Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/18—Growth factors; Growth regulators
  • A61K38/1875—Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
  • A61P15/18—Feminine contraceptives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
  • A61P5/24—Drugs for disorders of the endocrine system of the sex hormones

Definitions

• the invention relates generally to methods and compositions for the modulation of human fertility. More particularly, the present invention relates to methods and compositions the to enhance fertility, methods and compositions to delay the onset or alleviate symptoms of menopause, and methods and compositions to decrease fertility.
• Morphogens are members of the TGF- ⁇ superfamily that perform essential physiological functions in morphogenesis and organogenesis. Morphogens are expressed in a tissue-specific manner in many different cell types during embryonic and adult life in both vertebrates and invertebrates. The importance of morphogens in regulating crucial events in morphogenesis, organogenesis, and cytodifferentiation has been clearly established from studies of morphogen-deficient animals.
• Morphogens also referred to as osteogenic proteins (OPs) or bone morphogenic proteins (BMPs), are generally classified as a subgroup of the TGF- ⁇ superfamily of growth factors. Hogan, Genes & Development 10: 1580-1594 (1996).
• Members of the morphogen family of proteins include the mammalian osteogenic protein-1 (OP-1, also known as BMP-7, and the Drosophila homolog 60A), osteogenic protein-2 (OP-2, also known as BMP-8), osteogenic protein-3 (OP-3), BMP-2 (also known as BMP-2A or CBMP-2A, and the Drosophila homolog dpp), BMP-3, BMP-4 (also known as BMP-2B or CBMP-2B), BMP-5,
• morphogen receptors exist as two subtypes, the type I receptors and the type II receptors. Both types of morphogen receptors are structurally similar and both types possess intrinsic serine/threonine kinase activity. Two type I morphogen receptors, BMPR-IA (or ALK-3) and BMPR-IB (or ALK-6), have been identified. One type II morphogen receptor, BMPR-II, has also been identified. Individually, either type I morphogen receptors or type II morphogen receptors can bind morphogen as a ligand with low affinity. However, both receptor types are necessary to achieve high affinity binding and ligand-mediated signal transduction. After the ligand-receptor complex is formed, the type II receptor phosphorylates and activates the type I receptor. The type I receptor then triggers downstream events in the morphogen-signaling pathway.
• FSH follicle stimulating hormone
• LH luteinizing hormone
• ovarian follicle the female reproductive organ in which an oocyte (egg cell) is surrounded by one or more layers of granulosa cells, as well as other cells.
• oocyte egg cell
• a cavity forms in the ovary follicle; the ovarian follicle is then termed a Graafian follicle.
• FSH-dependent ovarian follicle growth is marked by increasing synthesis of estrogen, but not progesterone, in the cells of the developing ovarian follicle.
• luteinization This transformation of the mature ovarian follicle and its theca interna into a corpus luteum after ovulation, and the formation of luteal tissue is termed luteinization. It has been known for many years that a "luteinization inhibitor” plays an important role in inhibiting follicular progesterone production, but the molecular nature of the "luteinization inhibitor” remains a mystery.
• the invention provides a method for increasing fertility, by providing a "luteinization inhibitor" to a female subject in the form of a therapeuticaUy effective amount of a morphogen pharmaceutical.
• the morphogen is a peptide having an amino acid sequence selected from a sequence: (1) having at least 70% homology with the C-terminal seven-cysteine skeleton of human OP-1.
• the therapeuticaUy effective amount is nanomolar.
• the administration of the morphogen induces estrogen synthesis by the ovary of the subject, and can also attenuate progesterone synthesis by the ovary of the subject.
• the subject receiving the morphogen can have healthy ovary follicles, atretic ovary follicles, or both.
• the invention also provides a method for alleviating symptoms of menopause or for delaying the onset of menopause, in which a therapeuticaUy effective amount of a morphogen is administered to the subject.
• the morphogen induces ovarian follicle growth, which is marked by increasing ovarian synthesis of estrogen
• the administration of the morphogen can also attenuate progesterone synthesis by the ovary of the subject.
• Morphogens can thus be used to prolong promote follicular growth over the duration of a women's life when she can have menstrual cycles. Because menstruation results from a balance of hormonal factors, those factors that promote follicular growth can be used to delay the onset of menstruation.
• the invention further provides a method for contraception.
• the method reduces ovarian follicular growth, and thus ovulation and production of hormones in the ovary, by administering to a subject a compound which interferes with the binding of the morphogen and its receptor on oocytes or ovarian granulosa cells .is administered. Treating a subject with the compound results in a subdued ovulation and an increased luteinization of the follicles, decreasing the fertility of the woman receiving the compound.
• the compound that interferes the binding of the morphogen to its receptor is an anti-morphogen antibody.
• the compound that interferes the binding of the morphogen to its receptor is an anti-receptor antibody.
• the compound that interferes the binding of the morphogen to its receptor is a morphogen receptor antagonist.
• morphogen morphogen
• bone morphogen osteogenic protein
• OP bone morphogenic protein
• BMP morphogenic protein
• morphogenetic protein all embrace the class of proteins typified by human osteogenic protein 1 (hOP-1). Nucleotide and amino acid sequences for hOP-1 are provided in SEQ ID NOS: 1 and 2, respectively. For ease of description, hOP-1 is considered a representative morphogen. It will be appreciated that OP-1 is merely representative of the TGF- ⁇ subclass of true tissue morphogens and is not intended to limit the description Other known and useful morphogens include, but are not limited to, BMP-2, BMP-3, BMP-3b.
• useful morphogens include those sharing the conserved seven cysteine skeleton, and sharing at least 70% ammo acid sequence homology (similarity), withm the C-termmal seven-cysteme skeleton of human OP-1, residues 330-431 of SEQ ID NO 2 (hereinafter referred to as the presently-preferred reference sequence)
• the invention encompasses use of biologically active species (phylogenetic) variants of any of the morphogenic proteins recited herein, including conservative amino acid sequence variants, proteins encoded by degenerate nucleotide sequence va ⁇ ants, and morphogenically-active proteins sharing the conserved seven cysteine skeleton as defined herein and encoded by a DNA sequence competent to hybridize under standard stringency conditions to a DNA sequence encoding a morphogenic protein disclosed herein, including, without limitation, OP-1 or BMP-2 or BMP-4 Presently, however, the preferred reference sequence is that of residues 330-431 of SEQ ID NO 2 (OP-
• morphogens useful m methods and compositions of the invention are defined as morphogemcally-active proteins having any one of the generic sequences defined herein, including OPX (SEQ ID NO 3) and Generic Sequences 7 and 8 (SEQ ID NOS 5 and 6, respectively), or Generic Sequences 9 and 10 (SEQ ID NOS 7 and 8, respectively)
• OPX encompasses the observed variation between the known phylogenetic counterparts of the osteogenic OP-1 and OP-2 proteins, and is described by the amino acid sequence presented herein below and in SEQ ID NO 3
• Generic Sequence 9 is a 97 amino acid sequence containing the C-termmal six cysteine skeleton observed in hOP-1 (residues 335-431 of SEQ ID NO 2) and wherein the remaining residues encompass the observed ⁇ a ⁇ ation among OP-1, OP-2, OP-3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-8, BMP-9, BMP
• Generic Sequence 10 is a 102 ammo acid sequence which includes a five ammo acid sequence added to the N-terminus of the Generic Sequence 9 and defines the seven cysteine skeleton observed m hOP-1 (330-431 SEQ ID NO 2)
• Generic Sequences 7 and 8 are 97 and 102 ammo acid sequences, respectively, containing either the six cysteine skeleton (Generic Sequence 7) or the seven cysteine skeleton (Generic Sequence 8) defined by hOP-1 and wherein the remaining non-cysteme residues encompass the observed variation among OP-1, OP-2, OP-3, BMP-2, BMP-3, BMP-4, 60A, dpp, Vgl, BMP-5, BMP-6, Vgr-1, and GDF-1
• morphogens which, when provided to a specific tissue of a mammal, induce tissue-specific morphogenesis or maintain the normal state of differentiation and growth of that specific tissue
• the present morphogens induce the formation of vertebrate (e g , avian or mammalian) body tissues, such as but not limited to nerve, eye, bone, cartilage, bone marrow, ligament, tooth dentin, pe ⁇ odontium, liver, kidney, lung, heart, or gastrointestinal lining
• the present demonstrations can be carried out in the context of developing embryonic tissue, or at an aseptic, unscarred wound site in post-embryonic tissue
• Methods of identifying such morphogens, or morphogen receptor agonists are known in the art and include assays for compounds which induce morphogen-mediated responses (e g , induction of endochondral bone formation, induction of differentiation of metanephric mesenchyme, and the like)
• morphogen-mediated responses e g , induction of
• FIG 1 panels 1-A through 1-M are a tabular alignment of the ammo acid sequences of various naturally-occurring morphogens with a preferred reference sequence of human OP-1, residues 330-431 of SEQ ID NO 1
• FIG 2 is a tabular presentation of alternative ammo acids for "Xaa” positions in generic sequences SEQ ID NOS 5, 6, and 9 that represent amino acid variations in known morphogens
• FIG 3 is a tabular presentation of alternative ammo acids for "Xaa” positions in generic sequences SEQ ID NOS 5, 6, and 9 that represent ammo acid variations m known morphogens
• FIG 4 is a tabular presentation of alternative ammo acids for "Xaa” positions in generic sequences SEQ ID NOS 7, 8, and 10 that represent ammo acid va ⁇ ations in known morphogens
• FIG 5 is a set of graphs showing the effects of morphogens on estrogen and progesterone production by granulosa cells
• Granulosa cells (5 x 10 4 viable cells/well/200 ⁇ l) were cultured for 48 hours (hr) in serum-free medium containing androstenedione (1 ⁇ M), and either no additions (control), BMP-4 (3, 10, or 30 ng/ml), BMP-7 (3, 10, or 30 ng/ml), FSH (0 1, 0 3, 1, 3, or 10 ng/ml) or their combination
• estrogen levels Panels A and C
• progesterone levels Panels B and D
• FIG 6 is a set of graphs showing the time course effect of BMP-7 on estrogen and progesterone production by granulosa cells
• Granulosa cells (5 x 10 4 viable cells/well/200 ⁇ l) were cultured for 48 hr m serum-free medium containing androstenedione (1 ⁇ M), and FSH (3 ng/ml) m the absence or presence of BMP-7 (30 ng/ml) After culture, estrogen levels
• FIG 7 is a set of graphs showing that FSH ⁇ Luc expression is increased by morphogens and activm
• FIG 8 is a set of graphs showing dose-dependent inhibition of FSH ⁇ Luc expression using rabbit a i mOP-1 1
• FIG 9 is a set of bar graphs showing the neutralizing effects of mOP-1 and activm antibodies DETAILED DESCRIPTION OF THE INVENTION
• the invention provides methods for increasing fertility by inhibiting luteinization in the ovarian follicles Luteinization can be an important part of a healthy menstrual cycle when induced by the luteinization hormone (LH), an increase in the levels of which occur normally prior to ovulation
• LH luteinization hormone
• luteinization is a complex differentiation process involving the interaction of extrinsic and mtraova ⁇ an factors Undesirable luteinization caused by a low production of hormones by ovarian cells can result in a lowered fertility.
• the invention provides a method for increasing fertility, by providing a "luteinization inhibitor" to a female subject in the form of a morphogen pharmaceutical
• morphogens can act directly on the cells of the ovary to increase ovarian synthesis of estrogen
• morphogens act on the pituitary to increase synthesis of FSH FSH then binds to granulosa cells in the ovaries, stimulating estrogen production High FSH levels also advance the preovulatory stage of the dominant follicle in the early follicular phase of the cycle
• FSH controls follicle development in women at the recruitment-selection stage Messims et al , Hum Reprod 5(2) 153-6 (1990)
• morphogens can act on ovarian cells to potentiate the effects of FSH
• Recent findings from the study of the regulation of follicular dex elopment show that the potentiating effect of va ⁇ ous growth factors on ova ⁇ an sensitivity to FSH Lunenfeld et al , Bailheres Clin Obstet Gynaecol 4(3) 473-89 (1990)
• administration of morphogens can act in a more direct manner to potentiate the effects of FSH
• morphogens can act on ovarian cells to increase synthesis of FSH
• the invention provides a method for alleviating symptoms menopause
• Menopause results from the ovaries decreasing their production of the sex hormones estrogen and progesterone
• the drop in estrogen levels causes the most common symptoms during menopause.
• Current methods for treating menopause include estrogen replacement therapies, taken in the form of oral tablets, skin patches, or injections. Estrogen circulates through the body to reduce the short-term changes of menopause.
• the combination therapy of estrogen plus progesterone is called hormone replacement therapy.
• estrogen receptor modulators are used to treat menopause.
• the invention provides a method of using morphogens to induce FSH-dependent ovarian follicle growth, which is marked by increasing ovarian synthesis of estrogen.
• the invention thus provides another treatment, which can be an alternative to or a supplement for estrogen replacement therapies.
• Mo ⁇ hogens can be used to prolong promote follicular growth over the duration of a women's life when she can have menstrual cycles. Because menstruation results from a balance of hormonal factors, those factors that promote follicular growth can be used to delay the onset of menstruation. By promoting healthy follicular growth, the method of the invention also results in increased ovarian levels of estrogen.
• the invention also provides a method of contraception.
• Treating a female subject with a compound that prevents the binding of mo ⁇ hogen to a mo ⁇ hogen receptor on oocytes or ovarian granulosa cells will result m a subdued ovulation and an increased luteinization of the follicles. It is well known in the art that a woman's fertility is reduced as her ovulation diminishes, or when her ovaries' production of estrogen and other sex hormones decreases.
• the invention provides a method for reducing ovarian follicular growth and thus ovulation and production of hormones in the ovary, by administering compounds which interfere with mo ⁇ hogen activity in promoting follicular growth early in the menstrual cycle.
• the cellular sites of expression of the mo ⁇ hogen type IA, type IB, and type II receptors (BMPR-IA, BMPR-IB, BMPR-II) mRNAs and BMP-4 and BMP-7 mRNAs were characterized in the rat ovary, establishing for the first time the existence of a functional mo ⁇ hogen ligand-receptor system in the ovary of any species.
• the genes encoding mo ⁇ hogens and the genes encoding the famih of mo ⁇ hogen receptors are expressed in a cell-type-specific manner.
• BMP-4 and BMP-7 mRNAs indicate the coordinate regulation of these two mo ⁇ hogens during ovarian follicle development
• the expression of both mo ⁇ hogens is stage-specific in the cycle of fol culogenesis, being very high in healthy follicles but barely detectable in follicles undergoing atresia
• This pattern of expression shows that the coordinate expression of these two mo ⁇ hogens is subject to different patterns of regulation du ⁇ ng follicle development and atresia
• BMP-4 and BMP-7 with mo ⁇ hogen receptors causes marked stimulatory effects on FSH-mduced estiogen production and inhibitory effects on FSH-induced progesterone production, respectively
• mo ⁇ hogens appear to influence FSH signaling pathways to promote estrogen production, and decrease progesterone production
• Growth hormone and msuhn- ke growth factor-I are potent stimulators of
• IGF-I BMP-4 mRNA levels m human dental pulp fibroblasts cultured in vitro IGF-I is a potent stimulator of rat theca cell function Also, IGF-I expression is strong and weak m healthy and atretic follicles respectively Thus, IGF-I may be a physiological stimulus for mo ⁇ hogens expression du ⁇ ng ovarian follicle growth
• natural-sourced mo ⁇ hogen is a glycosylated dimer, typically having an apparent molecular weight of about 30-36 kDa as determined by SDS-PAGE
• the 30 kDa protein giv es rise to tw o glycosylated peptide subunits having apparent molecular weights of about 16 kDa and 18 kDa
• the unglycosylated protein which also has osteogenic activity, has an apparent molecular weight of about 27 kDa
• the 27 kDa protein gives rise to two unglycosylated polypeptide chains, having molecular weights of about 14 kDa to 16 kDa
• the naturally-occurring morphogens are translated as a precursor, hav mg an N-termmal signal peptide sequence typically less than about 30 residues, followed by a "pro" domain that is cleaved to yield
• Mo ⁇ hogens useful herein include any known naturally-occur ⁇ ng nativ e proteins including allelic, phylogenetic counte ⁇ art and other variants thereof, whether naturally-occurring or biosynthetically produced (e g , including “muteins” or “mutant proteins”), as well as new, osteogemcally active members of the general mo ⁇ hogemc family of proteins
• Particularly useful sequences include those comprising the C-termmal 97 or 102 amino acid sequences of dpp (from Drosophila), Vgl (from Xenopus), Vgr-1 (from mouse), the OP-1 and OP-2 proteins (see U S Patents 5,011 ,691 and 5,266,683, Ozkaynak et al , EMBO J 9 2085-2093 (1990)), as well as the proteins referred to as BMP-2, BMP-3, BMP-4 (see WO 88/00205, U S Patent 5,013,649 and WO 91/18098), BMP-5 and BMP-6 (see
• mo ⁇ hogens include any one of OP-1 , OP-2, OP-3, BMP-2, BMP-4, BMP-5, BMP-6, BMP-9, GDF-5, GDF-6, GDF-7, dpp, Vgl, Vgr, 60A protein, GDF-1, GDF-3, GDF-5, GDF-6, GDF-7, BMP-10, BMP-11, BMP-13, BMP-15, UNIVIN, NODAL, SCREW, ADMP or NURAL and ammo acid sequence variants thereof
• mo ⁇ hogens include any one of OP-1 , OP-2, OP-3, BMP-2, BMP-4, BMP-5, BMP-6, BMP-9, and ammo acid sequence variants and homologs thereof, including species homologs, thereof Publications disclosing OP-1 and OP-2 sequences, as well as their chemical and physical properties
• the candidate sequence and the reference sequence are aligned.
• the first step for performing an alignment is to use an alignment tool, such as the dynamic programming algorithm described in Needleman et al., J. Mol. Biol. 48: 443 (1970), and the Align Program, a commercial software package produced by DNAstar, Inc. the teachings of which are inco ⁇ orated by reference herein.
• an alignment tool such as the dynamic programming algorithm described in Needleman et al., J. Mol. Biol. 48: 443 (1970)
• Align Program a commercial software package produced by DNAstar, Inc. the teachings of which are inco ⁇ orated by reference herein.
• 1A through 1M which is a multiple sequence alignment of a family of known mo ⁇ hogens, including hOP-1. Once the alignment between the candidate and reference sequences is made and refined, a percent homology score is calculated. The individual amino acids of each sequence are compared sequentially according to their similarity to each other.
• Similarity factors include similar size, shape and electrical charge.
• One particularly preferred method of determining amino acid similarities is the PAM25O matrix described in Dayhoff et al., 5 ATLAS OF PROTEIN SEQUENCE AND STRUCTURE 345-352 (1978 & Supp.), inco ⁇ orated by reference herein.
• a similarity score is first calculated as the sum of the aligned pairwise amino acid similarity scores. Insertions and deletions are ignored for the pu ⁇ oses of percent homology and identity. Accordingly, gap penalties are not used in this calculation.
• the raw score is then normalized by dividing it by the geometric mean of the scores of the candidate compound and the seven cysteine skeleton of hOP-1. The geometric mean is the square root of the product of these scores.
• the normalized raw score is the percent homology.
• a functionally-equivalent mo ⁇ hogen sequence shares at least 60% amino acid identity with a reference sequence. That is, any 60% of the aligned amino acids are identical to the corresponding amino acids in the reference sequence.
• any one or more of the naturally-occurring or biosynthetic mo ⁇ hogens disclosed herein may be used as a reference sequence to determine whether a candidate sequence falls within the mo ⁇ hogen family.
• the reference sequence is the C-terminal seven-cysteine skeleton sequence of human OP-1 as shown in SEQ ID NO: 2.
• Examples of conservative substitutions for use in the above calculations include the substitution of one amino acid for another with similar characteristics, e.g., substitutions within the following groups are well-known: (a) valine, glycine; (b) glycine, alanine; (c) valine, isoleucine, leucine; (d) aspartic acid, glutamic acid; (e) asparagine, glutamine; (f) seine, threonine; (g) lysine, arginine, methionine; and (h) phenylalanine, tyrosine.
• substitutions within the following groups are well-known: (a) valine, glycine; (b) glycine, alanine; (c) valine, isoleucine, leucine; (d) aspartic acid, glutamic acid; (e) asparagine, glutamine; (f) seine, threonine; (g) lysine, arginine, methi
• conservative variant also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid in a given polypeptide chain, provided that antibodies having binding specificity for the resulting substituted polypeptide chain also have binding specificity (i.e., "crossreact” or “immunoreact” with) the unsubstituted or parent polypeptide.
• mo ⁇ hogens useful in the resent invention are defined by a generic amino acid sequence that represents variations in known mo ⁇ hogens.
• SEQ ID NOS: 4 and 5 encompass observed variations between preferred mo ⁇ hogens, including OP-1, OP-2, OP-3, CBMP-2A, CBMP-2B, BMP-3, 60A, dpp, Vgl, BMP-5, BMP-6, Vgr-1, and GDF-1.
• SEQ ID NO: 5 includes all of SEQ ID NO: 4, and also includes at its N-terminus the five amino acid sequence of SEQ ID NO: 8.
• the generic sequences include both the amino acid identity shared by these sequences in the C-terminal domain, defined by the six- and seven-cysteine skeletons (SEQ ID NOS: 5 and 6, respectively), and alternative amino acids for variable positions within the sequence. Positions that allow for alternative amino acids are represented by "Xaa”.
• FIG. 2 shows the alternative amino acids for each "Xaa” position in SEQ ID NOS: 5, 6 and 9.
• the "Xaa" at position 2 may be a tyrosine or a lysine.
• the generic sequences provide an appropriate cysteine skeleton for inter- or intramolecular disulfide bonding, and contain certain critical amino acids likely to influence the tertiary structure of the proteins.
• the "Xaa” at position 36 in SEQ ID NO: 5, or at position 41 in SEQ ID NO: 6, may be an additional cysteine, thereby encompassing the mo ⁇ hogenically-active sequences of OP-2 and OP-3.
• useful mo ⁇ hogens include those defined by SEQ ID NOS: 7 or 7, which are composite amino acid sequences of the following mo ⁇ hogens: human OP-1, human OP-2, human OP-3, human BMP-2, human BMP-3, human BMP-4, human BMP-5, human BMP-6, human BMP-8, human BMP-9, human BMP-10, human BMP-11, Drosophila 60A, Xenopus Vg-1, sea urchin UNIVIN, human CDMP-1 (mouse GDF-5), human CDMP-2 (mouse GDF-6, human BMP-13), human CDMP-3 (mouse GDF-7, human BMP- 12), mouse GDF-3, human GDF-1, mouse GDF-1 , chicken DORSALIN, Drosophila dpp, Drosophila
• SEQ ID NO: 8 includes all of SEQ ID NO: 7 and also includes at its N-terminus the five amino acid sequence of SEQ ID NO: 10.
• SEQ ID NO: 7 accommodates the C-terminal six-cysteine skeleton, and SEQ ID NO: 8 accommodates the seven-cysteine skeleton. Positions that allow for alternative amino acids are represented by "Xaa”.
• FIG. 4 shows the alternative amino acids for each "Xaa" position in SEQ ID NOS: 7, 8 and 10.
• useful mo ⁇ hogen sequences useful in this invention have greater than 60% identity, preferably greater than 65% identity, with the amino acid sequence defining the preferred reference sequence of hOP-1.
• These particularly preferred sequences include allelic and phylogenetic variants of the OP-1 and OP-2 proteins, including the Drosophila 60A protein, as well as the closely related proteins BMP-5, BMP-6 and Vgr-1.
• useful mo ⁇ hogens include proteins comprising the generic amino acid sequence SEQ ID NO: 3 (referred to herein as "OPX"), which defines the seven-cysteine skeleton and accommodates the homologies between several identified variants of OP-1 and OP-2. Positions that allow for alternative amino acids are represented by "Xaa”.
• FIG. 4 shows the alternative amino acids for each "Xaa" position in SEQ ID NO: 3.
• useful mo ⁇ hogens include those having an amino acid sequence encoded by a polynucleotide that hybridizes under high stringency conditions with DNA or RNA encoding a reference mo ⁇ hogen.
• Standard stringency conditions are well characterized in standard molecular biology texts. See generally, MOLECULAR CLONING: A LABORATORY MANUAL, (Sambrook et al, eds., 1989); DNA CLONING, Vol. I & II (D.N. Glover ed., 1985); OLIGONUCLEOTIDE SYNTHESIS (M.J. Gait ed., 1984); NUCLEIC ACID HYBRIDIZATION (B. D. Hames and S.J. Higgins eds., 1984); B. Perbal, A
• mo ⁇ hogens useful m the invention include the soluble complex form comprising a mature mo ⁇ hogen dimer linked to a mo ⁇ hogen pro domain or a solubihty-enhancmg fragment thereof
• a solubility-enhancing fragment is any N-termmal or C-termmal fragment of a mo ⁇ hogen pro domain that forms a complex with the mature mo ⁇ hogen dimer and increases the solubility of the mo ⁇ hogen dimer
• the soluble complex comprises a mo ⁇ hogen dimer and two pro domain peptides Mo ⁇ hogen soluble complex is described in published application WO 94/03600, mco ⁇ orated by reference herein
• useful mo ⁇ hogens include biologically active biosynthetic constructs, including novel biosynthetic mo ⁇ hogens and chime ⁇ c proteins designed using sequences from two or more known mo ⁇ hogens See U S Patent 5,011,691, inco ⁇ orated by reference herein (e g , COP-1, COP-3, COP-4, COP-5, COP-7, and COP-16)
• compositions of the present invention may be administered by any route which is compatible with the particular molecules and, when included, with the particular mo ⁇ hogen
• administration may be oral or parenteral, including intravenous and lntrape ⁇ toneal routes of administration.
• administration may be by periodic injections of a bolus of the composition, or may be made more continuous by intravenous or mtrape ⁇ toneal administration from a reservoir which is external (e g , an I v bag) or internal (e g , a bioerodable implant, or a colony of implanted, mo ⁇ hogen-producmg cells)
• compositions of the present invention may be provided to an individual by am suitable means, directly (e g , locally, as by injection, implantation or topical administration to a tissue locus) or systemically (e g , parenteraUy or orally) Where the composition is to be provided parenteraUy, such as by intravenous, subcutaneous, intramolecular, ophthalmic, mtrape ⁇ toneal, intramuscular, buccal, rectal, vaginal, mtraorbital, intracerebral.
• parenteraUy such as by intravenous, subcutaneous, intramolecular, ophthalmic, mtrape ⁇ toneal, intramuscular, buccal, rectal, vaginal, mtraorbital, intracerebral.
• the composition preferably comprises part of an aqueous or physiologically compatible fluid suspension or solution
• the carrier or ⁇ ehicle is phvsiologically acceptable so that in addition to delivery of the desired composition to the patient, it does not otherwise adversely affect the patient's electrolyte and/or volume balance
• the fluid medium for the agent thus can comprise nonrial physiologic saline (e g 9 85% aqueous NaCl, 0 15 M, pH 7-7 4)
• association of the mature mo ⁇ hogen dimer with a mo ⁇ hogen pro domain results in the pro form of the mo ⁇ hogen which typically is more soluble m physiological solutions than the corresponding mature form
• endogenous mo ⁇ hogens are thought to be transported (e g , secreted and circulated) in the mammalian body in this form
• This soluble form of the protein can be obtained from culture medium of mo ⁇ hogen-secretmg mammalian cells, e g cells transfected with nucleic acid encoding and competent to express the mo ⁇ hogen
• a soluble species can be formulated by complexing the mature, mo ⁇ hogemcally-active polypeptide dimer (or an active fragment thereof) with a mo ⁇ hogen pro domain polypeptide or a solubility-enhancing fragment thereof
• Solubility-enhancing pro domain fragments can be any N-terminal, C-termmal or internal fragment of the pro region of a member of the mo ⁇ hogen family that complexes with the mature polypeptide
• Useful solutions for parenteral administration may be prepared by any of the methods w ell known m the pharmaceutical art, described, for example, in REMINGTON'S
• Formulations of the therapeutic agents of the invention may include, for example, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes, and the like
• Formulations for direct administration may include glycerol and other compositions of high viscosity to help maintain the agent at the desired locus Biocompatible, preferably bioresorbable, polymers, including, for example, hyaluromc acid, collagen, t ⁇ calcium phosphate, polybutyrate, lactide, and glycohde polymers and lactide/glycohde copolymers, may be useful excipients to control the release of the agent in vivo
• Other potentially useful parenteral delivery systems for these agents include ethylene-vmyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and hposomes
• Formulations for inhalation administration contain as excipraric acid
• Formulations for topical administration to the skm surface may be prepared by dispersing the molecule capable of releasing mo ⁇ hogen inhibition (alone or in combination with a mo ⁇ hogen) with a dermatologically acceptable carrier such as a lotion, cream, ointment or soap Particularly useful are carriers capable of forming a film or layer over the skm to localize application and inhibit removal
• a dermatologically acceptable carrier such as a lotion, cream, ointment or soap
• the agent may be dispersed in a liquid tissue adhesive or other substance known to enhance adso ⁇ tion to a tissue surface
• tissue-coating solutions such as pectm-containmg formulations may be used
• composition is intended for use as a therapeutic for disorders of the CNS
• an additional problem must be addressed overcoming the blood-bram barner, the bram capillary wail structure that effectively screens out all but selected categories of substances present in the blood, preventing their passage into the bram
• the blood-bram barrier can be bypassed effectively by direct infusion of the molecule capable of releasing mo ⁇ hogen inhibition (alone or in combination with a mo ⁇ hogen) into the bram, or by mtranasal administration or inhalation of formulations suitable for uptake and retrograde transport by olfactory neurons D.
• Bioassav of Osteogenic Activitv Endochondral Bone Formation and Related Properties
• the assay consists of depositing test samples in subcutaneous sites in recipient rats under ether anesthesia A vertical incision (1 cm) is made under ste ⁇ le conditions in the skm over the thoracic region, and a pocket is prepared by blunt dissection In certain circumstances, approximately 25 mg of the test sample is implanted deep into the pocket and the incision is closed with a metallic skm clip
• the heterotropic site allows for the study of bone induction without the possible ambiguities resulting from the use of orthotopic sites
• the sequential cellular reactions occur ⁇ ng at the heterotropic site are complex
• the multi-step cascade of endochondral bone formation includes binding of fib ⁇ n and fibronectm to implanted matrix, chemotaxis of cells, proliferation of fibroblasts, differentiation into chondroblasts, cartilage formation, vascular invasion, bone formation, remodeling, and bone marrow differentiation
• Successful implants exhibit a controlled progression through the stages of protem-mduced endochondral bone development including (1) transient infiltration by polymo ⁇ honuclear leukocytes on about day one, (2) mesenchymal cell migration and proliferation on about days two and three, (3) chondrocyte appearance on about days five and six, (4) cartilage matrix formation on about day seven, (5) cartilage calcification on about day eight, (6) vascular invasion, appearance of osteoblasts, and formation of a new bone on about days nine and ten, (7) appearance of osteoblastic and bone remodeling on about days twelve to eighteen, and (8) hematopoietic bone marrow differentiation in the ossicle on about day twenty-one The time course of this process varies according to the matrix
• the assay is useful for quantitation and obtaining an estimate of bone formation very quickly after the test samples are removed from the rat.
• samples containing mo ⁇ hogen at several levels of purity have been tested to determine the most effective dose/purity level, in order to seek a formulation that could be produced on an industrial scale.
• the results as measured by alkaline phosphatase activity level and histological evaluation can be represented as "bone forming units".
• One bone- forming unit represents the amount of protein that is needed for half maximal bone forming activity on day 12.
• dose curves can be constructed for bone inducing activity in vivo at each step of a purification scheme by assaying various concentrations of protein. Accordingly, the skilled artisan can construct representative dose curves using only routine experimentation.
• In situ hybridization was used to determine the localization and level of expression of mo ⁇ hogens and mo ⁇ hogen receptors in adult rat ovaries. Probes to the mo ⁇ hogens, BMP-4 and BMP-7, and probes to the type IA, type IB, and type II mo ⁇ hogen receptors were used.
• Ovine FSH (NIDDK-oFSH-S 1 , 4453 IU/mg) was supplied by the National Hormone and Pituitary Program of the NIDDK (Rockville, MD). McCoy's 5a medium, Medium 199, and dinucleotide triphosphates were purchased from Gibco BRL (Grand Island, NY). Cell culture plates were purchased from Falcon (Lincoln Park, NJ). Reagents for RT-PCR were obtained from Perkin Elmer (Foster City, CA). Cell culture
• Sprague-Dawley rats (Harlan Industries, Indianapolis, IN) were implanted with silastic capsules containing 10 mg of diethylstilbestrol (DES) to increase granulosa cell number. Ovaries were removed and the granulosa cells isolated and cultured as previously described by Erickson and Hsueh, Endocrinology 102, 1275-1282 (1978).
• DES diethylstilbestrol
• Granulosa cells (5 x 10 4 viable cells) were pipetted into 96-well culture plates containing 200 : 1 (final volume) of tissue culture medium (McCoy's 5a Medium containing 100 U/ml penicillin, 100 mg/ml streptomycin sulfate, 2 mM L-glutamine, and 1 ⁇ M androstenedione).
• tissue culture medium McCoy's 5a Medium containing 100 U/ml penicillin, 100 mg/ml streptomycin sulfate, 2 mM L-glutamine, and 1 ⁇ M androstenedione.
• Granulosa cells were cultured for up to 48 hr at 37 ° C in water-saturated atmosphere containing 5% CO 2 in air with the indicated concentrations of FSH, BMP-4, BMP-7, or activin-A.
• the levels of progesterone and estrogen in the media were measured by radioimmunoassay as previously described by Wang et al.,
• RNA from 27-day old rat ovaries was prepared. Single-stranded cDNA was synthesized by reverse transcriptase and then subjected to PCR as described previously by Shimasaki et al, J Biol Chem 266, 10646-10653 (1991).
• DNA sequences of rat BMP-4 and BMPR-IA were obtained from GenBank.
• PCR primers for rat BMP-7, BMPR-IB, and BMPR-II were designed by choosing the homologous DNA sequence regions between human and mouse homologues of BMP-7, BMPR-IB and BMPR-II cDNAs which were available from GenBank.
• these primers are derived from the cDNA clones at nucleotides 737-757 and 1 181-1200 (accession number of the cDNA clone is Z22607) for BMP-4 (Chen et al, Biochem. Biophys.
• PCR was performed under the following conditions: 35 cycles, annealing at 50 ° C for 30 sec; extension at 72 ° C for 30 sec; denaturation at 94 C for 30 sec All PCR products were cloned into pBluesc ⁇ pt SK+ plasmid and their DNA sequences confirmed
• RNA probe 8 ⁇ m were cut from each ovary and mounted onto poly-L lysine coated glass slides The sections were digested with protemase K, acetylated, washed and dehydrated Each antisense and sense cRNA probe was prepared by means of in vitro transc ⁇ ption using T3 or T7 RNA polymerase Hybridization was carried out with the 3:, S-labeled RNA probe (4-6 x 10 6 cpm/ml) in a solution containing 50%> (vol/vol) deiomzed formamide, 0 3 M NaCl, 10 mM
• Tns (pH 8 2), 1 mM EDTA, 0 05% yeast tRNA, 10 mM dithiothreitol, 1 x Denhardt's solution and 10%o dextran sulfate Hybridization solution (20 ⁇ l) was placed over each section and covered with a 60 x 22 mm acid washed, sihconized covershp Covershps were sealed with liquid DPX Sections were hybridized for 16 hr at 58-60°C in a humidified chamber After hybridization, the sections were treated with ⁇ bonuclease A and washed in 15 mM
• BMP-7 The mRNAs foi BMP-4, BMP-7, and the BMPR-IA, BMPR-IB, and BMPR-II receptors weie expiessed in a tissue-specific manner in the adult lat ovary
• BMP-7 mRNA was present m the theca interstitial cells of healthy Graafian (dominant) follicles, but was undetectable in other ovarv cell types
• Hybridization with the control sense BMP-7 cRNA probe showed a nonspecific background signal, this was true for the other control sense probes used m these experiments
• BMP-4 mRNA was also expressed strongly in the theca cells of the dominant Graafian follicles, being present in both the theca interstitial and theca externa cells
• a weak but variable BMP-4 signal was observed in some co ⁇ ora lutea and surface epithelial cells BMP-4 mRNA was not detectable in the other ovarian cell types
• BMPR-IA and BMPR-IB are widely expressed in the rat ovary, with the strongest hybridization signals being observed in the granulosa cells and oocytes of developing follicles
• the intensity of the signals for BMPR-IB were higher than those for BMPR-IA
• Hybridization signals for BMPR-II were most intense m the granulosa cells of all growing follicles (healthy and atretic) after the secondary stage
• the BMPR-II message was weakly expiessed m some co ⁇ ora lutea
• a weak BMPR-II signal was observed in growing oocytes of primary follicles (those with a single layer of cuboidal granulosa cells), but none was observed in oocytes m late pre-antral and Graafian follicles No BMPR-II signal above background was observed m the other ovary cell types
• Mo ⁇ hogens are expressed strongly in the ovary, being prominent in thecal cells High levels of mo ⁇ hogen receptor expression found in granulosa cells Mo ⁇ hogen receptor (BMPR-IA, BMPR-IB, and BMPR-II) mRNAs are uniformly expressed at high levels m all granulosa cells m all follicles, healthy as well as atretic, suggesting that the granulosa cells are important targets for mo ⁇ hogen signaling These results suggest a parac ⁇ ne role for mo ⁇ hogens in regulating ovarian follicle growth
• Example 1 1 suggest a parac ⁇ ne role for mo ⁇ hogens whereby mo ⁇ hogens produced by thecal cells interact with mo ⁇ hogen receptors in the granulosa cells to regulate biological responses by a parac ⁇ ne mechanism
• BMP-4 and BMP-7 were assessed on basal and FSH-stimulated estrogen and progesterone production in primary cultures of rat granulosa cells grown in serum-free medium
• BMP-4 and BMP-7 also caused a significant increase
• BMPR-IA, BMPR-IB, and BMPR-II expression is strongest in the granulosa cells suggests that these cells are important targets for mo ⁇ hogen signaling.
• Mo ⁇ hogen receptor mRNAs are uniformly expressed at high levels in all granulosa cells in all follicles, healthy as well as atretic. These results implicate mo ⁇ hogens in normal follicle development and in the events leading to follicle death by apoptosis.
• SMAD proteins are negative regulator for the FSH signals that induce enzymes in the progesterone biosynthetic pathway, including StAR (steroidogenic acute regulatory protein), P450 scc (side-chain cleavage) or ⁇ -hydroxysteroid dehydrogenase.
• FSH ⁇ LUC Pituitary cells from transgenic mice harboring FSH ⁇ LUC were dispersed and cultured for two days before treatments.
• the FSH ⁇ LUC construct contains an ovine FSH ⁇ promoter driving a luciferase gene and functions in several transgenic mouse lines. Luciferase was expressed only in the pituitary and was regulated as if it were FSH ⁇ itself. Therefore, luciferase activity seems to reflect normal FSH expression.
• follistatin follicle stimulating hormone suppressing protein
• FSP follicle stimulating hormone suppressing protein
• OP-1 , BMP-6, or activm- A was added Four hours after addition of a mo ⁇ hogen or activin-A, the cells were harvested and assayed for luciferase activitv
• the data are representative of >3 individual experiments As illustrated m FIG 7, OP-1, BMP-6, and activm-A enhanced FSH expression in these mouse pituitary cell lines
• the mo ⁇ hogen effect on FSH expression was dose-dependent Cultured pituitary cells from FSH ⁇ LUC transgenic mice were treated with rabbit ant ⁇ -mOP-1 (0 1 to 10 ⁇ l/ml) or follistatin (250 ng/ml) for 24 hr After a 24 hr incubation, luciferase activity was measured As depicted in FIG 8, the ant ⁇ -mOP
• Example 2 1 In order to assess the specificity of the enhanced FSH expression by a mo ⁇ hogen and activin observed Example 2 1 , the cultured pituitary cells from FSH ⁇ Luc transgenic mice were treated for 24 hr with antibodies to mOP- 1 , activin- A, or activin-B and then assayed for luciferase activity
• the rabbit and sheep ant ⁇ -mOP-1 antibodies were both capable of blocking the FSH expression in mouse pituitary cultures
• neither anti-activin-A antisera nor anti-activin-B antisera significantly inhibit FSH ⁇ LUC in the mouse pituitary system
• FSH ⁇ LUC activity was mildly blocked by monoclonal antibody IB 12 at 100 ⁇ g/ml (weak inhibition) but not by the 12G3 monoclonal antibody
• Follicle-stimulating hormone is produced m pituitary gonadotropes as an ⁇ / ⁇ heterodimer, and synthesis of the ⁇ subunit is the rate-limiting step in overall FSH production
• FSH ⁇ is regulated by activm and inhibin, both of which are members of the transforming growth factor ⁇ (TGF ⁇ ) superfamily
• TGF ⁇ transforming growth factor ⁇
• BMPs Bone mo ⁇ hogenetic proteins
• BMP-7 or BMP-6 was found to stimulate oFSH ⁇ Luc expression by 6-fold Also transient expression of the oFSH ⁇ Luc m a transformed gonadotrope cell line, L ⁇ T2, was induced 4-fold by BMP-7 or BMP-6 treatment Both BMP-7 and BMP-6 increased FSH secretion from L ⁇ T2 cells, demonstrating for the first time that a functional BMP system is present m gonadotropes
• Two neutralizing antibodies to BMP-7 which cross-react with BMP-6 but not with activm A, decreased the basal expression of oFSH ⁇ Luc in transgenic mouse pituitary cultures by 80- 90%, suggesting an autocrme or paracrme role for BMP-7 or BMP-6 in FSH synthesis
• FSH folhcle-stimulatmg hormone
• GnRH gonadotropin releasing hormone
• GnRH can induce FSH ⁇ transcription by 2- to 3-fold in vivo and in tissue culture of primary gonadotropes or non- gonadotropes fortified with GnRH receptors
• mice were produced that express luciferase under control of 4 7 kb of the ovine FSH ⁇ promoter (oFSH ⁇ LUC) with or without functioning AP-1 sites (-120/-83) Luciferase was expressed in these mice (+/- AP-1 sites) only in the pituitary and regulated in vivo as if it were FSH ⁇ , itself (+/- AP-1 sites).
• oFSH ⁇ LUC ovine FSH ⁇ promoter
• Luciferase was expressed in these mice (+/- AP-1 sites) only in the pituitary and regulated in vivo as if it were FSH ⁇ , itself (+/- AP-1 sites).
• BMP6 and BMP7 could stimulate FSH ⁇ transcription 8- to 12-fold. Future studies will define activin/BMP response element(s) and use transgenic technology to determine the physiological relevance of activin/BMPs to reproductive function.

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