in detail in the Physician's Desk Reference. (See, for example, PDR (registered trademark), 57th Edn. 2003, page 2401). Both of these preparations are for intramuscular administration. Typically, this hormone is administered at a dose of about 5000 units to 10000 units to induce ovulation.

  Yet another hormone that can be used and packaged herein is GnRH. There are many commercial sources of this hormone. GnRH and its analogs are known as Cetrotide® (Serono; PDR®, 57th Edn. 2003, pages 3119-3121); Eligard® (Sanofl-Synthelabo, PDR®, 57th). Edn. 2003, page 2994); Lupron® (PDR®, 57th Edn. 2003, page 3185); and Zoladex® (AstraZeneca PDR®, 57th Edn. 2003, page 695). ) Commercially available. These substances are used to suppress LH / FSH production in women and are therefore used to delay ovulation. Typical dosages for these materials vary from about 0.25 mg to about 3 mg. Ovarian stimulation treatment with FSH typically begins on the second or third day of the menstrual cycle. GnRH or analogs thereof are administered once a day (eg, a dose of less than 0.25 mg) or as a single dose (eg, 3 mg) during the early to mid follicular phase. GnRH is administered until the day of hCG administration. Once ultrasonic testing reveals that the follicle is of an appropriate size, hCG is administered to induce ovulation and eventual maturation of the oocyte.

  In other embodiments where there are multiple co-administering therapeutic agents, the co-administered agents can be administered simultaneously with each other or separately after an appropriate time interval. For example, clomiphene can be administered simultaneously as a composition based on Kiss-1 derived protein. Commercial preparations of clomiphene are known to those skilled in the art and include, for example, Clomid® and Serophene®. In other embodiments, the Kiss-1 derived protein can be co-administered with a GnRH antagonist. Protocols for such GnRH antagonists (eg, Lupron®, Synarel®, Antigon®, or Cetrotide®) and their administration in infertility treatment are known to those skilled in the art. It is well known and is readily commercially available.

Patient Selection and Observation Patients receiving the treatment of the present invention are female patients in the range of 20-45 years old. Patient selection for the methods of the present invention can use parameters similar to those described in the PDR® section for use of the FSH-based therapy described above. For example, prior to treatment, patients are subjected to a thorough gynecological examination and endocrine assessment (eg, pelvic anatomy assessment). Primary ovarian disorders should be eliminated by measuring basal serum gonadotropin levels and the patient should be determined not to be pregnant.

  Through the treatment regimen of the present invention, patients should be evaluated and observed for signs of OHSS before, during and after therapy. Symptoms of OHSS include, but are not limited to, abdominal pain, abdominal distension, gastrointestinal symptoms (eg, nausea), diarrhea, severe ovarian enlargement, weight gain, dyspnea and oliguria. Clinically, symptoms develop in blood loss, blood concentration, electrolyte imbalance, ascites, intraperitoneal hemorrhage, pleural effusion, hydrothorax acute lung distress and thromboembolism. If symptoms of OHSS occur during administration of FSH-based therapy or any other substance administered for stimulation of follicular maturation, administration should be discontinued and subject should be under medical control And determine if hospitalization or other intervention is required. Other symptoms that can be used to observe FSH-based therapy include changes in vaginal cytology, the appearance and volume of the vaginal mucosa, spinnability, and fern crystal formation. These latter symptoms are indicative of the estrogenic effect of the treatment and must be observed because FSH administration stimulates estrogen production. Preferably, these estrogenic effects should be observed in combination with more direct measurements of follicular development (eg, serum estradiol and ultrasonographic measurements).

  Clinical symptoms of ovulation other than pregnancy can be obtained by either a direct or indirect measure of progesterone production. Such signs include: increased basal body temperature, increased serum luteinizing hormone, menstruation after temperature fluctuations. In conjunction with the above indicators of progesterone production, ovarian ultrasound visualization can be used to help determine if ovulation has occurred. Such monograph monitoring can include an assessment of the presence of fluid, ovarian stigma, and disrupted follicles in the cecum. Ultrasound measurements may also help in determining whether the ovaries are enlarged in OHSS.

Pharmaceutical Compositions Pharmaceutical compositions for administration of the present invention can comprise at least one Kiss-1 derived protein (eg, the peptide of SEQ ID NO: 3, which stimulates FSH production and / or release) Mutants or analogs or any other Kiss-1 derived protein). The pharmaceutical composition can also include another stimulant of FSH production (eg, clomiphene and / or at least one gonadotropin hormone, preferably FSH). Each of these preparations is preferably provided in a pharmaceutically acceptable form, optionally in combination with a pharmaceutically acceptable carrier. These compositions can be administered by any means that achieve their intended purpose. Individual amounts and schedules for administration of compositions for stimulating follicular maturation using the methods of the present invention are described in the use of such compositions in treating anovulatory diseases and their use in assisted reproduction techniques. It can be readily determined by one skilled in the art using guidance provided by Physician's Desk Reference for use. As discussed above, one of ordinary skill in the art can initially use the quantities and schedules of FSH, clomiphene, etc. currently used in such medical situations. For this effect, those skilled in the art will specifically refer to each item in the Physician's Desk Reference discussed above, and these items will be referred to as Serophene® as discussed herein above. , Clomid (R), Fertinex (R), Gonal F (R), Bravelle (R) and other teaching methods and compositions for the administration of substances such as all by reference Incorporated herein. Each of these items in the Physician's Desk Reference provides exemplary guidance regarding the type of formulation, route of administration, and treatment plan that can be used in the administration of FSH. Any protocol, formulation, route of administration, etc. described therein can be readily varied for use in the present invention.

  Compositions within the scope of the present invention are derived from at least one Kiss-1 of the present invention in an amount effective to achieve the intended purpose of stimulating, increasing or otherwise inducing ovulation. All compositions comprising proteins are included. Preferably, such induction is preceded by an increase in FSH production and / or an increase in LH compared to these parameters when these pharmaceutical proteins are not administered. In some cases, the number of ovulatory oocytes in an animal is the result of administration of these peptides (when administered either alone or in combination, preferably in combination with a small amount of FSH). Consider increasing as. The active substance used in the methods of the present invention can be administered by any means commonly used for such administration. Most preferably, the Kiss-1 derived protein composition used in the present invention is administered orally.

  It will be appreciated that the appropriate dose of the composition of the invention will depend on the age, health and weight of the recipient, the type of combination treatment (if any), the frequency of treatment, and the nature of the desired effect. Is done. However, the most preferred dosage can be adjusted for an individual subject as would be understood and determined by one of ordinary skill in the art without undue experimentation. This typically involves adjusting the standard dose (eg, reducing the dose if the patient is light in weight). If OHSS symptoms are observed, treatment should be discontinued.

  As discussed above, the total dose required for each treatment can be administered in multiple or single doses. The composition can be administered alone or in combination with other therapeutic agents directed at or related to the disease.

  The composition of the present invention should be formulated into a suitable pharmaceutical composition (ie, a form suitable for in vivo application in therapeutic intervention for infertility disease). In general, this requires preparing a composition (preferably for oral administration) that is essentially free of pyrogens and other impurities that may be harmful to humans or animals. right. FSH formulations of the same type as currently available FSH preparations discussed throughout this specification can be formulated. This peptide / protein formulation can be formulated like any other small molecule protein composition. Preferably, these formulations are for oral administration, but other routes of administration are contemplated (eg, infusion, subarachnoid administration, etc.). The results of kisspeptin's FSH-stimulating properties lead to the conclusion that the main site of kisspeptin action would be the central nervous system. The site of such action may be within the blood brain barrier (BBB). Thus, it is contemplated that formulations and routes of administration that facilitate this peptide / protein easily cross the BBB would be particularly useful. Receptor-mediated absorption across the BBB can be particularly useful.

  It would generally be desirable to stabilize the composition and allow absorption of the composition at the target site using appropriate salts and buffers. Generally, the hormone compositions of the present invention are provided in lyophilized form that is regenerated prior to administration, and the Kiss-1 derived protein composition appears to be formulated in tablet form. Buffers and solutions for hormone regeneration can be provided with pharmaceutical formulations to produce the aqueous compositions of the invention for administration. Such aqueous compositions will comprise an amount of each therapeutic agent effective to be used, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous solvent. Such a composition is also considered as an inoculation. The phrase “pharmaceutically or pharmacologically acceptable” refers to molecular moieties and compositions that do not cause side effects, allergies or other adverse reactions when administered to an animal or human. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion solvents, coatings, antibacterial, antifungal, isotonic, absorption delaying agents and the like. The use of such solvents and materials for pharmaceutically active substances is well known in the art. Except insofar as any conventional solvent or substance is incompatible with the therapeutic composition, its use in the therapeutic composition is contemplated. Additional active ingredients can also be incorporated into the compositions.

  The active compositions of the present invention include the classic FSH pharmaceutical preparations discussed herein and known to those skilled in the art. Methods of formulating proteins and peptides for therapeutic administration are also known to those skilled in the art. Administration of these compositions of the invention will be by any common route so long as the target tissue is available by that route. Most commonly, these compositions are formulated for oral administration. However, other conventional routes of administration (eg, subcutaneous, intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, intraocular, retrobulbar, intrapulmonary (eg, period release), aerosol, Sublingual, nasal, anal, vaginal, or transdermal delivery, or by surgical implantation at a specific site may also be useful, especially when oral administration is a problem. This treatment can consist of a single dose or multiple doses over a period of time.

  The active compound can be prepared for administration as a solution of the free base or pharmacologically acceptable salt in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and also in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

  Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved from the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. For example, the use of a coating such as lecithin, the maintenance of the required particle size in the case of a dispersion, and the use of a surfactant can maintain the proper fluidity. Inhibition of the action of microorganisms can be brought about by various antibacterial and antifungal agents (eg, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, it will be preferable to include isotonic agents, for example, sugars and sodium chloride. Prolonged absorption of injectable compositions can be brought about by the use of agents that delay absorption in the composition, such as aluminum monostearate and gelatin.

  Sterile injectable solutions are prepared by combining the required amount of the active compound in a suitable solvent with the various other ingredients listed above and, if necessary, filter sterilizing thereafter. Generally, dispersions are prepared by incorporating the various sterile active ingredients into a sterile excipient that contains a basic dispersion solvent and the required other active ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is a vacuum drying and lyophilization technique that produces a powder of the active ingredient and any additional desired ingredients from the previously sterile filtered solution. It is.

  As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion solvents, coatings, antibacterial, antifungal, isotonic, absorption delaying agents and the like. The use of such solvents and materials for pharmaceutically active substances is well known in the art. Except insofar as any conventional solvents and materials are incompatible with the active ingredients, their use in therapeutic compositions is contemplated. Additional active ingredients can also be incorporated into the compositions.

  The compositions of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (formation with free amino acids of the protein), which are inorganic acids (eg, hydrochloric acid or phosphoric acid), or organic acids (acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.) And form. Salts formed with free carboxyl groups are also inorganic bases (eg, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide), and organic bases (isopropylamine, trimethylamine, histidine, procaine, etc. ).

  For formulations, solutions are administered in a therapeutically effective amount in a manner compatible with the dosage formulation. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like. For parenteral administration in aqueous solution, for example, the solution should be appropriately buffered if necessary, and the liquid diluent should first be made isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.

  A “unit dose” is defined as an individual amount of a therapeutic composition dispersed in a suitable carrier. Parenteral administration of the therapeutic compound can be performed with an initial bolus followed by continuous infusion to maintain the therapeutic blood concentration of the formulation. Those skilled in the art will readily optimize effective doses and dosing regimens as determined by good medical practice and individual patient clinical symptoms.

  The frequency of dosing will depend on the pharmacokinetic parameters of the drug and the route of administration. The optimal pharmaceutical formulation will be determined by one of skill in the art depending on the route of administration and the desired dose. See, for example, Remington's Pharmaceutical Sciences, 18th Ed: (1990, Mack Publ. Co, Easton PA 18042), incorporated herein by reference. Such formulations can affect the physical state, stability, degree of release in vivo and degree of clearance in vivo. Depending on the route of administration, an appropriate dose can be calculated according to body weight, body surface area or organ size. Further improvements in the calculations necessary to determine the appropriate treatment dose are usually made by those skilled in the art without undue testing (especially the dose information and tests disclosed herein, as well as animals or humans). In light of pharmacokinetic data found in clinical trials).

  Appropriate doses can be ascertained by use of established tests for measuring blood levels in conjunction with relevant dose response data. The final dose depends on factors that alter the action of the drug (eg, specific activity of the drug, severity of injury and patient responsiveness, patient age, condition, weight, sex and diet, severity of any infectious disease Frequency, time of administration as well as other clinical factors) will be determined by the attending physician. As studies are conducted, further information regarding the appropriate dose level and duration of treatment for specific diseases and symptoms will become apparent.

  It will be appreciated that the pharmaceutical compositions and treatment methods of the present invention may be useful in the fields of human medicine and veterinary medicine. Thus, the subject to be treated can be a mammal, preferably a human or other animal. For veterinary purposes, subjects include, for example, livestock (eg, cattle, sheep, pigs, horses and goats), companion animals (eg, dogs and cats), exotic and / or zoo animals, laboratories Animals (eg, mice, rats, rabbits, guinea pigs and hamsters); and poultry (eg, chickens, turkeys, ducks and geese).

  The present invention also contemplates kits for use in the treatment of infertility diseases. Such a kit comprises at least a first composition comprising the protein / peptide described above in a pharmaceutically acceptable carrier. Another component can be FSH in a pharmaceutically acceptable carrier. The kit can further comprise a solution or buffer to effect delivery of the first and second compositions. The kit includes additional compositions comprising additional stimulants of FSH production / release, such as additional other Kiss-1 derived proteins, other stimulants such as clomiphene and / or additional hormones such as hCG, LH, etc. Can further be included. The kit can further comprise a catheter, syringe or other delivery device for delivery of the composition or compositions used in the methods of the invention. The kit can further comprise instructions including an administration protocol for the treatment plan.

  The following examples were included to demonstrate preferred embodiments of the invention. Those skilled in the art will recognize that the techniques disclosed in the following examples show that the techniques discovered by the inventor work well in the practice of the invention and therefore constitute a preferred mode for its practice. It should be. However, one of ordinary skill in the art appreciates that many changes can be made in the specific embodiments disclosed in light of the present disclosure and that similar or similar results can still be obtained without departing from the spirit and scope of the invention. Should be recognized.

Example 1 Exemplary Materials and Methods Animals All experiments were performed in female Sprague-Dawley rats (180-200 g) obtained from Charles River (MA), free of standard laboratory food and water. And kept light-dark for 12 hours in a temperature and humidity controlled room.

Surgical procedures All surgical procedures were performed under anesthesia with a single injection (ip) of a mixture of ketamine (100 mg / kg) and xylazine (15 mg / kg).

  i. c. v. Transventricular cannula transplantation for experiments. The anesthetized rat's head was placed in a stereotaxic apparatus and positioned so that Bregma and Lambda were horizontal. An incision was made from the front to the rear along the sagittal midline. A 1.5 mm diameter hole was drilled 0.9 mm behind the skull and 0.0 mm beside the bregma. A stainless steel cannula measuring 25 mm × 22 was lowered approximately 7 mm until cerebrospinal fluid could be aspirated from the third ventricle with a 1 cc syringe connected to the cannula by a 5 cm portion of a silastic tube. After sealing the outer opening of the cannula, it was fixed to the skull with three stainless steel screws and dental acrylic cement, and then the incision was closed. Rats were allowed to recover in 14 days.

  Ovariectomy and transplantation of estrogen-containing capsules. Both ovaries were removed from the animal's back incision under anesthesia to eliminate the diurnal variation in plasma estrogen concentration due to the variable estrous cycle. Just before closing the incision, two estrogen-containing capsules (0.5 mg / capsule, Innovative Research of America, FL) were inserted subcutaneously in the middle of the animal's back to maintain a consistent basal physiological concentration of plasma estrogen. Rats were allowed to recover in 7 days.

  Jugular cannula insertion and tethering to an automated blood sampler. The right jugular vein of anesthetized rats was exposed through a cervical incision. To handle the vein, a holding suture was tied to the exposed vein at the rostral tip. The tail end of the vein was fixed with small forceps to prevent bleeding, and at the same time a cannula (polyethene tube (OD 1.0 mm)) was inserted into the center of the vein through a 0.5 mm cut made with iris scissors. . After insertion of the cannula, a suture was tied around the vein at the tail end to secure the cannula in the vein. The neck incision was closed with a wound clip, and the outer end of the cannula was attached to a tube of an automated blood sampler (Bioanalytical Systems Inc., IN) filled with heparin-containing (20 IU / ml) sterile saline. Blood samples were collected using an automated blood sampler consisting of a rotating cage with a food box and water bottle, a balanced arm, a pump device, a fraction collector and a controller. Rats with jugular cannula inserted were tethered to the balance arm with a loosely attached plastic collar. The cage is rotated in the opposite direction of animal movement to prevent twisting of the jugular vein cannula connected to the sampler tube and also allows the rat to freely eat, drink and move during blood collection. Rats were allowed to recover in 24 hours.

Test compound GnRH, positive control, Antide, antagonist of GnRH receptor, and Kiss-1 (45-54; Tyr-Asn-Trp-Asn-Ser-Phe-Gly-Leu-Arg-Phe-NH2; SEQ ID NO: 3 ) Were obtained from Sigma (MO), Serono (MA) and Peptide International (KY), respectively. The fragment was used simply because it was previously reported that this 10 amino acid fragment of Kiss-1 showed the strongest binding affinity for GPCR54 (Kotani et al., J Biol Chem 276: 34631- 34636). Any other kisspeptin having receptor binding activity can be similarly used in the methods described herein.

Experimental Procedure All experiments were performed between approximately 13-14 hours. GPCR54 agonist, Kiss-1, dissolved in phosphate buffered saline, was passed through the jugular vein and intraventricular cannula in volumes of 200 ul and 10 ul, respectively, i. v. And i. c. v. Injected. Blood samples (100 ul) were removed using an automatic blood sampler before injection (−15 and 0 minutes) and after (15 minutes, 30 minutes and 45 minutes). Each blood sample was replaced with an equal volume of heparin-containing (20 IU / ml) sterile saline. Plasma was extracted and stored at −20 ° C. until measurement of LH and FSH by enzyme-linked immunoassay and labeled immunoassay, respectively.

Data analysis Changes in plasma LH and FSH concentrations (ng / ml) after infusion were obtained as the area under the concentration curve (AUC). It is calculated by first calculating the sum of hormone concentrations at 15 minutes, 30 minutes and 45 minutes after treatment by subtracting the basal level (average of pre-15 minutes and 0 minute concentrations) from each animal. And then averaged as a group. Statistical differences between treatment groups were determined by one-way ANOVA followed by LSD as a post hoc test. An initial data set is shown in FIGS. Further data is described in Examples 2 and 3 below.

Example 2 Kiss-1 i. v. Stimulation of the gonadal axis by injection This study aims to determine the effect of peripheral treatment of Kiss-1 on LH and FSH release. It also confirmed that the effect of Kiss-1 was mediated by the GnRH receptor in the pituitary gland by pretreatment with a GnRH receptor antagonist (Antide) 15 min prior to 400 ug / kg Kiss-1 infusion ( Established mechanism in the control of gonadotropins). These results are summarized in FIG. Except for the LH response at the highest dose, there was a dose-dependent increase in plasma LH and FSH concentrations with Kiss-1 treatment. Blocking the GnRH receptor successfully prevented GnRH- and Kiss-1-induced increases in blood gonadotropin concentrations. These results demonstrate that stimulation of the gonadal axis by Kiss-1 is mediated by the GnRH receptor in the pituitary gland.

Example 3 Kiss-1 i. c. v. Stimulation of the gonadal axis by injection Based on the observation that GnRH antagonists interfere with the promotion of Kiss-1 LH and FSH release, as shown in Example 2, Kiss-1 acts in the brain where GnRH neurons are located, It was assumed to stimulate their neural activity. To test this hypothesis, this test is based on the Kiss-1 i. c. v. The purpose was to see if the injection stimulated the gonadal axis. These results are summarized in FIG. Kiss-1 stimulated LH release in a dose-dependent manner. Furthermore, although high doses of Kiss-1 are not as effective as low doses, there was a significant increase in FSH concentration after Kiss-1 infusion. i. v. I. Of Kiss-1 resulting in LH and FSH responses in infusion studies. c. v. The dose is i. v. It was worth mentioning that it was as low as 1/25 and 1/100 of the dose. These results demonstrate that the primary site of action of Kiss-1 in promoting LH and FSH release is in the brain and that its regulatory mechanism can involve stimulation of GnRH neurons.

  All of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the present invention have been described in the context of preferred embodiments, without departing from the concept, spirit and scope of the present invention, the compositions and / or methods and methods described herein may be used. It will be apparent to those skilled in the art that changes can be applied in steps or sequence of steps. More specifically, it will be apparent that certain materials that are chemically and physiologically related can replace the materials described herein as long as the same or similar results are obtained. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

  Citations cited throughout this specification are all specifically incorporated by reference herein as long as they provide typical procedures or other details that supplement those described herein. It is.

  The following drawings form part of the present specification and are included to further demonstrate aspects of the present invention. The invention may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments herein.

FIGS. 1A-P show a comparison of the in vivo effects of GnRH and Kiss1 on the release of LH (FIGS. 1A-H) and FSH (FIGS. 1I-P) over 45 minutes. 1A and 1B show the effect of excipients alone on LH release. FIG. 1C and FIG. 1D show the effect of 90 ng / kg GnRH-I administration on LH release. FIGS. 1E and 1F show the effect of 9 μg / kg GnRH-I administration on LH release. FIGS. 1G and 1H show the effect of administration of 4 μg / kg Kiss1 on LH release. FIGS. 1I and 1J show the effect of excipients alone on FSH release. FIG. 1K and FIG. 1L show the effect of 90 ng / kg GnRH-I administration on FSH release. FIGS. 1M and 1N show the effect of 9 μg / kg GnRH-I administration on FSH release. FIG. 1O and FIG. 1P show the effect of administration of 4 μg / kg Kiss1 on FSH release. I. To plasma LH (FIG. 2A) and plasma FSH (FIG. 2B) levels in estrogen-treated ovariectomized rats. v. Effect of Kiss-1 by injection and suppression of these effects by pretreatment with GnRH antagonist (Antide). Exclusive letters indicate statistical differences at P <0.05, as determined by one-way ANOVA, following Fisher's LSD analysis as a post hoc test. I. To plasma LH (FIG. 3A) and plasma FSH (FIG. 3B) levels in estrogen-treated ovariectomized rats. c. v. Effect of Kiss-1 by injection. Exclusive letters indicate statistical differences at P <0.05, determined by one-way ANOVA, following Fisher's LSD as a follow-up test.

Claims (57)

  A method of stimulating ovulation in a mammal comprising administering to the mammal a first composition comprising a Kiss-1 derived protein.   2. The method of claim 1, wherein the Kiss-1 derived protein is a mature Kiss-1 protein comprising the sequence of SEQ ID NO: 2.   The method according to claim 1, wherein the Kiss-1 derived protein is a peptide obtained from a mature Kiss-1 protein.   The method according to claim 1, wherein the Kiss-1 derived protein is a Kiss-1 derived peptide capable of activating the GPCR54 receptor.   The method of claim 1, wherein the Kiss-1 derived protein is a peptide comprising the amino acid sequence of SEQ ID NO: 7.   The Kiss-1 derived protein is a peptide having the sequence of SEQ ID NO: 3, an analog of the peptide of SEQ ID NO: 3, a fragment of SEQ ID NO: 3, or an analog of the fragment of SEQ ID NO: 3. the method of.   7. The method of claim 6, wherein the analog of SEQ ID NO: 3 is a peptide that is a conservative variant of SEQ ID NO: 3.   2. The method of claim 1, further comprising administering a second composition that stimulates ovulation.   9. The method of claim 8, wherein the second composition for stimulating ovulation comprises an ovulation hormone or a chemical stimulant.   10. The method of claim 9, wherein the ovulation chemical stimulant is clomiphene citrate or letrazol.   The ovulatory hormone stimulant is a gonadotropin hormone selected from the group consisting of human menopausal urinary gonadotropin (hMG), follicle stimulating hormone (FSH), luteinizing hormone (LH), and human chorionic gonadotropin (hCG) the method of claim 9.   12. The method of claim 11, further comprising administering a composition comprising a GnRH antagonist.   12. The method of claim 11, wherein the gonadotropin is FSH.   14. The method of claim 13, further comprising administering a non-FSH gonadotropin hormone.   15. The method of claim 14, wherein FSH and LH are administered in equal amounts.   14. The method of claim 13, wherein the FSH is administered at a dose in the range of about 5-450 IU / day.   14. The method of claim 13, wherein the FSH is administered at a dose in the range of about 5-75 IU / day.   12. The method of claim 11, wherein the gonadotropin hormone is administered by infusion.   The method of claim 1, wherein the mammal is a human.   A method of stimulating FSH production in a mammal comprising administering to the mammal a composition comprising a Kiss-1 derived protein.   21. The method of claim 20, wherein the Kiss-1 derived protein is a mature Kiss-1 protein comprising the sequence of SEQ ID NO: 2.   21. The method of claim 20, wherein the Kiss-1 derived protein is a peptide obtained from mature Kiss-1 protein.   21. The method of claim 20, wherein the Kiss-1 derived protein is a Kiss-1 derived peptide capable of activating G-protein coupled receptor 54.   21. The method of claim 20, wherein the Kiss-1 derived protein is a peptide comprising the amino acid sequence of SEQ ID NO: 7.   21. The Kiss-1 derived protein is a peptide having the sequence of SEQ ID NO: 3, an analog of the peptide of SEQ ID NO: 3, a fragment of SEQ ID NO: 3, or an analog of the fragment of SEQ ID NO: 3. the method of.   26. The method of claim 25, wherein the analog of SEQ ID NO: 3 is a peptide that is a conservative variant of SEQ ID NO: 3.   21. The method of claim 20, further comprising administering a second composition that stimulates FSH production in the mammal.   28. The method of claim 27, wherein the second composition that stimulates FSH production comprises an anti-estrogen compound.   30. The method of claim 28, wherein the anti-estrogen compound is selected from the group consisting of clomiphene and letrazole.   A method of stimulating LH production in a mammal comprising administering to the mammal a composition comprising a Kiss-1 derived protein. a) inhibiting endogenous gonadotropin production in a mammal;
b) administering to the mammal an amount of a composition of a Kiss-1 related peptide effective to stimulate ovarian follicle growth in the mammal; and c) inducing ovulation and producing an egg in the mammal. A method for therapeutic intervention of infertility in a mammal, comprising:   32. The method of claim 31, wherein the method further comprises administering a gonadotropin preparation for stimulating ovarian follicle growth.   33. The method of claim 32, wherein the gonadotropin preparation comprises urinary or recombinant FSH or HMG, with or without recombinant LH.   35. The method of claim 32, wherein the gonadotropin preparation comprises recombinant FSH.   32. The method of claim 31, wherein the induction of ovulation comprises administering to the mammal a composition comprising HCG, natural LHRH, LHRH agonist or recombinant LH.   32. The method of claim 31, wherein the suppression of endogenous gonadotropin production in the mammal comprises administering a GnRH antagonist.   32. The method of claim 31, further comprising intrauterine insemination of the egg by sperm injection.   32. The method of claim 31, wherein the Kiss-1 derived peptide is administered in combination with a complex amount of an anti-estrogen agent effective to stimulate FSH production in the mammal.   32. The method of claim 31, wherein the mammal does not respond to standard gonadotropin stimulation of normal ovulation.   32. The method of claim 31, wherein the infertility disease is hypogonadotropic hypogonadism.   32. The method of claim 31, wherein the infertility disease is polycystic ovary syndrome.   32. The method of claim 31, further comprising collecting oocytes 12 days after the initial stimulation of ovarian follicle production.   43. The method of claim 42, further comprising fertilizing the collected oocytes in vitro and culturing the collected and fertilized oocytes to the 4-8 cell stage.   44. The method of claim 43, further comprising transferring a 4-8 cell stage fertilized oocyte to a mammalian uterus.   44. The method of claim 43, wherein the fertilized oocyte is transferred to the same mammalian uterus from which the oocyte was collected.   44. The method of claim 43, wherein the fertilized oocyte is transferred to a different mammalian uterus from which the oocyte was collected.   A method of alleviating one or more symptoms of hypogonadotropic hypogonadism in a subject comprising individually administering a therapeutically effective amount of a Kiss-1 derived peptide.   48. The method of claim 47, wherein the subject is a male subject.   48. The method of claim 47, wherein the subject is a female subject.   A composition comprising a Kiss-1 derived protein for use in the treatment of infertility. a. A first composition comprising a Kiss-1 derived protein in a pharmaceutically acceptable formulation, and b. A kit for treatment of infertility comprising a second composition for treatment of infertility.   52. The kit of claim 51, wherein the second composition for the treatment of infertility comprises the FSH preparation in a pharmaceutically acceptable formulation.   52. The kit of claim 51, wherein the FSH preparation is selected from the group consisting of a urinary FSH preparation and a recombinant FSH.   53. The kit of claim 52, wherein the FSH is human FSH.   53. The kit of claim 52, wherein the FSH is present in a unit dose of about 5 IU FSH to about 75 IU FSH.   52. The kit of claim 51, further comprising a third composition comprising LH in a pharmaceutically acceptable formulation.   57. The kit of claim 56, wherein LH is present in a unit dose of about 75 IU LH to about 150 IU LH.

Images