JP2008297313A - Methods of modulating apoptosis by administration of relaxin agonists or antagonists - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of modulating apoptosis by administration of relaxin agonists or antagonists to a subject. <P>SOLUTION: This is a method for modulating apoptosis, and the method includes a step of administrating relaxin agonists, to a subject who needs the same, in an effective amount and for a period of time sufficient to reduce apoptosis in a cell which develops a relaxin receptor. Accumulation of a relaxin responsive cell results in abnormalities of body tissues. Therefore, the method of modulating apoptosis in tissues by administrating relaxin agonists or relaxinantagonists is provided. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

(Cross-reference of related applications)
No. 60 / 238,232 (filed Oct. 4, 2000), U.S. Patent Provisional Application No. 60 / 241,991 (filed Oct. 20, 2000) and U.S. Provisional Patent Application No. 60 / 238,232. Claims priority from 60 / 242,037 (filed Oct. 20, 2000), the disclosures of which are incorporated herein by reference.

(Background of the Invention)
Normal tissue growth and development is achieved by programmed cell growth, differentiation and cell death. Cell proliferation and differentiation is required for the formation of new cells and tissues. Conversely, programmed cell death (also called apoptosis) is required to remove existing cells (including immature cells or damaged cells). Apoptosis occurs naturally in virtually all tissues of the body. Apoptosis plays an important role in tissue homeostasis, ie, ensures that the number of new cells produced is counterbalanced by an equal number of dead cells. For example, cells in the intestinal lining divide rapidly, so the body must eliminate cells after only 3 days to prevent overgrowth of the intestinal lining.

  Disruption of the genetic program, either by abnormally increasing or decreasing rates of cell proliferation and / or apoptosis, can result in abnormal tissue development. For example, a decrease in cell proliferation below normal levels can lead to immature tissue and other tissue abnormalities. Increased cell proliferation above normal levels is believed to be a major event in the progression of neoplasia and cancer, as well as other cell proliferative disorders. Abnormal increases in apoptosis can also lead to precancerous lesions. Precancerous lesions include breast lesions (which can develop into breast cancer), prostate lesions (which can develop into prostate cancer) or skin lesions (which can develop into malignant melanoma or basal cell carcinoma), Examples include colon adenomatous polyps (which can develop into colon cancer), and other such neoplasias. Such lesions show a strong tendency to develop into malignant tumors or cancers.

  Precancerous lesions can result from the accumulation of damage to existing cells in various tissues of the body. Such injury may include exposure to sunlight, radiation, mutagens and carcinogens normally found in the diet, chemicals (eg insecticides, herbicides, preservatives) and the like. These injuries can result in accumulation of mutations in the cells, which are hyperplastic (ie, an abnormal increase in the number of cells) (eg, excessive tissue in the liver, kidney, spleen, thymus, intestine, lung or prostate tissue). Like formation). Down-regulation of apoptosis can also lead to cell accumulation in these hyperplastic states.

  Abnormal increases in apoptosis can interfere with normal development and / or differentiation of tissues. For example, apoptosis is required during pregnancy and maturation of male genital tract tissue. An abnormal increase in apoptosis can also interfere with the formation of new cells and tissues, thereby preventing normal tissue maturation or development.

  Accordingly, there is a need for methods of modulating apoptosis by administering an agonist or antagonist of apoptosis. In particular, there is a need for methods of treating conditions associated with abnormally fast or slow apoptosis (directly or indirectly). The present invention satisfies this need by providing a method for the administration of relaxin agonists or antagonists to treat such tissue abnormalities by modulating apoptosis in relaxin-related tissues.

(Summary of the Invention)
The present invention relates to the discovery that relaxin is associated with the development of body tissue. Knockout of the gene encoding relaxin results in various abnormalities in the development of various body tissues (decreasing the size of such tissues, increasing collagen deposition in such tissues, and immaturity of such tissues) . Conversely, the accumulation of relaxin-responsive cells leads to abnormalities in body tissues. The present invention provides a method of modulating apoptosis in a tissue by administering a relaxin agonist or relaxin antagonist.

  In one aspect, the present invention provides a method of modulating apoptosis in a subject, which method is sufficient for a subject in need thereof to reduce apoptosis in a cell expressing a relaxin receptor. By administering an effective amount of a relaxin agonist over time. Tissues typically affected by administration of a relaxin agonist useful in the method according to the present invention include, for example, liver, kidney, spleen, thymus, brain, heart, intestine, skin, lung, male reproductive tract and female reproductive tract. Is mentioned. Examples of male genital tract tissues include prostate, epididymis, semen tissue, or testicular tissue. Examples of female genital tract tissues include uterus, cervix, pubic mesentery, or connective tissue in the lower limbs.

  In certain embodiments, administration of a relaxin agonist typically reduces the number of apoptotic cells. In other embodiments, relaxin agonists stimulate tissue maturation. For example, relaxin agonists can stimulate the maturation of male genital tract tissue (eg, prostate tissue, epididymis tissue, ganglia tissue, testis tissue or semen). In one embodiment, the maturation results in an increase in the number of cells in the underdeveloped testis (eg, an increase in the number of mature testis cells). In another embodiment, maturation of male genital tract tissue results in an increase in the number of live sperm cells compared to tissue not contacted with a relaxin agonist. In yet another embodiment, fibrosis can be reduced by a relaxin agonist and / or excessive collagen deposition can be reduced.

  A subject in need of administration of a relaxin agonist may have a relaxin deficient condition (such as immature tissue, excessive collagen deposition and / or low sperm count). For example, the immature tissue can be an immature male reproductive tract (eg, a testicular underdevelopment). Such immature tissue may be present in other mature animals or immature animals.

  The relaxin agonist can be relaxin, a relaxin analog, a small relaxin effector or a relaxin nucleic acid. Relaxin is typically vertebrate relaxin, and more typically human relaxin.

  A composition comprising a relaxin agonist useful in a method according to the present invention is for example administered by injection, injection, oral delivery, intranasal delivery, and administration by pulmonary delivery, rectal delivery, transdermal delivery, intra-tissue delivery or subcutaneous delivery Can be prescribed for. Such compositions can also be formulated for delayed release of relaxin agonists into the tissues and circulation of the subject. Certain embodiments include compositions formulated for infusion or injection or pulmonary delivery, subcutaneous delivery or transdermal delivery. In certain embodiments, a nucleic acid encoding a relaxin agonist can be formulated for administration to a subject with a vector encoding a relaxin agonist. For example, the vector can be an expression vector that expresses a relaxin agonist in a cell. Alternatively, relaxin or relaxin analog nucleic acids can be formulated for delivery to a subject. The subject can be before or after puberty.

  In another aspect, the present invention provides a method for modulating apoptosis in a subject in need thereof, wherein the method is effective for a time sufficient to increase apoptosis in a cell population expressing a relaxin receptor. By administering an amount of an antagonist of relaxin. In one embodiment, the relaxin antagonist inhibits the binding of relaxin to the relaxin receptor. In another embodiment, the relaxin antagonist reduces relaxin-related tissue remodeling.

  Tissues typically affected by administration of a relaxin antagonist useful in the method according to the present invention include, for example, liver, kidney, spleen, thymus, brain, heart, intestine, skin, lung, male reproductive tract, female reproductive tract Etc. Examples of male genital tract tissues include prostate, epididymis, semen tissue, or testicular tissue. In certain embodiments, the male reproductive tract tissue can be prostate tissue and can be mature or immature. Suitable target female reproductive tract tissues include, for example, the uterus, cervix, mesothelium, or connective tissue in the lower limbs. Alternatively, the cell population can include cells that express a relaxin receptor, such as fibroblasts, osteoblasts, monocytes, epithelial cells or endothelial cells.

The relaxin antagonist can be, for example, a relaxin binding factor, a relaxin receptor binding factor, a relaxin antisense nucleic acid, and the like. The relaxin binding factor can be, for example, an anti-relaxin antibody, a soluble relaxin receptor, a small molecule relaxin antagonist, and the like. The relaxin receptor binding factor can be, for example, an anti-relaxin receptor antibody, a relaxin analog, a small molecule relaxin receptor antagonist, and the like. Examples of relaxin or an antibody that binds to relaxin include a polyclonal antibody, a monoclonal antibody, Fab, Fab ′, F (ab ′) ₂ , Fv, single heavy chain, chimeric antibody, and the like.

  Compositions comprising relaxin antagonists useful in the methods of the invention can be administered by, for example, infusion, injection, oral delivery, intranasal delivery, and administration by pulmonary delivery, rectal delivery, transdermal delivery, intra-tissue delivery or subcutaneous delivery. Can be prescribed for. The composition may also be formulated for delayed release of a relaxin antagonist into the tissue and circulation of the subject. Certain embodiments include compositions formulated for infusion or injection or pulmonary delivery, subcutaneous delivery or transdermal delivery.

In certain embodiments, a nucleic acid encoding a relaxin antagonist can be administered to a subject with a vector encoding a relaxin antagonist. In one embodiment, the vector is an expression vector that expresses a relaxin antagonist in a cell population. Alternatively, relaxin or relaxin receptor antisense nucleic acids can be delivered directly to a subject according to any of the methods described above. In an exemplary embodiment, administration of a relaxin antagonist increases apoptosis and decreases unwanted cell accumulation. For example, these unwanted cells can be hyperplasia, hypertrophy, cancer or neoplasia.
In addition to the above, the present invention provides the following:
(Item 1) A method of regulating apoptosis, comprising:
Administering to a subject in need thereof an effective amount of a relaxin agonist for a time sufficient to reduce apoptosis in a cell expressing a relaxin receptor;
Including the method.
(Item 2) The method according to item 1, wherein the cell is derived from a liver, kidney, spleen, thymus, brain, heart, intestine, skin, lung, female reproductive tract or male reproductive tract.
(Item 3) The method according to item 2, wherein the male reproductive tract tissue is prostate, epididymis, seminal vesicle, or testis.
(Item 4) The method according to item 2, wherein the female reproductive tract tissue is connective tissue of the uterus, cervix, mesentery, or lower limbs.
(Item 5) The method according to item 2, wherein the number of apoptotic cells is decreased.
(Item 6) The method according to item 1, wherein tissue maturation is stimulated.
(Item 7) The method according to item 6, wherein fibrosis is reduced.
(Item 8) The method according to item 6, wherein the tissue is a male reproductive tract tissue.
(Item 9) The method according to item 8, wherein the tissue is prostate tissue, epididymis tissue, semen tissue, testis tissue or semen.
(Item 10) The method according to item 9, wherein the tissue is testis tissue.
(Item 11) The method according to item 10, wherein the maturation is an increase in the number of cells in a testis with developmental failure.
(Item 12) The method according to item 9, wherein the number of live sperm cells is increased.
(Item 13) The method according to item 1, wherein the subject has a relaxin-deficient state.
(Item 14) The method according to item 13, wherein the relaxin-deficient state is immature tissue, excessive collagen deposition or a low sperm count.
(Item 15) The method according to item 14, wherein the immature tissue is immature male reproductive tract tissue.
(Item 16) The method according to item 15, wherein the immature male genital tract tissue is a developmentally defective testis.
(Item 17) The method according to item 1, wherein excessive collagen deposition is reduced.
(Item 18) The method according to item 1, wherein the relaxin agonist is relaxin, a relaxin analog, a small molecule relaxin effector, or a relaxin nucleic acid.
(Item 19) The method according to item 18, wherein the relaxin is a vertebrate relaxin.
(Item 20) The method according to item 19, wherein the relaxin is human relaxin.
21. The method of claim 1, wherein the administering step is by infusion, injection, oral delivery, intranasal delivery, intrapulmonary delivery, rectal delivery, transdermal delivery, intratissue delivery or subcutaneous delivery.
22. The method of claim 21, wherein the administering step is by delayed release delivery.
23. The method of claim 21, wherein the subcutaneous delivery is by infusion or injection.
24. The method of claim 21, wherein the administering step is by pulmonary delivery, subcutaneous delivery or transdermal delivery.
25. The method of claim 1, wherein the administering step comprises delivery of a vector encoding the relaxin agonist.
26. The method of claim 1, wherein the vector is an expression vector, whereby the relaxin agonist is expressed in the cell.
27. The method of claim 1, wherein the administering step comprises delivery of a relaxin or relaxin analog nucleic acid.
(Item 28) The method according to item 1, wherein the subject is a subject after puberty.
(Item 29) The method according to item 1, wherein the subject is a subject before puberty.
30. A method for modulating apoptosis in a population of cells expressing a relaxin receptor, comprising:
Administering to a subject in need thereof an effective amount of an antagonist of relaxin for a time sufficient to increase apoptosis in a population of cells expressing the relaxin receptor;
Including the method.
31. The method of claim 30, wherein the relaxin antagonist inhibits the binding of relaxin to a relaxin receptor.
32. The method of claim 30, wherein the relaxin antagonist reduces relaxin-related tissue remodeling.
(Item 33) The method according to item 30, wherein the cell population is derived from heart, brain, liver, kidney, spleen, thymus, skin, female reproductive tract tissue or male reproductive tract tissue.
(Item 34) The method according to item 33, wherein the male reproductive tract tissue is prostate, epididymis, seminal vesicle or testis.
35. The method of claim 34, wherein the tissue is prostate tissue.
36. The method of claim 33, wherein the male genital tract tissue is mature.
37. The method of claim 33, wherein the female genital tract tissue is connective tissue of the uterus, cervix, mesentery, or lower limbs.
38. The method of claim 30, wherein the cell population comprises fibroblasts, osteoblasts, monocytes, epithelial cells or endothelial cells.
(Item 39) The method according to item 30, wherein the relaxin antagonist is a relaxin binding factor, a relaxin receptor binding factor, or a relaxin antisense nucleic acid.
40. The method of claim 39, wherein the relaxin binding factor is an anti-relaxin antibody, a soluble relaxin receptor, or a small molecule relaxin antagonist.
41. The method of claim 40, wherein the antibody is a monoclonal antibody, polyclonal antibody, single chain antibody, Fab, Fab ′, F (ab ′) ₂ , Fv, single heavy chain, or chimeric antibody.
42. The method of claim 39, wherein the relaxin receptor binding factor is an anti-relaxin receptor antibody, a relaxin analog, or a small molecule relaxin receptor antagonist.
43. The method of claim 42, wherein the antibody is a monoclonal antibody, polyclonal antibody, single chain antibody, Fab, Fab ′, F (ab ′) ₂ , Fv, single heavy chain, or chimeric antibody.
44. The method of claim 30, wherein the administering step is by infusion, injection, oral delivery, intranasal delivery, intrapulmonary delivery, rectal delivery, transdermal delivery, intra-tissue delivery or subcutaneous delivery.
45. The method of claim 44, wherein the administering step is by delayed release delivery.
46. The method of claim 44, wherein the subcutaneous delivery is by infusion or injection.
47. The method of claim 44, wherein the administering step is by pulmonary delivery, subcutaneous delivery or transdermal delivery.
48. The method of claim 30, wherein the administering step comprises delivery of a vector encoding the relaxin antagonist.
49. The method of claim 30, wherein the vector is an expression vector, whereby the relaxin antagonist is expressed in the cell population.
50. The method of claim 30, wherein the administering step comprises delivery of relaxin or a relaxin receptor antisense nucleic acid.
51. The method of claim 30, wherein increased apoptosis reduces unwanted cell accumulation.
52. The method of claim 51, wherein the unwanted cell accumulation is hyperplasia, hypertrophy, cancer or neoplasia.
53. Use of a therapeutically effective amount of a relaxin antagonist for the manufacture of a medicament for reducing or preventing relaxin-related abnormalities.
54. The use according to claim 53, wherein the relaxin-related abnormality is relaxin-responsive cell accumulation, hyperplasia, hypertrophy, cancer or neoplasia in a subject.
55. The use according to claim 53 or 54, wherein the relaxin antagonist is a relaxin binding factor, a relaxin receptor binding factor, or a relaxin antisense nucleic acid.
(Item 56) The use according to item 55, wherein the relaxin-binding factor is an anti-relaxin antibody, a soluble relaxin receptor, or a small molecule relaxin antagonist.
(Item 57) The use according to item 56, wherein the antibody is a monoclonal antibody, a polyclonal antibody, a single chain antibody, Fab, F (ab ′) ₂ , Fv, a single heavy chain, or a chimeric antibody.
58. The use according to claim 55, wherein the relaxin receptor binding factor is an anti-relaxin receptor antibody, relaxin analog, or small molecule relaxin receptor antagonist.
59. The use according to claim 58, wherein the antibody is a monoclonal antibody, a polyclonal antibody, a single chain antibody, Fab, F (ab ′) ₂ , Fv, a single heavy chain, or a chimeric antibody.
60. The use of claim 53 or 54, wherein the relaxin antagonist inhibits the binding of relaxin to a relaxin receptor.
61. The item 53 or 54, wherein the medicament is formulated for infusion, injection, oral delivery, intranasal delivery, intrapulmonary delivery, rectal delivery, transdermal delivery, intra-tissue delivery or subcutaneous delivery. use.
62. The use of claim 61, wherein the medicament is formulated for delayed release delivery.
63. The use of claim 53 or 54, wherein the medicament comprises a vector encoding the relaxin antagonist.
64. The use of claim 63, wherein the vector is an expression vector, whereby the relaxin antagonist is expressed in the cell population.
65. The use of claim 64, wherein the vector comprises relaxin or a relaxin receptor antisense nucleic acid.
(Item 66) The use according to item 53 or 54, wherein the tissue is prostate tissue, epididymis tissue, transfusion tissue or testis tissue.
(Item 67) The use according to item 53 or 54, wherein the subject is a subject after puberty.
(Item 68) The use according to item 53 or 54, wherein the subject is a subject before puberty.

(Definition)
Prior to presenting the present invention in more detail, it may be helpful to provide a further understanding of the present invention by providing definitions of certain terms as used herein below.

The term “nucleic acid” refers to a polymer composed of a plurality of nucleotide units (ribonucleotides or deoxyribonucleotides or related structural variants) linked through phosphodiester bonds. Nucleic acids can be of virtually any length, typically from about 6 nucleotides to about 10 ⁹ nucleotides, or more. Unless indicated otherwise, the conventional notation used herein for nucleic acids is as follows: the left-hand end of a single-stranded nucleic acid is the 5 ′ end; the left-hand direction of a double-stranded nucleic acid is 5 'Called the direction. The direction of 5 ′ to 3 ′ addition of the initial RNA transcript is referred to as the transcription direction. The sequence region of the DNA strand that has the same sequence as the RNA and that is 5 ′ to the 5 ′ end of the RNA transcript is referred to as the “upstream sequence”; it has the same sequence as the RNA and the coding RNA The sequence region of the DNA strand that is 3 ′ to the 3 ′ end of the transcript is referred to as the “downstream sequence”.

  As will be readily understood by those skilled in the art, nucleic acids include RNA, mRNA, cDNA, genomic DNA, synthetic forms, and mixed polymers (both sense and antisense strands), and nucleic acids are also chemically May be modified either chemically or biochemically, or may include non-natural or derivatized nucleotide bases. Such modifications include, for example, labeling, methylation, substitution of one or more naturally occurring nucleotides by analogs, internucleotide modifications (eg, uncharged linkages (eg, methylphosphonates, phosphotriesters, phosphoamidates). , And carbamates), charged linkages (eg, phosphorothioates and phosphorodithioates), pendant moieties (eg, polypeptides), intercalators (eg, acridine and psoralen), chelating agents, alkylators, and modified linkages ( For example, α anomeric nucleic acid)). Also included are synthetic molecules that mimic nucleic acids in their ability to bind to the indicated sequence through hydrogen bonding and other chemical interactions. Such molecules are known in the art, and include, for example, peptides in which peptide bonds are substituted in the backbone of the molecule instead of phosphate bonds.

  As used herein, the term “amino acid” or “amino acid residue” refers to an L amino acid or a D amino acid as further described below. Commonly used amino acid one-letter and three-letter abbreviations are used herein (eg, Alberts et al., Molecular Biology of the Cell, Garland Publishing, Inc., New York (3rd edition, 1994)).

  When described for a polypeptide, the term “conservative substitution” refers to a change in the amino acid composition of a polypeptide that does not substantially alter the activity of the polypeptide. Thus, “conservative substitutions” of specific amino acid sequences are those amino acid substitutions or similar properties that are not critical to polypeptide activity (eg, acidic, basic, positive or negative charge, polar or nonpolar, etc.) As a result, the substitution of amino acids by other amino acids that does not substantially alter activity even by substitution of important amino acids. Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, each of the following six groups includes amino acids that are conservatively substituted for each other: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E) 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) Phenylalanine (F), Threonine (Y), Tryptophan (W) (See also Creighton, Proteins, WH Freeman and Company (1984), incorporated herein by reference). In addition, individual substitutions, deletions or additions that alter, add or delete a single amino acid or a few percent of amino acids in an encoded sequence are also “conservative substitutions”.

  The term “relaxin analog” refers to a modified relaxin polypeptide that increases or decreases the functional activity of this molecule or its interaction with the relaxin receptor.

  The term “soluble” refers to the ability of a polypeptide to be dissolved (ie, molecularly or ionically dispersed) in an aqueous solution (eg, water, blood, or plasma) in the context of the polypeptide.

  The term “small molecule effector” refers to an agent that, in the context of relaxin or relaxin receptor, binds to relaxin or relaxin receptor and stimulates the activity of relaxin or relaxin receptor.

  The term “small molecule antagonist” refers to an agent that, in the context of relaxin or relaxin receptor, binds to relaxin or relaxin receptor and reduces or inhibits the activity of relaxin or relaxin receptor.

  The term “effective amount” refers to a dosage sufficient to provide relief from symptoms or treatment of an abnormal condition (eg, disease, disorder, or condition) that is to be treated. This dosage will vary depending on the subject, the abnormal condition being treated, and the treatment being effected.

  The terms “subnormal”, “subnormal” and “not fully developed” in the context of body tissue and / or cells refer to the tissues and / or cells of a normal subject of the same age and species of the subject. Refers to smaller size, decreased number of cells, and / or immature developmental state compared to size, cell number, and / or developmental state.

  The term “tissue” generally refers to a population of cells consisting of cells or cells of the same species that perform the same or similar function. The tissue can be part of an organ or bone, or can be a loose connection of cells (eg, cells of the immune system).

  The term “maturation” refers to the evolutionary progression and / or differentiation of a tissue to a fully developed state in the context of body tissue.

  The term “mature” refers to achieving full development, differentiation, and / or growth of tissue in the context of body tissue.

  The term “immature” refers to tissue that is not fully developed and / or differentiated in the context of body tissue.

  The term “pre-pubertal male” refers to a male who has not completely completed the process of puberty.

  The term “post-pubertal male” refers to a male who has completed the process of puberty.

  The term “puberty” refers to a series of phenomena in which a child becomes a young adult, characterized by gametogenesis, secretion of gonadal hormones, development of secondary sexual characteristics, and onset of reproductive function.

  The term “accumulation” refers to an increase in the number of normal (ie, non-mutated, non-malignant) cells in a tissue in the context of body tissue.

  The terms “biologically active” and “functionally active” refer to the ability of a molecule (eg, a relaxin agonist or relaxin antagonist) to bind relaxin or a relaxin receptor in body tissue, and apoptosis, cell accumulation, And / or ability to stimulate or inhibit tissue maturation.

  The term “relaxin deficient state” refers to a disease, disorder, or condition in a subject that has a relaxin level or relaxin receptor level that is less than normal in the associated tissue, cell, or subject.

  The term “relaxin responsiveness” refers to an increase in cell number or maturity status (ie, tissue development and / or differentiation) in response to the binding of relaxin to the relaxin receptor.

  The term “relaxin related” refers to a cell or tissue that is directly or jointly affected by an increase or decrease in functionally active relaxin levels or functionally active relaxin receptors. A characteristic, state, or response.

  The term “hyperplasia” or “hyperplastic tissue” refers to an increase in the number of cells in a tissue.

  The term “hypertrophy” or “hypertrophic” refers to an increase in tissue size.

  The terms “cancer” and “malignancy” generally refer to various types of malignant neoplasms, most of which invade surrounding tissues.

  The terms “cancerous” and “malignant” relate to cells or tissues having the characteristics of cancer or malignancy.

  The term “tissue remodeling” refers to the destruction of existing cells through the apoptotic pathway in response to the formation of new cells or tissues and signals mediated by relaxin or relaxin receptors.

  The term “apoptosis” induces suicide of selected forms of cells and is readily observable morphological and biochemical events (eg, deoxyribonucleic acid (DNA) fragmentation, chromatin condensation (for endonuclease activity)). Whether or not), chromosome movement, marginal orientation in the nucleus of the cell, formation of apoptotic bodies, mitochondrial swelling, mitochondrial crystal enlargement, mitochondrial permeability pore opening, and / or A regulated network of biochemical phenomena characterized by the mitochondrial proton gradient dissipation.

(Description of specific embodiments)
The present invention provides a method of administering a relaxin agonist or antagonist for modulation of apoptosis. Relaxin agonists and antagonists can be used to reduce, manage, treat, or prevent relaxin-related abnormalities. Relaxin-related abnormalities include a disease, disorder, or condition in a subject that is associated with an increased or decreased level of apoptosis compared to the level of apoptosis in a normal subject. Examples of relaxin-related abnormalities include relaxin-deficient states and relaxin-responsive states.

  In one aspect, a relaxin agonist is used to treat a relaxin-related disorder with increased apoptosis as compared to a corresponding cell from a normal subject (ie, a cell that does not have a relaxin-related disorder). Administered to the subject. Such relaxin-related abnormalities include, for example, immature body tissue, increased collagen deposition, fibrosis (ie, the presence of an abnormal amount of fibrous tissue compared to normal tissue), low tissue weight, Mention may be made of increased apoptosis of cells in a cell population. Relaxin-related abnormalities are typically associated with decreased levels of relaxin and / or relaxin receptors as compared to normal cells or tissues.

  In certain embodiments, the relaxin-related abnormality is a relaxin deficient state (eg, immature tissue, presence of excess collagen, low sperm count, etc.). The immature tissue can be, for example, immature male reproductive tract tissue (eg, immature prostate, epididymis, sperm, or testicular tissue or sperm). Immature tissue can be characterized, for example, by increased collagen deposition, low weight, and / or decreased cell number compared to a corresponding sample of normal tissue. Such tissue can also be characterized by lack of organization, incomplete development, and / or lack of differentiation or incomplete differentiation compared to a corresponding sample of normal tissue. In certain other embodiments, fibrosis (eg, skin fibrosis of these or other organs or tissues, pulmonary fibrosis, renal fibrosis, or fibrosis associated with aging) may be reduced.

  The relaxin agonist is administered in an amount effective to reduce, manage, treat, or prevent relaxin-related abnormalities. For example, administration of a relaxin agonist can reduce apoptosis of target cells, can stimulate maturation, development, and / or differentiation of immature tissue, can increase tissue weight, and increase the number of cells in the tissue. And can reduce collagen deposition.

  In another aspect according to the present invention, a relaxin antagonist is administered to a subject to reduce, manage, treat, or prevent relaxin-related abnormalities by increasing apoptosis in target cells. Such relaxin-related abnormalities can be, for example, accumulation of relaxin-related cells, decreased apoptosis relative to normal cells or tissues, hyperplasia, hypertrophy, cancer, neoplasm, and the like. In certain embodiments, relaxin antagonists can reduce remodeling of relaxin-related tissues, reduce unwanted cell accumulation, and reduce or prevent hyperplasia, hypertrophy, cancer, neoplasms, and the like.

  Examples of body tissues having an abnormality of relaxin-related tissue or relaxin-responsive tissue include, for example, brain, heart, liver, kidney, spleen, thymus, intestine, skin, lung, male reproductive tract (eg, prostate, epididymis, seminal vesicle , Or testis), or the tissues of the female reproductive tract (eg, connective tissue of the uterus, cervix, interpubic ligament, or lower limbs).

(Relaxin agonist)
In one aspect according to the present invention, a relaxin agonist is administered to a subject to treat a relaxin-related disorder. Relaxin agonists can be, for example, relaxin, relaxin analogs, small relaxin effectors, relaxin nucleic acids, and the like.

(Relaxin and relaxin analog)
In one aspect of the invention, the relaxin agonist is a relaxin polypeptide or a fragment or analog of a relaxin polypeptide. The term “relaxin” refers to a vertebrate relaxin polypeptide, including a full-length relaxin polypeptide or a portion of a relaxin polypeptide that retains biological activity.

  Relaxin is well defined in its natural human form, animal form, and its synthetic form. In particular, relaxin is widely described in US Pat. Nos. 5,166,191 and 4,835,251, both of which are hereby incorporated by reference. In this application, “relaxin” generally refers to the terms “relaxin”, “human relaxin”, “native relaxin”, and “synthetic relaxin” as described in US Pat. No. 5,166,191, and Refers to the terms “human relaxin” and “human relaxin analog” as described in US Pat. No. 4,835,251. In exemplary embodiments, relaxin can be used, for example, in US Pat. Nos. 5,179,195; 5,023,321; and 4,758,516, the disclosures of which are herein Human relaxin as described in US Pat. In this application, “relaxin” also used relaxin isolated in pigs, rats, horses, or other mammals or vertebrates, and cDNA clones of rat, pig, or other mammals or vertebrate relaxins. It refers to relaxin produced by recombinant technology.

  Methods for producing relaxin and analogs thereof are known in the art. In addition, methods for isolating and purifying relaxin are known in the art. Some sources of these methods are described in US Pat. No. 5,166,191 (see below: US Pat. No. 4,835,251, Barany et al., The Peptides 2: 1 (1980), Treager et al. , Biology of Relaxin and it's Role in the Human, including pages 42-55; EP 0251 615; EP 0107 782; EP 0107 045; and WO90 / 13659, all of which are incorporated herein by reference. Identified).

  Additional methods for producing relaxin are described in US Pat. No. 5,464,756 and PCT / US94 / 06997, the disclosures of which are hereby incorporated by reference. Relaxin is also described in EP 0 251 615 (published 7 January 1998, the disclosure of which is incorporated herein by reference) and the synthesis of A and B chains and their purification. And can be prepared by construction. For the in vitro construction of relaxin, a 4: 1 molar ratio of A chain: B chain is commonly used. The resulting product is then purified by any means known to those skilled in the art, including reverse phase HPLC, ion exchange chromatography, gel filtration, dialysis, etc., or any combination of such procedures. The Unprocessed or partially processed forms of relaxin (eg, preprorelaxin or prorelaxin) may also be used.

  In certain embodiments, relaxin polypeptides include H1 and H2 forms of human relaxin. The predominant species of human relaxin is the H2 relaxin form with a shortened B chain (ie, relaxin H2 (B29 A24) in which the four C-terminal amino acids of the B chain are deleted so that the B chain ends with serine at position 29. )). Any of this form (referred to as “short relaxin” or “long relaxin”, containing a 33 amino acid B chain) may be used.

  Relaxin agonists further include analogs (eg, naturally occurring amino acid sequence variants of relaxin). Relaxin analogs also include analogs that have been altered by substitution, addition, or deletion of one or more amino acid residues to provide a functionally active relaxin polypeptide. Such relaxin analogs include an amino acid sequence of a relaxin polypeptide (one or more functionally equivalent amino acid residues are substituted for residues in the sequence and silent functional changes (eg, conserved Including, but not limited to, those comprising all or part of the altered amino acid sequence) as primary amino acid sequences.

  In another aspect, the relaxin agonist is a polypeptide consisting of or comprising a relaxin polypeptide having at least 10 consecutive amino acids of the relaxin polypeptide. Alternatively, this fragment comprises at least 20-25 contiguous amino acids of the relaxin polypeptide. In another embodiment, the fragment is less than 20 or 30 amino acids.

  A relaxin analog is substantially similar to a relaxin polypeptide or fragment thereof (eg, in various embodiments, at least 60%, 70%, 75%, 80%, 90% or 95% identical or similar), or when compared, is substantially similar to an aligned sequence that is aligned by computer sequence comparison / alignment programs known in the art, or the encoding nucleic acid A polypeptide comprising a region that can hybridize to a relaxin nucleic acid under conditions of high stringency, moderate stringency, or low stringency (eg, Smith and Waterman, Adv. Appl. Math. 2: 482 (1981); Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970); Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444 (1988); GAP, BESTFIT, FASTA, and TEASTA in WI; see Ausubel et al. (Eds.), Current Protocols in Molecular Biology, 4th edition, John Wiley and Sons, New York (1999), for the disclosure of these (see specification). Incorporated by reference in the book)). Relaxin agonists further include functionally active relaxin polypeptides, analogs or fragments that bind to relaxin receptors.

  Relaxin agonists (eg, relaxin polypeptides, analogs, and fragments) can be produced by various methods known in the art. The operations to generate them can be performed at the gene level or the polypeptide level. For example, a cloned relaxin nucleic acid can be modified by any number of strategies known in the art (eg, generating conservative substitutions, deletions, insertions, etc.) (eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, New York (2001); Ausubel et al., Current Protocols in Molecular Biology, 4th edition, John W Incorporated herein by reference)). This sequence can be cleaved at an appropriate site by a restriction endonuclease and then subjected to further enzymatic modification, isolation, and ligation in vitro, if desired. In the production of relaxin nucleic acid encoding an analog or fragment, the modified nucleic acid is typically retained in an appropriate translation reading frame, so that this reading frame can result in a translational stop signal or synthesis of a relaxin analog or fragment. Blocked by other signals that block. Relaxin nucleic acids can also be mutated in vitro or in vivo to create and / or destroy translation initiation and / or termination sequences. Relaxin nucleic acids are also designed to create variations in the coding region and / or to form new restriction endonuclease sites or destroy existing sites, and to facilitate further in vitro modifications. Can be mutated. Any technique of mutagenesis known in the art (chemical mutagenesis, in vitro site-directed mutagenesis (see, eg, Hutchison et al., J. Biol. Chem. 253: 6551-60 (1978)), TAB (E.g., but not limited to the use of <RTIgt; (R) </ RTI> linkers (Pharmacia), etc.) can be used (see generally Sambrook et al., Supra; Ausubel et al., Supra).

  In certain embodiments, a relaxin analog is prepared from a relaxin-encoding nucleic acid that has been modified to introduce an aspartate codon at a specific site within at least a portion of the relaxin-encoding region (eg, US Pat. No. 5, No. 945,402 (the disclosure of which is incorporated herein by reference). The resulting analog can be treated with dilute acid to release the desired analog, whereby the protein can be more easily isolated and purified. Other relaxin analogs are described in US Pat. Nos. 4,656,249; 5,179,195; 5,945,402; 5,811,395; and 5,795. 807, the disclosures of which are incorporated herein by reference.

Manipulation of relaxin polypeptide sequences can also be done at the polypeptide level. Relaxin polypeptides, analogs, or fragments that are differentially modified during synthesis and after synthesis (eg, in vivo or in vitro translation) are within the scope of the present invention. Such modifications include conservative substitutions, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, antibody molecules, other polypeptides or other cellular ligands Connection to. Any number of chemical modifications include known techniques (specific chemical cleavage (eg, cleavage with cyanogen bromide), enzymatic cleavage (eg, cleavage with trypsin, chymotrypsin, papain, V8 protease, etc.); eg, NaBH ₄ , Including but not limited to acetylation, formylation, oxidation and reduction, metabolic synthesis in the presence of tunicamycin, and the like.

  Relaxin polypeptides, analogs, and fragments can be purified from natural sources by standard methods such as those described herein (eg, immunoaffinity purification). Relaxin polypeptides, analogs, and fragments can also be obtained by standard methods (including chromatography (eg, ion exchange, affinity, sizing column chromatography, high pressure liquid chromatography), centrifugation, differential lysis). Alternatively, it can be isolated and purified by any other standard technique for polypeptide purification. Relaxin polypeptides can be synthesized by standard chemical methods known in the art (eg, Hunkapiller et al., Nature 310: 105-11 (1984); Stewart and Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Co., Rockford, IL (1984), the disclosure of which is incorporated herein by reference.

  Furthermore, analogs of relaxin polypeptides can be synthesized chemically. For example, peptides that contain a desired domain or that correspond to a fragment of a relaxin polypeptide that mediates a desired activity in vivo can be synthesized, for example, by use of chemical synthesis methods using an automated peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the relaxin polypeptide sequence. Non-classical amino acids include D-isomers of common amino acids, α-aminoisobutyric acid, 4-aminobutyric acid, 2-aminobutyric acid, ε-aminohexanoic acid, 6-aminohexanoic acid, 2-aminoisobutyric acid, 3-amino Propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sacrocin, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, selenocysteine, fluoro-amino acids, designer amino acids (e.g. , Β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids, and general amino acid analogs, and the amino acids are D (dextrorotatory) or L (left-handed) obtain.

  In another embodiment, the relaxin agonist or relaxin polypeptide or fragment thereof (typically consisting of at least one domain or motif of a relaxin polypeptide, or at least 10 consecutive amino acids of a relaxin polypeptide). Or a fusion protein, which is linked to the amino acid sequence of a different protein at its amino terminus or carboxy terminus via a peptide bond. In one embodiment, such a chimeric protein is produced by recombinant expression of a nucleic acid encoding the chimeric polypeptide. A chimeric product is made by linking the appropriate nucleic acid sequences (encoding the desired amino acid sequence) to each other in the appropriate reading frame and expressing the chimeric product by known methods common in the art. Can be done. Alternatively, the chimeric product can be made by protein synthesis techniques (eg, by use of an automated peptide synthesizer).

  In certain embodiments, the fusion protein is a relaxin-ubiquitin fusion protein. For example, US Pat. No. 5,108,919 (the disclosure of which is incorporated herein by reference) discloses a method for preparing a relaxin chain and ubiquitin fusion protein.

  In preferred embodiments, the relaxin analog or fragment is functionally active (ie, may exhibit one or more functional activities associated with a full-length wild-type relaxin polypeptide). As one example, an analog or fragment that retains the desired relaxin properties of interest (eg, binding to a relaxin binding partner (eg, a relaxin receptor) and / or modulation (eg, inhibition) of apoptosis) is: It can be used as an inducer of such properties and its physiological related. Characteristic embodiments relate to relaxin analogs or fragments that bind to the relaxin receptor and induce a reduction associated with relaxin in apoptosis. Relaxin analogs or fragments can be tested for the desired activity by procedures known in the art (including but not limited to the functional assays described herein).

(Relaxin nucleic acid)
The present invention provides relaxin nucleic acid sequences for expression of relaxin polypeptides, fragments and analogs in vivo or in vitro. The relaxin nucleic acid may be a vertebrate or mammalian relaxin, including, for example, human, mouse, rat, pig, cow, dog or monkey relaxin. The relaxin nucleic acid may comprise a genomic nucleic acid, cDNA, relaxin coding region or fragment thereof. The relaxin nucleic acid further includes mRNA corresponding to the relaxin locus. Relaxin nucleic acids are also analogs (eg, nucleotide sequence variants) that encode the same amino acid, such as a naturally occurring allelic variant, or other codon choices possible for conservative amino acid substitutions thereof. )including. Due to the degeneracy of the nucleotide coding sequence, relaxin cDNA or other nucleic acid sequences encoding amino acid sequences that are substantially identical to the open reading frame can be used in the practice of the invention. These nucleic acid sequences include relaxins that are altered by substitution of different codons that encode the same or functionally equivalent amino acid residues (eg, conservative substitutions) within the sequence (thus resulting in silent changes). Examples include, but are not limited to, nucleic acid sequences that include all or part of a gene.

  The present invention further provides relaxin nucleic acid fragments (eg, hybridizable moieties) of at least 6 contiguous nucleotides. In other embodiments, the nucleic acid is at least 8 contiguous nucleotides of the relaxin sequence, at least 25 contiguous nucleotides, at least 50 contiguous nucleotides, at least 100 nucleotides, or at least 150 nucleotides or more Including more. In another embodiment, the nucleic acid is less than 150 nucleotides in length. Relaxin nucleic acids can be single-stranded or double-stranded. As will be readily appreciated, as used herein, a “nucleic acid encoding a fragment of a relaxin polypeptide” is not a contiguous sequence of other relaxin polypeptides, It is to be construed to refer to nucleic acids that encode only the listed fragments or portions of relaxin polypeptides. Also provided are fragments of relaxin nucleic acids encoding one or more relaxin domains.

  The relaxin nucleic acid or functionally active analog or fragment thereof can be inserted into an appropriate vector, such as an expression vector (ie, a vector containing factors essential for transcription and translation of the inserted polypeptide coding sequence). Essential transcriptional and translational signals can also be provided by the native relaxin gene and / or its flanking regions. A variety of host-vector systems may be utilized to express the relaxin nucleic acid sequence. These include, but are not limited to: mammalian cell lines infected with viruses (eg, vaccinia virus, adenovirus, adeno-associated virus, etc.), insect cells infected with viruses (eg, baculovirus). Systems, microorganisms (eg yeast containing yeast vectors or bacteria transformed with bacteriophage), DNA, plasmid DNA or cosmid DNA. The expression factor of a vector varies in its strength and specificity. Depending on the host-vector system utilized, any of a number of suitable transcription and translation factors can be used. In certain embodiments, the human relaxin nucleic acid or nucleic acid sequence encoding a functionally active portion of human relaxin is expressed in yeast or bacteria. In yet another embodiment, a fragment of relaxin comprising a domain of relaxin polypeptide is expressed.

  For insertion of the nucleic acid into the vector, any method known in the art can be used to construct an expression vector comprising a chimeric gene consisting of appropriate transcriptional / translational control signals and a relaxin nucleic acid sequence. These methods include in vitro recombinant DNA and synthetic techniques and in vivo recombination (genetic recombination). Expression of a nucleic acid sequence encoding a relaxin polypeptide, analog or fragment may be regulated by a second nucleic acid sequence so that the relaxin polypeptide, analog or fragment is transformed with a recombinant DNA molecule. Expressed in For example, relaxin polypeptide expression can be controlled by any promoter / enhancer element known in the art. Promoters that can be used to control gene expression include, but are not limited to: SV40 early promoter region (Benoist and Chamber, Nature 290: 304-10 (1981)), 3 of Rous sarcoma virus. 'Promoters containing long terminal repeats (Yamamoto et al., Cell 22: 787-97 (1980)), herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA 78: 1441-45 (1981)), metallothionein Regulatory sequences of (metallothenen) genes (Brinster et al., Nature 296: 39-42 (1982)), prokaryotic expression vectors (eg, β-lactamase promoter) (Villa-Komaroff et al., Proc. Natl. Acad. Sci. USA 75: 3727-31 (1978)) or tac promoter (deBoer et al., Proc. Natl. Acad. Sci. USA 80: 21-25 (1983)), Plant expression vector containing cauliflower mosaic virus 35S RNA promoter (Gardner et al., Nucl. Acids Res. 9: 2871-88 (1981)), photosynthetic enzyme ribulose diphosphate carboxylase promoter (Herrera-Estella et al., Nature 310: 115- 20 (1984)), promoter elements derived from yeast or other fungi (eg, Gal7 promoter and Gal4 promoter), ADH (alcohol dehydration) Enzyme) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter.

  Animal transcriptional control regions that exhibit the following tissue specificity have been utilized in transgenic animals: elastase I gene control regions that are active in pancreatic acinar cells (eg, Swift et al., Cell 38: 639-46 (1984)). Ornitz et al., Cold Spring Harbor Symp.Quant.Biol.50: 399-409 (1986); MacDonald, Hepatology 7 (1 Suppl.): 42S-51S (1987)); Regions (eg, Hanahan, Nature 315: 115-22 (1985)), immunoglobulin gene control regions that are active in lymphoid-like cells (eg, Grosschedl et al., Cell 38:64). -58 (1984); Adams et al., Nature 318: 533-38 (1985); Alexander et al., Mol. Cell. Biol. 7: 1436-44 (1987)), testicular cells, breast cells, lymphoid cells and fertile cells. Mouse mammary adenovirus virus control region (eg, Leder et al., Cell 45: 485-95 (1986)) active in the liver, and albumin gene control region (eg, Pinkert et al., Genes Dev. 1: 268-76) active in the liver. (1987)), α-fetoprotein gene regulatory regions that are active in the liver (eg, Krumlauf et al., Mol. Cell. Biol. 5: 1639-48 (1985); Hammer et al., Science 235: 53-58 (1987)). ;liver Α1-antitrypsin gene regulatory region (eg, Kelsey et al., Genes and Devel. 1: 161-71 (1987)) active in bone marrow cells (eg, Magram et al. , Nature 315: 338-40 (1985); Kollias et al., Cell 46: 89-94 (1986)); myelin basic protein gene regulatory region that is active in oligodendrocytes of the brain (eg Readhead et al., Cell 48). : 703-12 (1987)); a myosin light chain-2 gene regulatory region that is active in skeletal muscle (eg, Shani, Nature 314: 283-86 (1985)); and a gonadotropin releasing hormone gene that is active in the hypothalamus Control area For example, Mason, et al., Science 234: 1372-78 (1986)). In preferred embodiments, the tissue specific promoter is a prostate specific antigen promoter (see, eg, US Pat. No. 6,100,444, the disclosure of which is incorporated herein by reference). ).

  In another embodiment, a vector comprising a promoter operably linked to a relaxin nucleic acid, one or more origins of replication, and optionally one or more selectable markers (eg, antibiotic or drug resistance markers) is used. Is done. For example, an expression construct can be made by subcloning a relaxin nucleic acid into a restriction site of a pRSECT expression vector. Such constructs allow expression of relaxin polypeptides, analogs or fragments under the control of a T7 promoter with a histidine amino terminal flag sequence for affinity purification of the expressed polypeptide. In another specific embodiment, comprising a prostate specific antigen promoter operably linked to a relaxin nucleic acid, one or more origins of replication and optionally one or more selectable markers (eg, drug resistance markers) A vector is used.

  Expression vectors comprising relaxin nucleic acids can be identified by general approaches well known to those skilled in the art, including: (a) nucleic acid hybridization, (b) presence or absence of “marker” gene function, (C) expression of the inserted sequence, (d) polymerase chain reaction (PCR), etc. In the first approach, the presence of relaxin nucleic acid inserted into the expression vector can be detected by nucleic acid hybridization using a probe comprising a sequence homologous to the inserted relaxin nucleic acid. In the second approach, the recombinant vector / host system is responsible for certain “marker” gene functions (eg, thymidine kinase activity, antibiotic resistance, transformation phenotypes) caused by inserting a relaxin nucleic acid into the vector. , Occlusion bodies in baculoviruses, colorimetric changes, etc.) may be identified and selected based on the presence or absence. For example, if the relaxin nucleic acid is inserted within the marker gene sequence of the vector, a recombinant comprising the relaxin nucleic acid can be identified by the absence of marker gene function.

  In the third approach, recombinant expression vectors can be identified by assaying the recombinantly expressed relaxin polypeptide, analog or fragment. Such assays can be performed, for example, on the physical or functional properties of relaxin polypeptides, analogs or fragments (eg, binding to anti-relaxin antibodies, binding to relaxin receptors, etc.) in an in vitro assay system. Can be based. In the fourth approach, recombinant expression vectors can be identified by polymerase chain reaction. (For example, U.S. Pat. Nos. 4,683,202, 4,683,195 and 4,889,818; Gyllensten et al., Proc. Natl. Acad. Sci. USA 85: 7652-56 (1988). Ochman et al., Genetics 120: 621-23 (1988); Loh et al., Science 243: 217-20 (1989)). Once a particular recombinant vector has been identified and isolated, several methods known in the art can be used to amplify it.

  Once the appropriate host system and growth conditions are established, the recombinant vector can be amplified and prepared in large quantities. As explained above, vectors that can be used include, but are not limited to, the following vectors or their analogs to name a few: human or animal viruses (eg, vaccinia virus or Adenovirus); insect viruses (eg, baculovirus); yeast vectors; bacteriophage vectors (eg, λ); and plasmid and cosmid DNA vectors.

  Further, in the particular manner desired, host cell lines can be selected to modulate the expression of the inserted nucleic acid or to modify or process relaxin, relaxin analogs or fragments. Expression from a particular promoter can be increased in the presence of a particular inducer: thus, expression of a relaxin polypeptide, analog or fragment can be controlled. In addition, different host cells with characteristic and specific mechanisms for translation and post-translational processing and modification (eg, glycosylation and / or phosphorylation) can be used. Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the polypeptide, analog or fragment expressed. For example, expression in a bacterial system can be used to produce an unprocessed core protein product. Expression in mammalian cells can be used to ensure "native" processing of relaxin polypeptides or relaxin analogs or fragments of mammalian cells. Furthermore, different vector / host expression systems can affect the processing reaction to different degrees.

(Relaxin antagonist)
In another aspect of the invention, relaxin antagonists are provided for the regulation of apoptosis. For example, relaxin antagonists may include relaxin binding factors, relaxin receptor binding factors, antisense nucleic acids and the like.

(Relaxin antibody)
Relaxin antagonists include antibodies that immunospecifically recognize relaxin or a relaxin receptor polypeptide and antibodies that stimulate apoptosis and / or reduce or inhibit the accumulation of cells bound to relaxin in a cell population or tissue. May be included. Anti-relaxin antibodies and anti-relaxin receptor antibodies include, but are not limited to: polyclonal antibodies, monoclonal antibodies, chimeric antibodies (eg, fully humanized antibodies or human chimeric antibodies), single chain antibodies, antibody fragments ( For example, Fab, F (ab ′), F (ab ′) ₂ , Fv or hypervariable region), single heavy chain, and Fab expression libraries. In certain embodiments, full-length polyclonal and / or monoclonal antibodies, vertebrate or mammalian relaxin or relaxin receptor polypeptides are generated and selected for antibodies that selectively bind to relaxin or relaxin receptor polypeptides. Thereby functionally inactivating such polypeptides. In another embodiment, antibodies are generated against domains of vertebrate relaxin polypeptides or relaxin receptor polypeptides. In yet another embodiment, a vertebrate relaxin polypeptide or a fragment of a relaxin receptor polypeptide (identified as hydrophilic) is used as an immunogen for antibody production, and Selected for immunospecific binding to the peptide and inhibition of its biological activity.

  Various procedures known in the art can be used for the production of polyclonal antibodies against relaxin or relaxin receptor polypeptides or fragments or analogs thereof. For the production of such antibodies, a variety of host animals (including but not limited to rabbits, mice, rats, sheep, goats, etc.) can be used as native relaxin or relaxin receptor polypeptides or fragments thereof. Or it can be immunized by injecting an analog. Alternatively, transgenic animals with a human immune system can be immunized by injecting native relaxin or relaxin receptor polypeptide. (See, eg, US Pat. Nos. 6,114,598 and 6,111,166, which are hereby incorporated by reference). A variety of adjuvants can be used to enhance the immune response (depending on the host species), including but not limited to: Freund's adjuvant (complete or incomplete), minerals Gels (eg aluminum hydroxide), surface active substances (eg lysolecithin), pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol and potentially useful Human adjuvants such as BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum.

  For the preparation of monoclonal antibodies against relaxin or relaxin receptor polypeptides, fragments or analogs thereof, any technique provided for the production of antibody molecules by cell lines that continue in culture can also be used. Such techniques include, for example: hybridoma technology originally developed by Kohler and Milstein (Nature 256: 495-97 (1975)) and trioma technology, human B cell hybridoma technology (eg, Kozbor et al. , Immunology Today 4:72 (1983)) as well as EBV-hybridoma technology for generating human monoclonal antibodies (eg, Cole et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., 77-96). Page (1985)). Mammalian antibodies can be used and human B cells using hybridomas (see, eg, Cote et al., Proc. Natl. Acad. Sci. USA 80: 2026-30 (1983)) or EBV virus in vitro. It can be obtained by using transformation (see, eg, Cole et al. (1985), supra). Selection of hybridomas that produce antibodies with appropriate biological functions is well known in the art or is described herein below. Human monoclonal antibodies can also be prepared by preparing hybridomas from animals having a human immune system immunized by injecting native relaxin or relaxin receptor polypeptide (eg, US Pat. No. 6,114,598). No. and 6,111,166, which are incorporated herein by reference).

In addition, the present invention can prepare “chimeric” or “humanized” antibodies (eg, Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851-55 (1984); 604-08 (1984); Takeda et al., Nature 314: 452-54 (1985)). Such chimeric antibodies are typically prepared by splicing a non-human gene for an antibody molecule specific for relaxin or a receptor polypeptide together with a gene from a suitably active human antibody molecule. The antigen-binding region of a non-human antibody (eg, F (ab ′) ₂ , F (ab ′), Fv or hypervariable region) is transferred into the framework of a human antibody by recombinant DNA technology, and the human molecule is It may also be desirable to produce essentially. In a preferred embodiment, the antibody is fully humanized.

  Methods for producing such “chimeric” molecules are generally well known and are described, for example, in: US Pat. Nos. 4,816,567; 4,816,397; 693,762; 5,712,120; 5,821,337; 6,054,297; International Patent Publications WO 87/02671 and WO 90/00616; and European Patent Publication EP 0 239 400 (the disclosures of which are incorporated herein by reference). Alternatively, the human monoclonal antibody or portion thereof encodes an antibody that specifically binds to a relaxin or relaxin receptor polypeptide according to the general method described by Huse et al. (Science 246: 1275-81 (1989)). A human B cell cDNA library for the DNA molecule to be identified can be identified by first screening. The DNA molecule can then be cloned and amplified to obtain a sequence encoding an antibody (or binding domain) of the desired specificity. Phage display technology provides another technique for selecting antibodies that bind to relaxin or relaxin receptor polypeptides, fragments or analogs thereof. (See, eg, International Patent Publications WO 91/17271 and WO 92/01047; and Huse et al., Supra).

  In accordance with another aspect of the present invention, techniques for the production of single chain antibodies (see, eg, US Pat. Nos. 4,946,778 and 5,969,108) include relaxin or relaxin receptor. Can be adapted to produce specific single chain antibodies. (See also Riechmann and Muyldermans, J. Immunol. Methods 231: 25-38 (1999); Muyldermans and Lauwereys, J. Mol. Recognition. 12: 131-40 (1999)).

  A further aspect of the invention utilizes the techniques described for the construction of Fab expression libraries (see, eg, Huse et al. (1989), supra) and uses the desired relaxin polypeptides, fragments or analogs thereof. Identification of monoclonal Fab fragments with specificity for and biological activity can be facilitated quickly and easily.

Antibody fragments that contain the idiotype of the molecule can be generated by known techniques. For example, such fragments include, but are not limited to: F (ab ′) ₂ fragments that can be produced by pepsin digestion of antibody molecules, reducing disulfide bonds of F (ab ′) ₂ fragments. Fab 'fragments that can be produced by the method, Fab fragments that can be produced by treating antibody molecules with papain and reducing agents, and Fv fragments. Recombinant Fv fragments can also be produced in eukaryotic cells using, for example, the methods described in US Pat. No. 5,965,405.

  In the production of antibodies, screening for the desired antibody can be accomplished by techniques known in the art, such as ELISA (solid phase enzyme immunoassay) .In one aspect, the specifics of relaxin or relaxin receptor polypeptide Antibodies that recognize the domain can be used to assay hybridomas for products (eg, antibodies) that bind to relaxin or a fragment of relaxin receptor (including this domain). To select an antibody that specifically binds to a polypeptide but does not specifically bind to a second, different relaxin polypeptide, the antibody can bind to an antibody positive binding to the first polypeptide and a second Selected based on lack of antibody binding to different polypeptides Get.

(Soluble relaxin receptor)
In another aspect of the invention, the relaxin antagonist is a relaxin binding agent comprising a soluble relaxin receptor that binds relaxin, or a fragment or analog thereof. The term “soluble relaxin receptor” refers to a relaxin receptor polypeptide that does not bind to the cell membrane. This relaxin receptor is approximately 200 kilodaltons (see Palejwala et al., Endocrinology 139 (3): 1208-12 (1998), the disclosure of which is incorporated herein by reference). Soluble forms of relaxin receptors retain the ability to bind vertebrate relaxin, but typically lack the transmembrane and / or cytoplasmic domains. Soluble relaxin receptors can include additional amino acid residues (eg, affinity tags) that provide a means for purification of the polypeptide or attach the polypeptide to another polypeptide or immunoglobulin sequence. Provide a site for.

  The soluble relaxin receptor can optionally include a transmembrane domain that cannot bind to the cell membrane. By “transmembrane protein” is meant a domain of a relaxin receptor polypeptide that contains a sufficient number of hydrophobic amino acids, which allows the polypeptide to be inserted into the cell membrane and anchored. By “transmembrane domain that cannot bind to the cell membrane” is meant a transmembrane domain that is altered by mutation or deletion and as a result is not sufficiently hydrophobic to allow insertion or other binding into the cell membrane. Such a transmembrane domain prevents, for example, fusion of a relaxin receptor polypeptide or fragment thereof (which has a secretion signal sequence useful for polypeptide secretion from the cell). Amino acid sequence substitutions or alterations useful to achieve an inactive transmembrane domain include, but are not limited to, amino acid deletions or substitutions within the transmembrane domain. Methods for making soluble receptors are known in the art (see, eg, US Pat. Nos. 6,033,903; 6,037,450; and 5,925,549; These disclosures are incorporated herein by reference).

  Soluble relaxin receptors include naturally occurring amino acid sequence variants of soluble relaxin receptors. Soluble relaxin receptors further include receptors that are altered by substitutions, additions or deletions of one or more amino acid residues that provide a functionally activated relaxin receptor polypeptide. Such relaxin receptors include a relaxin receptor polypeptide amino acid sequence (one or more functionally equivalent amino acid residues are replaced with residues in the sequence, resulting in silent functional changes (eg, conservative Including, but not limited to, a primary amino acid sequence including all or a portion of (including sequences that cause substitution).

  In another aspect, the soluble relaxin receptor is a polypeptide consisting of or comprising a fragment of a relaxin receptor polypeptide having at least 10 consecutive amino acids of the relaxin receptor polypeptide. More typically, this fragment comprises at least 20 or at least 50 consecutive amino acids of a relaxin receptor polypeptide. In other embodiments, the fragment is greater than 100 amino acids or even greater than 200 amino acids.

  A relaxin receptor polypeptide may be a region substantially similar to a relaxin receptor polypeptide or fragment thereof (e.g., in various embodiments, at least 60%, 70%, 75%, over the same size amino acid sequence, Polypeptides, including 80%, 90%, or even 95% identity or similarity) or the sequences are aligned by computer sequence comparison / alignment programs known in the art or by visual inspection. A polypeptide when compared to an aligned sequence. (See, eg, Smith and Waterman, Adv. Appl. Math. 2: 482 (1981); Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970); Pearson and Lipman, Proc. Natl. Acad. Sci. USA. 85: 2444 (1988); GAP, BESTFIT, FASTA and TEASTA, Genetics Computer Group, 575 Science Dr., Madison, WI in Wisconsin Genetics Software Package; see Ausubel et al. Incorporated herein by reference). Sequence identity or similarity can also be determined by identifying nucleic acids that can hybridize to relaxin receptor nucleic acids under conditions of high stringency, moderate stringency, or low stringency. (See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, New York (2001); see Ausubel et al., (1996), supra; the present disclosure; Incorporated by reference in the book).

  Soluble relaxin receptor and fragments thereof can be produced by various methods known in the art. The manipulations that result in these productions can occur at the gene level or protein level. For example, a cloned relaxin receptor nucleic acid can be modified (eg, Sambrook et al., Supra) by any of a number of strategies known in the art (eg, performing conservative substitutions, deletions, insertions, etc.). See Ausubel et al., Supra). This sequence can be cleaved at an appropriate site using restriction endonuclease and then further enzymatically modified, isolated and ligated in vitro, if desired. In the production of relaxin receptor nucleic acids, the modified nucleic acid typically maintains its proper translation reading frame, so that this reading frame is a translation stop signal, or synthesis of a soluble relaxin receptor or fragment thereof. It is not disturbed by other signals that interfere with it. Relaxin receptor nucleic acids can also be mutated in vitro or in vivo to create and / or destroy translation initiation and / or translation termination sequences. Relaxin receptor nucleic acids can also be mutated to create variations in the coding region (eg, amino acid substitutions) and / or to form new restriction endonuclease sites or destroy existing sites, and to modify in vitro It can be made easier. Any mutagenesis technique known in the art can be used, including chemical mutagenesis, in vitro site-directed mutagenesis (eg, Hutchison et al., J. Biol. Chem. 253: 6551- 60 (1978)), use of TAB® linker (Pharmacia), and the like.

Manipulation of relaxin receptor polypeptide sequences can also be done at the polypeptide level. Within the scope of the present invention are relaxin receptor polypeptides that are differentially modified (eg, by in vivo or in vitro translation) during or after synthesis. Such modifications include conservative substitutions, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, to antibody molecules, proteins or other cellular ligands. Examples include bonding. Any of a number of chemical modifications can be performed by known techniques, including specific chemical cleavage (eg, with cyanogen bromide), enzymatic cleavage (eg, trypsin, chymotrypsin, papain, V8 Such as, but not limited to, NaBH ₄ , acetylation, formylation, oxidation and reduction, alteration by metabolic synthesis in the presence of tunicamycin, and the like.

  Relaxin receptor polypeptides and fragments thereof can be purified from natural sources by standard methods such as those described herein (eg, immunoaffinity purification). Relaxin receptor polypeptides and fragments can also be obtained by standard methods including chromatography (eg, ion exchange chromatography, affinity chromatography, sizing column chromatography, high pressure liquid chromatography), centrifugation, differential solubility, Alternatively, it can be isolated and purified by any other standard technique for polypeptide purification. Relaxin receptor polypeptides and fragments thereof can be synthesized by standard chemical methods known in the art (eg, Hunkapiller et al., Nature 310: 105-11 (1984); Stewart and Young, Solid Phase Peptide Synthesis). , 2nd edition, Pierce Chemical Co., Rockford, IL, (1984)). Moreover, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the relaxin receptor polypeptide sequence. Non-classical amino acids include D-isomers of ordinary amino acids, α-aminoisobutyric acid, 4-aminobutyric acid, 2-aminobutyric acid, ε-aminohexanoic acid, 6-aminohexanoic acid, 2-aminoisobutyric acid, 3 Aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, selenocysteine, fluoro-amino acid, designer ( designer) amino acids (eg, β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids), and common amino acid analogs, but are not limited to these. Furthermore, the amino acids can be D (dextrorotatory) or L (left-handed).

  In another embodiment, the soluble relaxin receptor is a relaxin receptor polypeptide, or fragment thereof (typically relaxin) that binds at its amino terminus or carboxy terminus to the amino acid sequence of a different protein via a peptide bond. A chimeric or fusion protein comprising at least a domain or motif of a receptor polypeptide, or consisting of at least 10 consecutive amino acids of a relaxin receptor polypeptide). In one embodiment, such a chimeric protein is produced by recombinant expression of a nucleic acid encoding the chimeric polypeptide. A chimeric product can be made by linking the appropriate nucleic acid sequences encoding the desired amino acid sequence together in an appropriate reading frame and expressing the chimeric product by methods commonly known in the art. Alternatively, the chimeric product can be made by protein synthesis techniques (eg, by use of an automated peptide synthesizer). In certain embodiments, the fusion protein is a relaxin receptor-ubiquitin fusion protein.

(Relaxin analog)
A relaxin antagonist may further be a relaxin analog (eg, a relaxin polypeptide that binds to a relaxin receptor but does not induce a response by that receptor). For example, relaxin analogs can be competitive inhibitors of relaxin binding or normal antagonists of relaxin receptors. The term “relaxin” refers to a vertebrate relaxin polypeptide, including a full-length relaxin polypeptide, or a portion of a relaxin polypeptide that retains biological activity. Relaxin is well defined in its natural human form, animal form, and synthetic form. In particular, relaxin is disclosed in US Pat. Nos. 5,179,195; 5,166,191; 5,023,321; 4,835,251; and 4,758,516. (The disclosures of which are hereby incorporated by reference). Methods for making relaxin and its analogs are known in the art (supra). Relaxin analogs can be prepared by modifying a relaxin polypeptide such that the relaxin analog maintains relaxin receptor binding activity but does not induce a response by the relaxin receptor. For example, a relaxin analog can be an amino acid sequence variant of relaxin that maintains relaxin receptor binding activity but does not induce a response by the relaxin receptor. Relaxin analogs further include relaxin polypeptides that are altered by the addition or deletion of one or more amino acid residues to maintain the relaxin receptor binding function but do not induce a response by the relaxin receptor.

  In various aspects according to the invention, the relaxin analog is a fragment of a relaxin polypeptide consisting of or comprising at least 10 consecutive amino acids of the relaxin polypeptide. Alternatively, the fragment comprises at least 20 or 40 consecutive amino acids of the relaxin polypeptide. In other embodiments, these fragments are not longer than 35 amino acids.

  A relaxin analog is a region substantially similar to a relaxin polypeptide (eg, in various embodiments, at least 60%, 70%, 75%, 80%, 90%, over the same size amino acid sequence). Or even 95% identity or similarity) or a polypeptide when aligned and compared to aligned sequences by computer sequence comparison / alignment programs known in the art Or the encoding nucleic acid can be a polypeptide that can hybridize to a relaxin nucleic acid under conditions of high stringency, moderate stringency, or low stringency (see above). .

  Relaxin analogs can be produced by various methods known in the art. The manipulations that result in these productions can occur at the gene level or protein level. For example, a cloned relaxin nucleic acid can be modified (eg, by making conservative or non-conservative substitutions, deletions, insertions, etc.) by any of a number of strategies known in the art (eg, See Sambrook et al., Supra; Ausubel et al., Supra). This sequence can be cleaved at an appropriate site using a restriction endonuclease and then further enzymatically modified, isolated and ligated in vitro if desired. In the production of relaxin analog nucleic acids, the modified nucleic acid typically maintains its proper translation reading frame, so that this reading frame can be a translation stop signal, or others that interfere with the synthesis of relaxin analogs. Is not disturbed by the signal. Relaxin nucleic acids can be mutated in vitro or in vivo to create and / or destroy translation initiation and / or translation stop sequences. Relaxin nucleic acids can also be mutated to create variations in the coding region and / or form new restriction endonuclease sites or destroy existing sites, and further facilitate in vitro modification. Any mutagenesis technique known in the art can be used, including chemical mutagenesis, in vitro site-directed mutagenesis (eg, Hutchison et al., J. Biol. Chem. 253: 6551- 60 (1978)), use of TAB® linker (Pharmacia), and the like.

  In certain embodiments, a relaxin analog is prepared from a relaxin-encoding nucleic acid that has been modified to introduce an aspartate codon at a specific position in at least a portion of the relaxin-encoding region (eg, US Pat. No. 5,945,402, the disclosure of which is incorporated herein by reference). The resulting analog can be treated with dilute acid to release the desired analog, thereby making the protein more easily isolated and purified.

Manipulation of relaxin polypeptide sequences can also be done at the polypeptide level. Within the scope of the present invention are relaxin analogs that are differentially modified (eg, by in vivo or in vitro translation) during or after synthesis. Such modifications include amino acid substitutions (either conservative or non-conservative), glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, antibodies Examples include binding to molecules, proteins or other cellular ligands. Any of a number of chemical modifications can be performed by known techniques, including specific chemical cleavage (eg, with cyanogen bromide), enzymatic cleavage (eg, trypsin, chymotrypsin, papain, V8 Such as, but not limited to, NaBH ₄ , acetylation, formylation, oxidation and reduction, alteration by metabolic synthesis in the presence of tunicamycin, and the like.

  Relaxin analogs can be purified from natural sources by standard methods such as those described herein (eg, immunoaffinity purification). Relaxin analogs can also be obtained by standard methods including chromatography (eg, ion exchange chromatography, affinity chromatography, sizing column chromatography, high pressure liquid chromatography), centrifugation, differential solubility, or of polypeptides. It can be isolated and purified by any other standard technique for purification. Relaxin analogs can be synthesized by standard chemical methods known in the art (eg, Hunkapiller et al., Nature 310: 105-11 (1984); Stewart and Young, Solid Phase Peptide Synthesis, 2nd edition, (See Pierce Chemical Co., Rockford, IL, (1984)). Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the relaxin polypeptide sequence. Non-classical amino acids include D-isomers of ordinary amino acids, α-aminoisobutyric acid, 4-aminobutyric acid, 2-aminobutyric acid, ε-aminohexanoic acid, 6-aminohexanoic acid, 2-aminoisobutyric acid, 3 Aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, selenocysteine, fluoro-amino acid, designer amino acid (For example, but not limited to, β-methyl amino acid, Cα-methyl amino acid, Nα-methyl amino acid), and general amino acid analogs. Furthermore, the amino acids can be D (dextrorotatory) or L (left-handed).

  In another embodiment, a relaxin analog is a relaxin polypeptide (typically at least a domain of a relaxin polypeptide or a relaxin polypeptide that binds to the amino acid sequence of a different protein via a peptide bond at its amino terminus or carboxy terminus. A chimeric or fusion protein comprising a motif, or consisting of at least 10 consecutive amino acids of a relaxin polypeptide. In one embodiment, such a chimeric protein is produced by recombinant expression of a nucleic acid encoding the chimeric polypeptide. A chimeric product can be made by linking the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other in the appropriate reading frame and expressing the chimeric product by methods commonly known in the art. Alternatively, the chimeric product can be made by protein synthesis techniques (eg, by use of an automated peptide synthesizer).

  In certain embodiments, the fusion protein is a relaxin analog-ubiquitin fusion protein. For example, US Pat. No. 5,108,919 (the disclosure of which is incorporated herein by reference) discloses a method for preparing a relaxin chain and ubiquitin fusion protein.

(Nucleic acid)
The invention further provides a nucleic acid for use as an antagonist or for expressing an antagonist according to the invention. Such nucleic acids include nucleic acids encoding soluble relaxin receptors or relaxin analogs for the synthesis of soluble relaxin receptors or relaxin analogs, respectively. Antisense nucleic acids are provided for inhibition of relaxin or relaxin receptor expression. Nucleic acids encoding relaxin polypeptides or relaxin receptor polypeptides are also provided for the preparation of antibodies (see above).

  In one aspect, the present invention provides a nucleic acid sequence encoding a relaxin receptor or relaxin analog for in vivo expression. Relaxin receptors or relaxin analogs, or antisense nucleic acids can be expressed in vivo for gene therapy. A relaxin receptor, relaxin analog or antisense nucleic acid is also used in vivo for the preparation of a recombinant soluble relaxin receptor, recombinant relaxin analog or recombinant antisense nucleic acid for exogenous administration to a subject. Or it can be expressed in vitro.

  The nucleic acid can be a vertebrate nucleic acid, for example, a relaxin receptor, relaxin, or relaxin analog derived from vertebrate relaxin of human, mouse, rat, pig, cow, dog, or monkey. These nucleic acids can include genomic DNA, genomic cDNA, or the coding region of a relaxin receptor, relaxin or relaxin analog. These nucleic acids may further comprise a relaxin receptor locus or mRNA corresponding to the relaxin locus. These nucleic acids may also be variants that encode other possible codon choices for the same amino acid, or variants that encode conservative amino acid substitutions thereof (eg, naturally occurring allelic variants). , Including nucleotide sequence variants. Due to the degeneracy of the nucleotide coding sequence, other nucleic acid sequences that encode substantially the same amino acid sequence as the relaxin receptor, or relaxin coding sequences may be used in the practice of the invention. These nucleic acid sequences include all relaxin genes that have been altered by substitution of different codons that encode the same or functionally equivalent amino acid residues within the sequence (eg, conservative substitutions), thus resulting in silent changes. Or a nucleotide sequence that includes a moiety, but is not limited thereto.

  The invention further provides a nucleic acid fragment of at least 6 contiguous nucleotides (eg, a hybridizable portion); in other embodiments, the nucleic acid comprises at least 8 contiguous nucleotides of a relaxin sequence, at least 25 Of contiguous nucleotides, at least 50 contiguous nucleotides, at least 100 nucleotides, 150 nucleotides or more. In another embodiment, these nucleic acids are less than 100 or 150 nucleotides in length. These nucleic acids can be single-stranded or double-stranded. As will be readily apparent, as used herein, a nucleic acid encoding a relaxin or a fragment of a relaxin receptor polypeptide encodes only the mentioned fragment or portion of a relaxin polypeptide, and It is to be understood as a nucleic acid that does not encode a relaxin receptor or other consecutive portion of a relaxin polypeptide as a contiguous sequence. Fragments of nucleic acids encoding one or more domains of relaxin or relaxin receptor are also provided.

  A relaxin receptor, relaxin analog or antisense nucleic acid, as desired in an appropriate vector (e.g., an expression vector containing the necessary elements for transcription or transcription and translation of the inserted polypeptide coding sequence), It can be inserted in either the sense direction or the antisense direction. The necessary transcriptional and translational signals can also be supplied by the native relaxin or relaxin receptor gene and / or its flanking regions. A variety of host vector systems may be utilized to express the polypeptide coding sequence. These include mammalian cell lines infected with viruses (eg, vaccinia virus, adenovirus, adeno-associated virus, etc.), insect cell lines infected with viruses (eg, baculovirus), microorganisms such as yeast including yeast vectors. Or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA, but are not limited thereto. The expression elements of vectors vary in their strengths and specificities. Depending on the host vector system utilized, any of a number of suitable transcription and translation elements can be used. In certain embodiments, the nucleic acid can be expressed or a nucleic acid sequence encoding a functionally active portion of a relaxin receptor or relaxin analog is expressed in a mammalian cell, yeast or bacterium. In yet another embodiment, a relaxin receptor or relaxin analog fragment comprising the domain of each polypeptide is expressed.

  Using any of the methods known in the art for inserting nucleic acids into vectors, an expression vector comprising a chimeric gene having appropriate transcriptional control signals, translational control signals, and / or polypeptide coding sequences. Can be built. These methods include in vitro recombinant DNA and synthetic techniques, and in vivo recombination (genetic recombination). Expression of a nucleic acid encoding a relaxin receptor or relaxin analog can be regulated by a second nucleic acid sequence such that the nucleic acid or polypeptide is expressed in a host transformed with the recombinant DNA molecule. For example, expression of a relaxin receptor or relaxin analog nucleic acid can be controlled by any promoter / enhancer element known in the art. Promoters that can be used to control expression include, but are not limited to, the SV40 early promoter region (see, eg, Benoist and Chamber, Nature 290: 304-10 (1981)), Promoters contained in the 3 'long terminal repeat of Rous sarcoma virus (see, eg, Yamamoto et al., Cell 22: 787-97 (1980)), herpes thymidine kinase promoters (see, eg, Wagner et al., Proc. Natl. Acad. Sci. USA 78: 1441-45 (1981)), regulatory sequences of the metallothionein gene (eg, Brinster et al., Nature 296: 39-42 (1982)). See), prokaryotic expression vectors such as the β-lactamase promoter (see, eg, Villa-Komarov et al., Proc. Natl. Acad. Sci. USA 75: 3727-31 (1978)) or the tac promoter. (See, eg, deBoer et al., Proc. Natl. Acad. Sci. USA 80: 21-25 (1983)), a plant expression vector comprising a cauliflower mosaic virus 35S RNA promoter (eg, Gardner et al., Nucl. Acids). Res. 9: 2871-88 (1981)), as well as the promoter of the photosynthetic enzyme ribulose diphosphate carboxylase (eg, Herrera-Estrella et al., Nature 310: 115- 20 (1984)), promoter elements from yeast or other fungi, such as the Gal7 and Gal4 promoters, the ADH (alcohol dehydrogenase) promoter, the PGK (phosphoglycerol kinase) promoter, the alkaline phosphatase promoter, and the like.

  The following animal transcriptional control regions (which exhibit tissue specificity) have been utilized in transgenic animals: elastase I gene control regions (eg, Swift et al., Cell 38: 639) active in pancreatic acinar cells. -46 (1984); See Ornitz et al., Cold Spring Harbor Symp. Quant. Biol. 50: 399-409 (1986); Insulin gene regulatory regions that are active in cells (see, eg, Hanahan, Nature 315: 115-22 (1985)), immunoglobulin gene regulatory regions that are active in lymphoid cells (eg, Grosschedl et al., ll 38: 647-58 (1984); Adams et al., Nature 318: 533-38 (1985); Alexander et al., Mol. Cell. Biol.7: 1436-44 (1987)), testis, chest, Mouse mammary carcinoma virus regulatory region (see, eg, Leder et al., Cell 45: 485-95 (1986)) active in lymphoid cells and mast cells, albumin gene regulatory region (eg, Pinkert et al., Genes Dev. 1: 268-76 (1987)), α-fetoprotein gene regulatory region active in the liver (eg, Krumlauf et al., Mol. Cell. Biol 5: 1639-48 (1985)). Hammer et al., Scien ce 235: 53-58 (1987)); α1-antitrypsin gene regulatory region active in the liver (see, for example, Kelsey et al., Genes and Devel. 1: 161-71 (1987)). A β-globin gene regulatory region that is active in myeloid cells (see, eg, Magram et al., Nature 315: 338-40 (1985); Kollias et al., Cell 46: 89-94 (1986)); Myelin basic protein gene control region active in oligodendrocytes (see, eg, Readhead et al., Cell 48: 703-12 (1987)); myosin light chain-2 gene control active in skeletal muscle Region (eg, Shani, Nature 314: 283) 86 (1985)); and activity in the hypothalamus, gonadotropin-releasing hormone gene control region (e.g., Mason, et al., Science 234: 1372-78 (1986) see). In preferred embodiments, the tissue specific promoter is a prostate specific antigen promoter (see, eg, US Pat. No. 6,100,444, the disclosure of which is incorporated herein by reference). ).

  In another embodiment, a vector is used that includes a promoter operably linked to a nucleic acid, one or more origins of replication, and optionally one or more selectable markers (eg, antibiotic or drug resistance markers). Is done. For example, expression constructs can be made by subcloning a relaxin receptor or relaxin analog nucleic acid into a restriction site of a pRSECT expression vector. Such constructs allow expression of relaxin analogs or relaxin receptor polypeptides under the control of the T7 promoter, having a histidine amino terminal flag sequence for affinity purification of the expressed polypeptide. In another specific embodiment, a prostate specific antigen promoter operably linked to a relaxin analog nucleic acid, one or more origins of replication, and optionally one or more selectable markers (eg, drug resistance markers). A vector containing is used.

  Expression vectors containing such nucleic acids can be identified by general approaches well known to those of skill in the art, which include: (a) nucleic acid hybridization or polymerase chain reaction, (b) “markers” Presence or absence of gene function, (c) expression of inserted sequences, or polymerase chain reaction (PCR). In the first approach, the presence of the nucleic acid inserted into the expression vector can be detected by nucleic acid hybridization or polymerase chain reaction using a probe comprising a sequence homologous to the inserted nucleic acid. In a second approach, the recombinant vector / host system is driven by a specific “marker” gene function (eg, thymidine kinase activity, antibiotics) caused by insertion of a vector comprising a relaxin receptor, relaxin or relaxin analog nucleic acid. Resistance, transformed phenotype, presence or absence of occlusion bodies in baculovirus, etc.) can be identified and selected. For example, if the nucleic acid is inserted into the marker gene sequence of the vector, the recombinant containing that nucleic acid can be identified by the absence of marker gene function.

  In the third approach, recombinant expression vectors can be identified by assaying the polypeptide expressed by the recombinant. Such assays are, for example, physical or functional properties of the polypeptide in in vitro assay systems (eg, binding to anti-relaxin antibodies, binding to relaxin or relaxin analogs, binding to relaxin receptors, etc.) Based on Once a particular recombinant vector has been identified and isolated, several methods known in the art can be used to propagate the vector. Once the appropriate host system and growth conditions are established, the recombinant expression vector can be propagated and prepared in large quantities. As previously described, expression vectors that can be used include, but are not limited to, vectors named below or derivatives thereof: human viruses or animal viruses (eg, vaccinia viruses or adenoviruses). ); Insect viruses (eg, baculovirus); yeast vectors; bacteriophage vectors (eg, λ); and plasmid and cosmid DNA vectors. In the fourth approach, PCR is used to detect nucleic acids in the vector (see above).

  In addition, a host cell strain may be chosen that modulates the expression of the inserted sequences, or modifies or processes the gene product in the specific fashion desired. Expression from a particular promoter can be increased in the presence of a particular inducer and thus the expression of the polypeptide can be controlled. In addition, different host cells with characteristic and specific mechanisms for translational processing, post-translational processing and modification (eg, glycosylation, phosphorylation) of polypeptides can be used. Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed. For example, expression in bacterial systems can be used to produce an unprocessed core protein product. Expression in mammalian cells can be used to ensure “native” processing of mammalian receptor polypeptides. Furthermore, different vector / host expression systems can affect the processing reaction to different degrees.

(Functional assays for relaxin and relaxin receptor agonists and antagonists)
Relaxin agonist and relaxin antagonist activities, and relaxin receptor agonist and antagonist activities can be determined by standard assays for relaxin and / or relaxin receptor activity. In one aspect of the invention, the activity of a relaxin agonist is assayed. For example, the ability of relaxin agonists to reduce apoptosis or stimulate tissue maturation is assayed.

In another aspect of the invention, the ability of relaxin antagonists to inhibit relaxin functional activity by binding to relaxin is assayed. Similarly, the ability of a relaxin antagonist to inhibit relaxin function or relaxin receptor function determines, for example, the inhibition of a relaxin receptor added to a relaxin receptor assay and compared to a control without a relaxin antagonist. Can be assayed. Appropriate measurement of the activity of a relaxin antagonist includes measurement of IC ₅₀ which is% inhibition.

  Suitable assays for measuring the activity of relaxin or a relaxin receptor agonist or antagonist include, for example, the assays described in the following references, which are incorporated herein by reference. : MacLennan et al., Ripening of the Human Cervix and Induction of Labor with Intracervical Purified Porcine Relaxin, Obstetrics & Gynecology 68: 598-601 (1986); Poisner et al., Relaxin Stimulates the Synthesis and Release of Prorenin From Human Decidual ells: Evidence For Autocrine / Paracrine Regulation, J. Clinical Endocrinology and Metabolism 70: 1765-67 (1990); O'Day-Bowman et al. III. Relaxin's Influenza on Certifical Biochemical Properties in Oblivionized Hormone-Treated Pregnant Gilts, Endocrinology Pand. 129: 196-76 (1991); J. et al. Obstetrics & Gynecology and Reproductive Biology 41: 197-201 (1991).

  Other assays include those disclosed by: Bullesbach et al., The Receptor-Binding Sites of Human Relaxin II, J. Biol. Biol. Chem. 267: 22957-60 (1992); Hall et al., Influence of Ovarian Steroids on Relaxin-Induced Uterine Growth in Ovarietized Gilts, Endocrinology 130: 3159-66; Otalyngol. Head Neck Surg. 118: 153-56 (1992); Lee et al., Monoclonal Antibodies Special for Rat Relaxin. VI. Passive Immunization with Monoclonal Antibodies Throughout the Second Half of Pregnancy Disrupts Histological Changes Associated with Cervical Softening at Parturition in Rats, Endocrinology 130: 2386-91 (1992); Bell et al., A Randomized, Double-Blind Placebo-Controlled Trial of the Safety of Vaginal Recombinant Human Relaxin for Mechanical Riping, Obstre cs & Gynecology 82: 328-33 (1993); Bryant-Greenwood et al., Sequential Appearance of Relaxin, Prolactin and IGFBP-1 During Growth and Differentiation of the Human Endometrium, Molecular and Cellular Endocrinology 95: 23-29 (1993); Chen The Pharmacokinetics of Recombinant Human Relaxin in Non-Prefectant Women After Intravenous, Intravaginal, and Intracural A ministration, Pharmaceutical Research 10: 834-38 (1993); Huang et al., Stimulation of Collagen Secretion by Relaxin and Effect of Oestrogen on Relaxin Binding in Uterine Cervical Cells of Pigs, Journal of Reproduction and Fertility 98: 153-58 (1993).

  Further assays are disclosed below: Saxena et al, Is the Relaxin System a Target for Drug Development Cardiac Effects of Relaxin, TiPS 14: 231 (June 1993, letter). IV. Relaxin Promotes Changes in the Histological Characteristics of the Cervix that are Associated with Cervical Softening During Late Pregnancy in Gilts, Endocrinology 133: 121-28 (1993); Colon et al., Relaxin Secretion into Human Semen Independent of Gonadotropin Stimulation, Biology of Reproduction 50: 187-92 (1994); Golub et al., Effect of Short-Term Infusion of Re. ombinant Human Relaxin on Blood Pressure in the Late-Pregnant Rhesus Macaque (Macaca Mulatta), Obstetrics & Gynecology 83: 85-88 (1994); Jauniaux et al., The Role of Relaxin in the Development of the Uteroplacental Circulation in Early Pregnancy, Obstetrics & Gynecology 84: 338-342 (1994); Johnson et al., The Regulation of Plasma Relaxin Levels During Human Preganc. , J. Endocrinology 142: 261-65 (1994); Lane et al., Decidualization of Human Endometrial Stromal Cells in Vitro: Effects of Progestin and Relaxin on the Ultrastructure and Production of Decidual Secretory Proteins, Human Reproduction 9: 259-66 (1994); Lanzafame et al. Pharmacological Stimulation of Sperm Mobility, Human Reproduction 9: 192-99 (1994); Petersen et al., Normal Ser. m Relaxin in Women with Disabling Pelvic Pain During Pregnancy, Gynecol. Obstet. Invest. 38: 21-23 (1994); Tashima et al., Human Relaxins in Normal, Benign and Neoplastic Breast Tissue, J. MoI. Mol. Endocrinology 12: 351-64 (1994); Winn et al., Individual and Combined Effects of Relaxin, Estrogens, and Progesterone in Oblivionated Gilts. I. Effects on the Growth, Softening, and Histologic Properties of the Cervix, Endocrinology 135: 1241-49 (1994); Winn et al., Individual and Combined Effects. II. Effects on Mammary Development, Endocrinology 135: 1250-55 (1994); Bryant-Greenwood et al, Human Relaxins: Chemistry and Biology, Endocrine Reviews 15: 5-26 (1994); Johnson et al., Relationship Between Ovarian Steroids, Gonadotrophins and Relaxin During the Menstral Cycle, Acta Endocrinology 129: 121-25 (1993).

  In yet another aspect of the invention, the activity of an agonist or antagonist is determined by measuring the ability of the agonist or antagonist to compete with a wild type relaxin polypeptide or relaxin receptor polypeptide for binding to an anti-relaxin antibody. ,It is determined. Various immunoassays known in the art can be used. Such assays include, but are not limited to: radioimmunoassay, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassay, immunoradiometric assay, gel diffusion precipitation reaction, immunodiffusion assay, In situ immunoassay (using colloidal gold, enzyme or radioisotope label, etc.), Western blot, precipitation reaction, agglutination assay (eg gel aggregation assay or hemagglutination assay), complement binding assay, immunofluorescence assay, protein A assay Competitive and non-competitive assay systems using techniques such as immunoelectrophoretic assays and the like. Antibody binding can be detected by measuring the amount of label on the primary antibody that bound the substrate or was prevented from binding to the substrate. Alternatively, primary antibody binding is detected by measuring the binding of a secondary antibody or reagent to the primary antibody. This secondary antibody can also be directly labeled. Many means for detecting binding in an immunoassay are known in the art and are considered to be within the scope of the present invention.

  The functional activity of an agonist or antagonist can also be determined in an in vivo system. For example, the ability of a relaxin agonist or relaxin antagonist to bind to a relaxin receptor or compete for binding to a relaxin receptor or to modulate apoptosis in a cell population and / or tissue can be measured. The above assay can be used to determine the activity resulting from expression of a relaxin agonist or relaxin antagonist in vertebrate cells. Alternatively, a relaxin agonist or relaxin antagonist can be expressed in a heterologous system and the activity of the relaxin agonist or relaxin antagonist can be assayed as a modulator of physiological changes in the system. For example, the ability of a relaxin agonist or relaxin antagonist to modulate apoptosis can be tested in vertebrate cells (eg, transfected mammalian cells).

(Administration of relaxin agonists and relaxin antagonists)
The present invention provides a method for administering to a subject an effective amount of a relaxin agonist or relaxin antagonist (collectively referred to as an “active agent”). Typically, the active agent is substantially purified prior to formulation. The subject can be a human or non-human vertebrate and is typically an animal including but not limited to cows, pigs, horses, chickens, cats, dogs and the like. More typically, the subject is a mammal, and in certain embodiments is a human.

  Various delivery systems (eg, by injection, by injection (eg, intradermal, intramuscular or intraperitoneal), oral delivery, nasal delivery, intrapulmonary delivery, rectal delivery, transdermal delivery, interstitial delivery or subcutaneous delivery) Known and can be used to administer the active agent. In certain embodiments, it may be desirable to administer the active agent locally to the area in need of treatment; such administration includes, but is not limited to, local infusion, local application, injection (eg, Within the testis or within the prostate), by a catheter, or by an implant, which is for example a porous material, a non-porous material, a gelatinous material or a polymer material, such as a silastic membrane Includes membranes or fibers. In one embodiment, administration can be by direct injection at the target site.

  A pharmaceutical composition comprising the active agent can be formulated according to the desired delivery system. Such pharmaceutical compositions typically comprise a therapeutically effective amount of the active agent and a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable” refers to a vertebrate (typically, as approved by a federal or state government agency or in the United States pharmacopoeia or other generally accepted pharmacopoeia. Means listed for use in animals, more typically humans). The term “carrier” refers to a diluent, adjuvant, excipient, stabilizer, preservative, viscogen or vehicle in which the active agent is formulated for administration. The pharmaceutical carrier can be a sterile liquid (eg, water and oils including petroleum, animal, vegetable or synthetic oils (eg, peanut oil, soybean oil, mineral oil, sesame oil, etc.)). Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, Examples include ethanol. If desired, the composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

  Suitable preservatives include, for example, sodium benzoate, quaternary ammonium salts, sodium azide, methyl paraben, propyl paraben, sorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, chlorobutanol, dehydroacetic acid, Examples include ethylenediamine, potassium benzoate, potassium metabisulfite, potassium sorbate, sodium bisulfite, sulfur dioxide, organic mercury salts, phenol and ascorbic acid. Suitable viscosity modifiers include, for example, carboxymethylcellulose, sorbitol, dextrose, and polyethylene glycol. Other examples of suitable pharmaceutical carriers are described, for example, in Remington's Pharmaceutical Sciences (Edited by Gennaro), Mack Publishing Co. , Easton, Pennsylvania (1990).

  The active agent can also be formulated as neutral or salt forms. Pharmaceutically acceptable salts include salts formed with free amino groups (eg, salts derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc.) and salts formed with free carboxyl groups ( Examples thereof include salts derived from sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, iron hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

  In one embodiment, the active agent is formulated as a pharmaceutical composition adapted for intravenous administration to humans according to conventional procedures. Water is a typical carrier for intravenous delivery. Saline solutions, aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition may also contain solubilizers or local anesthetics to relieve pain at the site of injection. In general, the ingredients are either separately or in a single dosage form (eg, as a dry lyophilized powder or in a water-free concentrate in a sealed container such as an ampoule or bag indicating the amount of active agent) ) Or are mixed together in Where the composition is to be administered by infusion, it can be dispersed in an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that these ingredients may be mixed prior to administration.

  Orally deliverable compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. Oral formulations can include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.

  For rectal administration, the composition is formulated according to standard pharmaceutical procedures. Typically, the composition is formed as a soluble composition (eg, a suppository). Suppositories can contain adjuvants that provide the desired consistency for the composition. Suppositories can also contain water-soluble carriers such as polyethylene glycol, polypropylene glycol, glycerogelatin, methylcellulose or carboxymethylcellulose. Suppositories can also contain wetting agents such as fatty acids, fatty acid glycerides, polyethylene sorbitan fatty acid esters, polyethylene sorbital fatty acid esters, polyethylene fatty acid esters, and higher alcohol esters of polyoxyethylene and esters of lower alkyl sulfonic acids. . Suppositories can also contain suitable emulsifying and dispersing ingredients, as well as ingredients for adjusting the viscosity, and coloring substances.

  Nasal administration is typically performed using the solution as a nasal spray and can be formulated by various methods known to those skilled in the art. Systems for intranasal administration of liquid as a spray are well known (see, eg, US Pat. No. 4,511,069, which is incorporated herein by reference). Preferred nasal spray solutions contain the active agent in a liquid carrier that increases the absorption of the drug and one or more buffers or other additives, if necessary, to minimize irritation of the nasal cavity. Includes non-ionic surfactants to limit. In some embodiments, the nasal spray solution further comprises a propellant. The pH of this nasal spray solution is typically between about pH 6.8 and pH 7.2.

  For intranasal administration, ingredients that improve absorption of nasally administered active agents and reduce nasal irritation are desirable, particularly when used in chronically administered treatment protocols. In this case, the use of surfactants is preferred in order to increase the absorption of this active factor (eg Hirai et al., Int. J. Pharmaceuticals 1: 173-84 (1981); British patent specification 1 527 605 (these Each of which is incorporated herein by reference). However, nasal administration of drugs augmented by surfactants can cause nasal irritation, including stinging, congestion and rhinorrhea. Thus, compositions that reduce irritation while increasing absorption through the nasal mucosa (eg, nonionic such as nonoxynol-9, lauret-9, poloxamer-124, octoxynol-9 and lauramide DEA) Surfactant) is desirable. Nonoxynol-9 (N-9) is an ethoxylated alkylphenol, a polyethyleneoxy condensate of nonylphenol and 9 moles of ethylene oxide. This surfactant is used in surfactant products and is SURFONIC® N-95 (Jefferson), NEUTRONYX® 600 (Onyx) and IGEPAL® (CO-630 (GAF) ). N-9 is considered a hard surfactant and has been used as a spermicide (see The Merck Index, 10th edition, Entry 6518). In order to minimize irritation due to the use of surfactants, one or more anti-irritant additives are included in the emulsion. In one example, polysorbate-80 has been shown to reduce irritation caused by intranasally administered drugs (this delivery is facilitated by using nonionic surfactants) (eg, See US Pat. No. 5,902,789, which is incorporated herein by reference).

  Intrapulmonary dosage forms containing the active agent can be administered intranasally to the respiratory tract, or can be administered to the respiratory tract by inhaling a spray or aerosol containing the active agent. This active agent is typically delivered directly into the lung in the form of small particle aerosols, which are specifically targeted to the minimal air pathway and alveoli.

  Intrapulmonary dosage forms are typically formed as particulate dispersed forms. This can be accomplished by preparing a solid particle aqueous aerosol containing the composition. Typically, an aqueous aerosol is made by formulating an aqueous solution or suspension of the composition together with conventional pharmaceutically acceptable carriers and stabilizers. These carriers and stabilizers typically include nonionic surfactants (eg, Tween, Pluronic or polyethylene glycol), harmless proteins (eg, serum albumin), sorbitan esters, oleic acid, amino acids (eg, glycine) ), Buffer, salt, sugar or sugar alcohol. The formulation may also include a mucolytic agent (such as that described in US Pat. No. 4,132,803, which is incorporated herein by reference), and a bronchodilator. This formulation is preferably sterile. Aerosols are generally prepared from isotonic solutions. The particles optionally contain normal lung surfactant protein.

  The aerosol of particles can be formed in the form of an aqueous suspension or a non-aqueous suspension (eg, a fluorocarbon propellant). The aerosol is preferably free of pulmonary stimulants (ie, surfactants that cause acute bronchoconstriction, cough, pulmonary edema or tissue destruction). Non-irritating absorption enhancers are also suitable for use herein.

  A sonic nebulizer can be used to prepare the aerosol. Sonic nebulizers minimize exposure of the composition to shear, which can cause degradation. A suitable device is a Bird Micronebulizer. Other suitable automated or nebulization systems or endotracheal delivery systems include, for example, the following as long as they are compatible with the composition to be administered and can deliver the desired size of particles: US Pat. No. 3,915,165; system disclosed in European Patent 0 116 476; jet nebulizer described by Newman et al. (Thorax 40: 671-76 (1985)); measuring dose inhaler (eg, Berenberg, J. Asthma-USA 22: 87-92 (1985)); Braunner (US Pat. No. 5,803,078) endotracheal catheter assembly or other device (see, for example, Sears et al., NZ. Med.J.96: 743II (1983); O'Reilly et al., Br. Med.J.286: 6377 (1983); or Stander et al, Respiration 44: 237-40 see (1982)). (The disclosures of these references are incorporated herein by reference).

  Particulate aerosol suspensions are typically fine dry powders containing active agents. Particulate aerosol suspensions are prepared by a number of conventional procedures. The simplest method for preparing such a suspension is to micronize the active agent (eg, as a crystal or lyophilized cake) and disperse the particles in a dry fluorocarbon propellant. In these formulations, the active agent is preferably suspended in the fluorocarbon. In an alternative embodiment, the active agent is stored in a separate compartment from the propellant. Spraying a propellant dispenses a predetermined dose from the storage compartment. Devices used to deliver active agents in this manner are known as measured dose inhalers (MDIs) (eg, Byron, Drug Development and Industrial Pharmacology 12: 993 (1986) (herein). See incorporated herein by reference)).

  The size of the aerosol or particle is generally in the range of about 0.5 μm to about 5 μm, typically about 2 μm to 5 μm, preferably about 2 μm to about 4 μm, or about 4 μm to about 5 μm. In some aspects, the smaller the particle, the less acceptable it is. This is because smaller particles do not accumulate and instead tend to diverge. Similarly, in other aspects, the larger the particles, the less preferred. Because larger particles are less likely to accumulate in the alveoli and are removed by insertion in the nasopharyngeal or oral cavity (see, eg, Byron, J. Pharm. Sci. 75: 433 (1986)). ) Aerosols or particulate compositions can be non-uniform in size distribution, but non-uniformities can be reduced by known methods (eg, screening units described in EP 0 135 390). Heterogeneity is typically not disadvantageous unless the proportion of particles having an average diameter greater than about 4 μm is large enough to interfere with delivery of a therapeutic dose by pulmonary inhalation. Suspensions containing more than about 15% of particles in the range of 0.5-5 μm may be used, but in general, particle portions having an average diameter greater than 5 μm will instead be about about the total number of particles. It is less than 25%, preferably 10% or less. The indicated diameter refers to the diameter of the particle when introduced into the airway.

  The amount of active agent effective in treating a particular subject is based on the particular abnormality being treated and can be determined by standard clinical techniques. In addition, in vitro assays can be used as needed to help identify optimal dosage ranges. The exact dose of active agent used in the formulation will also depend on the route of administration, and the severity of the condition, and must be determined according to the judgment of the physician and the circumstances of each subject. Suitable dosage ranges for administration are generally about 0.001 mg / kg to about 100 mg / kg of active agent per kg body weight. Effective doses can also be extrapolated from dose response curves derived from in vitro or animal model test systems. Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight; oral formulations typically contain 10% to 95% active ingredient.

  The present invention also provides a pharmaceutical pack or kit containing one or more containers filled with one or more components of the pharmaceutical composition of the present invention. Notifications in the form prescribed by the government that regulates the manufacture, use or sale of pharmaceuticals or biological products are optionally associated with such containers, which notifications are manufactured, used for human administration Or affect the authorization by the sales agent.

  In yet another embodiment, the active agent can be delivered in a sustained release system. In one embodiment, a pump may be used (eg, Langer, supra; Sefton, Crit. Ref. Biomed. Eng. 14: 201-40 (1987); Buchwald et al., Surgary 88: 507-16 (1980). Saudek et al., N. Engl. J. Med. 321: 574-79 (1989)). In another embodiment, polymeric materials can be used (Medical Applications of Controlled Release, Langer and Wise (ed.), CRC Pres., Boca Raton, Florida (1974); and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); Levy et al., Science 228: 190-92 (1985). See also; During et al., An n. Neurol.25: 351-56 (1989); Howard et al., J. Neurosurg.71: 105-12 (1989)) (the disclosures of which are incorporated herein by reference).

  Thus, in yet another embodiment, a sustained release system can be placed near a therapeutic target that requires only a systemic dose fraction (eg, Goodson, Medical Applications of Controlled Release, supra, No. 1). 2, pp. 115-38 (1984)). Other sustained release systems are discussed, for example, in a review by Langer (Science 249: 1527-33 (1990), which is incorporated herein by reference).

(Administration of nucleic acid)
The present invention relates to nucleic acids (eg, relaxin agonists or relaxin antagonists (relaxins, relaxin analogs, soluble relaxin receptors, relaxin binding agents, relaxin receptor binding agents) nucleic acids, relaxin antisense nucleic acids and / or Administration of relaxin receptor antisense nucleic acids, etc.) to provide additional methods of modulating apoptosis. Nucleic acids (both sense and antisense) can be used in gene therapy processes. Gene therapy refers to a process that provides for the expression of nucleic acid of exogenous origin, which includes subject antisense to regulate apoptosis (eg, to treat tissue abnormalities) within the subject. Examples include antisense nucleic acids that encode nucleic acids or relaxin agonists or relaxin antagonists. In certain embodiments, a nucleic acid encoding relaxin or a relaxin analog is administered into a cell having a relaxin receptor to reduce apoptosis. In other specific embodiments, a nucleic acid encoding a relaxin binding agent (eg, relaxin, a soluble relaxin receptor) or a relaxin receptor binding agent (eg, a relaxin analog) may be used to increase the relaxin receptor to increase apoptosis. Administration to expressing cell aggregates. Any method of gene therapy useful in the art can be used according to the present invention. An exemplary method is described below.

  For a general review of methods of gene therapy, see Steinberg and Raso (J. Pharm. Pharm. Sci. 1: 48-59 (1998)); Pantuc et al. (World J. Urol. 18: 143-47 (2000)). Prince (Pathology 30: 335-47 (1998)); Ledley (Curr. Opin. Biotechnol. 5: 626-36 (1994)); Goldspiel et al. (Clin. Pharm. 12: 488-505 (1993)); Wu; and Wu (Biotherapy 3: 87-95 (1991)); Tolstoshev (Ann. Rev. Pharmacol. Toxicol. 32: 573-96 (1993)); Mulligan (Science 2). 60: 926-32 (1993)); Morgan and Anderson (Ann. Rev. Biochem. 62: 191-217 (1993)); and May (TIBTECH 11: 155-215 (1993)).

  Methods that can be used and are generally known in the art of recombinant DNA technology are described in Ausubel et al. (1996, supra) and Kriegler (Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990)). The method to be mentioned is mentioned. In one embodiment, the nucleic acid comprises a sense nucleic acid (eg, a nucleic acid encoding a relaxin analog, soluble relaxin receptor, etc.) that is part of a vector that expresses the nucleic acid in a suitable host. In particular, such nucleic acids have a promoter operably linked to a coding region (eg, relaxin or relaxin receptor) in a sense orientation, the promoter being inducible or constitutive, and optionally It is tissue specific. In another embodiment, the nucleic acid comprises an antisense nucleic acid (eg, a relaxin antisense nucleic acid or a relaxin receptor antisense nucleic acid) that is part of a vector that expresses the nucleic acid in a suitable host. In particular, such nucleic acids have a promoter operably linked to a coding region (eg, relaxin or relaxin receptor) in an antisense orientation, the promoter being inducible or constitutive, and optionally It is tissue specific.

  In another specific embodiment, the nucleic acid (eg, a sense nucleic acid encoding a relaxin analog, a soluble relaxin receptor, or the like, or an antisense nucleic acid encoding a relaxin antisense nucleic acid, a relaxin receptor antisense nucleic acid, etc.) This nucleic acid and any other desired sequence are used when flanked by regions that promote homologous recombination at the desired site in the genome, thereby providing intrachromosomal expression of the nucleic acid (eg, Koller and Smithies, Proc. Natl.Acod.Sci.USA 86: 8932-35 (1989); No. 059; No. 5,487,9 No. 2; Nos and the 5,464,764; (the disclosures of which are incorporated herein by reference), reference).

For any of these embodiments, delivery of the nucleic acid into the subject can be direct (when the subject is directly exposed to the nucleic acid or nucleic acid transport vector) or indirectly (cells are Can be done either in vitro with a nucleic acid first and then transplanted into a subject). Each of these two approaches is known as in vivo gene therapy or ex vivo gene therapy. In certain embodiments, the nucleic acid is administered directly in vivo to express the nucleic acid and produce the encoded product. This can be accomplished by any of a number of methods known in the art: for example, a method that is made as part of an appropriate nucleic acid expression vector and administered into an intracellular nucleic acid; Methods by infection using retroviruses or attenuated retroviruses or other viral vectors (infra), direct injection of naked DNA (eg Asahara et al., Semin. Interv. Cardiol. 1: 225-32 (1996) Prazeres et al., Trends Biotechnol. 17: 169-74 (1999) (the disclosures of which are incorporated herein by reference), electroporation (see, eg, Muramatsu et al., Int. J .; Mol.Med.1: 55-6 2 (1998), (the disclosures of which are incorporated herein by reference), or the use of microparticle irradiation such as a gene gun (BIOLISTIC ^™ , Dupont) (see, eg, Biengenga et al., J. Neurosci. Methods 71: 67-75 (1997), the disclosures of which are incorporated herein by reference). Nucleic acids can also be inserted into cells by coating of naked nucleic acids with lipids or cell surface receptors or transforming agents, encapsulation in liposomes, derivatized liposomes, microparticles, or microcapsules (eg, De Smedt et al. 17: 113-26 (2000); Maurer et al., Mol.Membr.Biol.16: 129-40 (1999); Tarahovsky and Ivansky, Biochemistry 63: 607-18 (1998); Lassi, Trends Biotech 16: 307-21 (1998); Gao and Huang, Gene Ther. 2: 710-22 (1995); (the disclosures of which are incorporated herein by reference) Nucleic acids bound to peptides known to enter cells can also be administered, or nucleic acids bound to ligands that are affected by receptor-mediated endocytosis, which can be used to specifically bind receptors. Cell types that are expressed can be targeted (eg, Liang et al., Pharmazie 54: 559-66 (1999); Cristiano, Front. Biosci. 15: D1161-70 (1998); Guy et al., Mol. Biotechnol. 3: 237). -48 (1995); see Wu and Wu, J. Biol. Chem. 262: 4429-32 (1987), the disclosures of which are incorporated herein by reference. Nucleic acid ligand complex is a spindle that allows the ligand to disrupt the endosome It may be in a form containing cellular viral peptides and avoiding degradation of nucleic acids by lysosomes (eg, Phillips, Biologicals 23: 13-16 (1995), the disclosures of which are incorporated herein by reference) See)).

  In yet another embodiment, antisense nucleic acids can be targeted in vivo for specific uptake and expression of cells by targeting specific receptors (eg, Phillips, Biologicals 23: 13-16 (1995); International Patent Publications WO 92/06180; WO 92/22635; WO 92/20316; WO 93/14188, and WO 93/20221; the disclosures of which are incorporated herein by reference.

  In certain embodiments, the viral vector comprises a nucleic acid (eg, a sense nucleic acid encoding a relaxin analog, a soluble relaxin receptor, etc., or an antisense nucleic acid encoding a relaxin antisense nucleic acid, a relaxin receptor antisense nucleic acid). Etc.) is used. For example, retroviral vectors can be used (eg, Palu et al., Rev. Med. Virol. 10: 185-202 (2000); Buchschacher and Wong-Staal, Blood 15: 2499-504 (2000); Miller et al., Meth). Enzymol.217: 581-99 (1993), the disclosures of which are incorporated herein by reference. These retroviral vectors are typically modified to remove retroviral sequences that are not necessarily required for packaging of the viral genome and integration into host cell DNA. Antisense nucleic acids used in gene therapy are cloned in a vector to facilitate delivery of the antisense nucleic acid into the subject. Lentiviral vectors can also be used. (See, eg, Buchschacher and Wong-Staal, supra; Naldini et al., Science 272: 263-67 (1996), the disclosures of which are hereby incorporated by reference). Other references illustrating the use of viral vectors in gene therapy are Lundstrom (J. Recept. Signal. Transduc. Res. 19: 673-86 (1999)); Clowes et al. (J. Clin. Invest. 93: 644-51 (1994)); Kiem et al. (Blood 83: 1467-73 (1994)); Salmons and Gunzberg (Hum Gene Ther. 4: 129-41 (1993)); and Grossman and Wilson (Curr. Opin. Genet Dev. 3: 110-14 (1993)), the disclosures of which are incorporated herein by reference.

  Adenoviruses can also be used in gene therapy. Adenoviruses are particularly attractive vehicles for delivering genes to the prostate, liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson (Curr. Opin. Genet Dev. 3: 499-503 (1993), the disclosures of which are incorporated herein by reference) present a review of adenovirus-based gene therapy. To do. Herman et al. (Human Gene Therapy 10: 1239-49 (1999), the disclosures of which are incorporated herein by reference) describe a replication deficiency that includes a herpes simplex thymidine kinase gene in a human prostate. Intraprostatic infusion of adenovirus followed by intravenous administration of the prodrug ganciclovir in a phase I clinical experiment is described. Other examples of the use of adenovirus in gene therapy are Rosenfeld et al. (Science 252: 431-34 (1991)); Rosenfeld et al. (Cell 68: 143-55 (1992)); Mastrangeli et al. (J. Clin. Invest. 91: 225-34 (1993)); and Thompson (Onclol. Res. 11: 1-8 (1999)). Adeno-associated virus (AAV) can also be used in gene therapy (eg, Rabinowitz and Samulski, Curr. Opin. Biotechnol. 9: 475-85 (1988); Carter and Samulski, Int. J. Mol. Med. 6). 17-27 (2000); Tal, J. Biomed. Sci. 7: 279-91 (2000); Ali et al., Gene Therapy 1: 367-84 (1994); U.S. Pat. No. 4,797,368 and the like. No. 5,139,941; Walsh et al., Proc. Soc. Exp.Biol.Med.204: 289-300 (1993); Grimm et al., Human Gene Therapy 10: 2445-50 (1999)) (these Disclosure of which is incorporated by reference herein).

  Another approach to gene therapy involves transferring nucleic acids to cells in tissue culture by methods such as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Typically, the method of migration involves the transfer of a selectable marker to the cell. The cells are then harvested and placed under selection to isolate those cells that express the nucleic acid. The selected cells are then delivered to the subject.

  In one embodiment, the nucleic acid is introduced into the cell prior to in vivo administration of the resulting recombinant cell. Such introduction can be done by any method known in the art, including but not limited to: transfection, electroporation, microinjection, nucleic acid-containing viral or bacteriophage vectors Infection, cell fusion, chromosome-mediated gene transfer, micronucleus-mediated gene transfer, etc. Numerous techniques for introducing foreign genes into cells are known in the art (eg, Muramatsu et al., Int. J. Mol. Med. 1: 55-62 (1998); Liang et al., Pharmazie 54: 559-66 (1999); Loeffer and Behr, Meth. Enzymol. 217: 599-618 (1993); Cotten et al., Meth. Enzymol. 217: 618-44 (1993); Cline, Pharmacol. 92 (1985); the disclosures of which are incorporated herein by reference) and can be used in accordance with the present invention. The technique is typically given for the stable transfer of nucleic acid to a cell so that the nucleic acid can be expressed by the cell and is genetic and expressible by its cell progeny.

  The resulting recombinant cells can be delivered to a subject by various methods known in the art. Typically, cells are injected subcutaneously. In another embodiment, the recombinant skin cells can be applied to a subject as a skin graft. The amount of cells required for use depends on the desired effect, the condition of the subject, etc., and can be determined by one skilled in the art.

  Cells into which the nucleic acid can be introduced for gene therapy purposes encompass any desired useful cell type and include, but are not limited to: male reproductive tract (eg, prostate cells, testicular cells, seminal cells) Cells, or epididymal cells), female reproductive tract (eg, uterus, cervix, interpubic ligament, connective tissue in lower limbs, etc.), liver, kidney, spleen, thymus, brain, heart, intestine, skin A cell or collection of cells, such as lungs. Suitable cells further contain epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, and stem or progenitor cells. The cells used for gene therapy are generally autologous to the subject, but allogeneic cells that can be classified for compatibility with the subject can be used.

  In another aspect, the nucleic acid (eg, a sense nucleic acid encoding a relaxin analog, a soluble relaxin receptor, or the like, or an antisense nucleic acid encoding a relaxin antisense nucleic acid, a relaxin receptor antisense nucleic acid, etc.) directly into the cell. Administered. The nucleic acid is at least 6 nucleotides and typically an oligonucleotide (ranging from 6 to about 50 or more nucleotides). In certain aspects, the oligonucleotide is at least 10 nucleotides, at least 15 nucleotides, at least 100 nucleotides, or can be at least 200 nucleotides. The oligonucleotide can be DNA or RNA or a chimeric mixture or derivative thereof or an analog thereof, and can be single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone. The oligonucleotide is a peptide or an agent that promotes movement across the cell membrane (eg, Nielsen, Pharmacol. Toxicol. 86: 3-7 (2000); Somets et al., Front. Biosci. 1: D782-86 (1999)); Galderisi et al., J. Cell Physiol.181: 251-57 (1999); Lessinger et al., Proc.Natl.Acad.Sci.USA 86: 6553-56 (1989); Lemaitre et al., Proc.Natl. 84: 648-52 (1987); International Patent Publication WO 88/09810, see) Hybridization-induced cleavage agents (eg, Krol et al., BioTechniques 6: 958-76 (1). 88)) or intercalating agents (e.g., Zon, Pharm.Res.5: 539-49 (1988) refer), such as may include other additional radicals. (These disclosures are incorporated herein by reference).

In one example, the antisense oligonucleotide is provided as single stranded DNA. The oligonucleotide can be modified at any position in the structure with substituents generally known in the art. The oligonucleotide may comprise at least one modified base moiety such as: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetyl Cytosine, 5- (carboxy-hydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylamino-methyluracil, dihydrouracil, β-D-galactosyl queosine, inosine, N6- Isopentenyl adenine, 1-methyl guanine, 1-methyl inosine, 2,2-dimethyl guanine, 2-methyl adenine, 2-methyl guanine, 3-methyl cytosine, 5-methyl cytosine, N6-adenine, 7-methyl guanine, 5- Tilaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylcuocin, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N-6-isopentenyladenine, uracil- 5-oxyacetic acid (V), pseudouracil, cuocin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5 -Oxyacetic acid (V), 5-methyl-2-thiouracil, 3- (3-amino-3-N-2-carboxypropyl) uracil, 2,6-diaminopurine, and the like. In another embodiment, the oligonucleotide comprises at least one modified sugar moiety (eg, arabinose, 2-fluoroarabinose, xylulose, and hexose).
In yet another embodiment, the antisense oligonucleotide comprises at least one of the following modified phosphate backbones: for example, phosphorthionate, phosphorodithionate, phosphoramidothionate, phosphoramidate, phospho Rudiamidate, methylphosphonate, alkylphosphotriester, and formacetal or analogs thereof.

  In yet another embodiment, the antisense oligonucleotide is an α-anomeric oligonucleotide. α-anomeric oligonucleotides form specific double-stranded hybrids with complementary RNAs (its strands are parallel to each other as opposed to normal β-units) (Gautier et al., Nucl. Acids Res. 15: 6625-41 (1987)). The oligonucleotide can be conjugated to another molecule (eg, a peptide, a hybridization-induced cross-linking agent, a transport agent, a hybridization-induced cleavage agent, etc.).

  In certain embodiments, the antisense oligonucleotide comprises catalytic RNA, or a ribozyme (eg, Welch et al., Curr. Opin. Biotechnol. 9: 486-96 (1998); Norris et al., Adv. Exp Med. Biol). 465: 293-301 (2000); International Patent Publication WO 90/11364; Sarver et al., Science 247: 1222-25 (1990)). In another embodiment, the oligonucleotide is a 2′-0-methyl ribonucleotide (Inoue et al., Nucl. Acids Res. 15: 6131-48 (1987)), or a chimeric RNA-DNA analog (Inoue et al., FEBS Lett 215: 327-30 (1987)).

In another specific embodiment, double-stranded RNA directs sequence-specific degradation of mRNA by RNA interference (Hunter, Curr. Biol. 10: R137-40 (2000); Bosher and Labousee, Nat. Cell). Biol.2: e31-36 (2000); (the disclosures of which are hereby incorporated by reference). Briefly, double stranded nucleic acids are introduced into cells to selectively inhibit gene expression by causing degradation of mRNA. (See, eg, Zamore et al., Cell 101: 25-33 (2000), the disclosures of which are incorporated herein by reference)
Nucleic acids according to the present invention can be synthesized by standard methods known in the art. Enzymatic methods for nucleic acid synthesis frequently use Klenow, T7, T4, Taq or Escherichia coli DNA polymerase, as described in Sambrook et al. (Supra). Enzymatic methods for RNA nucleic acids frequently use SP6, T3, or T7 RNA polymerases as described in Sambrook et al. (Supra). Reverse transcriptase can also be used for RNA-derived DNA synthesis (Sambrook et al., Supra). Nucleic acids are typically prepared using a template nucleic acid enzymatically. The nucleic acid is either chemically synthesized or obtained as mRNA, genomic DNA, cloned genomic DNA, cloned cDNA, or other nucleic acid. Some enzymatic methods of DNA nucleic acid synthesis may require additional primers that can be chemically synthesized. Finally, linear nucleic acids can be prepared by polymerase chain reaction (PCR) technology (eg, described by Saiki et al. (Science 239: 487 (1988)).
Chemical methods can also be used to synthesize nucleic acids (eg, antisense oligonucleotides) (eg, by use of commercially available automated DNA synthesizers). For example, phosphorothioate nucleic acids can be synthesized by the method of Stein et al. (Nucl. Acids Res. 16: 3209-21 (1988)), and methylphosphonate nucleic acids can be synthesized with a controlled porous glass polymer support ( For example, see Sarin et al., Proc. Natl. Acad. Sci. USA 85: 7448-51 (1988), the disclosures of which are incorporated herein by reference. Other methods include those disclosed by: Usman et al. (J. Am. Chem. Soc. 109: 7845-54 (1987)), Scaringe et al. (Nucleic Acid Res. 18: 5433-41 ( 1990)); Caruthers (Oligoncleotides: Antisense Inhibitors of Gene Expression, pages 7-24, Cohen, (ed.), CRC Press, Inc. Boca Raton, Fla, 1989); , IRL Press, 1984); Oligonucleotides and Analogies, A Practi. al Approach (Eckstein, IRL Press, 1991); and U.S. Pat. Nos. 4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679; 5,026,838; 5,132,418; and Re. 34,069. All of the aforementioned references are incorporated herein by reference.

(Small molecule effector or small molecule antagonist)
Relaxin nucleic acids, polypeptides, analogs and fragments, and relaxin receptor nucleic acids, polypeptides, analogs and fragments and analogs are also screened to detect candidate compounds that specifically bind to relaxin polypeptides or relaxin receptors. Used in assays to modulate apoptosis. Such candidate compounds are typically small molecule effectors (agonists) or antagonists and can be identified by in vitro and / or in vivo assays. Such assays can be used to identify small molecule effectors or antagonists that are therapeutically effective as relaxin agonists or antagonists or as lead compounds for drug development. Thus, the present invention provides an assay for detecting a compound. The compound specifically affects the activity or expression of relaxin nucleic acids, relaxin polypeptides, relaxin receptor nucleic acids, relaxin receptors and the like.

  In a typical in vivo assay, recombinant cells expressing relaxin or relaxin receptor nucleic acid can be used to screen candidate compounds for compounds that affect relaxin or relaxin receptor nucleic acid expression. Agonist or antagonist effects on relaxin or relaxin receptor expression include stimulation or inhibition (eg, upregulation or downregulation) of mRNA transcription, increase or decrease of mRNA stability, translation of mRNA, relaxin or relaxor. Effects on the synthesis or synthesis of a toxin receptor polypeptide, relaxin or relaxin receptor polypeptide function (eg, binding to a relaxin receptor), and / or stability or localization of a relaxin or relaxin receptor polypeptide may be mentioned. Such effects on expression can be identified as physiological changes (eg, changes in relaxin-responsive tissue growth rate, division, viability, collagen deposition, apoptosis, etc.). In one embodiment, candidate compounds express relaxin or relaxin receptor polypeptides to identify those compounds that produce a physiological change (eg, stimulation or inhibition of relaxin or relaxin receptor polypeptide function). Administered to recombinant cells.

  Candidate compounds can also be identified by in vitro screening methods. For example, a recombinant cell expressing relaxin or a relaxin receptor nucleic acid can be recombinantly transferred to an in vitro assay to identify candidate compounds that bind to the relaxin or relaxin receptor polypeptide. Can be used to produce Candidate compounds (such as small molecules) are contacted with a polypeptide (or fragment or analog thereof) under conditions that lead to binding, and then candidate compounds that specifically bind to the polypeptide are identified. Methods that can be used to carry out the previous description are commonly known in the art, and can be random or combined peptides or non-peptides that can screen for candidate compounds that specifically bind relaxin or relaxin receptor polypeptides. Contains diverse libraries such as peptide libraries. Many libraries that can be used are known in the art, such as chemically synthesized libraries, recombinant phage display libraries, and in vitro translation-based libraries.

Examples of chemically synthesized libraries are described below: Fodor et al. (Science 251: 767-73 (1991)), Houghten et al. (Nature 354: 84-86 (1991)), Lam et al. (Nature). 354: 82-84 (1991)), Medynski (BioTechnology 12: 709-10 (1994)), Gallop et al. (J. Med. Chem. 37 (9): 1233-51 (1994)), Ohlmeyer et al. (Proc. Natl. Acad. Sci. USA 90: 10922-26 (1993)), Erb et al. (Proc. Natl. Acad. Sci. USA 91: 1422-26 (1994)), Hughten et al. (BioTechniques 13: 412-21 (1). 992)), Jaywickkreme et al. (Proc. Natl. Acad. Sci. USA 91: 1618-18 (1994)), Salmon et al. (Proc. Natl. Acad. Sci. USA 90: 11708-12 (1993)), International Patent. Publication WO 93/20242, and Brenner and Larner (Proc. Natl. Acad. Sci. USA 89: 5381-83 (1992)). (The disclosures of these references are incorporated herein.)
Examples of phage display libraries are Scott and Smith (Science 249: 386-90 (1990)), Devlin et al. (Science 249: 404-06 (1990)), Christian et al. (J. Mol. Biol. 227: 711). 18 (1992)), Lenstra (J. Immunol. Meth. 152: 149-57 (1992)), Kay et al. (Gene 128: 59-65 (1993)) and International Patent Publication WO 94/18318 (the disclosures of which are (Incorporated herein by reference).

  Libraries based on in vitro translation include those described in International Patent Publication WO 91/05058, and Mattheakis et al. (Proc. Natl. Acad. Sci. USA 91: 9022-26 (1994)). It is not limited to. As an example of a non-peptide library, a benzodiazepine library (see, eg, Bunin et al., Proc. Natl. Acad. Sci. USA 91: 4708-12 (1994)) can be adapted for use. Peptide libraries (see, eg, Simon et al., Proc. Natl. Acad. Sci. USA 89: 9367-71 (1992)) can also be used. Another example of a library that can be used, where the amide functionality in the peptide is palmitylated to produce a chemically transformed combinatorial library, is described by Ostresh et al. (Proc. Natl. Acad). Sci.USA 91: 11138-42 (1994)).

  Library screening can be accomplished by any of a variety of commonly known methods. See, for example, the following references disclosing the screening of peptide libraries: Parmley and Smith (Adv. Exp. Med. Biol. 251: 215-18 (1989)); Scott and Smith (1990, supra). Fowlkes et al. (BioTechniques 13: 422-28 (1992)); Oldenburg et al. (Proc. Natl. Acad. Sci. USA 89: 5393-97 (1992)); Yu et al. (Cell 76: 933-45 (1994)). Staudt et al. (Science 241: 577-80 (1988)); Bock et al. (Nature 355: 564-66 (1992)); Tuerk et al. (Proc. Natl. Acad. Sci. USA 89: 698). -92 (1992)); Ellington et al. (Nature 355: 850-52 (1992)); US Pat. Nos. 5,096,815, 5,223,409 and 5,198,346; Rebar And Pabo (Science 263: 671-73 (1994)); and International Patent Publication WO 94/18318, the disclosures of which are incorporated herein by reference.

  In certain embodiments, the screening comprises contacting a library member with relaxin, relaxin analog or relaxin receptor immobilized on a solid phase and binding of the relaxin, relaxin analog or relaxin receptor to the library. This can be done by collecting members. Examples of such screening methods, referred to as “panning” techniques, include, for example, Parmley and Smith (Gene 73: 305-18 (1988)); Fowles et al. (1992, supra); International Patent Publication WO 94/18318; Described by the references cited in them.

(Identification of subjects with relaxin-related abnormalities)
Relaxin nucleic acids (both sense and antisense), and fragments and analogs thereof, and anti-relaxin antibodies also have utility for identifying subjects with relaxin-related abnormalities. Such molecules can be used in assays (hybridization or immunoassays) to detect, prognose, diagnose or monitor various abnormalities, or relaxin expression or response to relaxin or relaxin analogs. Or not. Similarly, such molecules have utility for monitoring the treatment of cellular or tissue abnormalities. Specifically, methods such as immunoassays involve contacting a sample from a subject with an anti-relaxin antibody under conditions that lead to immunospecific binding, and any immunospecific binding of the protein by the antibody. This can be done by a process that involves detecting or measuring the amount. Detection of abnormal levels of relaxin and / or relaxin receptor polypeptide (eg, decreased level, absent or elevated) using binding of antibodies to relaxin or relaxin receptor polypeptide in tissue sections or from semen Can do. In certain embodiments, antibodies to relaxin or a relaxin receptor polypeptide can be used to assay a subject's tissue or semen for the presence of relaxin or a relaxin receptor polypeptide, wherein abnormal levels of relaxin are detected. Is an indicator of relaxin-related abnormalities. By “abnormal level”, an increase or relative to a level present in a similar sample from a part of the body that does not have an abnormality or from a subject, or a standard level indicative of the presence in those similar samples Means a reduced level.

  Immunoassays that can be used include, but are not limited to: competitive and non-competitive assay systems (using techniques such as Western blots), radioimmunoassays, ELISA (enzyme linked immunosorbent assays). ), “Sandwich” immunoassay, immunoprecipitation assay, precipitin assay, gel diffusion precipitation reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiometric assay, fluorescent immunoassay, protein A immunoassay and the like.

  Relaxin and relaxin receptor nucleic acids (both sense and antisense), including fragments and analogs thereof, can also be used in hybridization assays. Such nucleic acids comprising or consisting of at least 8 consecutive nucleotides can be used as hybridization probes or for polymerase chain reaction detection. Hybridization assays may be used to detect, prognose, diagnose or monitor diseases or conditions (such as those described above) associated with abnormal relaxin or relaxin receptor expression and / or activity. Specifically, the hybridization assay comprises contacting a sample containing polynucleotide with a nucleic acid probe capable of hybridizing to relaxin or relaxin receptor DNA or RNA under conditions that allow hybridization to occur, and any occurrences. It can be carried out by a method comprising the step of detecting or measuring the hybridization.

  In certain embodiments, abnormalities associated with over- or under-expression of relaxin can be diagnosed or suspected by detecting a decrease or increase in the level of relaxin polypeptide, relaxin RNA or relaxin functional activity Presence can be screened or a predisposition to develop such an abnormality can be identified. Further, overexpression of relaxin or increased relaxin functional activity may result in a mutation in relaxin RNA or DNA or relaxin polypeptide that causes an increase in relaxin polypeptide expression or activity (eg, translocation in relaxin nucleic acid, relaxin, respectively). Diagnosis by detecting shortening of the gene or relaxin polypeptide, changes in nucleotide or amino acid sequence relative to wild-type relaxin.

  For example, the level of relaxin polypeptide in a biopsy or from semen can be detected by tissue immunoassay; the level of relaxin RNA can be detected by a hybridization assay (eg, Northern blot or dot blot). Translocations or point mutations in relaxin or relaxin receptor nucleic acids include Southern blots, RFLP analysis, PCR using primers to detect point mutations, deletions or insertions, sequences of relaxin genomic DNA or cDNA obtained from samples It can be detected by determination or the like.

  In another embodiment, the level of relaxin or relaxin receptor mRNA or polypeptide is detected or measured in a sample of tissue isolated from a subject, wherein the elevated level is determined by the subject Indicates having or predisposition to related tissue abnormalities.

  In yet another embodiment, abnormal relaxin receptor activity in tissue is detected or measured using any of the functional assays described above. Abnormal relaxin receptor activity can include, for example, an increase or decrease in the number of relaxin receptors on relaxin-responsive cells, the presence of relaxin receptors on cells that are not normally responsive to relaxin, and relaxin receptor response times. It may include an increase or decrease, an increase or decrease in binding affinity for relaxin or relaxin receptor, or a change in the dissociation constant of relaxin from the relaxin receptor.

  Kits for diagnostic and / or prognostic applications are also provided, which include a relaxin agonist or antagonist and optionally a labeled binding partner for the antibody in one or more containers. Alternatively, the antibody can be labeled with a detection marker (eg, a chemiluminescent moiety, an enzymatic moiety, a fluorescent moiety, a radioactive moiety, etc.). Kits are also provided that include a nucleic acid probe capable of hybridizing to relaxin or relaxin receptor mRNA or DNA in one or more containers.

  In another embodiment, the kit can include a primer pair (eg, a size range of 6-30 nucleotides or more each) in one or more containers, wherein the primer pair comprises at least a portion of the relaxin nucleic acid. Amplification can be primed under suitable reaction conditions as amplified (eg, see polymerase chain reaction (see, eg, Innis et al., PCR Protocols, Academic Press, Inc., San Diego, CA (1989)). Ligase chain reaction (see, eg, EP 0 320 308), use of Qβ replicase, cyclic 5 ′ probe reaction, or other methods known in the art. The kit can optionally further include a predetermined amount of purified relaxin or relaxin receptor nucleic acid in a container, for example, for use as a standard or control.

  The following examples are merely provided as illustrations of various aspects of the invention and should not be construed as limiting the invention in any way.

Example 1
The effect of inactivation of one allele or both alleles of the mouse RLX gene was tested. The following methods and materials were followed.

(animal)
Wild-type, heterozygous and homozygous relaxin knockout mice were obtained from Howard Flory Institute of Experimental and Medicine (Parkville, Victoria, 3052, Australia) and were used to breed and program. Subsequent generations of RLX + / + (wild type), RLX +/− (heterozygous) and rlx − / − (null mutant) mice were made from RLX +/− parents. All animals are housed in a controlled environment and given access to Labdiet rodent experimental samples (Deans Animal Feed, San Bruno, Calif.) And water for 14 hours in the light, 10 hours in the dark Maintained on schedule. These experiments were approved by the Institute's Animal Experts Committee that adheres to the NIH Code of Practice for the care and use of laboratory animals.

(Genotyping by PCR)
Mouse DNA in 400 μl PCR lysis buffer (containing 50 mM Tris-HCl, pH 8.0, 0.5% SDS, 0.1 M EDTA and 1 mg / ml proteinase K (Gibco BRL, Gaithersburg, MD)) Isolated by lysing tail tissue (5-7 mm) overnight at 50-55 ° C. The digested sample is then mixed with 3M sodium acetate (40 μl), buffer saturated phenol (200 μl) and chloroform (200 μl) in a serum container tube, after which the sample is centrifuged at 3000 rpm for 10 minutes. did. The DNA (contained in the upper aqueous phase) was decanted into another microcentrifuge tube containing isopropyl alcohol (240 μl) to precipitate the DNA, after which the sample was vortexed, centrifuged and the supernatant removed . The remaining DNA pellet was dissolved in 40-50 μl of sterile water. For PCR, each DNA template (1 μl) was used in a 30 μl reaction mixture containing: PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl ₂ ), 2.5 mM. dNTPs, 2.5U Taq polymerase (PGC Scientific, Gaithersburg, MD), and 150 ng each of RLX + / + and rlx − / − primers (designed by Zhao and collaborators (Zhao Endocrinology 140: 445-53 (1999)) The amplification protocol consists of an initial denaturation step at 94 ° C. (3 minutes) followed by 35 consecutive cycles of 94 ° C. (60 seconds), 55 ° C. (60 seconds) and 72 ° C. (90 seconds), and 72 ° C. Extend for another 10 minutes at 15 μl of each sample was then analyzed by electrophoresis on a 2% (w / v) agarose gel and stained with ethidium bromide A 235 bp product was generated by primers designed from the wild type allele And a 170 bp product was generated from the mutant allele primer (Zhao (1999), supra).

(Organization collection and histology)
Relaxin wild-type, heterozygous and homozygous males and females (in each of the six groups, n is 20 or more) were obtained and weighed at 1 week, 1 month, 2 months and 3 months of age. . RLX + / + and rlx − / − male mice were then sacrificed under anesthesia with carbon dioxide for tissue collection. Male genital tracts (including testis, epididymis, prostate, seminal vesicles and associated fat) are collected from each animal at 1 week of age (n = 10 RLX + / + male, n = 11 − / − male); (N = 10 RLX + / + male, n = 10 − / − male); and 3 months of age (n = 10 RLX + / + male, n = 10 rlx − / − male). After weighing each tissue, they were placed in 10% formalin for histological analysis.

  Collected tissue was processed from 70% alcohol to paraffin (continuously dehydrated), then embedded, cut using an AO Spencer 820 microtome (4 μm sections), and poly-L-lysine coated Placed on a glass slide. Serial sections from each tissue were stained with H & E (hematoxylin and eosin) and for collagen, as described by the manufacturer, with Masson trichrome (staining kit, Richard-Allan Scientific, Kalamazoo, MI). . Stained slides were viewed with a Zeiss Axioplan-2 microscope, images were supplemented with a digital camera (Hamamatsu), and stored for retrieval and analysis. This image was digitally enhanced for maximum contrast and brightness using Adobe Photoshop (Adobe Systems Inc, Mountain View, CA).

(Antibody staining of paraffin-embedded tissue section)
Tissues from the genital tract of 1 month old and 3 month old male mice are placed on precoated slides and heated at 58 ° C. (about 30 minutes) to remove paraffin, then washed 3 times with xylene, anhydrous Washed twice with ethanol and twice with 95% ethanol, then dipped in water for a short time. These samples were then stained using an Immunocruz staining system utilizing a horseradish peroxidase (HRP) -streptavidin complex (Santa Cruz Biotechnology Inc, Santa Cruz, Calif.) In a humidified environment. Tissue sections were first treated with a peroxidase blocker (to quench endogenous peroxidase activity) (5 minutes) and then pre-blocked in goat serum (20 minutes). Subsequently, serial sections from each RLX + / + tissue sample and rlx − / − tissue sample were obtained from Bax monoclonal IgG primary antibody (4 μg / ml) (Santa Cruz Biotechnology Inc), caspase-9 polyclonal IgG antibody (4.5 μg / ml). ) (Santa Cruz Biotech.) Or proliferating cell nuclear antigen (PCNA) (Santa Cruz Biotech.) Monoclonal IgG antibody (4 μg / ml) (2 hours). Depending on the type of antibody used, either tissue mouse IgG control or rabbit IgG control (Santa Cruz Biotech.) Staining (2 hours) was also used in all experiments performed. Samples were washed with PBS (2 minutes), subjected to appropriate secondary antibody (goat anti-mouse IgG or goat anti-rabbit IgG) (30 minutes), washed as above (2 minutes) and HRP-streptavidin Treated with conjugate (30-45 minutes) and incubated with diaminobenzidine chromogenic substrate prepared according to manufacturer's instructions (2-10 minutes). The slide was then washed with distilled water (2 minutes), then dehydrated from 95% alcohol to xylene and mounted, then photographed as above.

(Statistical analysis)
Results were analyzed using the one-ANOVA test. All data in this document are presented as mean ± SEM, p <0.05 is considered statistically significant.

(Example 2)
(Effect of relaxin gene knockout on mouse growth)
Male and female relaxin wild type (+ / +), heterozygous (+/−) and null mutant (− / −) mice were weighed at 1 week, 1 month, 2 months and 3 months of age. Measured (n = 20-21 for each group). At 1 week of age, both male mice (RLX + / +: 3.87 ± 0.09 g; rlx − / −: 3.6 ± 0.13 g) and female mice (RLX + / +: 3.52 ± 0) .09g; rlx − / −: 3.37 ± 0.08 g), no significant difference in mean body weight was noted. However, the average body weights of both rlx-/-male (17.05 ± 0.65 g) and female (14.77 ± 0.42 g) in the 1 month old mice were higher than in each RLX wild type counterpart mouse. Were also significantly less (p <0.05) (RLX + / + M: 18.92 ± 0.61 g; RLX + / + F: 16.34 ± 0.51 g). Male and female null mutant mice remained significantly smaller (p <0.05) than RLX wild-type animals at 2 months of age (RLX + / + M: 25.42 ± 0.41 g; rlx− / −M: 24.23 ± 0.42 g; RLX + / + F: 20.96 ± 0.32 g; rlx − / − F: 19.94 ± 0.22 g); however, the size difference between the two groups is Less than the difference observed at 1 month of age. By adult (age 3 months), the average body weight of rlx − / − mice (rlx − / − M: 26.57 ± 0.47 g; rlx − / − F: 22.54 ± 0.31 g) Although slightly less than the average body weight of the / + mice (RLX + / + M: 27.30 ± 0.32 g; RLX + / + F: 23.03 ± 0.71 g), this difference was no longer significant. The average body weight of RLX +/− mice was between the average body weight of RLX + / + and the average body weight of rlx − / − mice. No significant difference was observed between the average body weight of RLX + / + animals and the average body weight of RLX +/− animals or between RLX +/− mice and rlx − / − mice.

(Effect of relaxin gene knockout on male genital tract size)
At 1 week of age, no significant difference was observed in male reproductive tract weight or size. This is mainly indicated by the testis (Table 1). By 1 month of age, the collected male genital tract consisted of the testis, epididymis, prostate, seminal vesicle, vas deferens and associated fat. At 1 month of age, no difference was observed in the weight of the entire genital tract, but the difference in testis and prostate size from 1 month old null mutant mice was approximately less than the corresponding tissue of RLX wild type animals, respectively. 20% and about 30% smaller. On the other hand, the size of epididymis derived from rlx − / − mice was significantly smaller than that of epididymis obtained from RLX + / + animals (p <0.05). However, no difference was noted in seminal vesicle size between tissue samples collected from RLX + / + mice and rlx − / − mice.

  By the time mice reached adulthood (3 months of age), significant differences (p <0.05) were observed in the overall weight of the male reproductive tract and the size of individual organs (Table 1). The weight of the genital tract in normal mice increased by 248.6% from 1 month of age to adults and corresponded to 3.37% of the total body weight. During this time, the weight of the genital tract from male RLX null mutant mice only increased by 187.9% and corresponded to 2.33% of total body weight. This latter finding means that the genital tract of male rlx − / − mice represents a 31% decrease in% body weight. Testis, epididymis, prostate and seminal vesicle sizes from rlx − / − mice were all smaller than their counterparts from RLX + / + mice (p <0.05).

  (Table 1.1 Weight and size of male reproductive tract from week-old to adult (6.5 months))

^１精巣および^３前立腺の各々のサイズを、組織の長さを幅で乗じて、その面積によって測定した。精巣上体および精嚢のサイズ（面積）は、正確には測定できなかったので、各組織の緊密な近似を、以下のように計算した：精巣上体（組織の長さに、精巣上体頭部の幅を乗じて測定した^２）；精嚢（各器官の長さに、組織の平均幅を乗じて測定した^４）。^＊は、ｐ＜０．０５を示す。

The size of each of the ¹ testis and ³ prostates was measured by their area multiplied by the length of the tissue by the width. Since the size (area) of epididymis and seminal vesicles could not be measured accurately, a close approximation of each tissue was calculated as follows: epididymis (tissue length, epididymis) Measured by multiplying the width of the head ² ); seminal vesicles (measured by multiplying the length of each organ by the average width of the tissue ⁴ ). ^* Indicates p <0.05.

(Effect of relaxin gene knockout on histology of male reproductive tract)
H & E & Masson trichrome staining: Male genital tract tissue sections from RLX + / + and rlx − / − mice were first observed for differences in sperm maturation, tube size / compactness (testis, epididymis) and collagen.

  Testis: At 1 week of age, the relaxin wild-type animal seminiferous tubule (the compartment containing germ cells / spermatocytes, Sertoli cells, where sperm maturation occurs) has a smaller, more cylindrical shape (Table 2). ), And mainly supported by a thin layer of collagen surrounding the white membrane of each organ (the membrane that covers the oval body). In contrast, tubules derived from the testes of rlx − / − mice were larger and enlarged, but were completely surrounded and supported by collagen within the testis. This immediately suggested a difference in the internal tissues of the testis even in the absence of relaxin, even immediately after birth. However, at 1 week of age, immature sperm was not detected in any group of tissue sections. By 1 month of age, testicular tubules from RLX + / + mice were larger in size (compared to 1-week-old tubule size), were slightly compact, and mainly contained immature sperm. In contrast, tubules from 1 month old RLX homozygous mice do not differ from the tubule size measured from 1 week old knockout mice and are less mature compared to samples from RLX + / + mice Not included sperm. This indicates that relaxin-null mutant mice undergo a delayed sperm maturation process that is further confirmed when compared to 3 month old tissue sections; sections from RLX + / + mouse tissues primarily produce mature sperm. It was further suggested to have larger tubules (compared to 1 month old tubule size). Conversely, sections from rlx − / − mice are slightly smaller tubules (compared to 3 month old RLX + / + tissue sections) with slightly mature sperm and still some immature sperm (this Was not different from tubule size from 1 week / month rlx − / − mice). Sperm maturation levels in testes from RLX + / + mice were shown to be significantly (p <0.05) higher than the sperm maturation levels observed in testes of rlx − / − mice (grading used) (See Table 2 for scales). It was also noted that the level of sperm maturation in the testes of adult RLX knockout mice was similar to the level observed in immature (1 month old) RLX wild-type mouse tissues. At 1 to 3 months of age, normal mouse testis remained surrounded by a thin film of collagen overlying the white membrane; in some tissues, a thin interstitial collagen lining may also support the seminiferous tubules. Observed. However, the inner layer of collagen was found to decrease with age. In rlx − / − mice, collagen detected in the middle of the tubule structure disappeared due to the structure of the larger tubule size in the testis, but compared to that observed in tissue samples from wild type animals. It was still more dense and solid. It is expected that the increased collagen observed in the reproductive tract of RLX null mutant mice at very young ages can make the tissue harder and stiffer, which is related to the tubule composition and sperm maturation discussed above. Can be related to change. Interestingly, the presence of relaxin in normal adult mice also induces relaxation of the tubule structure (increased stromal spacing) of the seminiferous tubules compared to the structure observed from normal immature (1 month old) mice did. In contrast, there was no difference in the organization (size / compactness) of testicular tubules from 1 month old and 3 month old rlx − / − mice. Furthermore, until 1 month and 3 months of age, testicular tubules from rlx − / − mice appeared to contain an increased number of dead cells compared to tubule cells from RLX wild type animals. The increased number of dead cells seen in immature tissues varied between animals but was consistently detected as mice matured with age.

  Epididymis: No significant differences in tubule size were noted in the epididymis of the RLX + / + and rlx − / − genital tracts at 1 month and 3 months of age. However, in adults, the compactness of tubules was more evident in tissues from RLX homozygous mice compared to tissues from RLX wild type animals. The epididymal tubules of rlx − / − mice were also supported to a greater extent by connective tissue. Tubular structures from 3-month-old RLX + / + mouse tissue were loose in shape and only partially maintained by collagen. Conversely, tissue sections obtained from RLX knockout animals were shown to have more compact tubules that were very nicely surrounded by a thin layer of collagen. In the testis, tissue from rlx − / − mice was supported by increased concentrations of collagen at all ages observed compared to tissue samples from RLX + / + mice. As shown in Table 2, the density of mature sperm (in epididymal tubules) was compared in each age group of 1 month and 3 months compared to tissue sections obtained from RLX wild type mice. It was also found to be lower in tissue sections obtained from RLX null mutant mice. However, this difference was not statistically significant. This decrease in mature sperm in the epididymis of mice lacking relaxin is probably due to the delayed sperm maturation process that occurs in the testes of these animals (Table 2).

表２：組織切片を、Ｈ＆Ｅで染色し、以下のように等級に分けた：
^ａ細管サイズ−１＝全ての細管が小さく、環状の形態である；２＝ほとんどの細管が１と比較してわずかに伸張しているが、いくつかのより小さい環状細管が観察される；３＝細管は１と比較して著しく伸張しているが、いくつかのより小さい環状細管および中サイズの細管が観察される；４＝全ての細管が１および２と比較して著しく伸張している。
^ｂ細管のコンパクトさ−１＝全ての細管の形が緩んでいる；２＝形が緩んでいる細管の割合のほうが、コンパクトな細管の割合よりも多い；３＝コンパクトな細管の割合のほうが、形が緩んでいる細管の割合よりも多い；４＝全ての細管がコンパクト。
^ｃ精子の成熟度−０＝精子は検出されず；１＝未成熟な精子のみが検出された；２＝未成熟な精子の割合のほうが成熟な精子の割合よりも多い；３＝成熟な精子の割合のほうが未成熟な精子の割合よりも多い；４＝成熟な精子のみが検出された。
示した数値は、測定した各パラメーターについて使用した等級分けスケールの平均±ＳＥ（ｎ）である。^＊はｐ＜０．０５を示す。

Table 2: Tissue sections were stained with H & E and graded as follows:
^a- tubule size-1 = all tubules are small and in an annular form; 2 = most tubules are slightly stretched compared to 1, but some smaller annular tubules are observed; 3 = Capillary stretches significantly compared to 1 but some smaller annular and medium sized capillaries are observed; 4 = all capillaries stretch significantly compared to 1 and 2 .
^b Compactness of tubules-1 = all capillaries are loose; 2 = the proportion of capillaries that are loose is greater than the proportion of compact tubules; 3 = the proportion of compact tubules is More than the percentage of tubules that are loose; 4 = all tubules are compact.
^c Sperm maturity-0 = no sperm detected; 1 = only immature sperm detected; 2 = percentage of immature sperm is greater than percentage of mature sperm; 3 = mature sperm Is higher than the proportion of immature sperm; 4 = only mature sperm detected.
The numerical values shown are the mean ± SE (n) of the grading scale used for each parameter measured. ^{* Indicates} p <0.05.

表３：組織切片を、Ｍａｓｓｏｎトリクロム染色によって染色し、以下のように等級に分けた：
^ａコラーゲン−１＝構造の外部層を取り囲むコラーゲンの薄い内層のみ；２＝組織の内部構成成分を取り囲むコラーゲンのさらなる内層；３＝組織の外部層および内部構成成分を取り囲むより厚いコラーゲンの内層；４＝３＋コラーゲンで充填された細管／構成成分との間の空間。

Table 3: Tissue sections were stained by Masson trichrome staining and graded as follows:
^a Collagen-1 = only a thin inner layer of collagen surrounding the outer layer of the structure; 2 = an additional inner layer of collagen surrounding the internal components of the tissue; 3 = inner layer of thicker collagen surrounding the outer and internal components of the tissue; 4 = 3 + space between tubules / components filled with collagen.

The numerical values shown are the mean of the grading scales ± SE (n). ^{* Indicates} p <0.05.

  Prostate: At 1 month of age, no significant difference in prostate structure was noted between RLX + / + and rlx − / − derived tissues. However, the space between the ducts of the prostate from normal animals was associated with only trace amounts of collagen. In contrast, prostate tubes from rlx − / − mice were mediated by a loosely shaped layer of connective tissue. In adults (3 months), rlx − / − mice included smaller prostates with smaller glands and ducts. Tubes of tissue from RLX knockout mice are more compact and fully supported by connective tissue, while tubes from RLX wild-type mice are more spread (loose in shape) and still by collagen Almost no support. Furthermore, adult prostate tubes from rlx − / − mice appear to have smaller epithelium (contain less cells), while RLX + / + prostate sample tubes have larger epithelial layer / glandular tissue Included. These results, in addition to our initial findings on the testis and epididymis, indicate that the delayed maturation process of male reproductive tissues occurs in mice lacking a functionally active relaxin gene. , And show that it is related to the accumulation of collagen in these tissues.

(Detection of cell apoptosis by antibody staining)
Based on observations made from H & E staining, in which tissue from rlx − / − mice causes reduced sperm maturation and increased cell death (testis) and includes a reduced epithelial (cell) layer (prostate) It was decided to investigate whether the observed increased cell death was a result of the apoptotic pathway.

  Bax antibody staining: Overexpression of Bax accelerates apoptotic death induced by cytokine blockade and also offsets death repressor activity of Bcl-2 (Krajewski et al., Am. J. Pathol. 145: 1323- 36 (1994)). Using the Bax monoclonal antibody, the in vivo distribution of Bax protein was evaluated in the male reproductive tract. Testis: Seminiferous tubules from immature (1 month) wild type animals showed weak immunostaining for Bax (dark brown cell staining), which was mainly observed in germ cells near the basement membrane. These findings are consistent with previous reports (Ben-Hur et al., Calcif. Tiss. Int. 53: 91-96 (1996)). In comparison, an increased number of cells stained positive for Bax in testis from rlx − / − mice. The number of Bax positive cells was counted using a hemocytometer and compared to the number of apoptotic cells observed from tissues from RLX + / + mice (13.1 ± 3.5 cells / testis (n = 6)). Significantly higher in the testis of rlx − / − mice (32.7 ± 4.8 cells / testis (n = 6)). Increased Bax staining was consistently observed in rlx − / − tissues, but the number of tubules that stained positive for Bax varied between tissue samples, and some tubules also did not contain Bax Was also observed. In the case of mature male genital tract, adult RLX wild-type mouse testis showed reduced staining for Bax (3.4 ± 1.4 cells / testis (n = 6)), which indicates that these animals Correlates with increased levels of sperm maturation and tissue maturity experienced. Conversely, the slower rate of tissue maturation observed in the rlx − / − mouse genital tract further increased Bax staining in 4 month old RLX knockout mice (44.4 ± 8.1 cells / testis (n = 6)). Many of the cells observed appear to be the final stage of apoptosis, possibly showing apoptotic bodies. However, Bax staining was not associated with mature sperm cells. Epididymis: More intense Bax staining was present in the epithelial cell epithelial cells of epididymal tubules obtained from 1 month RLX + / + mouse tissue. Tubular epididymal tubules obtained from 1 month rlx − / − mouse tissue contain a relatively stronger level of Bax staining, which is further maintained even when not increased in 3 month tissue sections. It was done. In contrast, the epididymis of adult wild type mice contained weaker Bax expression staining as this tissue matured as compared to tissue from 1 month normal mice. Prostate: Normal and 3-month-old animal prostate epithelial cells showed weak positive staining for Bax. Regarding the other tissues studied, especially cells in the epithelial layer of the rlx − / − animal (prostatic duct) showed increased staining for Bax in both immature (1 month) and adult (3 months) tissues. Indicated. These findings suggest that relaxin may play a new role in the regulation of cell apoptosis in the male reproductive tract, but further studies with another antibody to detect cell apoptosis to confirm the effect of relaxin Implemented.

  Caspase-9 (a central death protease) belongs to a unique family of cysteine proteases that differ in sequence, structure and substrate specificity from other described protease families. Members of this caspase family, which are usually involved in a cascade of proteolytic cleavage events, act as an important component of the apoptotic machinery by acting to destroy specific target proteins that are important for cellular activity Functions as an ingredient. Testis: Moderate caspase-9 staining is observed in 1 month RLX + / + testis (28.1 ± 5.6 cells / testis (n = 7)), which is either spermatogonia or spermatocytes It seems to be mainly associated with embryonic cells in the seminiferous tubule rather than cells. For Bax, a significantly (p <0.05) increased level of caspase-9 staining was observed in 1 month rlx − / − mouse testes (76.6 ± 10.2 cells / testis (n = 6)). Although detected, severe caspase-9 staining was hardly associated with tubules, while many tubules were observed to contain little or no caspase-9 protein. With respect to age, the level of caspase-9 detected in normal immature tissues was consistently found in older tissues at 3 months of age (26.9 ± 4 cells / testis (n = 6) ). However, as testis size increased, the number of apoptotic cells detected showed a small fraction of aged tissue. Testicular tubules from rlx − / − mice show slightly fewer apoptotic cells (61.6 ± 9.8 cells / testis (n = 5) at 3 months compared to the density of caspase 9 stained cells in 1 month tissue. )) Included. Despite the number of positively stained apoptotic cells from tissues of RLX knockout mice, the rest were significantly (p <0.05) larger than the wild-type counterparts of all ages studied. Epididymis: Caspase-9 staining was not detected from 1 month RLX + / + epididymal tubules, while traces of positive cells were observed in rlx − / − tissue sections. However, positively stained cells were sparsely scattered and detected in the epithelial layer of the tubule. With respect to age, no clear staining of caspase-9 was detected in 3 month old RLX + / + and rlx − / − mouse tissues. Prostate: Caspase-9 staining was not identified in RLX + / + and rlx − / − tissue prostates at all ages investigated (1-3 months of age). Nevertheless, the results obtained initially suggest that this relaxin is involved in the regulation of cell apoptosis. However, further studies were needed to establish whether relaxin is involved in the cell growth pathway.

(Detection of cell proliferation by antibody staining)
PCNA staining: Proliferating cell nuclear antigen (PCNA) antibodies were used to detect cell proliferation in the male reproductive tract based on the ability to associate with the nuclear region where DNA synthesis occurs. Testis: PCNA was detected in immature and mature seminiferous tubules, but a significant difference in PCNA staining was observed in testicular tracts from 1- and 3-month-old RLX + / + and rlx − / − mice. Not detected. In immature tissues, PCNA staining is usually highly expressed in mitotically active spermatogonia, and sometimes highly expressed in some Sertoli cells, these stainings correspond to proliferative activity. In this study, these cells are well and evenly labeled in PC adults for PCNA, which is likely to initiate both regeneration through the unrelenting activation of these proliferating cells. Reflect the ability of the group. Epididymis: No staining for PCNA was detected in epididymis from either 1- and 3-month-old RLX + / + mice or rlx − / − mice. These findings are consistent with the role of the epididymis acting as a reservoir for mature sperm. Prostate: Weak staining for PCNA was associated with epithelial cells of prostate ducts from 1 month RLX + / + and rlx − / − mice. However, no staining for PCNA was detected in any of the 3 month old groups. Leukocyte proliferation studies are limited to some degree of reproductive tissue, and this accumulated finding confirms that relaxin probably plays a more influential role in the regulation of cell apoptosis than on cell proliferation. It was done.

(Effect of relaxin gene knockout on histology of other somatic cells)
RLX wild type (RLX + / +) males and females and RLX gene knockout (rlx − / −) males and females were generated from the parent of the RLX heterozygote (RLX +/−) as described above. It was. Mice were weighed and sacrificed at 1 week, 1 month and / or 3 months for tissue recovery. The following tissues (including brain, heart, liver, kidney, thymus, spleen and male reproductive tract) were collected, weighed and placed in 10% formalin for detailed histological analysis. A summary of these tissue weights is shown in Tables 4-8.

  At 1 week of age, male rlx − / − mice contained significantly (p <0.05) smaller liver, kidney and spleen compared to RLX + / + animals. However, these weight differences were no longer apparent at 1 month of age. Instead, the thymus of 1 month old male rlx − / − mice was smaller than its RLX + / + counterpart (p <0.05). This weight difference was no longer apparent at 3 months of age. In the case of the male genital tract, no significant difference was noted between the RLX + / + group and the rlx − / − group at 1 week or 1 month of age. However, by 3 months of age, the genital tracts of rlx − / − mice were significantly (p <0.05) smaller than those obtained from RLX + / + animals.

  Notable differences were observed between female 1-week-old RLS + / + and rlx − / − mice. However, by 1 month of age, rlx − / − female mice had significantly smaller livers than their RLX + / + counterparts. By 3 months of age, this weight difference was no longer observed, and other significant differences in other organs were no longer notable.

  To determine whether organ and tissue differences in RLX − / − mice (derived from RLX +/− parents) are further emphasized when rlx − / − offspring are obtained from RLX − / − parents. At the same time, RLX + / + and rlx − / − mice were obtained from the corresponding parental set and sacrificed at 1 week, 1 month and 3 months of age. Brain, heart, liver, kidney, thymus, spleen, male reproductive tract, intestine, and lung were collected and weighed. The weights of these organs are shown in FIGS.

  It is further noted that although organ weights or tissue weights appear to be standardized as mice age, normal weights alone are not equal to normal organs or normal tissues. This data indicates that the ratio of organ (tissue) weight to body weight at any stage of the growth cycle indicates a change in potential organ or tissue development or cellular organization. In one example, a small liver infiltrated (modified) by fibrosis is the same or heavier than a larger normal liver or a liver filled with fat. Thus, the brain, heart, liver, kidney, thymus, spleen, intestine and lung (developing in the absence of relaxin) of female and male RLX − / − mice have increased apoptosis and extracellular matrix (collagen). Show evidence of accumulation.

  (Table 4: Tissue comparison of 1-week-old mice from RLX + / + parents and rlx − / − parents, expressed as mean ± SE (n), respectively)

（表５：平均±ＳＥ（ｎ）で示した、ＲＬＸ＋／−の親由来の、１ヶ月齢マウスの組織比較）

(Table 5: Tissue comparison of 1-month-old mice derived from RLX +/− parents, expressed as mean ± SE (n))

（表６：平均±ＳＥ（ｎ）で示した、それぞれ、ＲＬＸ＋／＋の親およびｒｌｘ−／−の親由来の、３ヶ月齢雄性マウスの組織比較）

(Table 6: Tissue comparison of 3-month-old male mice from RLX + / + parents and rlx − / − parents, expressed as mean ± SE (n), respectively)

（表７：平均±ＳＥ（ｎ）で示した、それぞれ、ＲＬＸ＋／＋の親およびＲＬＸ−／−の親由来の、１ヶ月齢マウスの組織比較）

(Table 7: Tissue comparison of 1 month old mice from RLX + / + parents and RLX − / − parents, respectively, expressed as mean ± SE (n))

（表８：平均±ＳＥ（ｎ）で示した、それぞれ、ＲＬＸ＋／＋の親およびｒｌｘ−／−の親由来の、３ヶ月齢マウスの組織比較）

(Table 8: Tissue comparison of 3-month-old mice from RLX + / + parents and rlx − / − parents, expressed as mean ± SE (n), respectively)

（皮膚の組織学に対するレラキシン遺伝子ノックアウトの影響）
組織リモデリングの調節におけるレラキシンの役割を、ｒｌｘ−ヌルマウス（ｒｌｘ−／−）における皮膚組織学を研究することによって調べた。これらのマウスは、ｒｌｘ−／−のオス親およびメス親の子孫であった。少なくとも５匹のオスマウスおよび５匹のメスマウスの、耳および背中からの連続した皮膚サンプルを、Ｈ＆Ｅ染色およびＭａｓｓｏｎトリクロム染色によって染色し、各時点で調べた。同じ時点の類似のサンプルを、同じ系統のＲｌｘ＋／＋マウスから得た。ｒｌｘヌルマウスの組織学的試験の際に、真皮が、時間の進行につれ厚くなること、および真皮一帯の増大した線維症を有することを見出した。ｒｌｘヌルマウスの皮膚サンプルは、１週齢において正常であった；しかし、１ヶ月までに初期の皮膚線維症が明らかとなった。３ヶ月齢までに、皮膚線維症の顕著な増大が存在し、これは、６ヶ月齢までに密度が増大した。これらの皮膚に関する知見は、雄性および雌性のｒｌｘヌルマウスにおいて類似していた。

(Effect of relaxin gene knockout on skin histology)
The role of relaxin in the regulation of tissue remodeling was investigated by studying skin histology in rlx-null mice (rlx − / −). These mice were offspring of rlx − / − male and female parents. Serial skin samples from the ear and back of at least 5 male and 5 female mice were stained by H & E staining and Masson trichrome staining and examined at each time point. Similar samples at the same time point were obtained from Rlx + / + mice of the same strain. Upon histological examination of rlx null mice, it was found that the dermis became thicker as time progressed and had increased fibrosis across the dermis. The skin samples of rlx null mice were normal at 1 week of age; however, early dermal fibrosis was evident by 1 month. By 3 months of age, there was a significant increase in dermal fibrosis, which increased in density by 6 months of age. These skin findings were similar in male and female rlx null mice.

  In these mice, the epidermis was normal and the hair was unchanged, except for the initial brighter coat color in rlx null mice, which became indistinguishable from Rlx + / + mice by 1 month of age. Serum chemistry, hematological parameters and urine tests from rlx mice were not noteworthy.

  These findings support previous observations that relaxin affects matrix turnover in vitro and in vivo by altering key matrix molecules, matrix degrading enzymes and growth factors. Many stromal cells that produce stromal collagen have a relaxin receptor, whether from a male or female source. Antibody-based assays can detect serum relaxin at low picogram levels during the luteal phase of the female menstrual cycle, and these levels are most apparent during pregnancy (at which time tissue remodeling is most apparent ). In contrast, serum relaxin in males has been reported to be undetectable.

  Rlx null mice provide the first direct evidence to associate relaxin with a universal alteration of collagen turnover in normal skin. These results indicate that relaxin can be produced or circulated at biologically relevant concentrations below the detected flow level in males and females. Relaxin, in cooperation with other matrix regulatory molecules, is involved in the regular maintenance of matrix turnover. Relaxin can affect tissue remodeling, promoting angiogenesis and stimulating vasodilation. These results also indicate that the relaxin synthesis pathway and the relaxin receptor are associated with fibrotic conditions (eg, scleroderma).

  The previous examples are provided for illustration and are not provided to limit the scope of the claimed invention. Other modifications of the invention will be readily apparent to those skilled in the art and are encompassed by the appended claims. All publications, patents, patent applications, and other references cited therein are hereby incorporated by reference.

Claims (1)

Invention described in the specification.