JP5867949B1 - Female hormone-dependent proliferative disease treatment / amelioration agent - Google Patents

Abstract

A low molecular weight peptide having an erythropoietin receptor antagonistic action, particularly an erythropoietin mimetic peptide (EMP). In particular, a female hormone-dependent proliferative disease treatment / amelioration agent comprising EMP9 is provided. EMP9 can be administered directly or intravenously to the foci of proliferative organ diseases such as adenomyosis, endometriosis, uterine fibroids, and prostatic hypertrophy, which are female hormone-dependent diseases, to improve or treat these diseases. [Selection figure] None

Description

  The present invention relates to a therapeutic / ameliorating agent for female hormone-dependent proliferative diseases.

  Conventionally, surgical resection, administration of hormone preparations such as GnRH agonists, aromatase inhibitors, estrogen preparations, or a combination thereof have been used as therapeutic means for adenomyosis and endometriosis. However, when these hormone preparations are applied, the symptoms are temporarily reduced, but they cannot be used for a long time due to side effects such as osteoporosis. Furthermore, detailed studies on the pathological pathogenesis of endometriosis / adenomyosis itself have not progressed. For this reason, no radical cure has been established.

  Erythropoietin (Epo) is involved in blood cell proliferation and differentiation and is produced in the kidney or liver and released into the blood. Erythropoietin acts on erythroblast burst-forming cells (BFU-E) and erythroid colony-forming cells (CFU-E) among erythroid progenitors, promotes their differentiation and proliferation, and induces erythrocyte production. (Non-patent document 1: Krantz SB, Blood, Vol. 77, pp 419-434 (1991)). When erythropoietin binds to the erythropoietin receptor (EpoR) present in the cell membrane of progenitor cells, phosphorylation of Janus Kinase 2 (JAK2) present in the cytoplasmic region of the receptor occurs, and then the substrate in the cytoplasm Phosphorylation is induced. One of its substrates, transcription factor signal transducer activator transcription-5 (STAT5), is phosphorylated (P-STAT5), which is transmitted into the nucleus and promotes proliferation of progenitor cells (non-patented) Document 2: Ihle JN, Nature, Vol. 377, pp 591-594 (1995)), erythrocyte differentiation, that is, intracellular accumulation of globin mRNA, hemoglobin production (Non-Patent Document 3: D) 'Andrea AD et al., Cell, Vol. 57, pp 277-285 (1989)). Furthermore, erythropoietin was found to be an essential factor for adult-type erythropoiesis from experiments with gene-deficient mice (Non-patent Document 4: Wu et al., Cell, Vol. 83, pp 59-67 (1995)). .

  As a site where erythropoietin expresses its gene in normal tissues other than the site related to erythropoiesis, the mouse embryo immediately after implantation (Non-Patent Document 5: Yasuda et al., Develop. Growth Differ., Vol. 35, pp 711-722 (1993)), human, monkey and mouse brains (Non-patent Document 6: Marti HH et al., Eur. J. Neu. Sci., Vol. 8, pp 666-676 (1996)), Human placenta (Non-patent Document 7: Conrad et al., FASEB. J., Vol. 10, pp 760-768 (1996)) and the like are known.

  When erythropoietin antibody or soluble erythropoietin receptor is injected into the uterine cavity of the mouse immediately after implantation to block erythropoietin information, 36.2% and 80% of embryos are stopped in the uterine cavity after 3 days, respectively. In addition, disorder of development of the placenta was observed in 88.2% and 85%, respectively (Non-Patent Document 8: Yasuda et al., Congenit. Anom. Vol. 47, pp 22-23 (2007)). This suggests that the development of embryos and placental primordia that express erythropoietin receptors depend on erythropoietin information. On the other hand, from the experiments of gene-deficient mice, the erythropoietin gene was found to develop myocardium (Non-patent Document 9: Wu et al., Development, Vol. 126, pp 3597-3605 (1999)) and brain development (Non-patent Document 10: Yu et al., Development, Vol. 129, pp 505-516 (2002)). Furthermore, it has been clarified that erythropoietin information is involved in myoblast proliferation but not myotube formation (Non-patent Document 11: Ogilvie et al., J. Biol. Chem., Vol. 275). , pp 39754-39761 (2000)), these suggest that erythropoietin is involved in the proliferation of smooth muscle cells.

  In addition, the present inventors expressed the erythropoietin gene in the human cervical canal, endometrium and ovary, and the erythropoietin receptor is a tissue composition of these female genital organs, that is, the cervical epithelium, vascular endothelial cells in the cervical matrix. Endometrial epithelial cells, uterine gland epithelial cells, decidual cells, ovarian embryonic epithelial cells, oocyte cytoplasm and follicular epithelial cells in primordial follicles, capillary endothelium intervening in inner follicular membrane cells of granulosa cells It was found to be expressed (Non-patent Document 12: Yasuda et al., Ital. J. Anat. Embryol., Vol. 106 (supplement 2) pp 215-222 (2001)). In other words, these erythropoietin receptor expression sites in female genital organs are involved in erythropoietin signaling, but the function itself has not been clarified.

When the erythropoietin gene becomes hypoxic, its gene expression is enhanced by a hypoxia-inducing factor (HIF-1α). As a result, it has been clarified that the number of red blood cells in the circulating blood increases and the hypoxic state is improved (Non-patent Document 13: Semenza GL and Wang GU, Mol. Cell. Biol., Vol. 12, pp 5447-5454 (1992); Non-Patent Document 14: Bunn HF and Poyton RO, Physiol. Rev., Vol. 76 (1996)). However, it has been clarified that the erythropoietin gene expressed in the endometrium is not enhanced by HIF-1α but is estrogen-dependent, and the erythropoietin protein is also secreted estrogen-dependent (Non-patent Document). 15: Yasuda et al., J. Biol. Chem., Vol. 273, pp 25381-25387 (1998)).
In addition, the present inventors orally administered a daily dose of 0.02 mg / kg body weight of ethinyl estradiol (EE ₂ ) olive oil solution for pregnant mice from the 11th to the 18th gestation, and the pregnancy create a group without a group ovariectomized the F ₁ female mice were obtained from mice at 16 weeks of age, were housed until subsequent 32 weeks of age, it was observed 32 weeks of the uterus. Of the 5 cases of endometrium of ovariectomized mice, 1 showed uterine gland hyperplasia and 4 showed a filamentous uterus. The endometrium of non-ovariectomized mice showed adenomyosis in 3 out of 3 cases (Non-Patent Document 16: Yasuda et al., Am. J. Obstet. Gynecol., Vol. 130, pp 506-508). (1978)). This embryonic EE ₂ exposure induces hyperplasia of the uterus glands after maturation, egg摘後also prenatal EE ₂ exposure to S. tiger die-ol (E ₂₎ content is high in the adult mouse endometrium Suggest to trigger. In addition, testosterone production was significantly lower in the testes of adult male mice treated in the same manner, whereas the E ₂ content was significantly higher. It was found that the E ₂ content was high in a dose-effective manner with respect to the EE ₂ exposure dose during the embryonic period. In other words, it was suggested that exposure to embryonic EE ₂ enhances the amount of aromatase and that the amount of testosterone converted to E ₂ is large (Non-patent Document 17: Yasuda et al., Am. J. Obstet. Gynecol., Vol. 159, pp 1246-1250 (1988)). This is because adenomyosis induced in the mouse endometrium after sexual maturation by EE ₂ embryonic administration is higher than normal in the conversion of progesterone, androstendion, testosterone, etc. to E _{2 due} to the increased amount of aromatase Show the potential. Thus, E ₂ is enhanced the erythropoietin gene in endometrium, induces excessive secretion of erythropoietin. Therefore, it is considered that it acts on the erythropoietin receptor expression site in the intima, glandular epithelium, decidual cells, smooth muscle cells, vascular endothelial cells and the like to promote the proliferation of those sites. From these results, the growth of ectopic uterine gland epithelium, stromal cells, and smooth muscle cells in adenomyosis, high local expression of aromatase gene induces local E ₂ overproduction, thereby causing endometrial Increases secretion of erythropoietin gene and erythropoietin. It was speculated that this erythropoietin is involved in the proliferation of endometrial epithelium, matrix cells, smooth muscle cells, and vascular endothelial cells expressing the erythropoietin receptor in the endometrium. In fact, it has been reported that the erythropoietin content in the ascites of endometriosis patients is significantly higher than that of normal patients (Non-patent Document 18: Matsuzaki et al., Hum. Reprod., Vol. 18, pp 52 -156 (2003)). In recent years, administered after further maturation to 0.01 mg / kg body weight prenatal exposure mice EE ₂ (8 weeks old) _EE 2 0.01 mg / kg body weight twice a week up to 20 weeks, 28 weeks Observations of the uterus and ovaries at age revealed that the incidence of invasive adenomyosis and the appearance of adenocarcinoma-like changes were significantly higher than those in the fetal administration group compared to the fetal administration alone ( Non-patent document 19: Koike et al., Congenit. Anom., Vol. 53, pp 9-17 (2013)).

The feature of the present invention is not intended to inhibit the production / secretion of highly expressed aromatase or E ₂ , but blocks the local function of erythropoietin involved in E ₂ , thereby causing symptoms such as adenomyosis. There is to improve.
The present inventors have heretofore prevented erythropoietin antagonists such as erythropoietin antibody and erythropoietin receptor protein from growing in the mouse uterine decidua after implantation, the mouse embryo and the decidua surrounding it. It was shown that the phenomenon of death or growth arrest has a dose-effect relationship with the dose of erythropoietin antibody or erythropoietin receptor protein. We found that β-galactosidase antibody and denatured erythropoietin receptor protein given as controls did not show these phenomena (β-galactosidase antibody) or showed a statistically significantly lower frequency (denatured erythropoietin receptor). (Non-Patent Document 20: Yasuda et al., Congenit. Anom., Vol. 47, pp 23-23 (2007)). However, these erythropoietin antagonists are all proteins, and low molecular weight compounds such as peptides that express the growth inhibitory effects of proliferating uterine epithelial cells, matrix cells, vascular endothelial cells and smooth muscle cells by blocking erythropoietin signal Etc. are not known at all.

  Patent Document 1 (Japanese Patent Laid-Open No. 10-101574) by the present inventors discloses a therapeutic / ameliorating agent for proliferative organ disease containing an erythropoietin antagonist as an active ingredient. Patent Document 2 (Japanese Patent Laid-Open No. 2002-326958) discloses a prophylactic / therapeutic agent for hypertrophic scar, keloid or joint inflammatory disease containing an erythropoietin antagonist as an active ingredient. 2003-201352) discloses a prophylactic organ disease, chronic arthritic disease, hypertrophic scar or keloid preventive / therapeutic agent comprising erythropoietin mimetic peptide.

JP-A-10-101574 JP 2002-326958 A Japanese Patent Laid-Open No. 2003-201252

Krantz S.B., Blood, Vol. 77, pp 419-434 (1991)) Ihle J.N., Nature, Vol. 377, pp 591-594 (1995)) D’ Andrea A. D. et al., Cell, Vol. 57, pp 277-285 (1989)) Wu et al., Cell, Vol. 83, pp 59-67 (1995)) (Yasuda et al., Develop. Growth Differ., Vol. 35, pp 711-722 (1993)) Marti H. H. et al., Eur. J. Neu. Sci., Vol. 8, pp 666-676 (1996)) Conrad et al., FASEB. J., Vol. 10, pp 760-768 (1996)) Yasuda et al., Congenit. Anom. Vol. 47, pp 22-23 (2007)) Wu et al., Development, Vol. 126, pp 3597-3605 (1999)) Yu et al., Development, Vol. 129, pp 505-516 (2002)) Ogilvie et al., J. Biol. Chem., Vol. 275, pp 39754-39761 (2000)) Yasuda et al., Ital. J. Anat. Embryol., Vol. 106 (supplement 2) pp 215-222 (2001)) Semenza G. L. and Wang G. U., Mol. Cell. Biol., Vol. 12, pp 5447-5454 (1992) Bunn H. F. and Poyton R. O., Physiol. Rev., Vol. 76 (1996)) Yasuda et al., J. Biol. Chem., Vol. 273, pp 25381-25387 (1998)) Yasuda et al., Am. J. Obstet. Gynecol., Vol. 130, pp 506-508 (1978)) Yasuda et al., Am. J. Obstet. Gynecol., Vol. 159, pp 1246-1250 (1988)) Matsuzaki et al., Hum. Reprod., Vol. 18, pp 52-156 (2003)) Koike et al., Congenit. Anom., Vol. 53, pp 9-17 (2013) Yasuda et al., Congenit. Anom., Vol. 47, pp 23-23 (2007)

  An object of the present invention is to provide a therapeutic / ameliorating agent for female hormone-dependent proliferative diseases such as adenomyosis, endometriosis and prostatic hypertrophy, which contains a low molecular compound such as a peptide as an active ingredient. .

  As a result of intensive studies to solve the above problems, the present inventors have found that a low molecular weight peptide having an erythropoietin receptor antagonistic activity has an unexpectedly excellent proliferation tissue suppressing effect. As a result of further investigation based on these findings, the present invention has been completed.

That is, the present invention
(1) A female hormone-dependent proliferative disease treatment / amelioration agent comprising a low molecular weight peptide having an erythropoietin receptor antagonistic action,
(2) The agent according to (1) above, wherein the low molecular weight peptide having an erythropoietin receptor antagonistic action is an erythropoietin mimetic peptide (EMP),
(3) The agent according to (1) above, wherein the low molecular weight peptide having erythropoietin receptor antagonistic action is selected from erythropoietin mimetic peptides 6, 7, 9, 12, 22, 23, 24, 25, 33 and 39,
(4) The agent according to (1) above, wherein the low molecular peptide having erythropoietin receptor antagonistic activity is erythropoietin mimetic peptide 9 (EMP9), and (5) female hormone-dependent proliferative disease is adenomyosis, intima The agent according to any one of the above (1) to (4), which is symptom, uterine fibroid or prostatic hypertrophy.

  According to the present invention, there is provided a therapeutic / ameliorating agent for female hormone-dependent proliferative diseases comprising the low molecular peptide having an erythropoietin receptor antagonistic activity of the present invention, in particular, erythropoietin mimetic peptide, especially erythropoietin mimetic peptide 9. These diseases can be ameliorated or treated by administration directly or intravenously to the lesions of proliferative organ diseases such as adenomyosis, endometriosis, uterine fibroids, and prostatic hypertrophy, which are female hormone-dependent diseases. it can.

It is an electrophoretic diagram of the EpoR protein fraction in an adenomyosis tissue specimen. It is a histology of an EpoR expression cell (glandular epithelium) and its surrounding tissue. It is a tissue image of an EpoR expression cell (smooth muscle fiber) and its surrounding tissue. It is a tissue image which shows an estradiol receptor (EstR) alpha expression cell. It is a tissue image which shows an EstR (alpha) expression cell. It is a tissue image which shows an aromatase expression cell. It is a tissue image which shows an aromatase expression cell. It is a structure | tissue image which shows coexistence of EpoR and EstR (alpha). It is a structure | tissue image which shows coexistence of EpoR and EstR (alpha). It is a structure | tissue image which shows coexistence of Epo and aromatase. It is a structure | tissue image which shows coexistence of Epo and aromatase. It is a histology which shows co-expression of STAT5 and PSTAT5 in a glandular epithelium. It is a tissue image which shows co-expression of STAT5 and PSTAT5 in a smooth muscle cell. It is a graph which shows the density of the blood vessel in an adenomyosis tissue. It is a H * E dyeing | staining image of the sample structure | tissue which shows destruction of a glandular structure. It is a H * E dyeing | staining image of the sample structure | tissue which shows destruction of a glandular structure. It is a H * E dyeing | staining image of the sample structure | tissue in which the defect | deletion site | part of a smooth muscle tissue occurs frequently. It is a TdT reaction image of a specimen tissue showing apoptotic death of glandular epithelium and matrix cells. It is a TdT reaction image of a specimen tissue showing apoptotic death of smooth muscle cells. It is a TdT reaction image of the specimen tissue showing gland drop cell necrosis. It is a histological image regarding the localization of the vascular endothelial cell by EMP9 treatment. It is a histology regarding the loss of the vascular endothelial cell by EMP9 treatment. It is a graph which shows the destruction of the blood vessel by EMP9 of an adenomyosis tissue. EEMP9 treated adenomyoma is shown. The thing after the formazan reaction of the cross section of the tumor of FIG. 23 is shown. The cross section of the glandular muscle species treated with saline is the one after formazan reaction. Shown are untreated glandular muscle species after transplantation. It is a tissue image before and after transplantation of the transplant material. It is a histology of adenomyoma after transplantation. It is a histological image which shows the glandular change and smooth muscle rupture in EMP9-treated adenomyomas. It is a histological image which shows destruction of the gland by EMP9 treatment. It is a histology which shows adenomyoma in the raw water treatment by aromatase dyeing | staining. It is a histological image showing apoptotic death of glandular epithelial cells by EMP9 treatment. The histology of the distribution of anti-FVIII positive microvessels is shown. It is a graph which shows the vascular collapse (ex vivo) of the adenomyoma by EMP9 treatment. It is an electrophoretic diagram which shows the expression of STAT5-tyrosine phosphate in an adenomyosis specimen. It is a histological image showing STAT5 and P-STAT5 by adenomyosis specimen in vitro EMP9 treatment. It is a histological image which shows STAT5 and P-STAT5 by adenomyosis specimen in vitro phosphate saline buffer treatment. It is a histological image which shows adenomyoma of EMP9 ex vivo treatment. It is a histology which shows adenomyoma of the raw water ex vivo treatment. It is a histological image which shows adenomyoma of EMP9 ex vivo treatment. It is an enlarged image of the glandular epithelial thin part of FIG. 38a. It is a histology of adenomyomas treated with saline. It is a strong expansion organization image of the arrow part of Drawing 38c. It is a histological image which shows the localization of EpoR and EstR (alpha) which express in a prostatic hypertrophy (BPH) tissue. It is the tissue image of the sample which shows coexistence of EpoR and EstR (alpha) in the glandular epithelium of BPH. It is a tissue image which shows a proliferative smooth muscle cell. It is an electrophoretic diagram which shows the expression of EpoR protein in a BPH sample. It is a histology which shows the histopathological finding of BPH by EMP9 treatment. It is a tissue image which shows destruction of the BPH tissue vascular network by EMP9 treatment. It is a graph which shows the change of the blood vessel density from which the blood vessel diameter differs in the EMP9 treatment tissue in a BPH tissue.

  The low molecular weight peptide having an erythropoietin receptor antagonistic activity used in the prophylactic / therapeutic agent for female hormone-dependent proliferative diseases of the present invention (hereinafter abbreviated as “the agent of the present invention”) is similar to the partial sequence of erythropoietin. A peptide having an amino acid sequence, such as a low molecular weight peptide consisting of at least 5 or more, preferably about 5 to 30, particularly preferably about 15 to 25 amino acid residues, is used. Among them, erythropoietin mimetic peptide (EMP) described in Biochemistry (Biochemistry 37, 3699, 1998), particularly EMP6, EMP7, EMP9, EMP12, EMP22, EMP23, EMP24, EMP25, EMP33, EMP39 are preferable. Of these, EMP9 is particularly preferable.

In SEQ ID NO: 1, the amino acid of EMP9 represents the sequence. As the peptide used in the agent of the present invention, a peptide containing an amino acid sequence substantially identical to the amino acid sequence is preferably used.
The peptide containing the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 is about 70% or more, preferably about 80% or more, more preferably about amino acid sequence represented by SEQ ID NO: 1. Examples thereof include peptides having an amino acid sequence having 90% or more homology, most preferably about 95% or more.
Furthermore, for example, one or more (preferably about 1 to 7, more preferably about 1 to 5, more preferably 1 to 3) amino acids in the amino acid sequence represented by SEQ ID NO: 1 A deleted amino acid sequence, one or more amino acids (preferably about 1 to 7, more preferably about 1 to 5, more preferably 1 to 3) in the amino acid sequence represented by SEQ ID NO: 1 1 or 2 or more in the amino acid sequence represented by SEQ ID NO: 1 (preferably about 1 to 7, more preferably about 1 to 5, even more preferably 1 to 3) Peptides containing an amino acid sequence in which amino acids are substituted with other amino acids, or an amino acid sequence combining them are also used.

In the sequence of SEQ ID NO: 1, the left end is the N-terminus (amino terminus) and the right end is the C-terminus (carboxyl terminus) according to the convention of peptide notation. The low molecular weight peptide having an erythropoietin receptor antagonistic action including the low molecular weight peptide having the amino acid sequence represented by SEQ ID NO: 1 is usually a carboxyl group (—COOH) or a carboxylate (—COO ⁻ ) at the C-terminus. However, the C-terminus may be an amide (—CONH ₂ ) or an ester (—COOR).
Here, as R in the ester, for example, a C _1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl or n-butyl, for example, a C _3-8 cycloalkyl group such as cyclopentyl, cyclohexyl, etc. C _6-12 aryl groups such as phenyl and α-naphthyl, for example, C _6-12 aryl C _1-2 alkyl groups such as benzyl, phenethyl and α-naphthylmethyl, and pivaloyloxymethyl ester It is done.
When the low molecular weight peptide having an erythropoietin receptor antagonistic activity has a carboxyl group (or carboxylate) other than the C-terminus, the low molecular weight peptide of the present invention includes those in which the carboxyl group is amidated or esterified. It is. As the ester in this case, for example, the above-mentioned C-terminal ester or the like is used.
Furthermore, for low molecular peptides having erythropoietin receptor antagonistic activity, for example, OH, COOH, NH ₂ , SH, etc. on the side chain of amino acids in the molecule are suitable protecting groups (for example, formyl group, acetyl group). those protected by C _1-6 an acyl group) such as also includes conjugated peptides such as glycopeptides which the sugar chains bound.

SEQ ID NO: 1
Gly Gly Thr Tyr Ser Cys His Phe Ala Pro Leu Thr Trp Val Cys Lys
1 5 10 15
Pro Gln Gly Gly
20

A small molecule peptide having an erythropoietin receptor antagonistic activity is a cell of a mammal (eg, guinea pig, rat, mouse, chicken, rabbit, dog, cat, pig, sheep, cow, monkey, human, etc.) [eg, ectopic growth Endometrial gland epithelium, proliferative intimal matrix cells, proliferative smooth muscle cells, intervening vascular endothelial cells, etc.] or cells derived from blood cells or cultured cell lines thereof, synthetic peptides, recombinant peptides Either may be sufficient.
Moreover, it can also manufacture according to a peptide synthesis method, and can also manufacture by culture | cultivating the transformant containing DNA which codes the said low molecular weight peptide.

  As a method for producing from a mammalian cell or tissue, for example, in the case of producing from a human or mammalian tissue or cell, the human or mammalian tissue or cell is homogenized and then extracted with an acid or the like. Can be isolated and purified by combining chromatography such as reverse phase chromatography and ion exchange chromatography.

As a method for producing according to the peptide synthesis method, for example, the low molecular weight peptide contained in the agent of the present invention can be produced according to a peptide synthesis method known per se, or by cleaving erythropoietin with an appropriate peptidase. it can.
As a peptide synthesis method, for example, either a solid phase synthesis method or a liquid phase synthesis method may be used. That is, the partial peptide or amino acid that can constitute the low molecular peptide contained in the agent of the present invention is condensed with the remaining part, and when the product has a protective group, the target low molecule is removed by removing the protective group. Peptides can be produced. Examples of known condensation methods and protecting group elimination include the methods described in the following documents.
M. Bodanszky and MAOndetti, Peptide Synthesis, Interscience Publishers, New York (1966)
Schroeder and Luebke, The Peptide, Academic Press, New York (1965)
Nobuo Izumiya et al., Basics and Experiments of Peptide Synthesis, Maruzen Co., Ltd. (1975)
Haruaki Yajima and Shunpei Sugawara, Biochemistry Experiment Course 1, Protein Chemistry IV, 205,
(1977)
Supervised by Haruaki Yajima, Development of follow-up medicines Volume 14 Peptide synthesis Hirokawa Shoten

More specifically, commercially available resins for peptide synthesis can be used for the synthesis of low molecular peptides contained in the agent of the present invention. Examples of such resins include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylphenyl. Examples include acetamidomethyl resin, polyacrylamide resin, 4- (2 ′, 4′-dimethoxyphenylhydroxymethyl) phenoxy resin, 4- (2 ′, 4′-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin, and the like. Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin according to various known condensation methods according to the sequence of the target low molecular peptide. At the end of the reaction, the low molecular weight peptide is cut out from the resin, and at the same time, various protecting groups are removed, and further an intramolecular disulfide bond forming reaction is performed in a highly diluted solution to obtain the target low molecular weight peptide or their amides.
For the above-mentioned condensation of protected amino acids, various activating reagents that can be used for peptide synthesis can be used, and carbodiimides are particularly preferable. As the carbodiimide, DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminopropyl) carbodiimide and the like are used. For activation by these, a protected amino acid is added directly to the resin together with a racemization inhibitor (for example, HOBt, HOBt), or the protected amino acid is activated in advance as a symmetric acid anhydride, HOBt ester or HOBt ester. After that, the resin can be added.

The solvent used for the activation of the protected amino acid and the condensation with the resin can be appropriately selected from solvents known to be usable for the peptide condensation reaction. For example, N, N-dimethylformamide, N, N-dimethylacetamide, acid amides such as N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, dimethyl sulfoxide, etc. Sulfoxides, ethers such as pyridine, dioxane and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, or appropriate mixtures thereof are used.
The reaction temperature is appropriately selected from a range known to be usable for peptide bond formation reaction, and is usually selected appropriately from a range of -20 ° C to 50 ° C. The activated amino acid derivative is usually used in an excess of 1.5 to 4 times. As a result of the test using the ninhydrin reaction, when the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. If sufficient condensation is not obtained even after repeating the reaction, the unreacted amino acid can be acetylated using acetic anhydride or acetylimidazole.

Examples of the protective group for the amino group of the raw material include Z, Boc, tert-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, C1-Z, Br-Z, adamantyloxycarbonyl, and trifluoroacetyl. , Phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like.
The carboxyl group is, for example, alkyl esterified (eg, linear, branched or cyclic alkyl ester such as methyl, ethyl, propyl, butyl, tert-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc. ), Aralkyl esterification, (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenanthyl esterification, benzyloxycarbonylhydrazide, tert- It can be protected by butoxycarbonyl hydrazide, trityl hydrazide and the like.
The hydroxyl group of serine can be protected, for example, by esterification or etherification. Examples of groups suitable for esterification include groups derived from carbonic acid such as lower alkanoyl groups such as acetyl groups, aroyl groups such as benzoyl groups, benzyloxycarbonyl groups, and ethoxycarbonyl groups. Examples of groups suitable for etherification include benzyl group, tetrahydropyranyl group, and tert-butyl group.
Examples of the protecting group for the phenolic hydroxyl group of tyrosine include Bzl, Cl ₂ -Bzl, 2-nitrobenzyl, Br-Z, tert-butyl and the like.
Examples of the protecting group for imidazole of histidine include Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc, and the like.
Examples of the activated carboxyl group of the raw material include a corresponding acid anhydride, azide, active ester [alcohol (eg, pentacrophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, And esters thereof with cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, HOBt) and the like. As the activated amino group of the raw material, for example, a corresponding phosphoric acid amide is used.

Examples of the method for removing (eliminating) the protecting group include catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd black or Pd-carbon, and anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethane. Acid treatment with sulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, and the like are also used. The elimination reaction by the acid treatment is generally performed at a temperature of about −20 ° C. to 40 ° C. In the acid treatment, for example, anisole, phenol, thioanisole, metacresol, paracresol, dimethyl sulfide, 1,4 -Addition of a chaotic supplement such as butanedithiol, 1,2-ethanediotil, etc. is effective. The 2,4-dinitrophenyl group used as the imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan is the above 1,2-ethanedithiol, 1,4-butanedithiol. In addition to the deprotection by acid treatment in the presence of the above, it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia and the like.
The protection of the functional group that should not be involved in the reaction of the raw material, the protection group, the removal of the protective group, the activation of the functional group involved in the reaction, etc. can be appropriately selected from known groups or known means.

As another method for obtaining the target low molecular peptide amide, for example, first, the α-carboxyl group of the carboxyl terminal amino acid is amidated and protected, and then the peptide chain is extended to the amino chain side to the desired chain length. Thereafter, a partial peptide except for only the protecting group of the α-amino group at the N-terminal of the peptide chain and a partial peptide from which only the protecting group for the carboxyl group of the C-terminal was removed were prepared. The condensation is performed in such a mixed solvent. The details of the condensation reaction are the same as described above. After purifying the protected peptide obtained by the condensation, all the protecting groups are removed by the above method, and the desired crude low molecular weight peptide can be obtained. This crude molecular peptide can be purified by using various known purification means, and the main fraction can be freeze-dried to obtain an amide of a desired low molecular peptide.
In order to obtain an ester form of a low molecular weight peptide, for example, the α-carboxyl group of the carboxyl terminal amino acid is condensed with a desired alcohol to form an amino acid ester, and then the desired low molecular weight peptide is obtained in the same manner as the low molecular peptide amide form. An ester of a peptide can be obtained.
Further, after the reaction, the desired low molecular weight peptide can be isolated and purified by combining ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization and the like. When the low-molecular-weight peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method. Conversely, when it is obtained by a salt, it is converted to a free form by a known method. be able to.
More specifically, the low molecular weight peptide contained in the agent of the present invention is produced by the method described in Biochemistry 37, 3699, 1998.

  Since the low molecular weight peptide contained in the agent of the present invention has a tumor growth inhibitory action and the like, the agent of the present invention can be used in mammals (for example, mice, rats, hamsters, rabbits, cats, dogs, cows, Sheep, monkeys, humans, etc.) are useful as prophylactic / therapeutic agents for adenomyosis, endometriosis, uterine fibroids and prostatic hypertrophy.

The low molecular weight peptide contained in the agent of the present invention has low toxicity and can be used as it is or mixed with a pharmacologically acceptable carrier according to a method known per se generally used in the production of pharmaceutical preparations. , For example, pharmaceutical preparations such as tablets (including sugar-coated tablets and film-coated tablets), powders, granules, capsules (including soft capsules), liquids, injections, suppositories, sustained-release preparations, orally or parenterally (Eg, topical, rectal, intravenous, ophthalmic administration, etc.) can be safely administered. In particular, dosage forms that can be administered directly or intravenously to the lesions of proliferative organ diseases such as adenomyosis, endometriosis, uterine fibroids, and benign prostatic hyperplasia, which are female hormone-dependent diseases, are preferred.
Examples of the pharmacologically acceptable carrier that may be used for the production of the agent of the present invention include various organic or inorganic carrier substances that are commonly used as pharmaceutical materials. For example, excipients, lubricants in solid formulations, Examples include binders and disintegrants, solvents in liquid preparations, solubilizers, suspending agents, isotonic agents, buffers and soothing agents. Further, if necessary, additives such as conventional preservatives, antioxidants, colorants, sweeteners, adsorbents, wetting agents and the like can be used in appropriate amounts.
Examples of the excipient include lactose, sucrose, D-mannitol, starch, corn starch, crystalline cellulose, light anhydrous silicic acid and the like.
Examples of the lubricant include magnesium stearate, calcium stearate, talc, colloidal silica and the like.
Examples of the binder include crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, starch, sucrose, gelatin, methylcellulose, sodium carboxymethylcellulose and the like.
Examples of the disintegrant include starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, carboxymethyl starch sodium, L-hydroxypropyl cellulose, and the like.
Examples of the solvent include water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil, olive oil and the like.
Examples of the solubilizer include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like.
Examples of the suspending agent include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glyceryl monostearate; for example, polyvinyl alcohol, polyvinylpyrrolidone, Examples thereof include hydrophilic polymers such as sodium carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, and hydroxypropyl cellulose.
Examples of the isotonic agent include glucose, D-sorbitol, sodium chloride, glycerin, D-mannitol and the like.
Examples of the buffer include buffer solutions of phosphate, acetate, carbonate, citrate and the like.
Examples of soothing agents include benzyl alcohol.
Examples of the preservative include parahydroxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.
Examples of the antioxidant include sulfite, ascorbic acid, α-tocopherol and the like.

In the agent of the present invention, the content of the low molecular peptide varies depending on the form of the preparation, but is usually about 0.1 to 100% by weight, preferably about 10 to 99.9% by weight, based on the whole preparation, Preferably, it is about 20 to 99% by weight.
In the agent of the present invention, the content of components other than the low molecular weight peptide varies depending on the form of the preparation, but is usually about 10 to 99.9% by weight, preferably about 20 to 90% by weight, based on the whole preparation. It is.
The dose of the agent of the present invention varies depending on the type of low molecular peptide, administration route, symptoms, patient age, etc., for example, when administered parenterally such as intravenous injection for the purpose of treating adenomyosis, About 0.005 to 50 mg, preferably about 0.05 to 10 mg, more preferably about 0.2 to 4 mg as low molecular weight peptide per kg body weight per day can be divided into 1 to 3 doses.

In addition to the low molecular weight peptide, the agent of the present invention can be used in combination with an appropriate amount of another drug or in combination with an appropriate amount.
Examples of such concomitant drugs include various hormone preparations that can be used for treatment of female hormone-dependent growth. Specifically, pharmaceuticals having a low immunosuppressive action, such as GnRH agonists, anti-estrogen preparations and aromatase inhibitors, secretory therapeutic agents (LH-RH agonists and antagonists, sex hormone antagonists, sex hormone synthesis inhibitors, etc.), etc. It is done.
In addition, autonomic nerve blockers such as phentolamine, which is an α ₁ -blocker, and atropine preparations can be used in combination with the agent of the present invention.

In the combined use of the agent of the present invention and the concomitant drug, the administration timing of the agent of the present invention and the concomitant drug is not limited, and the agent of the present invention and the concomitant drug may be administered simultaneously to the administration subject. Alternatively, administration may be performed with a time difference. The dose of the concomitant drug may be determined according to the dose used clinically, and can be appropriately selected depending on the administration subject, administration route, disease, combination and the like.
The administration mode of the agent of the present invention and the concomitant drug is not particularly limited as long as the agent of the present invention and the concomitant drug are combined at the time of administration. Examples of such administration forms include, for example, (1) simultaneous administration of two preparations obtained by separately formulating the agent of the present invention and a concomitant drug by the same administration route, and (2) the agent of the present invention. Administration of two kinds of preparations obtained by separately formulating a concomitant drug with a time difference in the same administration route, (3) Two kinds of preparations obtained by separately formulating the agent of the present invention and a concomitant drug Administration of the preparation at different time intervals in different administration routes (for example, administration in the order of the agent of the present invention → concomitant drug, or administration in the reverse order) and the like. Hereinafter, these administration forms are collectively abbreviated as the combination agent of the present invention.

The concomitant drug of the present invention has low toxicity. For example, the agent of the present invention or (and) the above concomitant drug can be mixed with a pharmacologically acceptable carrier according to a method known per se to obtain a pharmaceutical composition. it can.
As the pharmacologically acceptable carrier that may be used in the production of the concomitant drug of the present invention, the same carriers as those used in the pharmaceutical composition of the present invention described above can be used.
When the agent of the present invention and the concomitant drug are simultaneously formulated and used as a single agent, the content of the erythropoietin receptor antagonist or prodrug thereof in the concomitant drug of the present invention varies depending on the form of the preparation, The amount is about 0.1 to 100% by weight, preferably about 10 to 99.9% by weight, and more preferably about 20 to 90% by weight.
The content of the concomitant drug in the concomitant drug of the present invention varies depending on the form of the preparation, but is usually about 0.1 to 100% by weight, preferably about 10 to 99.9% by weight, based on the whole preparation, More preferably, it is about 20 to 90% by weight.
In the concomitant drug of the present invention, the content of components other than the erythropoietin receptor antagonist or its prodrug and concomitant drug varies depending on the form of the preparation, but is usually about 10 to 99.9% by weight based on the whole preparation. The amount is preferably about 20 to 90% by weight.
The compounding ratio of the erythropoietin receptor antagonist or the concomitant drug thereof in the concomitant drug of the present invention can be appropriately selected depending on the administration subject, administration route, disease and the like.
The dose of the concomitant drug of the present invention varies depending on the type of erythropoietin receptor antagonist or prodrug and concomitant drug, administration route, symptom, patient age, etc. When administered orally, about 0.005 to 50 mg, preferably about 0.05 to 10 mg, more preferably about 0.2 to 4 mg as an erythropoietin receptor antagonist or a prodrug thereof and a concomitant drug per kg body weight per day. Can be divided into 1 to 3 divided doses.

The same content may be used when the erythropoietin receptor antagonist and the concomitant drug contained in the agent of the present invention are formulated separately.
When the erythropoietin receptor antagonist and the concomitant drug contained in the agent of the present invention are separately formulated and administered together, the agent of the present invention and the pharmaceutical composition containing the concomitant drug are administered at the same time. However, after the pharmaceutical composition containing the concomitant drug is administered first, the agent of the present invention may be administered, or the pharmaceutical agent of the present invention is administered first, and then the concomitant drug is contained. The product may be administered. When administered at a time difference, the time difference varies depending on the active ingredient, dosage form, and administration method to be administered. For example, when a pharmaceutical composition containing a concomitant drug is administered first, a pharmaceutical composition containing the concomitant drug is selected. Examples thereof include a method of administering the agent of the present invention within 1 minute to 3 days after administration, preferably within 10 minutes to 1 day, more preferably within 15 minutes to 1 hour. When the agent of the present invention is administered first, the agent of the present invention is administered within 1 minute to 1 day, preferably within 10 minutes to 6 hours, more preferably from 15 minutes to 1 hour after the administration of the agent of the present invention. The method of doing is mentioned.

In the present specification and drawings, when bases and amino acids are represented by abbreviations, they are based on abbreviations by IUPAC-IUB Commission on Biochemical Nomenclature or conventional abbreviations in the field, and examples thereof are described below. In addition, when there is an optical isomer with respect to an amino acid, the L form is shown unless otherwise specified.
DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid Gly: glycine Ala: alanine Leu: leucine Val: valine Pro: proline Ser: serine Cys: cysteine Thr: threonine Tyr: tyrosine His: histidine Trp lysine Trp: tryptophan Gl : Phenylalanine Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

Effect of EMP9 on adenomyosis 1) Sample collection / treatment and search method a) Collection A resected tissue piece from a patient who had given consent by sufficient explanation. Immediately after excision, the tissue piece was transferred to a D-MEM culture medium (Sigma) mixed with streptomycin-penicillin and stored in a cool dark place (8 ° C.) until the start of the experiment. The number of specimens was 15 cases. When lesions and non-lesions were obtained, both were sampled. When non-lesions could not be obtained, only lesions were sampled. Untreated and in vitro treated tissue pieces and ex vivo adenomyomas (tumors from transplantation of adenomyosis tissue specimens into nude mice, hereinafter referred to as adenomyomas) were each washed with cold culture and then frozen with liquid nitrogen Or fixed with Zamboni fixative. The frozen material was subjected to biochemical search, and the fixed material was subjected to histopathological and immunohistochemical search.

b) Drugs EMP1 and EMP9 among erythropoietin mimetic peptides synthesized as peptides having erythrocyte proliferation activity similar to erythropoietin were commissioned to the peptide research institute (Mino, Osaka). EMP1 has an action similar to that of erythropoietin, and EMP9 has an erythropoietin receptor antagonistic action (Johnson DL et al., Biochemistry, Vol. 37, pp3699-3710 (1998)).

c) Treatment of adenomyosis specimen (Epo-EpoR information blocking) method
i) In vitro experiment 10 mg, 5 mg or 1 mg / ml of EMP9 treated under organ culture was dissolved in 0.025 M phosphate buffer and directly injected into the specimen. Furthermore, in order to induce erythropoietin-like action, 0.06 mg / ml of EMP1 equivalent to 10 mg / ml of EMP9 was used (Johnson DL et al., Biochemistry, Vol. 37, pp3699-3710 (1998)). As a control, a phosphate buffer was used. Using specimens from 4 cases (34, 47, 42, 48 years old), specimens from 1 to 24 hours after excision were cut to a size of 50 to 80 mg, and the drug was placed in a petri dish at 0.5 μl / mg tissue weight. Were injected into different parts of the tissue piece with a needle (Hamilton 32G) and syringe. Three and four injections were performed every hour. Except for the time of injection, the reaction was carried out in a petri dish without adding a culture solution under the conditions of 37 ° C. and 5% CO ₂ . After the final injection, the cells were cultured in 24-well culture blood (Falcon, 3047) by the method of using the D-MEM culture solution. Four to six tissue pieces were used as one treatment.

ii) Ex vivo experiment Nude mice (BalbC nu / nu, CLEA) were purchased at 5 weeks of age and used for transplantation after 1 week. Post-operative specimen tissue pieces were finely cut with a single blade (feather razor) and scissors (1 mm to 3 mm) on a rubber plate and in a petri dish. The fine slices were mixed well, and a 0.1 to 0.3 ml amount made wet with the culture solution was transplanted under anesthesia to the subscapular subcutaneous site. The time required from the postoperative excision to the start of transplantation was 30 minutes to 24 hours. Three to twelve mice were used per case specimen. Nude mice were bred for each cage. One week after transplantation, the drug was injected into the tumor (IT) or intraperitoneally (IP) from the body surface of the mouse. A solution of EMP9 in 10, 5 or 1 mg / ml physiological saline (saline water, Otsuka Pharmaceutical) was administered. Raw water was used as a control. At 1 week after drug administration or 1 week or 2 weeks after the start of administration, an incision was made in the dorsal scapula of a nude mouse under deep anesthesia, and the tumor was removed after observation of the outer surface of the tumor. In order to determine the viability of the tumor under macro observation, a cross section with a thickness of 1 mm was prepared from a tumor derived from 9 out of 11 cases using a brain slicer (NeuroScience Idea, Osaka). Observations were made. Adenomyomas from 2 cases and formazan-treated sections were frozen or fixed with Zamboni's solution and used for biochemical and histopathological examinations. Specimens used for the transplantation were 11 cases, and the animals used were 29 IT, 36 IP, and 8 untreated animals, for a total of 73 animals.

d) Histopathological and immunohistological search methods Post-operative specimens fixed with Zamboni's solution and specimens treated with in vitro and ex vivo systems were cryoprotected with 25% sucrose phosphate buffer solution and then frozen. Then, continuous frozen sections of 6 to 7 μm were prepared. As for the section, five sections at 70 to 98 μm apart were attached to one slide to prepare a plurality of slides. One of them was used for hematoxylin and eosin (HE) staining, and the other was used for immunostaining. The following primary antibodies were used. Anti-N-terminal EpoR antibody (Yasuda et al., Develop. Growth Differ., Vol. 35, pp711-722 (1993)), anti-Epo antibody (R & D), anti-estrogen receptor (Estrogen receptor, EstR) -α antibody ( Santa Cruz), anti-aromatase (Aromatase, Arom) antibody (abcam), anti-factor VIII (FVIII) antibody (DAKO), anti-PCNA antibody (Santa Cruz), anti-STAT5 antibody and anti-P-STAT5a / b antibody (Santa Cruz), anti-F4 / 80 antibody (Serotec), and anti-CD163 antibody (Santa Cruz) were reacted with each secondary antibody, then developed with DAB reaction, and colored or fluorescent secondary antibody was used. The fluorescent light was emitted for observation.

e) Biochemical search method
i) Search of transcription by quantitative RT-PCR method i) Epo mRNA, b) EpoR mRNA, c) aromatase mRNA, d) smooth muscle-myosin mRNA (MYH-11 mRNA), e) estradiol receptor (Estradiol) -receptor) The expression level of α mRNA was calculated as a ratio of the expression level of 18S rRNA mRNA in each tissue specimen. Total RNA was extracted from each tissue piece using Trizol (Gibco BRL) solution, cDNA was prepared using AMV-reverse transcriptase, and Quantitative PCR Supermix-UDG (Invitrogen) was used with ABI PRISM 7700 (Applied Biosystems). The PCR reaction was performed using Primers and probes such as Epo, EpoR, aromatase, Sm-myosin (MYH 11), estradiol receptor-α, 18S rRNA, etc., were applied by Applied Biosystem's Assay-on Demand Gene Expression product and Taq Man Ribosomal RNA control reagent. Each standard curve was determined using a 10-fold diluted plasmid solution of each cDNA. In the PCR reaction, denaturation was performed at 50 ° C. for 2 minutes and 95 ° C. for 10 minutes, annealing was performed at 95 ° C. for 40 cycles for 15 minutes, and extension was performed at 60 ° C. for 1 minute. Each data was from at least 3 independent experiments.

ii) Demonstration of EpoR by Western blotting Frozen tissue pieces were crushed with Polytron, and protein fractions were extracted using a solubilization buffer by a known method (Yasuda et al., 2003). After separating the membrane component fraction, 100 μg of protein amount of the membrane fraction extracted from each specimen was subjected to electrophoresis by SDS-PAGE and transferred to a PVDV membrane. Using anti-EpoR antibody (C-20, Santa Cruz 0.1 μml) or anti-N-terminal EpoR antibody (1: 100; Yasuda et al., Develop. Growth Differ., Vol. 35, pp711-722 (1993)) The cells were immunoreacted and visualized using secondary antibody and chemiluminescence (Amersham Bioscience).

iii) Measurement of estrogen content Frozen tissue pieces were crushed with polytron using a 25 mM phosphate buffer, and estradiol 17β in the tissue supernatant was measured by a method using ECLIA (electrochemiluminescence immunoassay). The protein content of each supernatant was measured using a protein quantification kit (Bio Rad) with bovine albumin as a standard.

2) Results a) Expression of erythropoietin signaling and estrogen response systems in adenomyosis tissue
i) Expression of EpoR protein in adenomyosis specimen FIG. 1 shows an electrophoretogram of the protein fraction. The PC-3 cell line membrane fraction was shown as a positive control. As shown in FIG. 1, the presence of 65 kDa EpoR protein was observed in all test specimens. In the figure, alphabets indicate patient identification and numbers indicate age. PC-3 is a prostate cancer cell line (Yasuda et al., Carcinogenesis, 24; 1021-1029, 2003).

ii) EpoR, EstRα and aromatase expression sites Since Epo is a secreted protein, normal immunostaining does not necessarily prove only Epo protein, so it is proved as an Epo response site as a protein localization site of EpoR. did. The expression site of EpoR, the expression site of EstRα and aromatase, and the coexisting protein site (Merge) in each tissue are shown in FIGS.

FIG. 2a shows EpoR expressing cells (glandular epithelium) and surrounding tissues. Positive reactions are observed in glandular epithelial cells (large arrows), vascular endothelial cells (small arrows), and matrix cells (arrowheads). The scale bar indicates 50 μm.
FIG. 2b shows EpoR expressing cells (smooth muscle fibers) and surrounding tissues. Positive reactions are observed in smooth muscle cells (large arrows) and peripheral blood vessels (small arrows). The scale bar indicates 50 μm.
FIG. 3 shows EstRα-expressing cells. Positive reactions are observed in glandular epithelial cells (arrowheads), vascular endothelial cells (small arrows), and smooth muscle fibers (large arrows). The scale bar indicates 50 μm.
FIG. 4 shows EstRα-expressing cells. Positive reactions are observed in medial smooth muscle fibers (large arrows) and vascular endothelial cells (small arrows). The scale bar indicates 50 μm.
FIG. 5 shows aromatase expressing cells. Positive reactions are observed in glandular epithelial cells (large arrows), smooth muscle fibers (small arrows), matrix cells (arrowheads), and dropped cells (double arrows). The scale bar indicates 50 μm.
FIG. 6 shows aromatase expressing cells. Positive reactions are observed in smooth muscle fibers (large arrows) and matrix cells (arrowheads). The scale bar indicates 50 μm.
FIG. 7 shows the coexistence (Mr) of EpoR and EstRα. Remarkably expressed in glandular epithelial cells (arrows). The scale bar indicates 50 μm.
FIG. 8 shows the coexistence (Yk) of EpoR and EstRα. Remarkably expressed in glandular epithelial cells (arrows) and matrix cells (stars). The scale bar indicates 50 μm.
FIG. 9 shows the coexistence (Mr) of Epo and aromatase. Remarkably expressed in glandular epithelial cells (arrows). A scale bar shows 10 micrometers.
FIG. 10 shows the coexistence (Mr) of Epo and aromatase. Remarkably expressed in smooth muscle fibers (arrows) and glandular epithelial cells (double arrows). A scale bar shows 10 micrometers.
FIG. 11 shows co-expression (Iz) of STAT5 and PSTAT5 in glandular epithelium. Remarkably expressed in glandular epithelial cells (arrows). The scale bar indicates 50 μm.
FIG. 12 shows co-expression (Iz) of STAT5 and PSTAT5 in smooth muscle cells. Remarkably expressed in smooth muscle cells (arrows). The scale bar indicates 50 μm.

iii) Expression levels of various mRNAs in adenomyosis resection specimens Table 1 shows the expression levels of various mRNAs in adenomyosis.
Expression levels of Epo mRNA and Sm-myosin mRNA in adenomyosis specimens were significantly higher than those in normal intima (P <0.001). The expression level of aromatase mRNA was higher than that of normal intima, but no significant difference was observed. On the other hand, the expression level of EpoR mRNA was significantly lower in the adenomyosis than in the normal intima (P <0.001).

iv) E ₂ content in adenomyosis, myoma and normal endometrial specimens Table 2 shows the E ₂ content in endometrial lesions.
Tissue content of E ₂ in adenomyosis tissue is higher than with the tissues of fibroids and normal endometrium showed significant differences between normal endometrium (P <0.05).

v) Distribution of blood vessels in adenomyosis For the purpose of seeing the blood vessel distribution density in adenomyosis tissue specimens, the number of blood vessels in a certain area in the sample section was added to the periphery of the gland epithelium, the smooth muscle region and their totals in the graph of FIG. The number of blood vessels is shown. The blood vessel density was significantly different depending on cases. In 10 out of 12 cases, the glandular lesion area had more blood vessels than the smooth muscle area (p <0.05, P <0.01, P <0.001).

vi) Summary of the nature of adenomyosis In the case of adenomyosis, Epo mRNA expression was significantly higher than that of normal endometrial tissue, and it was revealed that EpoR protein was expressed in all cases. In addition, EstRα coexists in this EpoR expression local area, that is, the Epo response site, and further, an aromatase protein coexists. The content of E ₂ is also high. These facts indicate that testosterone and androstender ion-binding protein transported from the blood are converted into estrogens by local aromatase, and this estrogen enhances Epo production in situ, and the pathway of STAT5-PSTAT5 is mediated by EpoR. Information transmission is enhanced in glandular epithelial cells, matrix cells, and smooth muscle cells, indicating that the proliferation of these cells is induced.

b) Induction of proliferative tissue disruption by EMP9 treatment of adenomyosis b-1. in vitro experiments
i) Pathological changes in EMP9 treatment in vitro The pathological changes in adenomyosis tissue by direct injection of EMP9 are shown in FIGS.
FIG. 14 is an H · E-stained image of the Hr (EMP9 15.0) specimen tissue showing the destruction of the glandular structure. Glandular destruction (large arrows, degree of destruction +3), matrix cell necrosis (small arrows), matrix cell thawing (marked *) and smooth muscle cell tissue defect (nucleusless / death **) . The scale bar indicates 50 μm.
FIG. 15 is a H · E-stained image of the Hr (EMP9 7.5) specimen tissue showing the destruction of the glandular structure. Gland epithelial cells dropped into the glandular cavity (*), epithelial flattening and cystization (*), epithelial lost cysts (**), matrix cell necrosis (small arrow) and dilution Is recognized. The scale bar indicates 50 μm.
FIG. 16 is an H / E-stained image of a Mh (EMP9 20.0) specimen tissue in which defective portions of smooth muscle tissue are frequently generated. The missing part is indicated by an arrow. The scale bar indicates 50 μm.
FIG. 17 is a TdT reaction image of Hr (EMP9 15.0) specimen tissue showing apoptotic death of glandular epithelium and matrix cells. The arrow indicates a positive nucleus, the arrowhead indicates chromatin concentration, and the double arrow indicates a necrotic image. A scale bar shows 10 micrometers.
FIG. 18 is a TdT reaction image of Mh (EMP9 20.0) specimen tissue showing apoptotic death of smooth muscle cells. Arrows are nuclei that show a positive reaction. A scale bar shows 10 micrometers.
FIG. 19 is a TdT reaction image of Hr (buffer solution) specimen tissue showing glandular cell necrosis. Arrow indicates apoptotic death, double arrow indicates necrosis, and scale bar indicates 10 μm.

  That is, in adenomyosis tissue by direct injection of EMP9, destruction of glandular structure (FIGS. 14 and 15), anuclear image of smooth muscle cells, rupture, frequent occurrence of defective sites (FIG. 16), degeneration / degeneration of substrate cells Apoptotic death / necrosis (FIG. 17), rupture of vascular endothelial cells, and vascular medial smooth muscle cell degeneration were observed. These cell degeneration / death showed apoptotic death (FIGS. 17 and 18). The dropped glandular epithelial cells in the control group were not apoptotic death but necrotic, and apoptotic death was observed in the matrix cells (FIG. 19). Moreover, there is little cell death compared with a treatment group. Among these pathological findings, Table 3 summarizes the degree and frequency of the destruction of the gland and the smooth muscle in the glandular epithelial proliferating part.

The frequency of high glandular destruction was also observed in buffer, needle injection, and no treatment, but the difference between the apoptotic death and necrosis could not be revealed by this analysis. Therefore, the frequency of those with zero decay image was compared between the EMP9 treatment group and the control group.
As shown in Table 3, the frequency of normal glandular images was significantly lower in the EMP9 treatment group than in the control group (P <0.05, P <0.001). On the other hand, the frequency of normal images was significantly higher in the EMP1-treated group (P <0.001). As for the damage to smooth muscle cells, the area occupied by nucleated smooth muscle cells in a certain compartment is 20% or more significantly less in the EMP9 treatment group than in the buffer treatment (P <0.001, P <0.01). , P <0.05) was observed in 2 cases (Ig and Yh), and on the contrary, the EMP1-treated group was significantly more (P <0.001, P <0.01). The frequency of leukocyte infiltration in the anucleated smooth muscle region was significantly higher in the EMP9 treated group than in the buffer treated group (P <0.001, P <0.01, P <0.05). Found in cases (Ig, Yh, Mh). In only one case of anucleated smooth muscle cells, the EMP9 treated group was more frequent than the buffer treated group (P <0.01, P <0.05). It has been suggested that EMP9 disrupts the gland and kills smooth muscle cells, inhibits the growth of adenomyosis and destroys the whole.

ii) Blood vessel destruction by EMP9 treatment FIGS. 20 to 21 are tissue images showing blood vessel destruction by EMP9 treatment.
FIG. 20 is a histological image relating to the localization of vascular endothelial cells by EMP9 treatment, where a is the localization of the matrix cell part and b is the localization of the smooth muscle cell part. A scale bar shows 10 micrometers.
FIG. 21 is a histological image relating to the loss of vascular endothelial cells by EMP9 treatment, and arrows indicate blood vessels lacking vascular endothelial cells. A scale bar shows 10 micrometers.
FIG. 22 is a graph showing the destruction of blood vessels by EMP9 in adenomyosis tissue.

  That is, the number of blood vessels within a fixed area is more in the former gland periphery and smooth muscle part than in the former (FIG. 20), and in the EMP9 treatment group, endothelial cell destruction is observed (FIG. 21). The number of blood vessels was significantly reduced (FIG. 22). Total blood vessel count was significantly reduced in the EMP9 treated group compared to the control group (P <0.001). With the same concentration of EMP9 treatment, the higher the number of administrations, the significant decrease was shown (P <0.01, P <0.001). In addition, EMP1 treatment with an equal concentration of EMP9 had significantly more blood vessels than EMP9 (P <0.001). In all cases, the number of blood vessels in the glandular lesion area was significantly lower in the EMP9-treated group than in the control group (P <0.001). In 1 of 3 cases in the smooth muscle proliferative area, the EMP9 total amount 10 and 20 μg / mg tissue weight treatment group significantly decreased compared to the control group (P <0.001). From these results, it was confirmed that EMP9 induces vascular disruption of adenomyosis, and that the gland growth lesion has a higher disintegration effect than the smooth muscle proliferation region.

iii) Biochemical analysis of adenomyosis tissue by EMP9 treatment in vitro Table 4 shows changes (in vitro) in the expression levels of various mRNAs in EMP9 or EMP1-treated adenomyosis tissue specimens. The values in Table 4 show the expression levels of Epo mRNA, EpoR mRNA, aromatase mRNA and Sm-myosin mRNA in each treatment specimen as a ratio of the expression levels of the respective 18S-rRNA mRNA.

  The expression level of Epo mRNA was not treated in Hr, Yh, and Mh, but was not significantly different from PBS treatment, but decreased. In addition, EMP1 treatment showed a significant increase compared to EMP9 (P <0.05). The expression level of EpoR mRNA was not significantly different in the EMP9 treatment group compared to the buffer treatment group, but decreased in 3 cases and the same value in 1 case. Regarding the aromatase mRNA expression level, one case in the EMP9 treatment group showed a significant decrease compared to the needle insertion and buffer treatment groups (P <0.05, P <0.01). Two cases showed a decrease. The expression level of Sm-myosin mRNA was significantly lower in the EMP9 treatment group than in the buffer treatment group in 2 cases (Ig, Mh) (P <0.05, P <0.001), the other 2 cases Shows a significant decrease (Hr) (P <0.01) or a decrease (Yh) although there is no significant difference from the EMP9 low concentration treatment group. Regarding the expression levels of the four types of mRNA, it was recognized that the mRNAs of aromatase and Sm-myosin were significantly decreased by EMP9 treatment compared to the control group. Epo mRNA was reduced in all cases compared to controls. Epo mRNA is strongly expressed in proliferative gland lesions. Therefore, it is considered that the individual specimens did not show a significant difference because there was a possibility that there was a difference in the degree of mixing of the glandular lesion, that is, the tissue structure, in each case and in the same case.

Table 5 shows the content of E ₂ in adenomyosis tissue pieces by EMP9 treatment. As shown in Table 5, it showed a dose effective reduction of E ₂ content relative EMP9. Although there was no significant difference, it was lower than the control group. In addition, EMP1 treatment showed a higher value than the control.

iv) Summary of in vitro treatment From the findings shown in Tables 3 to 5 and FIGS. 14 to 22, the adenomyopathic tissue is caused by local infusion of EMP9, proliferative glandular epithelium, smooth muscle tissue destruction and vascular system destruction. It is speculated that the lesion will be destroyed. Furthermore, biochemical analysis findings support these pathologic results.

b-2. Ex vivo experiment i) In vivo induction of tissue destruction of adenomyoma by EMP9 Adenomyoma in nude mice FIGS. 23 to 26 show specimens of glandular muscle species of nude mice.
FIG. 23 is a Mr 4.0 treated adenomyoma, where the arrow is the branch tumor feeding artery from the axillary artery.
FIG. 24 shows the cross section of the tumor of FIG. 23 after formazan reaction, and the red part is positive and indicates survival.
FIG. 25 shows a cross section of an adenomyomatous treated with a Mr. saline solution after formazan reaction.
FIG. 26 shows an untreated adenomyoma after Mr transplantation. 15 days after the transplantation.

That is, transplanted nude mice were sacrificed under deep anesthesia, and tumors were observed after incision. Tumors as shown in FIG. 23 were observed in all hosts. A formazan-stained macroscopic specimen of a 1 mm section of the excised tumor was taken into the image, and as shown in FIGS. 24, 25 and 26, the area occupied by the red surviving part and the white non-surviving part on the image Calculated by entry and expressed as a percentage of the total area. The results are shown in Table 6 below.
The stained specimen was fixed with Zamboni's solution or frozen with liquid nitrogen and subjected to histopathological and biochemical analysis, respectively.

ii) Survival suppression of adenomyoma by intra-tumor injection or intraperitoneal injection of EMP9 Table 6 shows the observation results of survival suppression (macro observation) of nude mouse transplanted adenomyoma by EMP9 treatment. As is clear from Table 6, in 3 cases out of 4 groups administered with a high concentration of IP (4.0 mg / animal), the survival part of adenomyomas was significantly lower than in the saline administration group or the non-administration group. (P <0.05, P <0.01). In contrast, in the E ₂ administration group, the surviving part of adenomyoma was significantly higher than the surviving part in the EMP9 and saline administration group (P <0.01, P <0.001). The local enhancement of E _2, suggesting increased survival portion by local Epo induction. On the other hand, in the case of IT, the survival part showed a low value in the dose effect of EMP9 in all four cases, but no significant difference was observed. The surviving parts of 2 cases of adenomyoma in the saline administration group were higher than or equal to those in the EMP9 administration group. Two cases in the non-administration group showed higher values than the two cases in the EMP9 administration group.
It should be noted that because a mixture of fine sections was transplanted, a difference in the morphology of the tumor was caused by the difference in the mixing of proliferative gland lesions even in specimens of the same patient in the formation of adenomyoma. This is considered to be reflected in the size of the missing part. The tumor in the EMP9 treatment group does not necessarily have a large defect compared to the control group. The size of the defect is not clearly related to EMP9 treatment (Iz, Ss, Mg, Mr). However, defects in the E ₂ dose group is smaller than the untreated group. That is, it is considered that the viability enhancement by E ₂ strengthened the bond between the pieces of tissue.
In conclusion, it was revealed that EMP9 high concentration intraperitoneal treatment suppresses the survival of adenomyoma by a macro search method.

iii) Histopathological change of adenomyosis due to EMP9 treatment Although the transplanted material is not uniform because it is a minced tissue of the specimen, a tumor composed of glandular tissue and smooth muscle tissue, which is characteristic of adenomyosis, was obtained.
A specimen of the transplant material and the tissue image of the adenomyoma are shown in FIG. 27, and the tissue images after transplantation are shown in FIGS.
FIG. 27 shows a tissue image before transplantation, a is a tissue before transplantation, b and c are tissue images of adenomyoma, an arrow in a indicates a gland surrounded by substrate cells, and a double arrow indicates a smooth muscle. (Iz). In b, glandular bodies (arrows) and smooth muscle parts (double arrows) treated with saline are observed, and in c, glandular bodies (arrows) that are collapsed, anucleated, and defective parts (*) are observed. A scale bar shows 20 micrometers.
FIG. 28 is a tissue image showing the glandular epithelium (Tb) of the adenomyoma after transplantation, a is a growth image of the glandular epithelium (arrow), and b to d are EpoR positive, aromatase positive, and Sm-myosin positive smooth, respectively. It is an image showing the localization of muscle fibers and glandular bodies (*). A scale bar shows 20 micrometers.
FIG. 29 is a histological image showing glandular changes and smooth muscle rupture (Hrt) in EMP9-treated adenomyoma. a is a cyst (*) adjacent to a cyst showing degenerative degeneration having EpoR positive epithelial cells (b), and b is a fallen epithelial cell showing EpoR positivity and showing cysts. c indicates cystization (arrow) and cyst (*) observed in the control group. d is a smooth muscle region of the same specimen as a and b, and indicates the intrusion of a defect (*) and blood cells.
The scale bar indicates 20 μm.
FIG. 30 is a histological image showing glandular body collapse (Mur) by EMP9 treatment. a and b are images showing glandular bodies degenerated in EMP-treated adenomyoma. c and d are aromatase-stained images of each gland, and the disappearance of aromatase is observed in the epithelial disruption (small arrow). The scale bar indicates 20 μm.
FIG. 31 is a histological image showing adenomyomas (Mr) in a saline treatment with aromatase staining. a is an image showing glandular cyst formation (arrow), b is a strongly magnified image of a, and shows aromatase localization (small arrow). The scale bar indicates 20 μm.
FIG. 32 is a histological image showing apoptotic death (Mr) of glandular epithelial cells by EMP9 treatment. a is an image showing glandular epithelial cells (arrow, TdT reaction) showing apoptosis, and b is an image showing glandular epithelium (arrow, TdT reaction) not showing apoptotic death of the saline treatment. The scale bar indicates 20 μm.

That is, in the tissue before transplantation, the glandular body is a single-layered epithelium surrounded by matrix cells, and smooth muscle tissue is also present (FIG. 27a). In the case of adenomyomas, the saline treatment showed a mixed form of smooth muscle tissue surrounded by glandular epithelial proliferation and matrix cells (FIG. 27b). With EMP9 treatment, shedding of glandular epithelium and cell death were observed, and defects in surrounding tissues were observed (FIG. 27c). After transplantation, immunostaining of untreated adenomyoma (Fig. 28a) showed EpoR positive reaction (Fig. 28b) in glandular tissue and surrounding matrix cells, the glandular epithelium showed aromatase positive reaction (Fig. 28c), Sm- Localization of myosin positive smooth muscle fibers was observed (FIG. 28d).
GMP with glandular epithelial shedding by EMP9 treatment (FIGS. 29a, b) were observed to progress to degenerative degeneration (FIG. 29a), cystization (FIG. 29b) and cysts (FIG. 29a) at that shedding degree. . This change is similar to the image observed with in vitro treatment (FIG. 15). Furthermore, nucleation of smooth muscle sites, tissue defects, and mononuclear cell invasion (FIG. 29d) were observed. Cysticization (FIG. 29c) and cysts (FIG. 29c) were also observed in the saline treatment.
In epithelial cells showing degenerative degeneration by EMP9 treatment, aromatase-positive reactive cells disappeared (FIGS. 30a, b, c, d). The thin, flattened glandular epithelial cells of the cysts observed with the saline treatment (FIG. 31a) showed aromatase positivity (FIG. 31b). In such a case, the normal gland was used. In addition, the glandular epithelial cells that dropped were markedly apoptotic death by EMP9 treatment, but differed by saline treatment (FIGS. 32a and b). These histological findings examined in individual adenomyomas are summarized in Table 7.

  As shown in Table 7, the presence of normal adenoids in EMP9 treatment was less in all adenomyoma cases than in saline and untreated adenomyomas, and in 4 cases significantly (P <0) compared with saline treatment. .05, P <0.01, Ht, Tb, Kg, Mr). In contrast, these tumors have glandular cystization (P <0.001, P <0.01, Ss, Kg, Mr) and cyst formation (P <0.001, P <0.01, Ht, Ss, Kw) is significantly higher. Regressive glands were significantly more abundant with EMP9 treatment (P <0.01, Mg). On the other hand, removal of the anucleated smooth muscle region by invasion of host mononuclear cells (macrophages and neutrophils) was significantly more observed in the EMP9 treated group than in the saline group except for one case (Kw). (P <0.05, P <0.01, P <0.001, Ht, Iz, Ss, Mg, Kg, Mr). From these phenomena, it was shown that EMP9 treatment degenerates glands with Epo and aromatase activity and induces changes to cysts that lost these protein activities. This glandular cystization and smooth muscle loss is similar to the changes observed with EMP9 treatment in vitro. Ex vivo further promotes the removal of anucleated smooth muscle by host phagocytic cells and predicts the disappearance of adenomyoma.

iv) Vascular destruction of adenomyoma by EMP9 treatment The number of microvessels in adenomyoma was calculated.
FIG. 33 shows a tissue image of the distribution (Ht) of anti-FVIII positive microvessels. a is a blood vessel (arrow) in which the vascular endothelium of an adenomyoma disappeared by EMP9 treatment, and b is an image of a blood vessel (arrow) of adenomyoma by saline treatment. The scale bar is 20 μm.
FIG. 34 is a graph showing vascular disruption (ex vivo) of adenomyoma by EMP9 treatment.
As shown in FIGS. 33a and b, vascular endothelial cells in EMP9-treated adenomyomas collapse and do not show a positive reaction with anti-FVIII antibody (FIG. 33a), but many of the blood vessels show a positive reaction in the control saline treatment It was observed (Figure 33b). The number of blood vessels in each adenomyoma is as shown in FIG.
In all cases observed, the number of blood vessels in the EMP9 treated group was significantly reduced (P <0.001, P <0.01) compared to the number of untreated and saline treated adenomoma. In two cases, it was recognized that there was a dose-effect relationship of EMP9 to vascular collapse.
v) Biochemical analysis in adenomyoma by EMP9 treatment The change in the expression level of 4 types of mRNA was examined, and the results are summarized in Table 8.

  As shown in Table 8, with regard to Epo mRNA, the expression level of one example (Mg) was significantly reduced (P <0.001) compared to the saline treatment group, and the other three cases (Ht, Kw, Kg) On the contrary, it showed a significantly high value (P <0.01, P <0.001) or hardly changed. Regarding EpoR mRNA, there was a significant decrease (P <0.05) in 2 cases (Mg, Yk) compared to the saline treatment, whereas in one case (Dc), the saline treatment was significantly more significant. A low value was indicated (P <0.001). As for aromatase mRNA, the saline treatment showed significantly lower values (P <0.05, P <0.01) in 2 cases (Mr, Dc). Concerning Sm-myosin mRNA, 2 cases (Kg, Dc) were contrary to the saline treatment, except that one case (Mg) showed a significantly lower value (P <0.001) than the saline treatment. The value was significantly high (P <0.001). Excluding aromatase from these, 14.3 to 28.6% of the EMP9 treatment group showed a significant decrease in the respective mRNA expression levels. Moreover, considering that EpoR mRNA in adenomyosis specimens was significantly lower than normal endometrial EpoR mRNA (Table 1), it was suggested that EMP9 treatment induced a near normal state. The

vi) Summary of ex vivo treatment Adenomyoma, which was prepared by transplanting a small section of tissue material from a specimen of adenomyosis, formed a gland similar to adenomyosis and became a tumor with smooth muscle tissue. EMP9 treatment induces degenerative degeneration and cystization of the gland due to apoptotic death of glandular epithelial cells, and the gland becomes a cyst without EpoR or aromatase activity. In addition, smooth muscle cell anucleation occurred, and these tissues were cleared by host macrophages and neutrophils, resulting in pathological findings suggesting that absorption and disappearance of collapsed adenomas . Cysticization of the glandular body and changes to cysts were also observed in the direct treatment of EMP9 on adenomyosis specimens in vitro. Therefore, it is judged that EMP9 treatment has an effect of inducing the decay and healing of adenomyosis in the human body.

b-3. Disruption of lesions in vitro and ex vivo by blockade of Epo-EpoR signaling induced by EMP9 treatment i) Expression of STAT5-tyrosine phosphate in adenomyosis specimens demonstrates STAT5 tyrosine phosphorylation in five adenomyosis specimens Therefore, STAT5 immunoprecipitation and P-Tyr western blotting were performed.
FIG. 35 is an electrophoretogram showing the expression of STAT5-tyrosine phosphate in adenomyosis specimens.
As shown in FIG. 35, except for one case (Kg), a STAT5 protein band was expressed, and in one case (Dg), expression of tyrosine phosphate (P-Tyr) was observed. This result suggests that it is difficult to obtain a positive reaction because the proportion of glandular bodies contained in the specimens of the cases used is small or weakened during storage and the amount of information is very small, but it is difficult to obtain a positive reaction. (FIG. 1) and the event that STAT5 and P-STAT5 were co-expressed at the expression site (FIGS. 11 and 12) reveal that adenomyosis is under the control of Epo-EpoR signaling.

ii) Expression of STAT5-PSTAT5 in adenomyoma Adenomyoma was immunohistochemically searched because the size of each tumor could not be extracted biochemically enough to perform STAT5 immunoprecipitation.
36 to 38 show the resulting tissue images.
FIG. 36a is a histological image showing STAT5 and P-STAT5 by adenomyosis specimen in vitro EMP9 treatment (Ig, total amount 1.5 mg). In this treatment example, destruction of the glandular epithelium (arrow) and destruction of matrix cells (*) that do not exhibit co-expression are observed. The scale bar indicates 50 μm.
FIG. 36 b is a histological image showing STAT5 and P-STAT5 by adenomyosis specimen in vitro phosphate saline buffer treatment (Ig, PBS). In this treatment example, co-expression glandular epithelium (arrow) and matrix cells (double arrow) are observed. The scale bar indicates 50 μm.
FIG. 37a is a histological image showing adenomoma of EMP9 ex vivo treatment (IT), and P-STAT5 is not expressed or is lowly expressed in the destroyed part. In the case of EMP9 administration (SS, total amount 0.45 mg), the arrow indicates the glandular epithelial destruction, and the expression of P-STAT5 is hardly recognized. The scale bar indicates 50 μm.
FIG. 37b is a histology showing adenoma of the saline ex vivo treatment (IT) showing co-expression in glandular epithelium and matrix cells. In the case of saline administration (SS. Control), the arrow indicates the nucleus of the glandular epithelium showing co-expression. The scale bar indicates 50 μm.
FIG. 38a is a histology showing EMP9 ex vivo treatment (IP) adenomyomas, where the dilute glandular epithelium undergoing destruction shows no or low expression of P-STAT5. In EMP9 administration (Mg, 0.4 mg), glandular epithelial detachment (stars) and dilution (arrows) are observed. The scale bar indicates 50 μm.
FIG. 38b is an enlarged image of the thin portion of the glandular epithelium of FIG. 38a. Fig. 38a shows low expression of P-STAT5 in the strongly enlarged region of Fig. 38a. The scale bar indicates 50 μm.
FIG. 38c is a histology of a saline myocardial treatment (IP) adenomyoma. Gland bodies and smooth muscle fibers in adenomyoma showing co-expression of STAT5 and P-STAT5 are observed. In the case of saline administration (Mg, 0 control), the arrow indicates the gland. The scale bar indicates 50 μm.
FIG. 38d is a strongly enlarged tissue image of the arrow portion in FIG. 38c. Arrows indicate individual glandular cells. Co-expressed in the cytoplasm and nucleus. A scale bar shows 10 micrometers.
As shown in FIGS. 36a, b, 37a, b, and 38a to 38d, coexpression of STAT5 and P-STAT5 is observed in the EMP9-treated example at the site showing the collapse / degeneration of glandular epithelium / matrix cells in which EpoR expression was observed. Was not recognized.

As described above, the significance of EMP9 treatment experiments in adenomyosis specimens in vitro and ex vivo is summarized. EpoR is also functional in adenomyosis tissue and its transplanted tumor. The expression was evident in the tissue of proliferating cells.
Thus, EMP9 treatment leads to EpoR dysfunction in the lesion, ie, induces proliferating tissue to collapse by blocking STAT5 phosphorylation.

Effect of EMP9 on benign prostatic hyperplasia 1) Sample collection, treatment, and retrieval method a) Collection Transurethral resection of patients with benign prostate hypertrophy (BPH) who have obtained consent with sufficient explanation Among the tissue pieces excised with the prostate, TUR-P), the remaining ones used for pathological examination were used. The excised specimen was frozen immediately after the operation or transferred to a D-MEM culture solution (Sigma) mixed with streptomycin / penicillin and stored in a cool dark place (8 ° C.) until the start of the experiment. The number of specimens was 15 cases. Those stored frozen were used for biochemical searches, and those stored in culture were used for histological analysis after in vitro treatment.

b) Drug EMP9 manufactured by Peptide Institute was used.
c) Treatment
In vitro organ culture was used. The EMP9 phosphate saline buffer or a phosphate saline buffer as a control was directly injected into the sample mass with an injection needle (Hamilton 32G). The organ culture method is the same as in Example 1.
d) Search method The specimen lump of 12-17 hours after drug treatment was fixed with Zamboni's solution to prepare a tissue specimen. In normal hematoxylin and eosin stained specimens, the effect of EMP9 on smooth muscle and acinar was searched. On the other hand, the influence on the distribution blood vessel was searched by immunostaining specimens with anti-FVIII antibody.

2) Epo-EpoR information expression in BPH a) Histopathological characteristics FIG. 39 is a histological image showing the localization of EpoR and EstRα expressed in BPH tissue. a is an image obtained by staining a gland and smooth muscle with an anti-EpoR antibody, an arrow indicates a glandular epithelial cell, and an * mark indicates a smooth muscle cell. b is an image of a smooth muscle cell group (arrow) expressing EpoR, c is an image of Sm-myosin of a smooth muscle cell (the staining is shaded, arrow), and d is an EstRα in a smooth muscle cell. It is an image which shows (arrow). A scale bar shows 20 micrometers.
FIG. 40 is a histological image of a specimen (IU, 70) showing coexistence of EpoR and EstRα in the BPH glandular epithelium. EpoR and EstRα coexisting in the glandular epithelium. Arrows indicate glandular epithelial cells, double arrows indicate vascular endothelial cells, and scale bar indicates 50 μm.
FIG. 41 is a histological image showing proliferative smooth muscle cells, and the arrows are nuclei showing a PCNA positive reaction. A scale bar shows 20 micrometers.

  As shown in FIG. 39a, BPH is composed of an acinus having an epithelium showing an EpoR positive reaction and smooth muscle fibers mainly showing an EpoR positive reaction (FIG. 39b) in the periphery. Furthermore, smooth muscle fibers showed positive reaction to smooth muscle myosin antibody (FIG. 39c) and estradiol receptor α (EstRα) antibody (FIG. 39d). As shown in FIG. 40, EpoR and EstRα are co-expressed, and these glandular epithelium and smooth muscle cells are considered to be proliferated by erythropoietin and estradiol, and actually show positive reaction to proliferative antibody (PCNA). It was. FIG. 41 shows a PCNA positive reaction in the nuclei of smooth muscle fibers.

b) Expression of EpoR in BPH specimen tissue EpoR protein was searched by membrane fraction of the tissue extract of 4 cases, and β-actin (actin) was searched by protein fraction in the extract by Western blotting. The results are shown in FIG.
FIG. 42 is an electrophoretogram showing the expression of EpoR protein in a BPH sample.
Each lane used 20 μg of protein. In all 4 cases, there was a light and dark expression, but an EpoR protein band was observed.
c) Expression of various mRNAs in BPH specimen tissues Table 9 shows the expression levels of various mRNAs.
Compared with the expression level in adenomyosis specimen tissue (Table 1 in Example 1), aromatase and Sm-
The expression level of myosin mRNA is high.

d) Histopathological findings of BPH treated with EMP9 FIG. 43 is a histological image showing the histopathological findings of BPH treated with EMP9. a to c are smooth muscle portions, and e to h are acini. a is a HE image of a smooth muscle tissue treated with EMP9 (SS, 5.0 μg / tissue mg). b is an anti-EpoR antibody reaction image of a neighboring section of a, c is a TdT treatment result in the neighboring section of a, and d is a positive control using the same EpoR antibody. Expression of EpoR is observed on the surface of mouse erythroblasts (small arrow) and yolk sac epithelial cells (large arrow), the arrows indicate smooth muscle fiber rupture (a, b), smooth muscle cell TdT positive nuclei (c ), And * indicates an unstructured site. e is a HE image of a collapsed acinus in an EMP9 treated (IU, 5.0 μg / mg tissue) example. f is a TdT-treated image of an adjacent section of e, g is a TdT-treated image of a collapsed acinus treated with EMP9 (IU, 2.5 μg / tissue), and h is a collective image of a small acinus treated with PBS (IU, control). It is. As for the arrow, e and f point to the same dead cell. f, g, and h are TdT positive nuclei (g to h). a to c and e to h are the same magnification, and the scale bar represents 20 μm.

When treated with two doses of EMP9 (10 mg / ml and 5 mg / ml, 5.0 and 2.5 μg per mg tissue), smooth muscle fibers expressing EpoR become anucleated and fragmented, eventually becoming unstructured It has been replaced by a substance (FIGS. 43a, b, c). Smooth muscle fiber and nuclear fragmentation showed apoptotic death (FIG. 43c). In the acinar, EMP9 showed the destruction of the glandular structure and dispersion of the contents (FIGS. 5e, g, f), and the dropped epithelial cells showed apoptotic death (435g, f). Those treated with PBS showed no destruction of the glandular structure and a few glandular epithelia showing apoptotic death (FIG. 43h).
Table 10 shows BPH tissue disruption images in EMP9 treatment. This is a summary of the results of analysis of all H / E stained specimens.

Glandular dispersion (FIG. 43e) appeared significantly more frequently in 6 of 7 cases with high EMP9 treatment compared to the respective controls and in 5 of 7 cases with low concentration treatment (P <0.001, P <0.05). Among those that did not show glandular dispersion, those that showed glandular epithelial shedding were significantly more common in 2 cases (KN, IA) at high EMP9 concentrations and 2 cases (KN, Nkt) at low concentrations than the respective controls. (P <0.001).
The compartments with smooth muscle fibers and nucleated cells were significantly fewer than controls in 4 cases at high and low concentrations of EMP9 treatment (P <0.001, P <0.05). In contrast, the number of cells having cells showing no nuclei was significantly higher in the EMP9-treated high concentration in 4 cases and in the low concentration in 3 cases compared with the control (P <0.001, P <0.01, P <0. 05). The number of smooth muscle fibers that were anuclear and showed fragmentation was 4 in the high concentration of EMP9 treatment and 5 in the low concentration, significantly higher than the respective controls (P <0.001, P <0). .01, P <0.05). In addition, one of these five cases showed a dose-effect relationship with EMP9 fragmentation. The frequency of appearance of unstructured regions was significantly higher in the three high-concentration treatments than in the controls (P <0.001, P <0.01, P <0.05).

e) Destruction of BPH tissue vascular network by EMP9 treatment FIG. 44 is a histological image showing destruction of BPH tissue vascular network by EMP9 treatment. a is an image showing vascular distribution (anti-FVIII antibody) without BPH treatment (IU), b is an image showing an example of EMP9 treatment (IU, 5.0 μg / mg of tissue), and c is an example of PBS treatment (IU, control). In the image shown, the arrows indicate collapsed (b) or normal (c) vascular endothelial cells that show a positive reaction. * Indicates an acinar cavity. a to c are the same magnification, and the scale bar represents 20 μm.
As shown in FIG. 44a, blood vessels distributed in the BPH surround the acinus and have different diameters. In the EMP9 10 mg / ml treatment group, many vascular endothelial cell-damaged blood vessels were observed around the collapsed acinus (FIG. 44b), whereas the control had little endothelium damage (FIG. 44c).

Tissue specimens stained with anti-FVIII antibody were divided into three groups (a> 10 μm, b = 10−3 μm, c <3 μm) in diameter, and the number of distributions was a constant area (5.7 × 10 ⁻³ mm ² ). Was counted as one section, and 50 sections were counted per specimen. Their blood vessel density is shown in FIG.
FIG. 45 is a graph showing changes in blood vessel density with different blood vessel diameters in EMP9-treated tissue in BPH tissue.
In addition, FIG. 45 shows in Table 11 a list of significant differences in the cases in which the blood vessel decrease was significantly between the respective diameters or the total numbers as compared with the EMP9 treatment and the PBS treatment.

  As shown in Table 11, 3 cases out of 8 cases were significantly decreased by EMP9 treatment by EMP9 treatment (P <0.001, P <0.01, P <0. 05). Six of the 8 cases showed a significant difference between the control group in a and b or b and c. In the total number of blood vessels, the EMP9 treatment group showed a significant decrease compared to the control group at both high and low concentrations, or only at the high concentration, except for one case. One case that did not show a significant difference in the total number showed a significant decrease in the number of blood vessels of size a and b in the high concentration treatment group. Thus, EMP9 treatment was found to damage the vascular network of BPH.

As described above, the effects of EMP9 treatment on BPH tissue specimens are summarized. In BPH, expression of Epo mRNA, EpoR mRNA, aromatase mRNA, EstRα mRNA and Sm-myosin mRNA is observed. Expression of EpoR protein and EpoR and EstRα are expressed in glandular epithelium and It became clear that it coexists with smooth muscle. Particularly, since aromatase mRNA is high, BPH suggests that testosterone, dehydroepiandrosterone, etc. that are transported via blood may be converted into estrogen in situ. Therefore, the mechanism of BPH is observed in adenomyosis and endometrium (Yasuda et al., JBC, Vol. 273, pp 25381-25387 (1998)), and this estrogen induces erythropoietin secretion, It is thought that this is because the proliferation of glandular epithelial cells, smooth muscle fibers and vascular endothelial cells expressing EpoR is promoted.
Thus, direct tissue injection of EMP9 blocks EpoR-mediated information, leading to apoptotic death in acinar epithelial cells and smooth muscle cells, and together with the destruction of the microvascular network, can lead to BPH disruption It became clear that it has sex.

  As described above, according to the present invention, it is possible to provide a medicament useful for the treatment and improvement of female hormone-dependent proliferative diseases such as adenomyosis, endometriosis, uterine fibroids, and prostatic hypertrophy.

Claims (5)

A therapeutic / ameliorating agent for adenomyosis, endometriosis, uterine fibroids or prostatic hypertrophy, comprising erythropoietin mimetic peptide 9 . The agent according to claim 1, which is a therapeutic / ameliorating agent for adenomyosis . The agent according to claim 1, which is a therapeutic / ameliorating agent for endometriosis . The agent according to claim 1, which is a therapeutic / ameliorating agent for uterine fibroids . The agent according to claim 1, which is a therapeutic / ameliorating agent for benign prostatic hyperplasia .